The Role of TIMP3 in Models of Inflammation and Immunity

David Smookler March 2010

A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy

Graduate department of Laboratory Medicine and Pathobiology

University of Toronto

ABSTRACT

The Role of TIMP3 in Models of Inflammation and Immunity by David Smookler a PhD thesis for the Department of Laboratory Medicine and Pathobiology University of Toronto, 2010

The inter-relation between inflammation, the immune system and leukocytes is multifaceted, with communication between stroma and immune cells mediated by cytokines, growth factors, chemokines, integrins and other molecules. Proteolysis plays

an important role in regulating these molecules. Proteolytic cleavage can not only destroy

some molecules but can activate or shed others, converting local juxtacrine signalling

proteins into effectors that act at a distance. Shedding can also convert membrane-bound

receptors into soluble ligand-binding inhibitors. Finally, cleavage can convert agonist

molecules into antagonists. As a wide-ranging inhibitor of metalloproteinases, tissue

inhibitor of metalloproteinase 3 (TIMP3) has the potential to down-regulate many of

these activities. We explore the role of TIMP3 in the regulation of inflammation,

revealing that loss of TIMP3 leads to a more rapid increase of soluble TNF, higher levels

of soluble TNF receptors and ultimately to increased TNF signalling in systemic

inflammation. We also demonstrate TIMP3 loss impacts local inflammation. In addition

we investigate the importance of TIMP3 in the expansion of hematopoietic cells.

ACKNOWLEDGMENTS

I would like to thank Dr. Rama Khokha for her unwavering support over the years.

Actually there has been a bit of wavering, but overall she has been a lighthouse to me and

my thanks are deep and sincere. I would also like to thank Marco DiGrappa who by

virtue of his intelligence and unstinting helpfulness coupled with a humorous state of

pessimism and self-deprecation has taught me never to take myself too seriously, and to

feel I will always have a sympathetic ear when something goes wrong. I would like to

thank Hart Jackson, Alison Aiken and Sam Molyneux for the respect they pay me,

probably as an older statesman of the lab but I hope respect earned by what I share with

them. I’d like to thank Alex Beristain for reminding me that “older statesman” essentially means “old man”. I’d like to thank Adi Murthy for reminding me of the

scientific enthusiasm that can be brought to my field, and I’d like to thank Virginie

Defamie for the unconditional warmth with which I am greeted whenever I appear at the

lab. I’d like to thank Purna Joshi for being an example of dogged focus and perseverance, not afraid to take on Herculean projects while other demands are made upon her. I would like to thank Iris Fang for her patience and generous helpfulness. I’d like to thank Trevor McKee for watching out for the post-docs’ welfare as I soon hope to join their ranks. I thank Gordon Duncan, whose practical advice was instrumental to my work, and Bill Sukloff who’s instrument, this laptop, made writing up my work practical.

I would also like to thank former members of the Khokha lab Zam Kassiri and Geoff

Wood for their guidance and support. I thank Swami Narala for reminding me that gratitude is payment in a currency that costs little but is worth much. And Linda, who put up with it all, I thank you.

iii

TABLE OF CONTENTS

Chapter 1: INTRODUCTION

The Extracellular Matrix------1

Collagens ------2

Glycosaminoglycans and Proteoglycans ------3

ECM glycoproteins ------4

Proteases in the Mammalian Genome ------6

Matrix Metalloproteinases ------6

MMP structure------6

Types of MMPs------7

Roles of MMPs in normal physiology ------10

MMPs target growth factor signalling in several ways ------12

Regulation of MMPs ------13

ADAMTSs------14

ADAMs ------14

TACE------16

Metalloproteinases in inflammation------19

Tissue Inhibitors of Metalloproteinases ------20

TIMP Structure------21

TIMP3 ------21

Structural interaction of TIMP3 with TACE ------22

Inflammation------26

Systemic Inflammatory Response Syndrome (SIRS) ------29

Lipopolysaccharide (LPS)------30

Tumour Necrosis Factor (TNF)------31

Expression of TNF and TNF Receptors ------34

TNF Signal Transduction ------34

TNF and IL-6------39

Nitric Oxide------42

TIMP3 and Hematopoiesis ------42

MPs targeted by TIMP3 affect hematopoietic growth factor signalling------43

MPs are involved in the trafficking of hematopoietic cells ------46

TIMPs influence hematopoiesis via MPi and non-MPi mechanisms------46

Rationale for the Thesis------47

Chapter 2: MATERIALS AND METHODS

Mice ------49

Reagents------50

ELISAs ------50

Statistical Analysis ------51

Tissue preparation for Western blots and TACE assay ------51

Protein Quantification------52

TACE assay------52

Western blot antibodies ------53

Quantitative PCR ------54

Alanine aminotransferase (ALT) analysis------55

Synthetic metalloproteinase inhibitors------56

Heart effluent------56

Nitric oxide analysis------56

Peripheral blood analysis ------57

Skin challenge ------57

Memory cytotoxic lymphocyte analysis ------57

Hematopoiesis ------58

Reproducibility and variation of results ------61

Chapter 3: TIMP3 Regulates TNF-Dependent Systemic Inflammation

ABSTRACT ------63

INTRODUCTION------64

RESULTS ------66

-/- Inactivated bacteria induces greater TNF release in Timp3 mice ------66

Timp3-/- mice are highly sensitive to LPS-induced septic shock ------71

-/- p55 pathway is required for heightened inflammation in Timp3 mice ------73

-/- -/- Timp4 mice do not share the sensitivity to LPS found in Timp3 mice ------75

DISCUSSION ------77

Chapter 4: Investigation of the Organ Response to Inflammatory Challenge in

Timp3-/- Mice

ABSTRACT ------81

INTRODUCTION ------82

RESULTS ------84

Timp3-/- livers show normal production of and sensitivity to IL-6 ------84

-/- In response to LPS, JNK activation is greater in Timp3 livers ------86

Comparison of TNF levels in Timp3-/- versus wild-type liver ------86

TACE activity does not change in liver tissue following LPS exposure ------88

-/- Transcription levels of TACE are unaltered in the Timp3 livers ------88

Early liver injury does not differ between wild-type and Timp3-/- mice ------90

Unchallenged livers show constitutive activation of inflammatory pathway ------90

Little differential response to LPS found in spleen ------93

TACE activity is moderately increased in the spleens of Timp3-/- mice ------93

Timp3-/-hearts subjected to ischemic stress release more TNF than wild-type hearts-96

Synthetic metalloproteinases inhibitors protect Timp3-/- mice against LPS-induced

systemic inflammation ------98

DISCUSSION ------104

Chapter 5: The Effect of TIMP3 Loss on Leukocyte Development, Local Inflam-

mation and Acquired Immunity

vii

ABSTRACT ------107

INTRODUCTION ------108

RESULTS ------110

Contact hypersensitivity model shows a difference between wild-type and TIMP3-/-

mice ------110

Loss of TIMP3 does not affect the expansion of CD8+ T cells ------110

-/- Primary inflammatory reaction to DNFB appears greater in the Timp3 mice ------114

Loss of TIMP3 alters hematopoietic regeneration in peripheral blood ------114

Total bone marrow cell numbers in Timp3-/- are comparable to wild type------118

- -/- No difference in Lin levels between wild-type and Timp3 animals ------119

KSL-MP cell numbers double in Timp3-/- at day 9, compared to control mice ------121

DISCUSSION------123

Chapter 6: CONCLUSION 126

REFERENCES 133

viii

TABLES

Chapter 1

Table 1: Ligands and receptors cleaved by TACE/ADAM-17.------18 Table 2: Hematopoietic growth factors and receptors and metalloproteinases that target them.------45

FIGURES

Chapter 1

Figure 1. MMP domains------8 Figure 2. Proteolytic activators of MMPs------9 Figure 3. Structure of ADAMs and ADAM-TSs ------15 Figure 4. TIMP3 structure ------23 Figure 5. LPS triggers TNF expression via several pathways ------32 Figure 6. TNF signalling pathway ------35 Figure 7. TNF acts on almost all cells of the body ------38 Figure 8. IL-6 signalling pathway ------40 Figure 9. Development of hematopoietic cells------44

Chapter 3

Figure 1. Serum levels of TNF are dramatically higher in Timp3-/- mice after B. pertussis exposure ------67 Figure 2. Loss of Timp3 alters levels and kinetics of cleavage products of TACE in response to LPS ------69 Figure 3. Loss of Timp3 leads to 3x increase of IL-6 at 150 minutes following LPS exposure ------70 Figure 4. Loss of Timp3 leads to reduced survival of LPS-induced septic shock ------72 -/- Figure 5. The susceptibility to LPS of Timp3 mice is dependent on TNF signalling ---74 Figure 6. Timp4-/- mice do not share the sensitivity to LPS found in Timp3-/- mice ------76

Chapter 4

-/- Figure 1. Acute phase response is similar in wild-type and Timp3 mice ------85 Figure 2. JNK signalling in Timp3-/- livers increases minutes after exposure to LPS ----87 Figure 3. TACE is equally active in wild-type and Timp3-/- livers ------89 Figure 4. Serum ALT levels do not differ between wild-type and Timp3-/- mice------91 Figure 5. Timp3-/- livers show signs of low-level inflammation ------92 Figure 6. Spleens of LPS-treated Timp3-/- mice show moderately increased TACE activity ------94 Figure 7. Effluent from isolated Timp3-/- hearts has elevated levels of TACE cleavage products------97 Figure 8. Increased susceptibility to septic shock of Timp3-/- mice is metalloproteinase dependent------99 Figure 9. Metalloproteinase inhibitor PD166793 is less potent at inhibiting TACE activity than a general MPi ------101 Figure 10. Timp3-/- mice are rescued from LPS injury PD166793, an MMPi that weakly inhibits TACE ------102

Chapter 5

Figure 1. Timp3-/- mice show a greater response to DNFB than wild-type mice ------111 Figure 2. Memory T cells of LCMV-infected Timp3-/- mice expand at similar rate to wild-type cells ------113 -/- Figure 3. Timp3 animals have a stronger primary reaction to DNFB ------115 Figure 4. Dampening of hematopoietic kinetics in Timp3-/- mice compared to wild-type

following 5-FU exposure ------117 Figure 5. 5FU-induced loss of bone marrow cells is similar for wild-type and Timp3-/- mice ------120 Figure 6. Timp3-/- and wild-type live KSL cells incubated with Hoechst 33342------122

THE ROLE OF TIMP3 IN MODELS OF INFLAMMATION AND IMMUNITY

a PhD thesis in the department of Laboratory Medicine and Pathobiology. University of Toronto by David Smookler 2010

Chapter 1: INTRODUCTION

The Extracellular Matrix

All structures, including such dynamic ones as human beings, are composed of materials

that provide either compressive or tensile strength. In an office tower, for example,

compressive strength is provided by concrete, which resists downward pressure, whereas tensile strength is provided by concrete-embedded steel rebar or by steel beams, both of which resist pulling. Similarly compression in animals is resisted by massive hydrated molecules called glycosaminoglycans and proteoglycans whereas tension is resisted by collagens, laminins and elastin, which are equivalent to the steel beams and bars in a building. These structural molecules in animals which provide compressive and tensile strength, along with connecting proteins such as fibronectins, make up the external framework in which cells function. This framework is known as the extracellular matrix

(ECM).

There are several kinds of ECM throughout the body. The various connective tissues such as bone; articular cartilage; and areolar (loose connective) tissue each contain different compositions of dozens of ECM molecules to form a three-dimensional network

embedded with fibroblasts. In addition, the ECM can be a basement membrane, a two-

dimensional structure associated with epithelial cells. Epithelia tightly pack to form

sheets which line surfaces both inside and out, including the inner lining of vessels and body cavities. Blood vessel endothelia, for example, are a form of epithelial cell [1]. The

basement membrane provides a structure for these cells to attach to, as well as creating a

normally impregnable barrier, and a source of factors which regulate epithelial activity.

In general, the ECM has a dynamic role beyond its mechanical properties, communicating with the surrounding cells to alter their behaviour while itself subject to remodelling by cellular activities, a topic to be covered in greater detail later. It should also be noted that mobile cells may express molecules of the ECM on their surface, for example the chondroitin sulfate proteoglycan NG2 is expressed on the surface of certain macrophages [2], and syndecans, a family of four transmembrane heparan sulphate proteoglycans, are widely expressed by cells throughout the body [3]; this means ECM- binding proteins may be carried or presented as cell surface proteins. Thus the molecules of the ECM can surround cells; they can be a barrier between compartments; or they can in effect be carried along as part of the exterior of mobile cells.

Collagens

There are 29 distinct collagen genes in the human genome [4] [5]. All contain regions allowing for triple helix formation in either a portion or the entirety of the protein known as the collagenous domain. As mentioned, collagens are the proteins primarily responsible for resisting tensile stress. Fibrillar collagen, which makes up as much as

90% of the collagen in the body [6] is composed almost entirely of protein triple helices

covalently linked by lysine/hydroxylysine bonds. These residues can also form cross-

links between parallel adjacent trimers, creating an extracellular macromolecule larger

than any single cell could produce. Most fibrillar collagen is made of collagen I but can

include types II, III, V and XI as well [7] [8]. The basement membranes of all tissues

contain collagen IV, a heterotrimer composed of combinations of the six isoforms of the

gene. Unlike fibrillar collagens which link in parallel, Collagen IV trimers bind end-to-

end with multiple binding sites at each end to form a large net [9] on which other

molecules of the basement membrane can attach. In addition, there are a number of

collagen proteins, i.e. proteins containing collagenous domains, which are also

proteoglycans, these are know as fibril associated collagens with interrupted triple helices

(FACIT) collagens and include collagens IX, XII, XIV, XVI, XIX, XX, XXI, AND XXII

[10].

Glycosaminoglycans (GAGs) and Proteoglycans

GAGs are long unbranched chains of repeated disaccharides. All sugars are highly hydrophilic, GAGs are rendered more so by the presence of an acetylated amine in one of the sugar pairs and a carboxyl group in the second of the pair. Additionally, all but one of the GAGs are heavily sulphated, increasing their negative charge. Most GAGs are assembled in the Golgi where they grow perpendicularly from serine residues at multiple sites along a core protein. This GAG/protein combination is known as a proteoglycan, a subset of glycoproteins. The number of GAG attachments on the core protein can range from one to more than 100 [11]. One exceptional GAG is hyaluronan (HA). HA does not link to a core protein and thus does not form a proteoglycan. HA is also not made in the Golgi but produced extracellularly by enzymes attached to the exterior cell surface,

creating a massive chain of up to 25 000 repeated disaccharide units [12]; HA is also

unique in being the only GAG that has no sulphates. HA itself can provide a linear

scaffold onto which proteoglycan monomers non-covalently assemble, creating huge

structures. Hundreds of copies of the proteoglycan aggrecan, for example, can bind to

HA to form a quaternary structure of 100 million Daltons [13] [14].

Proteoglycans provide bulk to the ECM and resistance to compression, aggrecan for

example is a major component of joint cartilage. However, proteoglycans can have

essentially non-mechanical roles as well, and are not even necessarily part of the ECM.

For example, the highly charged proteoglycan heparin is released from mast cells and

acts as an extremely potent anti-coagulant by binding to and activating antithrombin [15].

Aside from heparin, there are four other classes of proteoglycans, based on the types of

disaccharides in the GAGs. They are heparan sulphates (similar in structure to heparin,

but having fewer sulphate groups [16]—a fine example of misleading nomenclature);

chondroitin sulphates, of which aggrecan is a member, these are a major component of

cartilage; dermatan sulphates, which are the most predominant proteoglycans in skin

[17]; and finally keratin sulphates, which are primarily found in the cornea [18].

ECM glycoproteins

Many proteins bind to proteoglycans [11]. These include, of course, other components of

the ECM: collagens [19]; laminins, which are large multi-chained glycoproteins found between epithelial cells and their underlying basement membrane [20]. Other proteoglycan-binding ECM proteins include fibronectins, which link collagens to

proteoglycans [21] [22] as well as linking cells to the ECM; vitronectin, which is both a

serum and an ECM glycoprotein, and which plays an important role in regulating serine

proteases involved in matrix degradation (primarily fibrin) by stabilizing plasminogen

activator inhibitor (PAI) [23]; and thrombospondin, a glycoprotein which binds many

other matrix molecules in addition to the proteoglycans, and which plays a role in angiogenesis, platelet aggregation and smooth muscle migration [24]. Further

proteoglycan-binding proteins include tenascins, which inhibit cell binding to the ECM

by interfering with cell/fibronectin interactions [25]; and von Willebrand factor, a

glycoprotein which forms a covalently linked complex of dozens of copies, binds to a

number of ECM molecules and soluble proteins, and is involved in promoting blood clotting both by binding to platelets and by stabilizing Factor VIII, which promotes fibrin

formation [26] [27].

In addition to the above matrix glycoproteins, hundreds of other proteins bind to the

proteoglycans [28]. Of especial significance to this work, several growth factors bind to

proteoglycans [29]; tissue inhibitor of metalloproteinase 3 (TIMP3) has been shown to

strongly bind to the GAGs of proteoglycans [30]; and the matrix metalloproteinases

(MMPs), which are targets of TIMP3, can also bind to proteoglycans [31].

Proteases in the Mammalian Genome

The MMPs are part of a larger protease group, a subclan known as the metzincins, which

all share a structural topology at their catalytic site. The metzincins are in turn part of the

larger class of proteases with a metal ion at their catalytic site: the metalloproteinases.

The other four classes of mammalian proteases are named after amino acid active at their

catalytic sites: aspartic, cysteine, serine or threonine. Collectively the 641 proteases in

the mouse genome are known as the degradome [32], of which the MMPs make up only a

small fraction.

Matrix Metalloproteinases

The MMPs are a family of proteases that collectively can degrade all elements of the extracellular matrix. Because several growth factors and morphogens adhere to ECM components, MMP cleavage can affect the availability of these signalling proteins, and thus their activity. In addition, the MMPs target many bioactive substrates directly,

including growth factors [33], chemokines [34], cytokines [35] and cell adhesion proteins

[36] and thus are key regulators of cell-to-cell and cell-to-matrix interactions. Of the

greater than 600 murine proteases, the 23 MMPs [37] are the enzymes primarily

responsible for degrading and remodelling tissue [38] [39]. Outside of the MMPs, the

few other proteases involved in matrix proteolysis include the cysteine proteinase

cathepsin K, involved in bone resorption [40] and the more closely related ADAMTS

proteins, involved in cartilage degradation.

MMP structure

The MMPs are expressed as inactive zymogens, containing a prodomain which folds to

bridge an unpaired cysteine with a zinc ion found in the catalytic domain. Cleavage of

the prodomain activates the protein. In addition to the catalytic zinc, MMPs also require

calcium to maintain proper structure and activity [41]. The prodomain and the catalytic

domain are common to all MMPs, however several other domains are found in various

MMPs (Fig. 1). These include the hemopexin domain which appears to assist in the binding to and unwinding of collagen triple helices [42], as well as being one of the binding sites for TIMPs [43]; three fibronectin-like repeats found in both MMP-2 and 9, which form hydrophobic pockets that assist binding to denatured collagen fibres [43]; a short peptide sequence RX[R/K]R known as the furin-like recognition sequence is a target for intracellular cleavage, which if cleaved removes the pro-domain region and allows certain MMPs to be secreted in an active form [44]. Extracellular cleavage of the

MMP prodomains and thus activation of these proteins is effected by both MMPs and proteases from other families (Fig. 2), suggesting a complicated interplay of activity between proteases. Further domains found in MMPs include an immunoglobulin-like domain, unique to MMP-23; and finally a transmembrane domain or a GPI anchor signal sequence, either of which tether a group of MMPs to the outer cell surface.

Types of MMPs

The MMPs were originally classified based on their ECM targets or on their cellular localization. Subsequently, it was found that the MMPs have multiple overlapping targets; as well some of the more recently discovered MMPs did not fit neatly into the original categories, so this system has largely been replaced with a numerical nomenclature. It is still instructive, however, to recognize these general categories. They are the collagenases, the gelatinases, the stromelysins and the membrane-type matrix metalloproteinases. The collagenases degrade intact fibrillar collagens, the most abundant proteins in the ECM and indeed in the body [45]. Hydrolyzed collagen is

7 MMPs sharing same Average Structural Domains structural domains # of aa’s*

MMP-7 264

MMP-23 391

MMP-1,3,8,10,11, 490 12,13,19,20,27,28 MT4,6 MMP 596 (MMP-17,25) MT1,2,3, 5 MMP 616 (MMP-14,15,16,24) MMP-2,9 696

Signal peptide Pro-domain Furin-like recog. sequence (also found in MMP-11) Catalytic domain Fibronectin-like repeats Hemopexin domain Ig-like domain Transmembrane domain Glycosyl phosphatidyl- inositol linkage (GPI) signal

Figure 1. MMP domains. The 24 murine MMPs all share three domains: a signal peptide ensuring export from the cell, an inhibitory pro-domain and a catalytic domain. Most MMPs also have one or more of the 6 additional domains shown above.

Data from Brinckerhoff & Matrisian Nature Reviews Molecular Cell Biology 3, 207-214 (March 2002) and Nagase, Visse & Murphy, Cardiovascular Research 2006 Feb 15;69(3):562-73. *aa size from Jax Mouse Genome Informatics http://www.informatics.jax.org/javawi2/servlet/WIFetch?page=searchTool&query=mmp&selectedQuery=Genes+and+Markers A collagenases

7 1 8 13

14 3 15 MT- 16 10 stromelysins MMPs 17 11 24

25 2 9 12 gelatinases

kallikrein, plasmin kallikrein, B chymase chymase, tryptase, plasmin 7 1 8 13 furin, collagenases 14 plasmin 3 15

furin* 16 MT- 10 cathepsin G MMPs 17 11 furin stromelysins 24 gelatinases plasmin 25 2 9 12

Figure 2. Proteolytic activators of MMPs. A) MMP activators of MMPs. Coloured arrows indicate an entire family, i.e. all MT-MMP family members can activate MMP-2; MMPs 3 & 10 can activate all members of the collagenase family. B) Non-MMP proteases known to activate MMPs. Note: for MT-type MMPs what is shown is furin activation based on published results, however all MT-MMPs contain furin-recognition sequences and therefore all are potentially processed by furin or other subtilisin-like proprotein convertase family members.

Based on Chakraborti, S Mandal, M Das, S Mandal, A Chakraborti, T. Molecular & Cellular Biochemistry 2003 Nov;253(1-2):269-85. *Additional information from Kang, Nagase, Pei Cancer Research 2002 Feb 1;62(3):675-81.

known as gelatin, because the denatured hydrophilic fibers form a three-dimensional gel

with water. These single strands are cleaved by the two gelatinases MMP-2 and MMP-9.

Activity of either of these two MMPs is especially easy to detect, using gelatin as a

substrate in a process known as gelatin zymography. The stromelysins are named for

their discovery in stromal cells and their ability to degrade many ECM components other

than collagen [46]. The six membrane-type MMPs, known as MT-MMPs, are anchored to the surface of cells that express them, unlike the other MMPs which are found as soluble extracellular proteins.

Roles of MMPs in normal physiology

MMPs are active in a variety of normal physiological processes involving not only tissue remodelling through targeting ECM proteins but also cell migration, proliferation and

apoptosis via targeting of growth factors, cytokines, chemokines and other bioactive

molecules [47]. Morphogenesis of many tissue types has been shown to involve MMP

activity, for example formation of islets of Langerhans; of ureteric buds in the embryonic

kidney; and tubule formation by endothelial cells—all of these employ MMPs [48] [49]

[50]. Beyond embryogenesis, menstruation, implantation, mammary development, and

wound repair all involve MMPs, which have multiple roles in these complex remodelling

activities.

Taking wound repair as an example, we can follow the many stages that involve MMPs.

During re-epithelialization healthy epithelial cells at the edges of the injury begin to lose their connections and migrate to and proliferate within the wound. This mobilization involves the degradation of both cell-to-matrix bonds, utilizing MMPs 1, 3, 9, 10, 14 and

28 [51], and of cell-to-cell bonds such as E-selectin, degraded by MMP-7 [52].

Proliferation of epithelial cells immediately distal to the wound is promoted by MMP-14

[53]. Protection of the wound from infection requires the influx of leukocytes, which

follow a chemokine gradient to the injury. MMP-1 and MMP-3 expressed by epithelia

and stroma at the site of damage cleave chemokines to regulate this gradient [51].

Another aspect of wound repair is the formation of new blood vessels, sprouting from

existing vessels, a process known as angiogenesis. This complex activity itself employs a

host of MMPs. The simplest blood vessels, capillaries, consist of an internal layer of

endothelial cells and an exterior layer of support cells know as pericytes. The basement

membrane is found between the two types of cells. The membrane consisting primarily

of collagen IV, the proteoglycan perlecan, and the glycoproteins nidogen and laminin

[54]. Initially, nearby endothelial cells are activated by factors such as VEGF, a growth

factor released from the wound site by platelets [55]; the normally quiescent endothelial

cells begin to divide rapidly and release proteases such as MMP-2 and MMP-9 which

dissolve the basement membrane. Degradation of collagen IV exposes so-called cryptic

sites, which further promote angiogenesis [56] by revealing binding sites for the

integrin αvβ3, which is expressed on the tips of motile endothelial cells. MT1-MMP is

associated with this integrin and is apparently essential for angiogenesis [57]. Further

requirements for angiogenesis include migration of the endothelial cells into the wound,

folding of the endothelia into tubes and organization into loops and recruitment of

supporting cells known as pericytes. The shaping of endothelia into functioning vessels

requires local signals of both pro and anti-angiogenesis. As cleavage of the basement

membrane by MMPs continues, it creates products that are anti-angiogenic; thus MMP

degradation of surrounding matrix regulates both the activation and the arrest of angiogenesis. These products include endostatin, arrestin, canstatin and tumstatin, all formed by collagen cleavage [58]. In this one aspect of wound healing, angiogenesis, we can see the complexity of the role that MMPs play in normal physiology. Many, perhaps most, non-infectious diseases include restructuring and remodelling of tissue, and unsurprisingly MMPs have been linked to them. Arthritis, chronic congestive heart

failure, atherosclerosis, stroke, ulcerations, tumour growth, tumour metastasis, kidney

disease, emphysema, cirrhosis, psoriasis and a variety of other diseases show an alteration in MMP activity.

MMPs target growth factor signalling in several ways

MMPs regulate growth factors via multiple mechanisms. Cleavage can liberate growth

factor sequestered in the ECM, for example MMP-2 releases bFGF from the matrix of the

lens capsule [59]. Alternatively, MMPs can shed active ligands from the cell surface, for

example HB-EGF is shed by MMP-3 and MMP-7 [33, 60]. As well, proteolysis by

MMPs can destroy a ligand, for example IL-1β cleavage by MMP-1, MMP-3 or MMP-9

[61]. A fourth mechanism by which MMPs can regulate growth factor signalling is by

the shedding of receptors, such as the destruction of IL-2-Rα by MMP-9 [62]. Finally,

MMPs can make growth factors biologically available by degrading proteins that

sequester these factors, for example the insulin-like growth factors IGF-1 and IGF-2 are

regulated by several IGF binding proteins (IGFBPs), limiting their bioavailability, the

IGFBPs are cleaved by several MMPs [63].

Regulation of MMPs

MMPs are tightly controlled. The expression of most MMPs is minimal under normal conditions, typically only activated by cytokine or growth factors during tissue remodelling [64]. As mentioned above, the proteins contain a pro-domain which inhibits activity unless cleaved. In the blood, MMP activity is predominantly arrested by α2- macroglobulin (α2M), a large homo-tetramer that is a general protease inhibitor in the serum and which binds a multitude of other soluble proteins [65]. Cleavage of a bait polypeptide region triggers a conformational change in α2M, trapping the protease [66].

In the tissue, specific inhibition of the MMPs is predominantly provided by the tissue inhibitor of metalloproteinases (TIMPs) [67].

As mentioned earlier, the MMPs are members of the metzincin superfamily of metalloproteinases. The metzincins include three other mammalian families: A

Disintegrin And Metalloproteinase family (ADAM); the ADAM with ThromboSpondin motifs (ADAMTS) family; and the astacin family[68]. The metzincins are zinc- dependent endopeptidases that share a common structure of three histidines arranged to hold the metal ion in place. The 3D structure of ligands holding the zinc is reflected in the consensus sequence HEXXHXXGXXH found in all metzincins. In addition, the metzincins have a methionine-containing loop 20 to 40 residues further on [69] that also interacts with the zinc (thus the name) [70]. Three of the metzincin families are relevant to this thesis, as they contain members inhibited by TIMP3, they are the MMPs, the

ADAMs and the ADAMTSs.

ADAMTSs

The most recent group of metalloproteases known to contain members inhibited by

TIMP3 is the ADAMTS family. These are similar in structure to the ADAM proteins, described below, but are not membrane-anchored, and are distinguished by at least one thrombospondin-like repeat (Fig. 3) (thus the name ADAMTS). The thrombospondin

domain is a highly structured, positively charged motif which can promote strong binding

to the negatively charged GAGs of proteoglycans [71]. There are 20 murine ADAMTSs

[[72] supplementary Table S3], many of which are know to cleave extracellular proteins

[73]. ADAMTS-4 and ADAMTS-5, also know as aggrecanase-1 and -2 respectively,

bind to and degrade aggrecan, the large proteoglycan found in articular cartilage. Both of

these ADAMTSs are inhibited by TIMP3. ADAMTS-1 has also been shown to degrade

aggrecan and to be inhibited by TIMP3 [74].

ADAMs

In mice the ADAMs are a family of 40 cell-surface bound proteins (Fig. 3) [75], [[72]

supplementary Table S3]. Several of the ADAMs are not catalytically active [76].

Family members were first identified as important for sperm-egg fusion [77].

Subsequently, it has been found that many of the ADAMs are predominantly or entirely

expressed in the testes [78]. Of the remainder, 10 ADAM proteins have been shown to

be proteolytically active and involved in shedding signalling ligands and their receptors from the surface of cells [79].

14 A B

ADAM structure ADAMTS structure

Signal peptide Signal peptide Pro-domain Pro-domain

Metalloprotease-like Metalloprotease catalytic domain catalytic domain

Disintegrin-like Disintegrin-like domain domain Thrombospondin-type repeat Cysteine-rich domain Cysteine-rich domain EGF-like repeat

Transmembrane Spacer domain domain Thrombospondin-type repeats (multiple) Cytosolic domain Various C-terminal modules

Figure 3. Structure of ADAMs and ADAMTSs, two other metzincins families with members inhibited by TIMP3. Both ADAMs and ADAMTSs contain an MMP- like catalytic domain and disintegrin domain, similar to an integrin-binding domain found in snake venom. A) ADAMs are membrane-bound proteins, most of which are catalytically inactive. There are 10 proteolytically active ADAMs outside the testes: ADAMs 8, 9,10, 12, 13, 15, 17, 19, 28, 33. B) ADAMTSs are extracelluar proteases, they lack a transmembrane domain and they contain an isolated well-conserved thrombospondin repeat (TSR type 1), followed by multiple (0 to 14) TSR repeats after a cysteine-rich domain and spacer region.

Based on The ADAM Family of Proteases edited by Nigel Hooper & Uwe Lendeckel, pg. 3 Springer 2005 and the lab homepage of Suneel Apte http://www.lerner.ccf.org/bme/apte/adamts/domain_organization.php

The cleaving of receptors and ligands by ADAMs often leaves the targets functionally intact so that the cleaved, soluble molecules are themselves important biological mediators: the shedding of ligands can expand the region of influence of a cell beyond cell-cell contact; alternatively, shed receptors can act as decoys by binding to and inhibiting ligands. In the context wherein the cleaved molecule is biologically active, these proteases are known as sheddases. The first ADAM characterized as such was

ADAM-17, also know as TNF-alpha Converting Enzyme or TACE [80]. TACE appears to be the most important sheddase in terms of the range of its targets (Table 1). A second

ADAM, ADAM 10, the mammalian homologue of the fly protease Kuzbanian (kuz), is also quite important having been shown to be essential for development, via cleavage/activation of Notch, and for activation of the EGF receptor, via cleavage/activation of HB-EGF [81] and other ligands. TIMP3 strongly inhibits both

TACE and ADAM-10 sheddases.

TACE

Regarding regulation of immunity and inflammation, TACE is by far the most important of the ADAMs, and perhaps of all metzincins. TACE was originally identified as the protease responsible for converting TNF from a cell-membrane-bound molecule to a soluble cytokine [82, 83]. Tumour Necrosis Factor is a master regulator of inflammation.

TACE potentially has a significant impact on inflammation as it is the sheddase which controls the availability of TNF. In addition, TACE targets the receptor of another major regulator of inflammation, IL-1 [84]. Specifically, TACE targets the inhibiting decoy receptor of IL-1 known as IL-1R II [85, 86]. During systemic inflammation, there is a major change in homeostasis to cope with the putative infection that triggers the

inflammation; this change is known as the acute phase response. The cytokine primarily responsible for initiating the acute phase response is IL-6 [87]. TACE cleaves the 80 kDa

IL-6 receptor [88]. Thus TACE is involved in regulating at least three apical points in inflammation: TNF signalling, IL-1 signalling and IL-6 signalling. In addition, TACE sheds a number of other proteins involved in more specific aspects of inflammation and immunity, including leukocyte adhesion molecules, a chemoattractant and various growth factors and receptors involved in hematopoiesis (Table 1). TACE mRNA is expressed throughout the body to varying degrees in multiple tissues. Anti-TACE antibodies have shown the protein to be expressed on the surface of several cell types including monocytes, T-cells, neutrophils and endothelial cells [82].

Role of TACE in development

The expression of proteolytically active TACE is necessary for development, as is revealed by the lethality of mutant mice lacking the catalytic portion of TACE [89].

These animals either die in utero, or more typically within a few hours of birth.

Occasionally (approximately 5% of the homozygous mutants), the mice will survive a week or more. These survivors were cited as having inflamed corneas. This keratitis suggests that TACE is not essential for inflammation, even if it is an important regulator of the process. As Table 1 shows, TACE regulates the shedding of several of the EGFR ligands. Its importance in EGFR signalling is suggested by the phenotype of the TACE mutant, which is similar to the phenotype of EGF ligand mutants. For example, with either loss of TGFα or loss of active TACE both lead to abnormal hair growth and inflamed corneas [90]. Loss of HG-EGF or loss of TACE leads to heart valve enlargement in developing embryos [91]. Mutation of HB-EGF to a non-cleavable form,

a more relevant mutation in view of the role of TACE as a sheddase, also produces mice

with severe heart valve defects [92].

Table 1. Ligands and receptors cleaved by TACE/ADAM-17*

Chemokines Fractalkine Glycoproteins: MUC1, GP1b-α Adhesion molecules: VCAM-1 L-selectin EGF ligands: proTGFα proHB-EGF proamphiregulin proepiregulin, Other growth factors and GF receptors: TRANCE/RANKL erbB4/HER4M-CSFR c-Kit Notch1 GHR Neuron & neurological environment: TrkA APP PrPc pro-NRG-α2c Cytokines and cytokine receptors: proTNF p55 p75 IL-1R II [85] IL-6Rα IL-15Rα Other: MICB [93] CD30 CD40 CD44 Collagen XVII *Based on The ADAM Family of Proteases ed. by Hooper & Lendeckel, p3 Springer 2005

Activation of TACE

TACE is expressed with its pro-domain removed, i.e. no pro-TACE is detected at the cell

surface [94]. Despite the lack of its pro-domain, the cell-surface TACE is not necessarily

active. Phorbol 12-myristate 13-acetate (PMA also known as TPA) triggers TACE

activity in cultured cells within 30 minutes and up to at least 60 minutes. Surprisingly, it

does so without increasing the amount of TACE protein [95]. On the contrary, PMA appears to induce the loss of cell-surface expressed TACE [94]. In addition, PMA also induces transcription of TIMP3 [96], which once translated and expressed would inhibit

TACE activity. These findings underscore that the mechanisms which induce the activity of the protease are still poorly understood.

PMA is a powerful inflammatory stimuli (it is the active ingredient in croton oil, a blistering agent that has been studied for almost 200 years [97]) and a common promoting agent in tumourigenic studies [98]. PMA is an exogenous analogue of diacylglycerol (DAG), which binds to and activates members of the protein kinase C family (PKC) [99] [100]. PMA indirectly leads to rapid phosphorylation of TACE at threonine 735 by ERK1/2 MAP kinase [101]. Activation by another reagent, GRP, causes serine phosphorylation of TACE within minutes [102]. The details of how phosphorylation might lead to TACE activation are as yet unknown [103].

The role of other metalloproteinases in inflammation.

The emphasis on TACE and inflammation is due to the number and importance of the proteins that TACE acts upon which are involved in the inflammatory process. However,

TIMP3 inhibits many proteases, the key to understanding its effect on inflammation may lie with one of these less obvious proteins. MT1-MMP, for example, and not TACE, appears to be central to monocyte transendothelial migration in response to TNF via cleavage of ICAM-1 [104]. The LPS-induced increase in inflammation in the Timp3-/- mice could be driven by an increased accumulation of leukocytes in tissue due to unregulated MT1-MMP activity. Another candidate, MMP-8, also know as neutrophil

collagenase, is released from storage granules in neutrophils at the site of inflammation

[105]. As neutrophils are the primary leukocytes to respond to inflammation, and there

are 10x to 60x more neutrophils in the blood of mice than monocytes [106] any alteration in the sensitivity to MMP-8 may have a dramatic effect on inflammation. Higher MMP-8 activity would cause greater degradation of surrounding collagen; the cleaved collagen

products themselves potentially promote an increase of IL-1 expression [107] and thus

further inflammation. It has been recently discovered that ADAM10 can cleave vascular

endothelial cadherin (VE-cadherin) [108], an adhesion molecule essential for the

formation of blood vessels [109], and that by cleaving VE-cadherin ADAM10 can

increase vascular permeability; since loss of blood pressure is one of the key factors in

shock, an increase in vessel permeability could be directly responsible for the increased

mortality of the Timp3-/- mice exposed to LPS.

Tissue Inhibitors of Metalloproteinases

TIMPs are small extracellular proteins of approximately 200 amino acids. Together the

four TIMPs are able to inactivate all MMPs. Unlike α2M which only targets active

proteases, some TIMPs can form tight bonds with either the zymogen or active form of

certain MMPs. Although TIMPs are seen primarily as MMP inhibitors most are known

to have secondary properties: TIMP1 and 2 can act as growth factors for some cell types,

TIMP1 was characterized as a leukocyte factor that can promote expansion of erythroid precursor cells [110] [111, 112] as can TIMP2 [113]; TIMP2 also acts as an adaptor

between MT1-MMP and pro-MMP-2 leading to activation of MMP-2 [114]; TIMP3 has

been shown to have apoptotic effects on cells in culture and in vivo [115] [116], and there

is evidence it can bind to and block the activation of the vascular endothelial growth

factor receptor 2 (VEGFR-2) [117]. As well, TIMP3 has been shown to inactivate several other members of the metzincins from both the ADAMs and the ADAMTSs.

TIMP structure

TIMPs contain two domains, simply named the N and C terminal; the N terminal domain is twice as large as the C terminal domain [118]. Both domains have six cysteine residues forming three disulphide bonds each. The four TIMPs are structurally very similar, with over 40% identity in amino acid sequence [119]. The first five residues of all TIMPs are C(S/T)CXP. The shape of the TIMPs is characterized as a wedge [120].

These five residues, on the edge of the wedge, insert into the catalytic site of the target protease, blocking activity [121]. This major contact site is one of five segments in the N terminus which interact with the target protease [122]. The C-domain of TIMPs can assist in the binding to target enzymes, for example the C-domain of TIMP2 binding to the hemopexin domain of MMP-2 [123].

TIMP3

TIMP3 is unique among the four TIMPs in being insoluble, due to strong binding to the

ECM. As mentioned above, TIMP3 adheres to the highly negatively charged sulphated sugars of proteoglycans. There is some debate about what portion of the protein is responsible for this binding. It has been proposed the ECM binding is due to the numerous positively charged peptide residues in the N-domain (Fig. 3) [30]. However, the N-terminus of TIMP3 alone is a soluble protein [124], suggesting the C-terminus is responsible for this unique affinity of TIMP3 amongst TIMPs. One lab which initially supported the hypothesis of C-terminus binding [125] has since suggested that the N-

terminus of TIMP3 is responsible for ECM binding but requires the C-terminus for

proper conformation to bind to the proteoglycans [126]. Functionally, the N-terminal

domain alone is sufficient to inhibit metalloproteinase activity, for example the N-domain

of TIMP3 is as effective as the entire protein against ADAM17 [118].

Structural interaction of TIMP3 with TACE

According to crystal analysis, the N portion of TIMP3 binds with the catalytic region of

TACE at five points in a manner identical to known TIMP/MMP interactions [122]. In a

“front view” orientation of a TIMP ribbon model the N-domain is to the left and the C- domain is to the right (Fig. 4). In the N-domain the upper portion of this shape forms a wedge that interacts with the catalytic portion of proteases [120]. The N-domain contains a 5-strand β-pleated sheet that curls into a barrel. In the front view orientation the first three strands are vertical and are named ABC from left to right. Strands A and B are joined by a bridge of amino acids known as the AB-loop. The AB-loop is the first of the five contacts points with the TACE catalytic domain, going from left to right. Although all TIMPs have this loop and use it for contact with their protease targets, the AB-loop in

TIMP3 is especially suited for binding with TACE. The AB-loops of TIMP2 and 4 are

considerably longer and the loop in TIMP1 is slightly shorter than the TIMP3

22 A N domain C domain

cc c c c c cccc c c N

ar B sug

PF C AB-loop

C B A

Figure 4. TIMP3 structure shown in increasing detail. A) TIMP3 like all TIMPs consists of two domains, the N domain is twice as large as the C domain; each contains 6 cysteine residues, forming 3 cystine bonds per domain. Specific to TIMP3 is an asparagine (N) near the far end of the C terminus where the protein is a glycosylated. B) Multiple positively charged amino acids (K, R) in the N domain of TIMP3 may contribute to TIMP3’s unique ability amongst the TIMPs to bind to the negatively charged glycosaminoglycans of the ECM. Grey sequences, labelled ABCDE, represent regions involved in a 5-strand β-barrel found in all TIMPs. C) 3D wedge-shaped structure of the N domain of TIMP3. Proline33 and Phenylalanine34, shown as PF at the top of the AB-loop, contribute to TIMP3’s unique ability to inhibit TACE by tightly fitting into a hydrophobic grove in the ADAM. The first six amino acids of TIMP3, shown as CTCSPS, form the wedge’s thin edge, which inserts into the catalytic site of the protease, inhibiting activity.

Figure 4B originally published by David Smookler in Annals of the Rheumatic Diseases 2003;62:43-47ii F Mohammed, D Smookler, R Khokha Metalloproteinases, Inflammation, and Rheumatoid Arthritis, based on Yu, Yu, Meng, Brew & Woessner J. Biol. Chem. Oct. 2000 & Langton, Barker and McKie J. Biol. Chem. July 1998.

Figure 4C is taken directly from Wisniewska M, Goettig P, Maskos K, Belouski E, Winters D, Hecht R, Black R, Bode W. Journal of Molecular Biology 2008 Sep 19;381(5):1307-19.

AB-loop. The TIMP3 AB-loop has a proline at position 33 followed by phenylalanine.

The kink of P33 orients the bulky, hydrophobic F34 to fit into a hydrophobic groove in

TACE. P33 and F34 provide the direct molecular contacts of the TIMP3 AB-loop with

TACE [122]. Mutational analysis has shown the AB-loop to have a dominant role in

TACE inhibition [127] through maintaining stability of binding between the two proteins.

The top of the next strand in the β-barrel, strand C, provides the next contact region. The third segment, the major contact between TIMP3 and the catalytic domain of TACE, is six consecutive amino acids running along the edge of the wedge from C1 to S6 (Fig.

4C), the first cysteine forming two ligands with the catalytic zinc. The final two contact points are provided by the sD-sE loop and the sE-sF loop [122]. Aside from the insolubility of TIMP3, the protein stands out among the TIMPs for the breadth of proteases it can inhibit, including members from the ADAM family and the ADAMTS family.

In addition Timp3, far more so than other TIMP genes, has been shown to be hypermethylated in a number of tumour tissues. A September 2008 survey of Pubmed contains only three papers showing hypermethylation of TIMP-2 in tumour tissue [128]

[129] [130], with no methylation described for TIMP1 or TIMP4. In contrast, dozens of analyses of tumour tissue show hypermethylation, and thus inactivation, of Timp3 in a number of cancers including esophageal [131], lung [132], bladder [133], hepatic [134], breast [135], cervical [136], pancreatic [137], colorectal [138], renal[139], gastric [140] and both primary [141] and secondary[142] brain tumours. This suggests that Timp3 is a

tumour suppressor gene, i.e. a gene that normally limits the growth of cancer, and that

TIMP3 plays a particular role, separate from the other TIMPs, as a tumour suppressor.

It is worth speculating on what differences lead to TIMP3 having the more significant

tumour suppressor activity. Differences between the TIMPs include: 1) the microenvironment in which they work; 2) the targets they inhibit; and 3) the factors

which promote their expression. Regarding the microenvironment, TIMP3 tightly

adheres to the proteoglycans, whereas the other TIMPs are soluble. In cancer

development and metastasis, one possibility is that TIMP3 is located more effectively for

preventing proteolysis of the basement membrane proteoglycans. TIMP3 produced by

pericytes has been shown to stabilize the BM of blood vessels [143]. An intact BM can

act either as a physical barrier to mobile mutant epithelial cells, or as a source of signals

that promote order and quiescence in the epithelium. Proteolysis of the BM could assist

tumour spread and development. TIMP3 in direct contact with the matrix proteoglycans

may be more able to protect the BM than other TIMPs and thus maintain epithelial

control.

In terms of target differences, several proteases targeted by TIMP3 are involved in

regulating cell growth, differentiation and mobility. However, almost all of these

proteases are also inhibited by other TIMPs as well: MT1-MMP by TIMP2 [144]; the

soluble MMPs by various TIMPs; ADAM-10 by TIMP1 [145]. Two notable exceptions

to this are ADAM-12 and TACE/ADAM-17 [146], which are not inhibited by the other

TIMPs. ADAM-12 cleaves HB-EGF and the IGF binding proteins IGFBP3 and IGFBP5

[147]. The IGFBPs regulate IGFs which are active in many developing cancers [148].

TACE targets a considerable number of proteins involved in inflammation, including

cytokines, chemokines and growth factors (Table 1). Thus, since inflammation is

implicated in many cancers [149] TIMP3 may primarily act as a tumour suppressor by regulating TACE.

Finally, putative tumour suppression may not be linked to the unique targets of TIMP3 among the TIMPs, but rather linked to differences in what promotes TIMP3 expression.

TIMP3 may be preferentially activated to inhibit tumour growth. This is difficult to confirm, however. Inducers of TIMP3 include TGFβ, EGF and the exogenous PKC activator PMA; yet each of these also activates TIMP1, [96]. The IL-6 family member

Oncostatin M strongly activates TIMP3 in chondrocytes [150] whereas in other cells it downregulates TIMP3 [151]. TIMP3 is activated by the steroid dexamethasone [96], which is anti-inflammatory, but the combined activity of the inflammatory cytokines IL-

1β and TNF strongly down-regulate TIMP3 expression in mouse brain endothelial cells

[152]. Thus, the present understanding of TIMP3 transcription does not clearly show a single role or simple interpretation of how or when it is activated. However, the fact that several agents involved in promoting or regulating inflammation also regulate TIMP3 suggests the inhibitor plays a role in this process, and inflammation plays a profound role in tumour progression [149].

Inflammation

Inflammation can be acute or chronic, local or systemic. As a condition it was recognized and documented over 2000 years ago in De Medicina by the Roman encyclopaedist Celsus who described inflammation as having four characteristics: rubor, calor, tumor and dolor, i.e. redness, heat, swelling and pain. Rather than these being signs of the host overcome by pathogens or injury, inflammation is a defensive response

to a variety of insults: infection, a cut, a blow or a burn can all elicit inflammation. The

four classic indications of inflammation are all due to a response by the blood.

Specifically, capillary beds open near the site of injury, making the inflamed skin red and

warm; the tissue swells as the vessels begin to leak in response to bradykinin—a cleavage

product of a serum protein—and due to the local extravasation of leukocytes; bradykinin and the release of prostaglandins from local cells stimulate neuroreceptors, causing pain.

Internal organs have corresponding indications of inflammation, increased blood flow to the area, swelling, pain and, histologically, an effux of leukocytes into the tissue (these are such common signs of organ dysfunction that the suffix ‘-itis’, meaning

“inflammation of the”, is perhaps the most familiar medical term to laypeople, e.g. hepatitis, laryngitis, tonsillitis).

While inflammation is initiated as a defensive response to an injury or infection, it can itself become harmful if the inflammatory reaction is overly powerful or prolonged.

Swelling, for example, can impair the function of several organs: meningitis can produce dangerous pressure in the confines of the skull, pericarditis can interfere with the pumping of the heart, and pneumonia, an inflammation of the lung alveoli, impairs the proper exchange of gasses from the air to the blood. With chronic inflammation other problems include tissue damage due to proteases and reactive molecules released by macrophages and neutrophils, as well as impaired function due to fibrosis from proteins in the exudate. Chronic inflammation can also contribute to cancer. One estimate suggest 15% of malignancies are caused by infection and the related inflammation [149].

Cancer requires both genetic mutation and cellular proliferation; inflammation can

contribute to both. Reactive oxygen and reactive nitrogen species produced by

neutrophils and macrophages during inflammation can induce DNA damage [153]. In

addition inflammation induces the expression of numerous local cytokines which can

promote cellular growth [154].

Systemic inflammation can be triggered by the same agents that induce local

inflammation: a bacterial or viral infection, a burn, certain cytokines, if presented in larger doses or introduced intravenously or intraperitoneally. The idea of systemic

inflammation at first glance appears paradoxical, since inflammation suggests an increase of blood flow to a particular area. With systemic inflammation no specific area becomes inflamed. Instead capillary beds open and the small vessels become more permeable throughout the body.

An additional feature of systemic inflammation is the activation of the acute phase response. In 1930, it was noted that a purified fraction of pneumococcus bacteria,

Fraction C, is precipitated by an unknown protein found in the sera of acutely ill pneumonia patients [155]. This so-called C-reactive protein subsequently disappears from the serum a few days after the acute phase of pneumonia. It was later established that this ‘acute phase protein’[156] is induced by a variety of conditions which induce inflammation. Ultimately, dozens of serum proteins manufactured by the liver were discovered to be involved in the acute phase response [87]. Despite the potential for many of these proteins to protect against bacterial infection, and their presumed role in controlling disease, systemic inflammation and the acute phase response can be extremely harmful to the host.

Systemic Inflammatory Response Syndrome

Systemic Inflammatory Response Syndrome (SIRS) is the name given to disease due to large scale inflammation, regardless of what induces the inflammation. The term was developed to avoid confusion caused by the more commonly used ‘sepsis’, which of course refers to a live infection. It is often the case that the overwhelming danger from a bacterial infection is the inflammatory response, and in fact “When the process is due to

infection, the term sepsis and SIRS are synonymous” [157].

The early stages of severe SIRS include a drop in blood pressure due to the following:

peripheral vasodilation; an increase in the permeability of the microvasculature; and a

leakage of serum proteins from the capillaries, which reduces the osmotic pressure in the

vessels. The collapse of blood pressure impairs tissue oxygenation, while at the same

time the body experiences hypermetabolism, thus a second feature of severe sepsis in the

early stages is global hypoxia [158]. In addition, inflammatory cytokines concomitantly

trigger activation of clot formation [158]. This widespread clotting leads to what is

called disseminated intravascular coagulation (DIC). To some degree the other

symptoms can be countered, vasopressors and the administration of fluid can increase

blood pressure; patients can be given oxygen; DIC, on the other hand, is not easily

reversed. With the clotting cascade activated throughout the body, blood vessels are clogged and circulation is compromised.

Sepsis, or SIRS, is a leading cause of death in hospitals [158]. Death is not due to the

failure of any one organ. Rather, widespread activation of inflammation can compromise

many organs at once. The whole system of organs struggle to maintain homeostatsis

under hypoxic conditions. At such a point if any one of the organs reaches a crisis, the

other quickly succumb in their weakened state and the patient or animal dies. Simply

put, infection causes SIRS, which can cause Multiple Organ Dysfunction Syndrome

(MODS) and result in death [159] [160]. The universality of MODS can make it difficult to study, as no single tissue or organ is responsible.

Lipopolysaccharide (LPS)

LPS, originally called endotoxin [161], is a complex molecule found in the cell walls of

gram negative bacteria, consisting of a long polysaccharide region linked to a lipid component known as lipid A [162]. LPS is not inherently toxic, that is, it does not interfere with metabolic functions like many toxins; rather it is a pathogen-associated molecular pattern (PAMP), i.e. an essential molecule of a pathogen, in this case gram negative bacteria, that the innate immune system has evolved to recognize so as to rapidly react to infection [163]. LPS is a powerful inducer of both local and systemic

inflammation. The potency is such that the induced inflammation can cause serious and

even deadly disease [164].

The biology of LPS activation is intricate. LPS unites with a soluble mammalian

molecule, LPS binding protein (LBP) a member of the acute phase proteins. LBP

delivers the LPS to CD14 [165] which exists in both a soluble and a GPI-linked

membrane-bound form, either of which can be involved in activating LPS signalling.

Soluble CD14 associated with LPS/LBP can activate endothelial cells [166], whereas membrane bound CD14 is found on macrophages [167]. LPS is then presented to Toll-

like receptor 4 (TLR4) on the surface of responsive cells, primarily macrophages, which

carry the membrane-bound version of CD14. MD-2, another cell membrane molecule,

binds to TLR4 and is also involved in the LPS activation of the receptor [168]. Once

TLR4 is activated the surface signal passes through the cell via the adaptor molecule

MyD88. Signal transduction can induce the expression of Tumour Necrosis Factor by various pathways [169, 170] (Fig. 5).

Tumour Necrosis Factor (TNF)

Serum levels of the cytokine TNF rise minutes after LPS injection. Most if not all of the

phenotype caused by LPS can be recreated by TNF alone [171]. TNF is predominantly

made by macrophages and monocytes but it is also produced in a variety of cells

including T cells [172], B cells [173], astrocytes, basophils, eosinophils, mast cells,

keratinocytes, cardiac myocytes, microglial cells, NK cells, neutrophils, Langerhans cells

[174] and fibroblasts. In addition, there are various stimuli other than LPS that induce

TNF. These include other cytokines, growth factors, exotoxins, phorbol esters, calcium

ionophores, the complement protein C5a, hypoxia, nitrites, oxygen radicals, leukotrienes and viruses [174].

31 TrX B, JNK or B, JNK Κ TrX ASK1 LPS signallingLPS is P ROS ASK1 TRAF6 TNF p38 iption. In addition stability stability of addition TNF iption. In RNA is TRAF6 MEKK3 NADPH oxidase B (TLR4). Intracelluar activationof NF- κ JNK

TAB1 NF-

γ IKK TAB2 TRAF6 TRAF6 Ub β IRAK1 α P IKK TAK1 IKK MD2 MyD88 P TLR4 IRAK4

LPS CD14 IRAK3 LBP LPS TRAM LBP p38 signalling can all pathways lead to TNF transcr tightlypost-transcriptionalby controlled processing (not shown). activated via complex receptor activity involving membrane-bound and soluble proteins soluble activated and via complexmembrane-boundinvolving receptoractivity deliveringthe ligand to Toll-like receptor4 Figure5. LPS triggersTNF pathways.expressionseveral via Based partially on Matsuzawa A, Saegusa on Matsuzawa Based partially Sadamitsu T, Noguchi K, Nishitoh C, S, Koyasu H, Nagai S, Ichijo K, Takeda K, Matsumoto Jun;6(6):587-92. 2005 Immunology Nature H.

In the case of LPS, however, by far the majority of TNF is made by macrophages,

illustrated by the following two pieces of evidence. Firstly, transfer of wild-type

macrophages into LPS-insensitive mice is necessary and sufficient to confer lethal sensitivity to LPS [175], and this is because of the release of TNF from the macrophages

[176]. Secondly, op/op mice, which lack macrophages due to a mutation in the gene for

M-CSF, are completely insensitive to a dose of LPS that would kill wild-type mice [177].

T cells, which can produce considerable TNF, are not responsive to LPS [178] (citation

specifically refers to CD4+ cells). T cells manufacture TNF in toxic quantities in vivo in

response to so-called superantigens. Whereas macrophages create large amounts of TNF as part of the innate immune response to infection, activated via TLR-4, the mechanism by which T cells release large quantities of TNF is quite different. T-cells are normally activated in a highly selective manner. The individuality of T cells is ensured by the genetic recombination of the genes for two proteins, α and β, in the T cell receptor

(TCR). Three regions (V, D and J) of the β gene, for example, collectively have dozens

of alternative exons to choose from to provide thousands of unique VDJ combinations

(vastly increased by the addition of random nucleotides by terminal deoxynucleotidyl

transferase between the regions). A superantigen such as staphylococcal enterotoxin B

(SEB) works by binding to one specific region of the β protein, the V region. The VDJ

combination may be unique, but there are only 22 V exons in the mouse genome [179].

SEB specifically binds to Vβ8 [180]. In certain strains of mice as many as 12% of all

thymocytes express this particular V region [181]. Instead of activating a few specific T

cells amongst millions, a superantigen could activate 1/8th of all the T cells in the body,

causing a massive cytokine release.

Expression of TNF and TNF receptors

TNF is expressed on the cell surface as a type II 26 kD membrane-bound protein [182].

TACE cleaves the membrane-bound TNF releasing a 17 kD soluble fragment [83] [82].

Both the membrane-bound and the soluble forms of TNF are biologically active And function as homotrimers. TNF activates receptors by aggregation as homo-trimers (Fig.

6). There are two types of TNF receptors, p55 and p75 (aka TNFR1, TNFR2; or

TNFRSF1A, TNFRSF1B respectively). Whereas TNF production is primarily restricted to hematopoetic cells, TNF receptors are expressed on virtually every cell except red blood cells [183]. P55 is considerably more important of the two with regards to almost all TNF activity and is more widely expressed [184]. P75 plays a role in thymocyte and

CTL proliferation [185] and is primarily activated by membrane-bound, rather than soluble TNF [186].

TNF signal transduction

There are several alternative signalling pathways potentially activated by p55, some of which act in direct opposition to each other. For example, p55 trimerization can lead to the formation of the so-called death inducing signalling complex (DISC), a collection of adaptor proteins that ultimately recruit and activate the protease caspase-8. Activation of caspase-8 leads to a cascade of caspase activation and rapid destruction of the cell by

34 TNF

TNFR1

RIP TRADD DISC T R FADD A F γ Caspase-8 2 IKKα IKKβ IKK programmed cell death MAP4K2

MEKK1 ROS

ASK1 ASK1 MKK7 TrX NF-κB TrX JNK IL-6

Figure 6. TNF signalling pathway. TNF activates intracellular signalling via receptor trimerization. Receptor activation can lead to dramatically different cellular responses, depending on intracellular pre-conditions. Left: TNFR1 signalling can induce apoptosis via formation of the death inducing signalling complex (DISC) and activation of the caspase cascade. Middle: The JNK signalling pathway, associated with cellular stress, can be activated through the TRAF2 adaptor protein via several avenues. Right: The NF-κB signalling pathway, associated with cell survival and the production of pro- inflammatory proteins is activated via RIP.

Details from Wajant H, Pfizenmaier K, Scheurich P Cell Death and Differentiation. 2003 Jan;10(1):45-65 and from Hsu H, Huang J, Shu HB, Baichwal V, Goeddel DV Immunity 1996 Apr;4(4):387-96.

proteolysis (Fig. 6, upper left portion). Alternatively, p55 signalling can lead to NF-κB activation (Fig. 6 right), a transcription factor responsible for a large number of inflammatory and immune genes as well as genes that prevent cell death by proteolysis: the cellular inhibitor of apoptosis proteins (CIAP) which directly bind to and inhibit the caspases. In addition, TNF signalling can trigger the mitogen activated protein kinase

(MAPK) pathways JNK and p38, which also lead to transcriptional activation [187] resulting in cell growth, differentiation and/or cytokine expression.

The expression of TNF is tightly controlled, for example, even in the persistent presence of LPS, monocyte synthesis of TNF stops after 3 to 4 hours [174]. Production of TNF is inhibited by anti-inflammatory cytokines such as IL-10, TGFβ and IL-12 [174]. TNF is removed from circulation rapidly, the biological half-life of TNF is only 6 to 7 minutes

[188]. Studies in mice show soluble TNF is primarily absorbed by the liver (31%), and the skin (30%), with considerably less absorption by the GI tract (9%), kidneys (8%)

lungs (2%) and the spleen (1%) [188].

The effects of TNF are manifold. It has specific local as well as global effects. It acts on various cells in dramatically different ways. Local effects include: promoting the release of chemokines from fibroblasts [189] keratinocytes [190] myocytes [191] and other cells as well as promoting the expression of selectins on endothelial cells, the combined result being TNF induces the recruitment of a large number of leukocytes to a target tissue and subsequent inflammation (Fig. 7). TNF can be directly cytotoxic, inducing apoptosis; indirectly cytotoxic by priming neutrophils; or it can be mitogenic, for example, being required for liver regeneration.

A systemic effect of circulating TNF is the induction of insulin resistance. Adipocytes

can regulate energy intake by releasing TNF [192]. TNF, via p55 signalling, triggers

phosphorylation of serine 307 of the insulin receptor substrate-1 (IRS-1), an intracellular

protein normally activated when insulin binds the insulin receptor. Phosphorylation of

this serine prevents the phosphorylation of a tyrosine in IRS-1 that is normally induced

by insulin [193]. By inhibiting the signal transduction of insulin, TNF prevents cellular

uptake of glucose, and thus promotes a rise in blood glucose levels [194]. Various

morbid conditions and pathologies which elevate TNF levels lead to insulin resistance,

such as chronic obesity and severe burn injury [195]. In addition, TNF inhibits fat

synthesis in adipocytes and promotes the production and release of high levels of

triglycerides into the blood [194].

A seemingly unrelated global effect of TNF is cachexia, an ongoing loss of fat and

muscle tissue observed in many chronic diseases. The mechanisms by which TNF induce

cachexia are poorly understood [196], although this was one of the first observed effects

of TNF, (the cytokine was originally known as cachectin [197]). TNF-induced cachexia

may in part be due to the upregulation of proteins involved in converting stored energy into heat, known as uncoupling proteins (UCP) [198] [199].

37 chemokines leukocytes elia oth end

myocyte NO

NO fibrobast NO NO

TNF mast cell

Figure 7. TNF acts on almost all cells of the body, orchestrating an inflammatory response. For example injury can cause mast cells to release TNF (left) into tissue. TNF will then act on stromal and parenchymal cells such as fibroblasts and myocytes (above) causing them to release chemokines, attracting leukocytes to the area. TNF also acts on the endothelia of local blood vessels (above, right) triggering the expression of leukocyte adhesion molecules such as E-selectin, ICAM-1 and VCAM-1*. As well it indirectly promotes vascular leakage by down-regulation of adhesion molecules between endothelia**. TNF also acts on circulating leukocytes promoting the production of nitric oxide (far right) which triggers vasodilation of blood vessels. Therefore the cardinal signs of inflammation, swelling due to vascular leakage, redness due to vasodilation, influx of leukocytes, can all be triggered by TNF.

*DD Henninger, J Panes, M Eppihimer, J Russell, M Gerritsen, DC Anderson and DN Granger Journal of Immunology 1997;158;1825-1832. ** Josef Friedl, Markus Puhlmann, David L. Bartlett, Steven K. Libutti, Ewa N. Turner, Michael F. X. Gnant, and H. Richard Alexander Blood, 15 August 2002, Vol. 100, No. 4, pp. 1334-1339

TNF and IL-6

TNF also causes systemic effects more directly related to inflammation. It promotes the

production of IL-6 in a number of cells including monocytes/macrophages, fibroblasts,

endothelial cells and keratinocytes [200]. IL-6 induces fever by acting on the

hypothalamus [201]. As well, IL-6 acts on the liver to initiate the acute phase response

[202], a reorganization of liver activity to downregulate several housekeeping genes and

upregulate a variety of antimicrobial proteins that are part of the innate immune system

such as C reactive protein [203], mannose-binding lectin [204], and the aforementioned

LBP.

The IL-6 receptor is made of two components: IL-6Rα, a ligand-specific protein with no

signal transduction properties, and gp130, common to all IL-6 receptor family members

[205]. Activation by IL-6 leads to the formation of a hexamer of two each of the IL-6Rα,

IL-6 and gp130 (Fig. 8). Bound to the cytoplasmic tail of each gp130 protein is a member of the Janus kinases (Jaks) [206] which trans-phosphorylates its opposite partner

[207]. The phosphorylated Jaks act as tyrosine kinases, phosphorylating STAT3 proteins

which then dimerize and translocate to the nucleus [208] to activate transcription of

acute-phase genes (Fig. 8). STAT3 was initially identified in a search for the transcription factor binding to response elements of several acute-phase genes, following

IL-6 stimulation [209].

39 IL-6 Signalling

IL-6

IL-6Rα g g p p SHP-2 SHP-2 1 1 P GRB2 3 3 P SOS 0 0 J J J SHC P GRB2 A A A K K P ras K P P GDP GRB2

ras SHC GTP

STAT3 STAT3 P STAT3 Raf-1 P P

MEK P

ERK P

LBP STAT3 P STAT3 P

Figure 8. IL-6 activates the acute phase proteins via phosphorylation of the STAT3 transcriptional activator. Dimerization of the gp130 molecules induces transphosphorylation of JAK proteins attached to the cytoplasmic tail of gp130. Phospho- JAK proteins then phosphorylate STAT3 proteins which dimerize and translocate to the nucleus on phosphorylation. Right: other signalling pathways such as MAPK cascade are also activated by IL-6.

Based on Skiniotis G, Boulanger MJ, Garcia KC, Walz T Nature Structural and Molecular Biology 2005 Jun;12(6):545-51.

One of the genes downregulated in liver production during the acute phase response is albumin, a serum protein critical for maintaining osmotic pressure within blood vessels.

Thus Tumour Necrosis Factor promotes the transcription of IL-6 which activates the acute phase response, which includes down-regulation of albumin, the loss of which leads to reduces osmotic pressure in the blood and an increase of vascular leakage. This contributes to the dangerous drop in blood pressure seen with systemic inflammation. IL-

6 is both a pro- and anti-inflammatory agent. IL-6 is pro-inflammatory because of its role in fever and the acute phase response. In addition, at the cellular level IL-6 promotes the differentiation of monocytes into macrophages, rather than dendritic cells [210].

Dendritic cells are more involved with presenting antigen to the acquired immune system, and less involved with inflammation than macrophages are. Incidentally, IL-6 affects several other cell types, promoting the differentiation of T and B cells and the proliferation of T cells, keratinocytes, mesangial cells and megakarayocytes [211].

IL-6 also appears to be anti-inflammatory: in a model of toxic shock the administration of IL-6 protects mice against LPS-induced death and, conversely, antibodies to IL-6 increase mortality [212]. Furthermore, mice lacking the IL-6 gene tend to produce higher levels of TNF in response to LPS [213] suggesting IL-6 is a negative regulator of this inflammatory cytokine.

TNF can trigger hypotension by three further mechanisms: TNF promotes the exit of leukocytes from the vasculature, which also increases leakage; TNF by disrupting calcium release within myocytes, impairs cardiac contractility which reduces the

pumping efficiency of the heart [214]; and finally, TNF is an inducer of nitric oxide

[215], which is a vasodilator as well as an inducer of vascular leakage.

Nitric oxide

Nitric oxide (NO) has multiple biological functions. It is released as a bactericide during

infection, but is also a signalling molecule targeting both neurological and vascular

tissue. It has a half life of a few seconds in the body and must therefore be generated by

one of the three isoforms of NO synthase: eNOS, nNOS or iNOS. The eNOS and nNOS

isoforms are constitutively expressed, whereas iNOS is activated by cytokines as part of

the inflammatory response [216]. NO induces smooth muscle relaxation, causing

vasodilation and inhibits platelet function [217]. Studies using iNOS-deficient mice

found that NO generated by iNOS is responsible for the vascular hypotension caused by

sepsis or LPS [218] [219]. A third study showed iNOS-/- mice were completely protected

from LPS-induced death [220]. However, one study showed iNOS-/- mice to be even

more vulnerable to LPS-induced death [219]. As well, LPS induction of NO is not solely

dependent on TNF signalling, as p55-deficiency reduces serum NO levels in mice by

30% to 50%, rather than by 100% [221].

TIMP3 and Hematopoiesis

Another aspect of the immune system that TIMP3 may be involved in is the development

of leukocytes. The expansion, differentiation and migration of immune cells is regulated

by a number of proteins, many of which are targeted by metalloproteinases inhibited by

TIMP3. Hematopoietic cells undergo multiple transitions as they progress from stem

cells to their terminal states throughout the body. The forces governing cell fate involve

both internal stochastic mechanisms and a variety of extracellular factors. These

regulating factors may be soluble, membrane-bound or matrix bound, acting individually

or synergistically. In the mature animal this development is initiated and progresses in

the bone marrow.

The architecture of the bone marrow niche includes a solid substrate of trabeculae; a

network of microvasculature; macrophages and an extracellular matrix of collagens,

glycoproteins and proteoglycans produced by stromal cells consisting of fibroblasts and

adipocytes—all interacting with the hematopoietic cells via adhesion and growth factors

[222]. It is estimated that there are 275 million bone marrow cells in the average mouse,

of which 27 600 are long term reconstructing stem cells, or one in 10 000 cells [223].

Figure 9 shows the stages of hematopoietic cell development along with the growth

factors involved in their differentiation and expansion.

MPs targeted by TIMP3 affect hematopoietic growth factor signalling

Several hematopoiesis factors which determine cell fate are cleaved by MMPs and

ADAM sheddases that are targeted by TIMP3. Table 2 lists growth factors and receptors

acting in the bone marrow that are known to be targeted by MMPs and ADAMs . It

appears that metalloproteinases can directly influence half of the signalling pathways

listed in Figure 9, by either converting the ligand to a soluble form, as for SCF, Jagged,

Dll1, FLT3L and M-CSF, or by cleaving the receptor, as for c-Kit, Notch, c-fms, IL-2Rα

and IL-15Rα.

43 multipotent stem cell

Self-renewal

Notch

SCF, TPO IL-7, FLT3 primitive progenitors myloid stem cell lymphoid stem cell IL-3, SCF, TPO GM-CSF IL-7 IL-7 committed precursors

MEP GM TNK BP

IL-7 TPO EPO IL-7 IL-2 lineage committed precursors

erythroblastthrobla GP MP TP NKP

mega- G-CSF, IL-5, SCF M-CSF IL7, IL-2 IL-15 IL-4 karyocyte

TPO EPO granulocytes: T cell B cells neutrophils, eosinophils, basophils plateletserythrocytes monocytes NK cells leukocytes

Figure 9. Stoichiometric and signalling factors combine to direct the development of hematopoietic cells. MEP: Megakaryocyte/erythroid precursor; GM: granulocyte/monocyte precursor; TNK: T cell/NK cell precursor; BP: B cell precursor; GP: granulocyte precursor aka myoblast; MP: monocyte precursor; TP: thymocyte precursor; NKP: NK cell precursor; SCF: Stem Cell Factor aka Steel Factor aka c-Kit Ligand; TPO: thrombopoietin aka THPO, aka megakaryocyte growth and development factor; EPO: erythropoietin; CSF: colony stimulating factor.

From Kaushansky, New England J. of Medicine 2006 May 11;354(19):2034-45 review. With supplemental information from Borge OJ, Adolfsson J, Jacobsen AM Blood. 1999 Dec 1;94(11):3781-90 and Duncan AW, Rattis FM, DiMascio LN, Congdon KL, Pazianos G, Zhao C, Yoon K, Cook JM, Willert K, Gaiano N, Reya T Nat Immunol. 2005 Mar;6(3):314-22.

Table 2: Hematopoietic growth factors and receptors and metalloproteinases that target them

Ligand Ligand MP Receptor Receptor MP SCF MMP-9 [224] c-Kit TACE [225] (Steel Factor, Kit-ligand, KL) (CD117) Jagged TACE [226] Notch TACE [227] Delta-like 1 ADAM10 [228] Notch TACE [227] Delta-like 4 ADAM9,12,17 [229] Notch TACE [227] FLT3L unknown MP [230] FLT3 ------M-CSF TACE [231] c-fms TACE [232] IL-2 ------IL-2Rα MMP-9 [62] IL-15 ------IL-15Rα TACE [233]

Typically receptor cleavage reduces signal transduction for that pathway. In the case of

Notch, however, cleavage activates the signalling pathway. Specifically, extracellular

cleavage of Notch by TACE—induced by ligand binding—precedes intracellular

proteolysis by γ-secretases, allowing the cytosolic fraction of Notch to translocate to the

nucleus as a transcription factor [227] where it inhibits hematopoietic stem cell

differentiation [234]. If TIMP3 normally inhibits TACE in this process, its absence could

lead to increased Notch cleavage and impair the exit of hematopoietic stem cells from

their undifferentiated state.

Comparing Figure 9 and table 2 we see that metalloproteinases inhibited by TIMP3,

specifically, TACE, MMP-9 and ADAM10 cleave growth factors or their receptors

involved in myeloid cell development (SCF/c-Kit); the formation of monocytes ( M-

CSF/c-fms); and the differentiation of T and NK cells (IL-2/IL-2Rα, 15/1L-

15Rα). Disruption of any one of these pathways might block or skew the expansion of

these blood cell types.

MPs are involved in the trafficking of hematopoietic cells

MMPs also play a role in mobilizing hematopoietic stem cells [235]. It has been shown

that exogenous MMP-9 releases leukocytes from the bone marrow ECM [236]. As well,

several MMPs can cleave and thus inactivate the chemokine SDF-1 [34]. SDF-1, also

known as CXCL12, is the major chemoattractant acting to retain hematopoietic stem cells

in the bone marrow [237]. This chemokine also plays a role in keeping primitive

hematopoietic cells in the G0 state [237]. By inhibiting MP activity, TIMP3 may

normally help retain hematopoietic progenitor cells in the bone marrow and maintain

them in a quiescent state by preventing cleavage of SDF-1. In such a case the loss of

TIMP3 could accelerate this release.

TIMPs influence hematopoiesis via MPi and non-MPi mechanisms

TIMP3 may have functional properties outside of its role as a metalloproteinase inhibitor

to influence blood cell development. Even before discovery as a protease inhibitor,

TIMP1 was characterized as a leukocyte factor that promotes the expansion of erythroid precursor cells [110] [111] [112], (initially identified using an in vitro assay adapted from Iscove et al. [238]). The basis for the ability of TIMP1 to double erythroid growth remains unknown, though it has been established it is separate from its MMP inhibitory

activity [239] and that the effect can be seen in vivo [240]. Subsequently, TIMP2 has

also been shown to have similar erythroid-promoting ability in vitro [113]. As of

recently no study has been published exploring any ability TIMP3 may have to affect

erythroid growth.

TIMP3 could potentially influence hematopoiesis indirectly via TNF or IL-6 regulation.

TNF triggers expression of the hematopoietic factor M-CSF [241], which is necessary for

the development of monocytes. Chapters 3 and 4 of this present work show TIMP3

moderates TNF signalling in inflammation and ischemic stress, thus TIMP3 could alter

hematopoietic development by regulating TNF signalling. In addition, we found that IL-

6 levels are dysregulated in the Timp3-/- mice, and it is known that TACE can target the

IL-6Rα. Several hematopoietic growth factors trigger the expression of IL-6, which then promotes differentiation towards a particular lineage [242].

Rationale for the Thesis

The discovery that TNF is cleaved to a soluble form by a metalloproteinase [243], and that this metalloproteinase is inhibited by TIMP3 [146] opened up a new avenue of research in the field of metzincins and TIMPs. This thesis represents some of the initial investigations into the physiological importance of this relationship, by exposing Timp3-/- mice to infectious and inflammatory conditions that normally elicit or involve TNF responses. In Chapter 3 I examine the role of TIMP3 in systemic inflammation, demonstrating that the loss of TIMP3 exacerbates the severity of the innate immune response to LPS, as well as increasing the expression of a key activator of the acute phase response, IL-6.

In Chapter 4 I investigate the mechanisms by which loss of TIMP3 increases the inflammatory response to LPS. I conclude that despite the importance of the acute phase response it does not appear to be central to the effects observed in Timp3-/- after LPS

treatment. The chapter also explores the relative activity of TACE in two major organs

of TNF in response to LPS, and the activity in a third organ, the heart, in response to

ischemic stress. I also show that there are subtle signs of increased inflammation in

unchallenged livers in the Timp3-/- mice. Finally, I show that the effects of LPS on the

Timp3-/- mice can be ameliorated with synthetic inhibitors of metalloproteinase, and that,

surprisingly, TACE inhibition may not be the only metalloproteinase that is key to the

Timp3-/- susceptibility to LPS.

Chapter 5 expands the range of challenges I used to explore the role of TIMP3 in inflammation and immunity. Challenges include a chemical immunogen that normally promotes delayed-type hypersensitivity; inoculation with the virus LCMV; and hematopoietic ablation by the antiproliferative drug 5-FU. Collectively these chapters underscore the role that TIMP3 plays directly and indirectly in regulating TNF, other

cytokines, leukocyte expansion and local and systemic inflammation, and points to new

directions future research should take in this field.

Chapter 2: MATERIALS AND METHODS

Mice. All animal experiments were undertaken according to Canadian Council of Animal

Care guidelines; protocols were reviewed and approved by the Animal Care Committee

of the Ontario Cancer Institute. Timp3-/- mice were previously generated in our lab [244]

and were backcrossed at least seven times into the FVB or C57BL/6 background. FVB

and C57BL/6 strains originally obtained from Jackson labs. Timp3-/- typing used the

following primers: wild-type primer: agt tgc aga agg cat cct ggg gat ggc t; mutant

primer: cca aat taa ggg cca gct cat tcc tcc ca; and a shared reverse primer: caa gaa tct tct

tct, ccc, gct, tct, ccg ctt, with annealing at 68° 35 cycles; wild-type band ~380 bp, mutant

band ~280 bp.

P55-/- animals (C57BL/6 background) were provided by Jackson Labs from a strain

generated in the lab of Tak Mak at the University Health Network in Toronto [245]. P55

animals were typed by PCR using the following: wild-type primer: tgt gaa aag ggc acc ttt

acg gc; mutant primer: att cgc caa tga caa gac gct gg; common primer: ggc tgc agt cca

cgc act gg. PCR of p55-/- generated a 300 bp product, wild-type mouse PCR generated a

470 base pair allele, under the following conditions: hot start, annealing at 63°, 30 cycles.

Timp4-/- mice were kindly provided by Ilpo Koskivirta, generated in the lab of Eero

Vuorio, Department of Medical Biochemistry and Molecular Biology, University of

Turku, Finland and backcrossed at least 7 times into FVB background. Timp4-/- typing used wild-type primer: ccc gtc cag ggg gcc aca gca aa; mutant primer: tcc tcg tgc ttt acg

gta tc; and a shared reverse primer: act tcc caa ctg ggt ttg ttg gtc tgg tca ga. Primers were

annealed at 58° for 35 cycles, wild-type band ~300 bp, mutant band ~470 bp.

Reagents Heat-killed Bordetella pertussis (2 x 109 cells i.p.; Lee Laboratories, Grayson,

GA) was administered intraperitoneally (i.p.) concurrent with subcutaneous injection of methylated bovine serum albumin (mBSA) and Freund’s complete adjuvant (Life

Technologies, Inc., Gaithersburg, MD) in an experiment to generate immune sensitivity to the mBSA [246].

LPS, obtained from Sigma, serotype 0111:B4 (catalogue # L-2630), was reconstituted from lyophilized stock to 10 mg/mL in sterile PBS. Thawed stock was injected undiluted using a 250 µL glass Hamilton Syringe (part # 81101). LPS was injected i.p. into mice for a total of 100 µg into C57BL/6 or 200 µg into FVB mice, with two exceptions: in

Chapter 3 Figure 4C mice received 600 µg; and in Chapter 4 Figure 10 animals received

180 µg, as indicated in the figures. Blood was collected by tail-vein bleeds at indicated time-points or from heart puncture when obtained from animals at time of sacrifice.

Blood was allowed to sit for 20 minutes at room temperature to allow clotting before serum was isolated using Becton Dickinson serum separator tubes (catalogue # 365956).

ELISAs. TNF, p75/TNFR2 and IL-6 ELISA kits were from BD Pharmingen catalogue numbers 558874, 558857 and 555240 respectively. In all cases the assay diluent was

PBS with 10% sterile FBS stored at 4°, the wash was PBS with 0.05% Tween-20 stored indefinitely at room temperature and the coating buffer was 0.1% Sodium Carbonate pH

9.5 kept indefinitely at room temperature. P55/TNFR1 ELISA was from R & D Systems

using the following reagents: anti-mouse TNFR1 antibody for capture, biotinylated anti- mouse soluble TNFR1 antibody and recombinant mouse soluble TNFR1 catalogue numbers MAB425, BAF425 and 425-R1 respectively. Serum was diluted 25x for TNF

ELISAs, 250x for p55/TNFR1 ELISAs and 1000x for p75/TNFR2 and IL-6 ELISAs to obtain maximum values well within standard curves. Procedures for all ELISAs followed manufacturers’ directions unless otherwise noted.

Statistical Analysis. Statistical differences in TNF levels between Timp3-/- and control

animals were calculated using two-tail Student’s t-test. Differences in the kinetics of

serum protein levels were analyzed by two-way ANOVA, using GraphPad Prism

(p<0.05). The area under the curve was calculated by the trapezoid rule, using GraphPad

Prism. Survival differences were calculated using the Fisher Exact Test. All error bars

indicate standard error of the mean.

Tissue homogenate for Western blots was prepared in RIPA buffer with the following

recipe: 0.1% SDS, 1% Deoxycholate Na, 1% Triton X-100, 1% Nonidet P40, 50 mM

Tris-HCl pH 7.4, 150 mM NaCl, and included the following inhibitors: 5 mM EDTA,

Complete Mini EDTA-free protease inhibitor cocktail (Roche, cat # 11836170001), 1

mM NaF and 1mM PMSF.

Homogenate for TACE assay was prepared in 1% Triton X-100 (a mild non-ionic

detergent chosen to minimize disruption of enzyme activity), 10mM Tris-HCl pH 7.4,

and Complete Mini EDTA-free protease inhibitor cocktail (Roche, cat # 11836170001),

experiment.

All tissues were homogenized using a hand-held sonicator in 1.5 mL Eppendorf tubes

cooled in ice or ice-water. Tissue was stored at -70° before use. Pieces of spleen

between 3 and 50 mg in size were lysed in 20 µL/mg buffer to tissue; aliquots of

homogenate were diluted 50x for protein quantification. For liver samples 50 to 100 mg

of tissue were lysed in 5uL/mg buffer to tissue; aliquots of homogenate diluted 80x for protein quantification.

Protein quantification was performed using the Detergent Compatible (DC) Protein

Assay Kit (Bio-Rad catalogue # 500-0116) based on the Lowry technique (Lowry, NJ

Rosebrough, AL Farr, RJ Randall - Journal of biological chemistry, 1951).

The TACE assay used a fluorescence-quenching substrate for TACE originally developed by Jin et al. [247] provided by Peptides International (catalogue # SDP-3818-

PI). Briefly the substrate is a peptide with the fluorophore o-aminobenzoyl (Abz) attached to one end and the fluorescence-quenching dinitrophenyl diaminoproionic amide

(Dpa) group at the other. The peptides of substrate Abz-AQAVRSSSR-Dpa correspond to human TNF amino acids 74-82. The corresponding mouse TNF sequence is

AQTLTLRSSSQ. We ensured that the mouse TACE enzyme could cleave the fluorescent substrate by comparing recombinant mouse TACE (R & D Systems catalogue

# 2978-AD) with recombinant human TACE (R & D Systems catalogue # 930-ADB).

Interestingly the mouse recombinant TACE was, by weight, more than 3x more effective at cleaving the substrate than the human recombinant TACE, even though the substrate was based on the human TNF sequence; both TACE enzymes were inhibited by

recombinant human TIMP3 (data not shown). The substrate was used according to the

manufacturer’s suggestions [247] with the following modifications: for greater accuracy

comparisons were made of the rate of change of florescence during the linear phase of

enzyme activity (typically 0-2000 seconds) rather than of absolute levels of fluorescence

after 1 hour. NaCl was eliminated from the TACE reaction buffer because salt

powerfully inhibits the TACE enzyme activity, as stated in the rhTACE R&D data sheet

http://www.rndsystems.com/pdf/930-adb.pdf ; independently we have confirmed the

inhibitory effects of NaCl, (data not shown). Samples were read on a SpectraMax M5 microplate reader (Molecular Devices) using black 96-well plates (Perkin Elmer Life

Sciences OptiPlate-96F catalogue # 6005270), excitation 320nm, emission 420nm.

Samples were analyzed in duplicate. Analysis began within two minutes of addition and

mixing of the reagents and read every 1 minute for 5 hours, well beyond the linear range

of enzyme activity. Calculation of the rate of enzyme activity used the first 25 or 30

minutes of readings, during the linear phase. Each type of tissue and each recombinant

protein was titrated to ensure the final concentration used was in a linear range, i.e. that

half or double the amount of tissue or recombinant protein halved or doubled the rate of enzyme activity, different tissues giving dramatically different results. For example 1.5

µg of kidney tissue was only slightly less effective at cleaving the TNF peptide substrate

as 100 µg of liver tissue (28 RFU/min versus 33 RFU/min).

Western blot analysis antibodies against pSTAT3, pJNK and JNK were from Cell

Signaling, catalogue #s 9131, 9251, and 9252 respectively. Silver staining of gels to

compare relative amounts of total protein used Silver Stain Plus from Bio-Rad (catalogue

# 161-0449).

Though not presented in the thesis it should be mentioned that several antibodies against mouse TIMP3 were tested but failed negative control tests against Timp3-/- tissue. These included two anti-human antibodies, one from Fuji, catalogue # AF-8502; and one from

Chemicon, catalogue # MAB3318; and an anti-mouse antibody from the lab of Roy

Black, all of which showed bands in the Timp3-/- tissue homogenate. In addition

Millipore’s AB19027, an anti-TACE antibody raised against a human TACE peptide and advertised to cross react with the mouse protein, showed only non-specific bands in

Western blots of mouse kidney tissue, while showing no band at all in a lane with 100 ng of recombinant human TACE (data not shown).

Quantitative PCR RNA samples were isolated from 12 to 30 mg of frozen tissue homogenized in 500 µl of Trizol (Invitrogen). Samples were ground in Kimble Konte’s

1.5 mL tubes with pestles, catalogue # 749520-0090, with the tubes sitting in wet ice to preserve RNA. After purification following the Trizol protocol, RNA pellets were resuspended in 30 µL DEPC water and treated with Ambion’s DNAse kit, catalogue #

1906, using 1 µL DNAse I according to the kit protocol. After DNAse inactivation samples were quantified on an ND-1000 NanoDrop Spectrophotometer. 1 µg of RNA was then used to make cDNA with the following procedure: RNA plus 2 µL random hexamers (Amersham Pharmacia, catalogue # 27-2166-01) plus water to a final volume of 11 µL, incubated at 70° for 10 minutes. This was then treated with Superscript II reverse transcriptase (Invitrogen catalogue #18064-022) according to kit instructions to make 1 µg cDNA in 20 µL, an aliquot of which was then diluted 100x for the PCR

reaction. All samples loaded in triplicate in 384 well plates. Samples were run and read

on an ABI Prism 7700 sequence detection system from Applied Biosystems. A standard

curve for each gene was generated to ensure primers and reagents were functioning

properly for every plate. Template for the standard curves was wild-type mouse brain

RNA processed as above, diluted to provide 6 logs of concentration from 20 to 0.625 ng

cDNA. Sample results were plotted on each standard curve and expressed as cycle

thresholds (CT), i.e. number of cycles required for each sample to reach the same stage of

fluorescence in the exponential phase of expansion. Real-time PCR can provide either

absolute or relative quantification of gene expression. We used relative quantification,

comparing genes of interest with a reference gene in the same sample. This provides

greater reliability, normalizing for any differences in loading. Target gene expression

levels were quantified relative to 18S ribosomal RNA as the reference gene. Due to the

abundance of 18S in the cell only 1 µL of the diluted cDNA was used per well, versus

5µl used for each well of the target gene. Final results were CT values of target genes

divided by 18S CT values. The following primers were used: SOD2 primer set and probe and LBP primer set and probe purchased from Applied Biosystems, assay ID #s

Mm00449726_m1 and Mm00493139_m1 respectively. ADAM17/TACE forward

primer: 5’-AAGTGCAAGGCTGGGAAATG-3’; reverse primer: 5’-CACACGGG

CCAGAAAGGTT-3’; probe: 5’-CCTGCGCATGCATTGACACTGACAAC-3’. All

probes were labelled with the fluorophore 6-carboxyfluorescein (FAM) at the 5’ end and

the quencher tetramethylrhodamine (TAMRA) at the 3’ end.

Alanine aminotransferase (ALT) levels in serum were analyzed by Vita-Tech (now

IDEXX Laboratories) from tail bleeds 3 hours after i.p. injections of 200 µg LPS.

Synthetic metalloproteinase inhibitor AG3340, obtained from Agouron

Pharmaceuticals (now part of Pfizer) was administered in vivo i.p. 90 minutes prior to

LPS treatment, following the protocol of Price et al. [248] with the modification that all animals were administered 2.5 mg (a dose approximately equivalent to 100 mg/kg).

PD166793, from Pfizer, previously described as selectively inhibiting MMPs and not

TACE [249] was administered as an oral dose by gavage, 30mg/kg of mouse, three times

to animals: two days before LPS injection, the day before and the day of injection. The

MPi was dissolved in water at 4mg/mL with 0.005g/mL carboxymethylcellulose.

Control animals were gavaged with water plus the carboxymethylcellulose. For in vitro

analysis both synthetic inhibitors were dissolved in DMSO.

Heart effluent was collected during isolated heart perfusion with 1 mL of PBS delivered

antegrade through the aorta.

Nitric oxide levels in the heart perfusate were inferred from levels of nitrite, a stable

primary breakdown product of nitric oxide. Nitrite was analyzed using the Griess

Reagent System (Promega product # G2930). Briefly, triplicates of 50 µL of each

exudate was incubated with 50 µL of Griess reagent A (sulfanilamide) followed by 50 µL

of Greiss reagent B (N-1-napthylethylenediamine dihydrocholoride) and absorbance was read at 550 nm and compared to a standard solution of nitrite ranging from 100 µM to

1.5625 µM (i.e. the original concentration and 6 two-fold serial dilutions).

Peripheral blood cell concentrations were analyzed from ~25 µL tail bleeds of female

wild-type and Timp3-/- FVB mice. Animals were i.p. injected with 150 µg 5-FU per gram

of mouse; the 5-FU was first dissolved by heating to 65° for 10’ in PBS at a

concentration of 15 mg/mL. Tail bleeds were drawn into EDTA-coated capillary tubes

(Drummond catalogue # 1-000-2000) and analyzed with a Beckman Coulter AcT Diff

analyzer with a veterinary software card.

Skin challenges employed 2,4-dinitrofluorobenzene (DNFB) from Sigma, catalogue #

D1529. Initial exposure was 25 µL of a 0.5% solution of DNFB diluted in a 4:1 mix of

acetone to olive-oil applied to the shaved abdomen of anesthetised mice; for animals that

received a second exposure 20 µL of a 0.2% solution of DNFB, diluted as before, was

applied to back of the right ear of each mouse 5 days after primary exposure [250], and the left ear received 20 µL of the acetone/olive-oil diluent alone. Differences between left and right ear swelling was measured using digital thickness callipers (Mitutoyo product # 547-300) 24 hours after secondary application of DNFB.

Memory cytotoxic lymphocyte analysis was initiated with 50 µL of the murine virus

LCMV-Armstrong, with a titre of 30 000 PFU/mL, injected into the right rear footpad of

Timp3-/- and wild-type C57BL/6 mice. The Armstrong strain causes acute infection and

local swelling [251]. Footpad sizes were measured in triplicate at day 0 and daily from

day 4-21 following injection. After 21 days animals were killed and splenocytes re-

stimulated in culture and allowed to expand for 5 days. Specifically, splenocytes were

plated in triplicate in 24-well plates, 3 million cells per well in 2 mL Iscove’s media with

10% FBS and stimulated with one of two peptides. Cells received either GP33, a peptide

from the GP protein of LCMV that is one of the major epitopes of the virus, restricted to

the H-2Db MHC receptor; or a control peptide AV, an epitope with similar affinity for the

H-2Db receptor selected from an adenovirus [252]. Peptides were added for a final

concentration of 10-6M, incubated at 37° for 1 hour and washed. Cells were grown with

or without IL-2 (5U/mL recombinant mouse protein), however the exogenous IL-2 had

no effect in the following assay, therefore the IL-2 data is not shown. After the 5 days

the re-stimulated cells were tested for cytotoxic activity in a chromium release assay in

the following manner. Cells were tested from two separate 24 well plate wells for each

condition. All but 500 µL of media was removed from the wells of the re-sensitized cells, the wells were then triturated and 50µL from each well was added to the first row of 96-well plates and threefold serially diluted down each column. The 96-well plates were previously plated with 104 target EL-4 cells per well in 100 µL of media. The EL-4

cells were incubated beforehand for 2 hours at 37 ° with 400 µCi/mL of 51Cr

(PerkinElmer) and either GP33 or AV, as above and washed 3 times [253]. The re-

sensitized effector cells were incubated with the EL-4 cells for 5 hours at 37° to allow

lysis of the target cells. 70µL of the media was then removed from each well and

counted using a Wallac Wizard counter (Perkin Elmer).

To study hematopoiesis in the bone marrow 150 mg/kg of 5-FU was injected i.p. into 5

wild-type and 5 Timp3-/- female C57BL/6 mice, i.e. for a 25 g mouse we injected 250 µL

of a 15 mg/mL solution previously dissolved in PBS by heating at 65° for 10 minutes.

This dose corresponds to one used in an earlier study [254]. Animals were killed with

cervical dislocation and the bone marrow of the fibulas, tibias and the humeri was flushed

with HBSS + 2% FCS + 10 mM HEPES. Bone marrow cells from the six bones were

pooled, triturated to separated cells and diluted to 10 mL. A small aliquot was incubated

in AfCS, a red blood cell lysis buffer, and the white blood cells were counted in triplicate with a hemocytometer. Primitive hematopoietic cells were isolated using the Miltenyi lineage cell depletion kit for mouse (catalogue # 130-090-858). The kit employs streptavidin coated magnetic beads which bind via the streptavidin to biotin-labelled antibodies against six antigens expressed by terminally differentiated hematopoietic cells and their committed precursors. These were: anti-CD5, which detects mature thymocytes, T cells and B cell precursors [255] [256], it is worth noting that double negative T cells, which are an immature form of T cell, expresses very little CD5 [257]); anti-CD45R (B220) which targets B cells [258]; anti-CD11b (Mac-1) which targets neutrophils, NK cells and monocytes [259]; anti-Gr-1 (Ly-6G/C), which detects granulocytes and early monocytes [260]; the antibody 7/4 which binds to neutrophils

[261]; and finally anti-Ter-119, which targets erythroid cells [262]. The reagents were used according to the manufacturer’s protocol and the cells passed through Miltenyi MS columns (catalogue # 130-042-201). For the unchallenged mice the flow-through, known as lineage negative cells, was typically 3% of the total WBC count of the bone marrow.

An aliqot of lineage positives cells was subsequently recovered by washing the column in

PBS in the absence of the magnet. These cells were to use in calibrating the FACS

machine, typically 1 million cells per staining condition.

Cells were stained with Hoechst dye 33342 (Sigma-Aldrich catalogue # B2261). The

powdered Hoechst dye was previously resuspended in 1 mg/mL water, filter sterilized

and frozen in small aliquots. Staining procedure followed the protocol of Goodell et al

1996 [263] with details listed on the Margaret Goodell lab website

6 http://www.bcm.edu/labs/goodell/protocols/goodell_hoechst.pdf . Specifically, cells were resuspended at 10 cells per mL in DMEM + 2% FCS + 10 mM HEPES buffer. Hoechst was then added to a final concentration of 5 µg/mL, i.e. diluted 200x. Cells were immediately incubated in a

37° waterbath for 90 minutes and immediately placed on ice. Samples are kept cold for the remainder of the experiment to prevent the Hoechst dye from leaking out of the cells.

Leakage would falsely increase the number of side-population (SP) cells, i.e. those cells that actively exclude the dye. Before addition of the Hoechst, for each FACS session, a small aliquot of the Lin- cells was treated with verapamil (Sigma). The verapamil inactivates calcium-dependent pumps that remove the dye from SP cells. Disappearance of the side population in verapamil-treated cells confirms that the side population consists of active pumping cells only. Verapamil is dissolved in 95% ethanol at 5 mM as a 100x stock and added for a final concentration of 50 µM; treatment was done for all FACS session for which there was an adequate number of Lin- cells, which included all but day

3, post-5-FU exposure. After Hoechst staining cells were washed once and labelled with cKit-FITC and Sca-1-PE antibodies (Pharmingen) (0.5 and 1.0 µl of each antibody respectively added per million cells, or per tube, when cell numbers were less than 1 million). To calibrate the FACS machine Lin+ cells were stained with B220-FITC and

GR1-PE (0.5 and 0.2 µl of each antibody respectively added to each tube) as well as with

Hoechst. After antibody staining cells were incubated at 4° for 30 minutes and washed in

PBS. Immediately prior to FACS analysis 0.2 µl of 1 mg/mL propidium iodide was added to the cells, final concentration ~2 µg/mL, to stain for dead cells. Dead cells were gated out first; then cells were gated for cKit+ Sca-1+, therefore all FACS figures are cKit+, SCA-1+, PI- and the final display is of Hoechst emission in two channels. The

Hoechst is excited at 350 nm and viewed through a 450/20 BP filter (Hoechst Blue) and a

675 EFLP filter (Hoechst Red). Cells were analyzed in a Becton Dickinson LSR six colour flow cytometer. Samples were chilled during FACS analysis with ice-water pumped through a sleeve surrounding the tubes to keep the cells cooled at all times. No side population was detectable in any cells treated with verapamil (data not shown).

Estimates of total numbers of KSL-SP and KSL-MP cells from the bone marrow were calculated by multiplying the populations in regions P4 and P5 of the dot plots by the following ratio (total number of Lin- cells)/(total number of events recorded by FACS).

In the KSL experiment the total number of events recorded by FACS were as follows: wild-type, days 0,3,6,9: 863,772; 81,986; 263,748; and 610,211 respectively; Timp3-/- days 0,3,6,9: 624,876; 76,628; 299,000; and 713,357 respectively.

Reproducibility and variation of results was as follows: treatment with the broad MPi

AG3340 was performed twice, the result shown (Chpt. 4, Fig. 8) being a compilation of the two independent experiments. Treatment with the MMP inhibitor (Chpt. 4, Fig. 10) was performed once. LPS experiments with Timp3-/- C57BL/6 mice crossed with p55-/-

C57BL/6 animals were performed twice, the results presented being a compilation from the two independent experiments (Chpt. 3, Fig. 5). The kinetics of serum levels of cytokines and receptors (Chpt. 3, Figs. 2, 3) in LPS treated wild-type and Timp3-/- mice were performed twice. Skin response to the irritant DNFB (Chpt. 5, Fig. 3) was performed five times, three times showing a marked difference in the erythematic appearance between genotypes, and twice showing little or no difference.

Chapter 3

TIMP3 Regulates TNF-Dependent Systemic Inflammation

A version of this chapter appeared as Smookler DS, Mohammed FF, Kassiri Z, Duncan GS, Mak TW and Khokha R Tissue inhibitor of metalloproteinase 3 regulates TNF- dependent systemic inflammation. Journal of Immunology 2006 Jan 15;176(2):721-5.

Figure 1 was originally published as Mahmoodi M, Sahebjam S, Smookler D, Khokha R, Mort JS. Lack of tissue inhibitor of metalloproteinases-3 results in an enhanced inflammatory response in antigen-induced arthritis. American Journal of Pathology 2005 Jun;166(6):1733-40.

Generation of Timp4-/- mice and analysis of their response to LPS (Figure 7) was performed by Ilpo Koskivirta. Mice generated in the lab of Eero Vuorio, Department of Medical Biochemistry and Molecular Biology, University of Turku, Finland.

ABSTRACT

Host response to infectious agents must be rapid and powerful. One mechanism is the release of presynthesized membrane-bound TNF. TNF shedding is mediated by TNF- alpha Converting Enzyme (TACE), which is selectively inhibited by the tissue inhibitor of metalloproteinase 3 (TIMP3). We show that loss of TIMP3 impacts innate immunity, dysregulating cleavage of TNF and its receptors. In Timp3-/- mice LPS causes serum

levels of TNF and its receptors to rise more rapidly and remain higher compared with

wild-type mice. The altered kinetics of ligand and receptor shedding enhances TNF

signalling in Timp3-/- mice, indicated by elevated serum IL-6. Physiologically, Timp3-/-

mice are more susceptible to LPS-induced mortality. Ablation of the TNF receptor gene

TNFR1/p55 rescues Timp3-/- mice from the altered response to LPS. These effects are specific to TIMP3 loss, in that ablation of a similar protein, TIMP4, causes no change in

LPS response. Thus, TIMP3 is essential for normal innate immune function.

INTRODUCTION

More than any other cytokine TNF is central to the initiation and orchestration of

inflammation. It directs circulating leukocytes to sites of infection by inducing selectin

expression on endothelial cells and integrin expression on leukocytes. It promotes

dendritic cell migration to lymph nodes and leukocyte migration to infected tissue via the

induction of chemokine expression. It is involved in the formation of granulomas around

unresolved infections. TNF also triggers clots in microvasculature, which is important

for the containment of local infections and can promote cell growth, survival or death

[264]. Excessive or prolonged release of TNF underlies many human diseases. Although

the pathophysiological importance of TNF is well illustrated by the success of anti-

inflammatory therapies that rely on binding to and inactivating this cytokine [265],

limitations in current therapies still remain. Understanding all aspects of TNF regulation

is thus critical for developing novel therapies to control TNF activity.

TNF is expressed as a membrane-bound molecule and is released from the cell surface by proteolytic cleavage. Membrane-bound and cleaved TNF have distinct physiological effects as demonstrated in models of heart disease, arthritis, and systemic shock [266-

268]. Biochemically, p75 is more easily activated by membrane-bound TNF, whereas p55 readily responds to soluble TNF [268]. The TNF receptors, like TNF itself, are subject to proteolytic cleavage and this process has also proved to be physiologically important. Prevention of p55 proteolysis promotes liver inflammation and enhances sensitivity to septic shock [269]. The major metalloproteinase responsible for TNF cleavage is a disintegrin and metalloprotease (ADAM) known as ADAM17 or TNFα- converting enzyme (TACE) [82], which also cleaves both TNF receptors [85, 89].

Additionally, TACE processes a number of other molecules involved in immunity and inflammation [85, 270, 271]. TACE can swiftly alter the availability of TNF by cleaving it from myeloid and T cells, allowing the shed molecule to diffuse and act on the surrounding tissue, vasculature and at distant sites. Thus the regulation of TACE may be an important checkpoint for the magnitude of an inflammatory response.

RESULTS

Inactivated bacteria induces greater TNF release in Timp3-/- mice

Timp3-/- animals were given heat-killed Bordetella pertussis bacteria as an adjuvant to enhance the response to an antigen in a model of induced arthritis [246]. B. pertussis is a

Gram-negative bacterium, meaning it is a member of a large class of bacteria that do not retain the crystal violet dye used in the Gram stain test. B. pertussis, like all Gram- negative bacteria, carries the endotoxin LPS, as well it carries the pertussis toxin; these contribute to a powerful inflammatory response [272]. One of the features of an inflammatory reaction is the appearance of TNF in the blood. We hypothesized that the killed bacteria would elicit greater levels of soluble TNF in response in the Timp3-/- mice,

since TIMP3 is an established biochemical inhibitor of TACE, and TACE can transform

the membrane-bound TNF into a soluble protein. One hour after injecting the killed B.

pertussis bacteria, we found that serum levels of TNF in Timp3-/- mice were

approximately twice the values in the wild-type mice (Fig. 1).

Subsequent experiments used LPS, which is the major inflammatory component of gram

negative bacteria and is a conventional trigger for inflammation mediated by an innate

immune response. The kinetics of serum cytokines were dramatically altered in Timp3-/- mice after LPS injection. Normally soluble TNF levels appear and disappear over a period of hours following LPS challenge. Blood was collected from groups of mice at staggered intervals, with repeat bleedings every hour per mouse so that serum was available at 20-minute intervals over 190 minutes after LPS injection. Baseline levels of

TNF were undetectable in both Timp3-/- and wild-type serum. In wild-type mice, TNF

66 8 Timp3+/+ -/- 6 Timp3

TNF ng/mL 2

Figure 1. Serum levels of TNF are higher in Timp3-/- mice after B. pertussis exposure. levels in wild-type and Timp3-/- BL6 mice were measured in blood removed 1 hour after immunization with 2x109 heat killed B. pertussis, Freund’s complete adjuvant and the antigen mBSA. TNF levels are significantly higher in Timp3-/- mice; n=15 for wild- type mice, n=12 for Timp3-/- mice p<0.01.

levels peaked sharply at 90 minutes, with a second attenuated peak at 150 minutes. In

contrast, TNF levels rose more rapidly in Timp3-/- mice and failed to return to the

baseline levels during this time. Overall, the serum TNF levels remained elevated by

~35% over the 3-hour period, as determined by the area under the curve (Fig. 2A). These

data suggest that TIMP3 deficiency leads to accelerated TNF shedding and higher soluble

TNF levels in response to LPS, presumably due to increased proteolysis by enzymes normally inhibited by TIMP3.

Biochemical studies show that in addition to cleaving TNF, TACE converts membrane-

bound TNF receptors to their soluble forms [85]. Both TNFRs are shed in response to

LPS, and p55 release is considered essential for normal down-regulation of the

inflammatory response [269]. Analysis of serum from LPS-stimulated mice revealed

consistently elevated levels of both TNFRs over the course of 190 min following LPS

injection in timp3-deficient mice (Fig. 2, B and C). The temporal pattern of soluble p75

mirrored that of p55 in Timp3-/- mice, showing similar dysregulation compared with wild-

type mice. Thus, TIMP3 deficiency also affects the shedding of both TNF receptors.

Higher serum TNF increases TNF signalling, whereas greater shedding of TNF receptors can reduce TNF signalling by either binding soluble TNF or reducing the number of intact receptors available for this cytokine. Since Timp3-/- mice have increased shedding

of both the TNF ligand and its receptors following LPS stimulation, we sought to

determine the net outcome for TNF signalling. Serum IL-6 levels were used as a measure of TNF signalling, since the rise in IL-6 in response to LPS is partially TNF-dependent

[273, 274]. We measured the levels of IL-6 in the serum from the same timed bleed

68 A Timp3+/+ 35 Timp3 -/- 30 25 20 15

TNF ng/mL 10 5 0

250 200

150 100 p55 ng/mL 50 0

250

200 150 100 p75 ng/mL 50 0 10 30 50 70 90 110 130 150 170 190

LPS minutes

Figure 2. Loss of Timp3 alters levels and kinetics of cleavage products of TACE in response to LPS. Timp3+/+ and TIMP3-/- FVB mice were injected with 200 µg of LPS. Values represent protein levels in serum from timed bleeds. Each mouse was tail-vein bled at either 10, 30, or 50' after LPS injection and re-bled at 60' intervals thereafter (n=3 mice/timepoint). A) TNF. B) P55/TNFR1. C) P75/TNFR2. Significant differences were found betwen genotypes in all panels by two-way ANOVA (p<0.05). 1000 Timp3+/+ Timp3 -/- 800

600 400 IL-6 ng/ml

200 0 10 30 50 70 90 110 130 150 170 190

LPS minutes

Figure 3. Loss of Timp3 leads to a 3-fold increase of IL-6 at 150' following LPS exposure. IL-6 levels were analyzed in Timp3+/+ and TIMP3-/- FVB mice after i.p. injection with 200 µg of LPS. Values represent protein levels in serum from timed bleeds. Sample are from the same experiment as in figure 2. Significant differences were found betwen genotypes as tested by two-way ANOVA (p<0.05).

experiment. IL-6 levels were substantially increased in Timp3-/- mice beginning at 70

min, with a 3-fold elevation by 150 min (Fig. 3), demonstrating an overall increase in

LPS-stimulated TNF signalling in Timp3-deficient mice.

Timp3-/- mice are highly sensitive to LPS-induced septic shock

TNF is a principal mediator of the lethal effects of LPS [275]. We therefore exposed the

animals to LPS for a greater period of time to investigate the physiological importance of

increased TNF signalling found in the Timp3-/- mice following LPS challenge. Timp3-/-

mice injected with 200 µg of LPS showed significantly higher mortality than Timp3+/- control littermates (Fig. 4). Only 20% of the Timp3-/- mice survived this dose compared

with 80% or more of the control mice. This increased sensitivity to LPS was gender-

independent, similarly affecting male and female mice (Fig. 4A, 4B). A dose of 600 µg

of LPS was equally lethal for Timp3 null and wild-type mice (Fig. 4C).

A dose of 200 µg of LPS was tested on wild-type, Timp3+/- and Timp3-/- concurrently to

confirm that there was no intermediate effect with the loss of a single allele of Timp3 i.e.

that the heterozygous mice were no more sensitive to LPS than wild-type animals.

Timp3+/- mice showed no signs of haploinsufficiency in this model. There was no

intermediary effect in the heterozygous animals in either the serum levels of IL-6 three

hours after exposure to LPS or in the survival of the animals (data not shown). Both

wild-type and Timp3+/- animals were used as control littermates to Timp3-/- mice in later

studies.

71 A 100 +/- Timp3 n=8 80 60 40 Timp3 -/- 20 n=14

0 0 123456 B

100 +/- Timp3 n=7 80 60 40 % survival -/- 20 Timp3 n=10

0 0 123456 C 100 80 60 40 +/- Timp3 n=7 20 -/- 0 Timp3 n=7 0123456 days

Figure 4. Loss of TIMP3 leads to reduced survival of LPS-induced +/- -/- septic shock. Timp3 and Timp3 FVB mice were i.p. injected with LPS and monitored for signs of morbidity. A) Females injected with 200 µg LPS. B) Males injected with 200 µg LPS. C) Females injected with 600 µg of LPS. Survival was significantly higher in +/- -/- Timp3 versus Timp3 mice at 200 µg LPS in A and B, but equally poor at the higher dose.

P55 pathway is required for heightened inflammation in Timp3-/- mice

LPS-mediated septic shock is not always dependent on TNF signalling. For example, even in the absence of p55 very high doses of LPS can cause septic shock [276]. We asked whether increased susceptibility to septic shock in the absence of TIMP3 is dependent on TNF signalling, or occurs through a TNF-independent pathway. We generated double-deficient Timp3-/-/p55-/- mice in the C57BL/6 background. We first

tested wild-type C57BL/6 for their response to LPS and found this strain to be generally

more sensitive to LPS than the FVB strain. Therefore, a lower dose of LPS (100

µg/mouse) was used for additional experiments involving the BL6 strain. Ablation of

p55 rescued Timp3-/- mice from septic shock; specifically, Timp3-/-/p55-/- double-

knockout mice were significantly less sensitive to 100 µg of LPS compared to Timp3-/- mice (Fig. 5A). As expected, p55-/- mice were completely resistant to LPS-induced shock

at this dose. Furthermore, similar to the FVB mice, we found significantly higher serum

TNF levels (Fig. 5B) in the Timp3-/- C57BL/6 mice (black bar) than in the control mice

(white bar), taken 90 min after LPS injection.

73 A +/- -/- 100 Timp3 , p55 n=4 -/- -/- Timp3 , p55 n=18 80

+/- +/ 60 Timp3 , p55 * n=5 -/- +/ Timp3 , p55 * n=16 40 % survival 20

0 0 12345 days

B Timp3+/* , p55+/*

5 * * -/- +/ 16 Timp3 , p55 * +/ -/- 8 Timp3 * , p55

-/- -/- 17 Timp3 , p55

0 0.5 1 1.5 2 2.5 3.0 relative TNF levels

-/- Figure 5. The susceptibility to LPS of Timp3 mice is dependent on TNF signalling. Ablation of TNF receptor p55 in C57BL/6 mice -/- rescues Timp3 mice from LPS induced septic shock. A) survival of - - -/- p55 mutant, Timp3 mutant and Timp3 / /p55 double mutant mice all in the C57BL/6 background, following exposure to 100µg LPS. Survival of -/- double knock-outs (triangles), is significantly greater than of Timp3 mice (diamonds). B)TNF levels in thes mice at 90 min following LPS injection. * indicates + or - as control groups included +/+ and +/- animals. **p < 0.01.

It has been previously reported that in response to LPS, serum TNF levels are

significantly higher in p55 null mice compared to wild-type controls [277], suggesting this receptor is involved in lowering serum TNF levels. We also found that soluble TNF levels in the Timp3-/-/p55-/- double-deficient mice were significantly higher than those in controls (Fig. 5B, black striped bars vs open bar). IL-6 levels for both Timp3+/- and

Timp3-/- groups dropped when combined with p55 loss, 30% in Timp3+/ mice (p = 0.089) and 43% in Timp3-/- mice (p = 0.024). These data show that an intact TNF signalling

pathway is necessary for the heightened LPS-induced inflammatory response in the

Timp3-/- mice.

Timp4-/- mice do not share the sensitivity to LPS found in Timp3-/- mice.

Since the increased sensitivity to LPS may not be due specifically to the loss of TIMP3,

but to a reduction of the total level of TIMPs in general, we tested the sensitivity to LPS in another TIMP knockout. TIMP4 has been shown in human disease to be upregulated during inflammation [278], making it a reasonable candidate to compare with TIMP3.

As expected, considering TIMP4 does not inhibit TACE, TNF levels were not significantly different between wild-type and Timp4-/- animals after LPS injections (Fig.

6A). More significantly, there was no increase in mortality for Timp4-/- mice, compared

to wild-type mice, in response to LPS (n=13 for both groups) (Fig. 6B). There was no

statistical difference in the survival of the two groups, nor was there any in the rate of

recovery of the surviving mice (Fig. 6C). These data show that the loss of the Timp4

gene does not have the same effect of increasing sensitivity to LPS that the loss of Timp3

caused.

75 A 20 Timp4+/+ Timp4-/- 15

TNF ng/ml 5

0 10 3 5 7 9 1 1 1 0 0 0 0 20 50 80

minutes

B 100

80 Timp4+/+ n=13 60 Timp4-/- n=13 40 % survival 20 0 0 1 2 3 4 5 6 7 days

C 5 4 3 Timp4+/+ n=10 -/- 2 Timp4 n=8 1 day of recovery 0

Figure 6. TIMP4 loss does not lead to increased sensitivity to LPS. A) TNF ELISA of serum taken 10 minutes after 200µl LPS injection and at subsequent 20’ intervals to 90 min, and then 30’ intervals to 180 min. n≥ 3 for all values except Timp4-/- at 10’ (n=1) and wild-type at 50’ (n=2). B) Survival graph of FVB male mice following LPS injection. There is no difference in survival for wild-type versus Timp4-/- mice, using Logrank test. C) Recovery of surviving mice of panel B, defined according to first day animals begin to gain weight. No statistical difference was found in the recovery time between the two groups of animals.

DISCUSSION

Inflammation must be tightly controlled due to its damaging and potentially lethal effect

on the host. An animal initiating an inflammatory reaction balances the need for an

appropriately strong response against the harm it will cause. Nowhere is this as critical as in the measured release of the cytokine TNF. Once TNF signalling is activated, a seemingly irreversible process of events begins to rapidly unfold that have both local and systemic consequences. Considerable research has gone into what activates TNF, what regulates it at the transcriptional level and how it is regulated at the translational level.

Another level of control, the processing of the protein as an external cell-membrane bound molecule, has only more recently been investigated. Reduction of TNF cell- membrane cleavage has been studied using transgenic animals that express uncleavable

TNF, these studies have shown that TNF must be shed to have its full effect as an inducer of inflammation [268].

The converse is revealed here, that excessive cleavage of TNF results in a dramatic increase in the inflammatory response. In this study we show that the loss of TIMP3, an inhibitor of the TNF sheddase TACE, results in an increase of soluble TNF levels in response to gram-negative bacteria. Furthermore, we show that in Timp3-/- mice TNF

signalling is increased in response to LPS, as shown by increased IL-6, and that these

animals are more sensitive to LPS-induced systemic inflammation. LPS is a major

component of gram-negative bacteria and a conventional tool for triggering an innate immune response. While resting serum levels of TNF in Timp3-/- mice are undetectable,

as in wild-type mice, within minutes following LPS injection TNF levels in the Timp3-/- mice rise more rapidly and peak earlier. The rapidity of the response indicates it is pre-

formed TNF that is released into the serum. At 150 minutes a second peak of TNF

appears in the wild-type and Timp3-/- mice, which may be due to freshly transcribed and

translated TNF. Although concurrent, the second peak appears to be higher in the Timp3-/-

mice. Within 70 minutes of the initial stimulation we see a significant difference in TNF

signalling in the Timp3-/- mice; by 150 minutes soluble IL-6 levels in the serum are three

times greater in the Timp3-/- mice than in the wild-type mice. By two days, both wild-

type and Timp3-/- mice showed signs of acute systemic shock (weight loss and lethargy,

data not shown) but only the wild-type mice were able to recover. Additionally, we show

here that the difference in the in Timp3-/- mice is gender-independent, and that a single

copy of the gene is sufficient to maintain a normal response to LPS. This is worth noting as two other disease models from our lab using Timp3-/- mice revealed a haplo-

insufficient phenotype: Timp3+/- mice have decreased heart function after aortic banding

Kasseri et al. [279]; and Timp3+/- mice show delayed onset of tumour growth in two

models of breast cancer (Hojilla et al. unpublished data).

The innate immune system is an ancient response to infection, some elements of which

are shared by all multicellular species [280]. The TIMPs are also an ancient family of

genes, strongly conserved throughout evolution [198]. The loss of TIMP in Drosophila

melanogaster leads to autolysis and premature death in the adult fly [281]. The

Drosophila TIMP can inhibit human TACE, and therefore has some functional similarity

to TIMP3 [198]. TIMP3 is unique among the mammalian TIMPs in its ability to inhibit

TACE, the enzyme primarily responsible for releasing TNF [82, 282]. In this capacity

TIMP3 is ideally situated to protect the organism against an over-active innate immune

response, by inhibiting the wide release of a powerful initiator of inflammation.

The incidence of severe sepsis remains very high in hospitals, and has been steadily increasing by approximately 9% annually in the United States for the last 20 years [283].

Sepsis often leads to a dangerously powerful systemic inflammatory response causing widespread microvascular coagulation and shock. As TNF is implicated in these processes we postulated that TIMP3 is an important regular of the innate immune response to sepsis. Our study investigates an extracellular point of control in acute systemic inflammation, specifically focusing on TNF signalling, which plays a principal role in initiating inflammation. TNF regulation has become a focus for a new generation of anti-inflammatory therapies and we postulate that TIMP3-based therapies can be developed to control inflammation.

Chapter 4

Investigation of the Organ Response to Inflammatory

Challenge in Timp3-/- Mice

Figure 6 was originally published by Fedak PW, Smookler DS, Kassiri Z, Ohno N, Leco KJ, Verma S, Mickle DA, Watson KL, Hojilla CV, Cruz W, Weisel RD, Li RK, Khokha TIMP-3 deficiency leads to dilated cardiomyopathy. Circulation 2004 Oct 19; 110(16):2401-9.

Chapter 4

ABSTRACT

Chapter 3 demonstrated that LPS elicits strikingly different responses in Timp3-/- versus

control mice. The liver plays a major role in orchestrating the response to endotoxin as it

is the major source of LPS-induced TNF and the site of the acute phase response. We

investigated whether TNF shedding and subsequent cytokine activity is altered in Timp3-/-

livers after LPS treatment. Tissue levels of the cytokine IL-6 were significantly higher

although only moderately elevated in Timp3-/- livers following LPS challenge. IL-6 signalling in the liver showed no difference between genotypes, as reflected in comparable STAT3 phosphorylation. TACE activity and expression levels were also comparable in the liver, and no evidence of greater liver injury was seen in Timp3-/-

livers. Analysis of the spleen showed no difference in the Timp3-/- mice in the levels of

p75 or TNF, suggesting no alteration of TACE activity in this organ. There was,

however, a slight but consistent increase in TACE activity in the homogenate of the

Timp3-/- spleens according to a biochemical assay of TACE activity. Use of either a

synthetic broad-spectrum metalloproteinase inhibitor or a synthetic inhibitor generally

considered to be MMP-specific rescued the Timp3-/- from the increased LPS sensitivity.

Our results suggest that the hypersensitivity to inflammatory stimuli found in Timp3-/- mice is not due to a change in any one organ, and is not solely dependent on unrestricted

TACE activity. It is more likely a result of increased reaction to inflammatory signalling

molecules throughout the entire animal.

INTRODUCTION

Endotoxin causes multiple organ failure by compromising the circulation in the body. It

leads to vascular hypotension and leakage; to adhesion of leukocytes to the endothelium

and damage by them to endothelial cells; and it activates the clotting cascade, which can lead to the blocking of small vessels. Despite the generalized nature of the resulting

pathology which affects many organs, the majority of cells initially responsive to LPS are found in a single organ, the liver. The Kupffer cells of the liver are a necessary link in the pathology of systemic inflammatory response syndrome (SIRS); inactivation of the

Kupffer cells, which are the primary source of LPS-induced TNF, is sufficient to completely protect animals from LPS-induced death [284]. The importance of the liver as a source of endotoxin-induced TNF is revealed with partial hepatectomy: when two- thirds of the liver is removed from rats who are then administered LPS, the endotoxin elicits two-thirds less soluble TNF in the blood [285]. Thus, even though LPS-induced death is a multi-organ phenomenon, characterized by disseminated vasodilation and blood coagulation, the origins of the disease can be linked, in part, to a specific organ.

In addition, the liver is the source of the acute phase response (APR), a reorganization of plasma protein expression involving a reduction of normal serum proteins, such as albumen, and the generation of many proteins involved in battling infectious agents, so- called acute phase proteins (APPs). IL-6 is a key inducer of the APPs [286]. As well, levels of IL-6 correlate with the severity of septic shock and the mortality induced by endotoxin [287]. In the previous chapter we revealed a differential response in IL-6 levels in Timp3-/- mice. Here, we investigate whether this difference can be traced to the

liver, whether there is a corresponding change in the APR in the Timp3-/- animals, and

whether activity of the TNF sheddase TACE in the liver is responsible for the Timp3-/- phenotype.

The spleen, along with the liver, is a major site of localization of LPS after its introduction into the body [288]. The role of the spleen in endotoxic injury is unclear, although it is evident that it is secondary to the liver. Early research in the 1970s showed splenectomy of mice could protect against endotoxin-induce death [289]; however, a later paper from another lab showed the contrary, that splenectomy can increase LPS sensitivity [290]. More recent work has demonstrated a surprising role of the spleen as a suppressor of sepsis-induced inflammation, regulated by the parasympathetic nervous system [291]. Thus, the spleen is an important alternative organ to consider in testing whether TACE, its substrates and the signalling pathways they induce are altered in the

Timp3-/- mice.

Finally, TACE has the potential to be active in many organs. We investigated whether

TACE appears more active in the heart as an example of an organ involved in systemic inflammatory response syndrome (SIRS) that is not itself a central regulator of inflammation. As well, to effectively distinguish the importance of this sheddase in the

Timp3-/- phenotype throughout the body rather than in specific organs, we treated wild-

type and Timp3-/- animals with metalloproteinase inhibitors that differentially inhibit

TACE.

RESULTS

Timp3-/- livers show normal production of and sensitivity to IL-6

Kupffer cells are a major source of IL-6. In a comparison of several types of resident macrophages, Kupffer were the most productive of this cytokine [292]. In addition, in a model of haemorrhage-induced trauma, Kupffer cells were shown to be the major source

of IL-6 in response to the treatment [293]. We therefore assessed the level of IL-6 in the

liver using ELISA following LPS challenge. In agreement with our earlier studies [294],

we found a base-line elevation of IL-6 in the tissue of Timp3-/- livers (Fig. 1A). Levels

dropped in both wild-type and Timp3-/- animals ten minutes after stimulation with LPS.

Subsequently, IL-6 levels fluctuate over the next few hours. The levels of IL-6 were

higher in the Timp3-/- liver tissue, however, the differences were not significant at any

one time-point.

Regardless of where the IL-6 originates from, the liver is the primary destination for IL-6.

Injections of radioactively labelled IL-6 into rats show 80% of circulating IL-6 is retained

in the liver [295]. Since the liver is the target of IL-6, we determined whether IL-6

signalling was elevated in the livers of Timp3-/- mice, following LPS challenge. STAT3 is part of the IL-6 intracellular signalling pathway; activation of STAT3 is instrumental in transducing the acute phase response [296], and in fact STAT3 was originally known as

the acute phase response factor (APRF) [297]. Engagement of the IL-6 receptor triggers

phosphorylation of STAT3 whereupon it translocates to the nucleus and acts as a

transcription factor, activating genes for a variety of plasma proteins expressed in

response to acute inflammation(see Introduction, Fig. 8).

84 A 160 Timp3+/+ 140 Timp3-/- 120 g µ 100 80 60

pg IL-6 per 4 40 20

0’ 10’ 30’ 60’ 3hrs B

10’ 30’ 60’ -/- +/+ -/- +/+ -/- +/+ -/- +/+ -/- +/+ -/- +/+ -/- pSTAT3

silverstain

60’ 180’ -/- +/+ -/- +/+ -/- +/+ -/- +/+ -/- +/+ -/- +/+ -/- +/+ pSTAT3 silverstain

C 35 35 Timp3+/+ 30 Timp3-/- 30 25 25

20 20 /18S /18S 15 15 LBP SOD2 10 10 5 5

0 0

Figure 1. Acute Phase Response appears similar in wild-type and Timp3-/- mice. Liver tissue from mice sacrificed at indicated time-points after LPS injection. A) IL-6 levels measured by ELISA from 4µg of liver protein. n=4,5,3,3,5 for wt and 5,5,3,3,4 forTimp3-/- for each successive time-point. 2-way ANOVA indicates significant overall differences between genotypes, but no significant differences between genotypes at any one time-point. B) Phospho-STAT3 Western blot of liver tissue. Note upper blot is over-exposed to allow detection of faint 30’ signal. C) Quantitative RT-PCR of two acute phase protein genes from liver RNA of the above tissue, obtained from livers at 3 hour time-point post-LPS injection; n=5 for wild-type RNA , n=3 forTimp3-/- RNA, p>0.05 between genotypes for both SOD2 and LBP.

After LPS administration the levels of phosphorylated STAT3 steadily increased over 60 minutes (Fig. 1B). The increase, however, was similar between the genotypes. An independent indication of IL-6 signalling is activation of the acute phase response. Real- time PCR was used to assay the transcription levels of two acute phase protein genes,

LPS-Binding Protein (LBP) and MnSOD/SOD2, both known to be activated by STAT3

[298] [299]. We observed no difference between wild-type and Timp3-/- livers in the expression of either of these genes 3 hours after LPS injection (Fig. 1C).

In response to LPS, JNK activation is greater in Timp3-/- livers than in wild-type livers

The stress activated protein kinase, also know as c-Jun NH2-terminal kinase,

(SAPK/JNK) is one of the three MAPK signalling pathways. SAPK phosphorylates and activates transcription factors (such as c-Jun) and cytosolic proteins. SAPK is involved in inflammation, T-cell activation and apoptosis [300]. In contrast to the pSTAT3 levels which showed no relative change in the livers between wild-type and Timp3-/- animals in response to LPS, pSAPK levels are markedly higher in Timp3-/- livers at 10 minutes and

30 minutes following LPS injection (Fig. 2). The difference was transient, disappearing at the 60’ and 3 hour time-points.

Comparison of TNF levels in Timp3-/- versus wild-type livers

In Chapter 3 we charted the kinetics of TNF in serum following LPS challenge (Chapter

3, Fig. 2A). These differences in TNF levels between control and Timp3-/- mice could be due to differences in shedding of the cytokine, or differences in total cytokine production, or a combination. The liver is a major source of TNF production in response to LPS. If

86 LPS 0’ 10’ 30’ 60’ 3hr -/- +/+ -/- +/+ -/- +/+ -/- +/+ -/- +/+ -/- +/+

pJNK total JNK

Figure 2. JNK signalling in Timp3-/- livers increases minutes after exposure to LPS, returning to wild-type levels by 3 hours. Western blots of total and phosphorylated JNK, centred on 50 kD band. At 0 minutes, before LPS exposure, there is little or no phosphorylation of JNK in either wild-type or Timp3-/- liver tissue. Ten minutes after LPS exposure, levels are dramatically higher in the Timp3-/- livers. This increase remains up to 30 minutes. Differences between the genotypes disappears by 60 minutes.

the rapid appearance of serum TNF in Timp3-/- mice was due to accelerated cleavage of

TNF by TACE from Kupffer cells, one may expect to see a drop in TNF levels in liver

tissue in the Timp3-/- mice, relative to the wild-type mice. However, no reduction was

observed, while an increase in TNF production was seen at 60 minutes (Fig. 3A).

TACE activity does not change in liver tissue following LPS exposure in either wild-type or Timp3-/- mice

We used a commercially available synthetic substrate, one based on the TNF peptide

sequence targeted by TACE, to assay TACE activity. The substrate peptide contains a

fluorophore at one end and a quencher at the other and thus is designed to fluoresce after

cleavage by the metalloproteinase. Clearly the substrate is processed by enzymes in the

homogenate of both Timp3-/- and wild-type livers; however, no difference was observed

between genotypes. Further, there was no difference in cleavage after LPS exposure

(Fig. 3B).

Transcription levels of TACE are unaltered in the Timp3-/-mice

We tested whether the loss of Timp3 triggered either a positive or negative feedback to

the expression of TACE. If TACE were under-expressed, it would account for equivalent

TACE activity in the enzyme assay in the absence of the endogenous inhibitor of the

protein. If it were over-produced in response to LPS it would help account for the greater

sensitivity of the Timp3-/- to inflammatory stimuli. However, there was no difference in

the liver tissue (Fig. 3C).

88 A 200 Timp3+/+ 160 Timp3-/- g µ 120

pg 3 TNF per pg 40

0 0’ 10’ 30’ 60’ 3hrs

50 Timp3+/+ -/- 40 Timp3

RFU/minute 10

0 0’ 10’ 30’ 60’ 3hrs

C 4 Timp3+/+ -/- 3 Timp3

/18S 2

TACE 1

Figure 3. TACE appears equally active in wild-type and Timp3-/- livers. Animals sacrificed at indicated time-points following LPS injection. A) TNF ELISA of 3µg of liver protein n=4,5,3,3,5 for wild-type and 5,5,3,3,4 forTimp3-/-. According to 2-way ANOVA there are significant changes over time and significant differences between genotypes, but no significant differences between genotypes at any one time-point. B) Enzyme assay of TACE activity in 100 µg of liver tissue. There is no difference in TACE activity over time or between genotypes. Sample numbers as in panel A. C) Quantitative PCR of liver tissue for TACE expression 3 hrs post-LPS.

Early liver injury does not differ between wild-type and Timp3-/- mice

Increased serum level of alanine aminotransferase (ALT) is a common indicator of liver

injury, as damage to hepatocytes leads to leakage of this cytosolic protein into the

circulation [301]. ALT levels 10 minutes after LPS exposure correspond to known baseline concentrations in mice [302] confirming there is no constitutive ongoing damage

to the livers in the Timp3-/-mice. Three hours after LPS induction ALT levels more than

double (Fig. 4), indicating liver injury. However, the damage did not differ between

control and Timp3-/- livers, suggesting they are equally affected by the LPS at 3 hours.

Unchallenged livers show constitutive activation of inflammatory pathway

Intriguingly, we did find signs of a moderate, ongoing inflammation in the livers of

Timp3-/- in the form of faint but clearly detectable constitutive activation of the STAT3 pathway (Fig. 5A). To determine whether there was any long term effect from this low level STAT3 activation, we examined the livers of aged (1.5 year-old) Timp3-/- mice.

These livers contained foci of white blood cells, particularly around portal veins (Fig. 5B

upper panel). Some foci could be seen in wild-type livers, but these were smaller and far

less frequent than in the Timp3-/- mice (Fig. 5B middle panel). Immunohistochemical

analyses of these white blood cells showed them to be primarily T lymphocytes, both

CD4+ cells and CD8+ [294]. These signs of hepatic inflammation disappeared when

Timp3-/- animals were crossed with p55-deficient animals, suggesting dysregulation of

TNF signalling was responsible for the inflammation in the aged livers (Fig. 5B lower

panel, 5C). The presence of phosphorylated STAT3 in the liver suggests a shift in

90 120 Timp3+/+ 100 Timp3-/-

units/litre 40

0 10 min 3hr

Figure 4. Serum ALT levels do not differ between wild-type and Timp3-/- mice. ALT levels significantly rise after 3 hours in both wild-type and Timp3-/- livers following LPS injection, however there is no significant difference between genotypes at either time- point. n=5,5 for wild-type and 5,4 for Timp3-/- livers for 10 minutes and 3 hours respectively . A T3 -/- +/+ -/- +/+ -/- +/+ -/-

B T3−/− p55+/− 200 µm

50 µm

T3+/+ p55+/−

T3−/− p55−/−

C p55+/ p55-/- Timp3+/ 1/4 ----

Timp3-/- 4/7 0/4

Figure 5. Timp3-/- livers show signs of ongoing low-level inflammation. A) Upper band: Western blot of pSTAT3, lower band: silverstain of corresponding gel, 20 µg protein/well. Young adult Timp3-/- mice display constitutive activation of the STAT3 signalling pathway, undetectable in the wild-type mice. B) The livers of agedTimp3-/- mice (18-19 months old) contain clusters of leukocyte infiltration (upper panel) rarely seen in wild-type mice (middle panel). Clusters are completely absent in the livers of Timp3-/- mice also lacking the TNF receptor p55 (lower panel). C) Fraction of livers found to have high leukocyte infiltration in aged mice with or without the Timp3 and p55 genes.

homeostasis to a chronic low level of inflammation which may be responsible for a

cumulative effect over time resulting in an increase of lymphocytes in the aged liver.

Little differential response to LPS found in spleen

Potentially tissues other than liver could be responsible for the elevated levels of TNF we

found in the serum of Timp3-/- mice. Therefore we tested the spleens of animals after

LPS injection. Spleens were removed at 0’, 10’, 30’, 60’ and three hours after challenge

and analyzed for TNF concentration. Although TNF levels in the spleen rose over time, as they do in serum (Chapter 3 Fig. 2A) there was no difference of TNF between genotypes (Fig. 6A). Note that the concentrations of TNF were considerably less in the

spleen than in the liver. For example, 3 µg of liver tissue had approximately 140 pg of

TNF while 10 ug of spleen contained only about 30 pg at 3 hours. This is in keeping with

the expectation that the liver is the major source of TNF in response to LPS.

TACE activity is moderately increased in the spleens of Timp3-/-mice

TACE activity in spleen was next tested using the fluorescent substrate. There was no

significant difference between genotypes at any single time-point. However, according

to 2-way ANOVA there was a significant difference overall between the genotypes, with

a slight increase in TACE activity in the Timp3-/- spleens. Comparing the genotypes at

individual time-points the differences was never more than 10% (Fig. 6B).

An independent measure of TACE activity is the shedding of p75, therefore spleen tissue of LPS-treated mice was analyzed by ELISA for p75 protein levels. There was no significant difference in the amount of shedding between wild-type and Timp3-/- spleens

93 A B Timp3+/+ 80 40 g Timp3-/- µ

60 30

40 20 RFU/min pg TNF per 10 20 10

0 0 0’ 10’ 30’ 60’ 3h 0' 10' 30' 60' 3h

C D 200 25

20 g

g 150 µ µ 15 100 10 pg IL-6 per 4

pg p75 per 10 50 5

0 0 0’ 10’ 30’ 60’ 3h 0' 10' 30' 60' 3h

Figure 6. Spleens of LPS-treated Timp3-/- mice show slight increase in TACE activity. A) TNF ELISA of 10µg spleen protein. No difference between genotypes. B) Enzyme assay of TACE activity from 100 µg spleen. There is a small (~5%-10%) but consistently greater cleavage in Timp3-/- spleen; significant according to 2-way ANOVA. At three hours TACE activity drops by ~14% in both groups. C) ELISA of p75 in 10µg spleen protein. No difference between genotypes at any time-point. P75 levels significantly drop over time, rising again at 3 hours. D) IL-6 ELISA of 4µg spleen protein. No difference between genotypes from 0 to 60 minutes. Levels are significantly higher in Timp3-/- spleens at 3 hours. For all panels: animals sacrificed at indicated time-points following LPS injection; n=4,5,3,3,5 for wild-type spleens and n=5,5,3,3,4 forTimp3-/- spleens for each time-point respectively. For all panels white bars represent wild-type, black bars represent Timp3-/-.

(Fig. 6C). Both groups showed a steady drop of p75 levels from the tissue for the first 60

minutes following LPS induction, commensurate with a steady increase in soluble p75

levels found in the blood over that time-period (Chapter 3, figure 2C). Overall, aside

from the modest difference in the TACE activity, as measured by fluorescent substrate

cleavage, there is no indication that TACE is more active in the Timp3-/- spleens.

We also examined the level of IL-6 in the spleen. Unlike in the liver (Fig. 1A), IL-6

levels in the spleen were virtually undetectable before LPS induction (Fig. 6D), and

remain so for the first 30 minutes. Even though the levels rise considerably by 3 hours

the levels found in 4µg of spleen never reach more than a fraction of the levels found in

4µg of liver tissue (maximum 20pg in the spleen at 3 hours versus a minimum of 60pg in the liver at 3 hours). The kinetics of IL-6 in the spleen exactly matches that seen in the blood (Chpt. 3, Fig. 3) where levels are initially undetectable and climb steeply after 60 minutes, with significantly greater amounts in the Timp3-/- versus the wild-type serum at

3 hours. Because of the low levels in the spleen, a highly vascular organ, and the

matching pattern to the serum it is possible that source of IL-6 found in the spleen comes

from the blood, rather than originating in the spleen. It should also be noted, however,

that the concentration of IL-6 in the spleen is considerably higher than found in the blood.

The peak of IL-6 for Timp3-/- serum at three hours is approximately 600 ng/mL (Chpt. 3

Fig. 3), whereas the concentration in the Timp3-/- spleen at 3 hours is approximately 20

pg/4µg, equivalent to 5µg/gram (Chpt. 4 Fig. 6D). That is, the concentration of IL-6 in

the spleen is more than eight times greater than in the blood, but as the spleen contains

IL-6 receptors, the splenic tissue may be sequestering IL-6 from the blood.

Timp3-/- hearts subjected to ischemic stress release more TNF than wild-type hearts

We then investigated the inflammatory reaction in the heart, another organ involved in

SIRS. In this case, an inflammatory response was triggered with temporary ischemia rather than LPS. Hearts from mature Timp3+/ and Timp3-/- mice were removed and

compared for soluble indicators of inflammation. By isolating the hearts, we ensured that

any soluble TNF came primarily from the local tissue, and not from leukocytes or from

circulating molecules produced elsewhere in the body. One mL of buffer was injected

through an aortic cannula and the outflow gathered. This exudate, having passed through

the coronary arteries, the capillaries and into the cardiac veins, contained soluble

molecules released by the myocytes and cardiac endothelia. These hearts were still

viable, as confirmed by the resumption of beating minutes later when reperfused with

oxygenated media. Levels of soluble TNF were significantly elevated in the exudates

from ex vivo Timp3-/- hearts compared to control hearts (Fig. 7 left panel). We also determined levels of soluble p75. These were also significantly elevated in the Timp3-/- hearts (Fig. 7, middle panel). Increased soluble p75 levels is an independent indication of

TACE activity, as TACE targets both proteins. The increase of both soluble TNF and p75 suggested TACE is responsible for the increased shedding activity in the absence of

TIMP3 inhibition. In addition, the ischemic Timp3-/- hearts showed signs of increased

nitric oxide activity (Fig. 7, right panel). Nitric oxide production is activated by TNF; it

96 8 300 * 15 * 6 200 10 4

100 mM nitrite 5 2 soluble p75 pg/mL soluble TNF pg/mL TNF soluble 0 0 0

Figure 7. Effluent from isolated Timp3-/- hearts has elevated levels of TACE cleavage products. Soluble p75 and TNF were both elevated >2-fold in Timp3–deficient mice (n=4) compared with age-matched wild-type littermates (n=3, P=0.03 and P=0.05, respectively). In addition nitrite, a break-down product of nitric oxide, was also elevated (P=0.06). Nitric oxide is a downstream product of TNF activation. For all panels white bars represent wild-type, black bars represent Timp3-/-.

is, amongst other things, an inducer of vasodilation [303]. These results suggest that loss

of TIMP3 in the heart is sufficient for an increase in pro-inflammatory activity in an organ not primarily involved in inflammation.

Synthetic metalloproteinases inhibitors protect Timp3-/- mice against LPS-induced

systemic inflammation

TIMP3 inhibits many metalloproteinases including TACE. We intended to study whether

TACE was the primary metalloproteinase involved in the LPS sensitivity of Timp3-/- mice. However, there is no metalloproteinase inhibitor available that exclusively targets

TACE. Therefore, we used a synthetic broad inhibitor of metalloproteinases that includes

TACE as one of its targets, as well as another synthetic inhibitor described as not inhibiting TACE [279]. With these tools we could also investigate whether or not the effect of Timp3 ablation was due to the property of TIMP3 as a metalloproteinase inhibitor, since it has been reported that TIMP3 can also exert effects independent of its role as a metalloproteinase inhibitor [117].

A single dose of the broad-spectrum synthetic metalloproteinase inhibitor (MPi) AG3340 was administered to mice before LPS injection. This treatment completely rescued the

Timp3-/- mice from increased susceptibility to LPS, restoring the incidence of septic shock to levels found in control animals (Fig. 8A). Furthermore when the AG3340- treated Timp3-/- mice were administered LPS (Fig. 8B, black stripes), the level of TNF in

their blood at 90 minutes was comparable to the level in the untreated wild-type mice.

These data suggest that TIMP3 functions as a metalloproteinase inhibitor during the

inflammatory response.

98 A 100

80 +/ MPi + Timp3 * n=10 -/- MPi + Timp3 n=10 60 +/ vehicle + Timp3 * n=9

40 % survival

-/- 0 vehicle + Timp3 n=11 0 12345678 days

B +/ vehicle + Timp3 * 9 ** -/- 11 vehicle + Timp3

+/ 10 MPi + Timp3 *

-/- 10 MPi + Timp3

0 0.5 1 1.5 2 2.5 3.0 relative TNF levels

-/- FIGURE 8. Increased susceptibility to septic shock of Timp3 mice is metalloproteinase-dependent. A) Mutant and control FVB mice were given MPi 90 minutes before i.p. injection of 200 µg of LPS and monitored for signs of -/- morbidity. The MPi treatment completely reversed the LPS sensitity of Timp3 mice. B) Serum TNF levels at 90 minutes from these mice. Values are expressed relative to the control: vehicle-treated Timp3+/ . Number of mice per group is indicated within the bars. * indicates + or - as control groups included both +/+ and +/- animals. ** p < 0.05.

A second synthetic inhibitor, PD166793, characterized as MMP-specific [279], was administered to investigate the necessity of TACE inhibition in rescuing the Timp3-/- mice from LPS-induced injury. We first tested the relative ability of PD166793 to inhibit

TACE compared to AG3340. Homogenate from Timp3-/- tissue rapidly cleaves the

fluorescent TACE substrate. (Fig. 9A, upper line labelled ‘none’; Fig. 9B, black bar).

Cleavage by the homogenate was almost entirely inhibited with the addition of AG3340

(Fig. 9A, lower line labelled ‘AG’; Fig. 9B, white bar). PD166793 was also able to

significantly inhibit the ability of the homogenate to cleave the fluorescent substrate.

However the ability of PD166793 to inhibit cleavage of the TACE substrate was ¼ that

of the broad-spectrum MMP inhibitor (Fig. 9A middle line labelled ‘PD’, Fig. 9B spotted

bar).

PD166793 protected all mice, both wild-type and Timp3-/-, from the toxic effects of LPS

(Fig. 10A); none of the animals given the drug succumb to LPS-induced death. For the

surviving mice another indication of the effect of LPS was weight loss and recovery. The surviving Timp3-/- mice took an average of 5.5 days to recover, whereas the wild-type

mice took only 4 days (Fig. 10B). Pre-treatment with PD166793 accelerated recovery

time to 3 days for both Timp3-/- and wild-type animals. Interestingly, in the two groups

of Timp3-/- mice, i.e. animals given PD166793 versus animals given vehicle alone, the

serum levels of TNF at 90 minutes were no different (data not shown). That is,

PD166973, being a relatively poor inhibitor of TACE, did not reduce the level of soluble

TNF in the blood, and yet it clearly protected animals against LPS. Thus although TACE

100 A

2000 none

1000 PD RFU

AG 0 0 2000 50005000 10,0001000 time in seconds

B 60 50 * 40 30

RFU / min 20 10 0 e D n P M AG M µ µ

kidney alo -/- T3 kidney + 1 kidney + 1

Figure 9. Metalloproteinase inhibitor PD166793 is considerably less potent at inhibiting TACE activity than a general MPi. 3 µg of kidney protein from an untreated FVB Timp3-/- mouse was tested for TACE enzyme activity under three conditions: by itself; with 1 µM AG3340; or with 1µM PD166793. A) Graph of relative fluorescence for kidney alone (labelled ‘none’); kidney plus AG3340 (labelled ‘AG’); or kidney plus PD166793 (labelled ‘PD’). B) Rate of increase of fluorescence during linear phase of reaction (0-2000 seconds). The general MPi AG3340 is over 4x more effective at inhibiting TACE activity than PD166793, p<0.0001. A 1.0

0.8

0.6

0.4 MPi + Timp3+/+ MPi + Timp3-/-

fraction of survivors 0.2 vehicle + Timp3+/+ vehicle + Timp3-/- 0.0 0 1 2 3 4 5 6 days

B *

3 2 day of recovery 1

0 wtT3-/- wt T3-/- vehicle alone + MMPi

Figure 10. Timp3-/- mice are rescued from LPS injury with an MMPi that only weakly inhibits TACE. Adult female FVB wild-type and Timp3-/- mice were injected with 180ug LPS. All mice were pre-administered either the synthetic MMPi PD166793 or vehicle alone. A) Timp3-/- (and wild-type) animals given PD166793 were completely rescued from LPS toxicity. n=10 for all 4 groups. B) Surviving mice were monitored for recovery, measured by first day of weight gain. Of animals given vehicle alone, wild-type mice recovered significantly faster than Timp3-/- mice (white bar versus black bar). Treatment with PD166793 significantly accelerated recovery time in both genotypes (dotted bars), p<0.05. Untreated wt n=8; treated wt n=10; untreated T3-/- n=5; treated T3-/- n=10.

may be an important trigger for the inflammatory reaction, other metalloproteinases are more than likely to be essential for implementing the full response to LPS.

103

DISCUSSION

Data presented in Chapter 3 showed that the serum of LPS-treated Timp3-/- mice have

elevated levels of TNF, followed by elevated levels of IL-6 and finally that these animals

have greater mortality relative to control animals. Here we tested whether the loss of

TIMP3 inhibition of TACE-mediated processing of TNF in the liver is the key controlling event in this response, and whether the liver is the primary organ regulating

the over-reaction to LPS. In keeping with this hypothesis, I expected TNF levels in the

livers of LPS-treated Timp3-/- mice to fall as soluble TNF levels in the serum rose. In

other words TNF levels would be lower in the liver tissue of Timp3-/- mice as TACE more quickly sheds TNF from the surface of Kupffer cells, liberating the cytokine into the blood. As well, we anticipated greater TACE activity in an independent enzyme assay of liver tissue.

Strikingly, neither expectation was correct. TNF was elevated, not reduced in the liver tissue of Timp3-/- mice (Fig. 3A), and no increase in TACE activity in the Timp3-/- livers

was observed (Fig. 3B) . Furthermore, we observed little evidence of any difference in

IL-6 signalling in the liver: the phosphorylation of STAT3, responsible for the acute

phase response, following LPS activation is no greater in the Timp3-/- background, and

two key acute phase genes activated by phospho-STAT3 are expressed at wild-type levels. We found only modest differences in splenic TACE activity between wild-type

and Timp3-/- animals; in addition p75, TNF and IL-6 levels (Fig. 6) were no different

between the two groups, with the exception of a small difference in IL-6 levels at 3 hours.

104

Possibly no one central organ is responsible for TIMP3 loss having the effect it has on

LPS sensitivity. Instead, minor effects throughout the body may cumulatively result in

the increased reaction. For example, we observed that the heart, which is not a major

regulator of inflammation, sheds significantly more TNF under stress in the Timp3-/- background than in the control background. The increased TNF we observed in the serum of LPS challenged Timp3-/- mice could be due to shedding from all of the organs.

If there is a major organ responsible for increased mortality in this model it may be the

entire body vasculature. This would be difficult to confirm in vivo. Interestingly, there was some suggestion that metalloproteinases other than TACE are involved in the increased sensitivity, as the synthetic MPi that only weakly inhibits TACE was at least as effective as the broad spectrum inhibitor at rescuing the Timp3-/- mice from LPS

challenge. This is not to say TACE is not involved in the process, but that other

metalloproteinases may ultimately be responsible for executing those changes that change

a moderate reaction into a grave illness or death.

105

Chapter 5

The Effect of TIMP3 Loss on Leukocyte Development,

Local Inflammation, and Acquired Immunity

106

ABSTRACT

Given my previous observations that TIMP3 loss had a profound effect on acute systemic

inflammation we broadened our investigation to examine other aspects of immunity and

the inflammatory response that may be affected by TIMP3. These included leukocyte expansion, local inflammatory response, and re-establishment of the hematopoietic

population after ablation. We observed alteration in hematopoiesis in both the bone marrow and the periphery in Timp3-/- animals after exposure to the antimitotic agent 5-

fluorouracil, suggesting a role for TIMP3 in the bone marrow niche. Using 2,4-Dinitro-

1-Fluorobenzene (DNFB) in a model of contact hypersensitivity we observed greater

sensitivity in Timp3-/- animals. On the other hand, we did not observe a role for TIMP3 in the expansion of memory CD8 T cells, following infection with the lymphocytic

choriomeningitis virus (LCMV). Collectively, these findings reveal a wide range of

circumstances where TIMP3 influences local, systemic, acute and chronic inflammation

as well as inflammatory cells that participate in the immune and inflammatory response.

These systems are implicated in numerous human disorders. In recent years,

metalloproteinases have emerged as important mediators of inflammatory diseases, this

work suggests that TIMP3 plays an important role in regulating this important class of

enzymes during the progression of those diseases.

107

INTRODUCTION

The onset of inflammation or an immune response typically begins with signals from

damaged or challenged tissue, followed by the recruitment of leukocytes, their activation

and, depending on cell type, their expansion. With the emerging role of

metalloproteinases in immunity [282] there are multiple checkpoints at which TIMP3

may normally operate to moderate the immune or inflammatory reaction.

An early response comes from the tissue itself, which includes the release of TNF from

resident macrophages or mast cells [304]. Cytokines induce the production of

chemokines by endothelial cells, attracting leukocytes, as well as inducing the expression

of selectins on the surface of local endothelium which bind to these leukocytes,

promoting an influx of neutrophils and monocytes to the area [305]. Small blood vessels dilate in response to the expression of nitric oxide. Fluid and leukocytes enter the tissue from the blood. In the case of infection, pathogen-associated molecular patterns

(PAMPs) activate innate immune cells, which further release cytokines; dendritic cells in the tissue migrate to the site of inflammation and then away to local lymph nodes [306], where they present antigens to naive T cells. Antigen-specific lymphocytes expand in the lymph nodes, and then pass into the circulation.

TIMP3 may affect many of these processes. The release of TNF and its receptors; the cleavage of L-selectin, involved in the homing of T cells to lymph nodes; the release of fractalkine, a TNF-activated chemokine produced by endothelium [307]; all of these are targeted by TACE, which is inhibited by TIMP3. As well, TIMP3 inhibits MT1-MMP-

108

induced cleavage of the intracellular molecule ICAM-1 [104], involved in the

extravasation of monocytes through the endothelium into the tissue [308].

Beyond these early responses, TIMP3 has the potential to impact many different branches

of hematopoiesis, immunity and inflammation, primarily as a regulator of TACE, but also

as an inhibitor of other proteases which target growth factors, cytokines and adhesion

molecules. Examples include MMP-9 which cleaves the growth factor KIT ligand/Stem

Cell Factor (SCF), as well as the receptors for IL-2 and for IL-8 [309]; ADAMs 9 10 and

12 which cleave Dll1, an activator of the Notch signalling pathway; and MMP-7 which targets the IL-6 receptor. Thus there are multiple avenues by which the loss of TIMP3 may impact the various branches of immunity and inflammation, some of which are investigated here.

109

RESULTS AND DISCUSSION

Contact hypersensitivity model shows a difference between wild-type and TIMP3-/- mice.

We challenged animals with the hapten 2,4-dinitrofluorobenzene (DNFB) to investigate

whether there was a differential response in the Timp3-/- mice in a model of acquired

immunity. This a typical assay for delayed-type hypersensitivity (DTH), wherein the

animals are initially exposed to a hapten, followed by a delay of several days to allow the

expansion of antigen-specific lymphocytes. The reaction involves an interplay of

inflammation, innate immunity and acquired immunity, the latter including both CD4 and

CD8+ T cells. In the case of the hapten DNFB, earlier research has shown that acquired

immune response is primarily mediated by CD8+ cells [310]. A differential response is

indicated by comparing the degree of swelling at the site of a second exposure to the

hapten. The second application of DNFB was onto the right ear of each mouse five days

after the initial exposure. The day following the second application, the ears of the

Timp3-/- mice appeared more red and were found to have swollen significantly, with an increase in thickness more than double that seen with the wild-type mice (Fig. 1).

Loss of TIMP3 does not affect the expansion of CD8+ T cells

There are several potential causes of the difference found in the Timp3-/- in the DTH

experiment. To investigate whether the greater reaction indicated a deregulation of

CD8+ T cells, we used a model which specifically monitors their increase. We challenged animals with the lymphocytic choriomeningitis virus (LCMV) and subsequently measured the relative amount of LCMV-specific memory CD8+ T cells remaining after recovery from exposure to the virus.

110 25 Timp3+/+ 20 Timp3-/- -2 15

mm x 10 10

Figure 1. Timp3-/- mice show a greater response to the irritant DNFB than wild- type mice. Twenty micrograms of DNFB was administered to the right pinna of each mouse following a primary exposure 5 days before. Ear swelling: values are the difference in thickness between the treated (right) and untreated (left) pinna. Timp3-/- animals have more than double the swelling of wild-type animals. N=5 for each group, p<0.001.

After footpad injection of LCMV, mice were observed over three weeks for the onset and

reduction of swelling of the injected foot, to confirm the activity of the virus and its

subsequent clearing by the mice (Fig. 2A). Swelling peaks at day 8 in both genotypes, and appears slightly greater in the Timp3-/- footpads, however this is not statistically significant. The kinetics of swelling in Fig. 2A are as expected for this model. The activation of CD8+ T cells by this virus is well-characterized, with specific epitopes known to be antigenic in C57BL/6 mice. After three weeks the mice were sacrificed and their spleens removed. Splenocytes were cultured with antigen-presenting cells, which selectively activates CTL cells sensitive to LCMV. The activating cells were from an H-

2b mouse thymoma cell line [311] known as EL-4, loaded with GP33, an H-2Db-binding

epitope from the LCMV glycoprotein [312]. The activated lymphocytes were then

incubated with 51Cr filled EL-4 cells, loaded with either GP33 or a control peptide.

Release of the chromium indicates lysis of the target cells. The experiment provides a

sensitive assay of any difference in the original number of memory CD8+ T cells that

remain in the spleen after infection. Target cells are mixed with lymphocytes in several

ratios to ensure several readings between maximum and minimum lysis.

Target cells bearing the random peptide showed very little chromium release above

baseline (Fig. 2B upper panel). Target cells loaded with GP33 were more than twice as

likely to be lysed (Fig. 2B lower panel) showing that the lymphocytes were antigen-

specific and that the assay was working properly. We found no difference between the

GP33-reactive T cells from Timp3-/- mice versus those from the wild-type animals; the amount of chromium released was virtually the same in both cases (Fig. 2B, lower panel).

112 A

2.9 2.7 2.5 wt means T3-/- means 2.3 mm 2.1 1.9 1.7 1.5 0 3 6 9 12 15 18 21 days

3000 wt mice n=3 T3-/- mice n=3 2000

CPM 1000

0 1:1 1:3-1 1:3-2 1:3-3 1:3-4 1:3-5 1:3-6

3000 wt mice n=3 T3-/- mice n=3 2000

CPM 1000

0 1:1 1:3-1 1:3-2 1:3-3 1:3-4 1:3-5 1:3-6

Figure 2. Memory T cells of LCMV-infected Timp3-/- mice expand at similar rate to wild-type cells. Chromium release assay of antigen-specific CD8+ T cells reveals no difference between genotypes. A) Footpad swelling monitored over 21 days following injection of LCMV confirms development and resolution of viral infection. B) Radioactive chromium released from target EL-4 cells incubated with splenocytes from LCMV-infected mice. Splenocytes were incubated with either a random epitope (upper panel) or the LCMV- specific epitope gp33 (lower panel). Cells were incubated in the presence of IL-2 to promote greater expansion of activated lymphocytes. The x axis indicates serial dilutions of CTLs mixed with a fixed number of target cells. Higher values in the lower panel confirm that lysis was antigen specific. There is no statistical difference between genotypes.

These results show that TIMP3 is not involved in regulating the expansion of CD8+

cells, at least in the LCMV model, and suggests that the difference between the genotypes

found in the DTH reaction is not due to differences in T cells, at least not CD8+ T cells

which are the predominant mediator of the DNFB DTH reaction.

Primary inflammatory reaction to DNFB appears greater in the Timp3-/- mice

Although DNFB is used to study delayed hypersensitivity, we found it can also elicit an

initial inflammatory reaction. We returned to this model to see if the initial activation

stage of DNFB, one that does not involve acquired immunity, differs in the Timp3-/- mice.

We applied DNFB to an area of specific size to ensure an equally dense distribution for each animal. After 5 days there was a dramatic difference between Timp3-/- and wild-

type mice in the reaction to the DNFB, with marked redness and scabbing more prevalent

in the Timp3-/- animals (Fig. 3). This effect was transitory, by the seventh day lesions of

Timp3-/- mice had resolved to the same degree as wild-type mice (data not shown). As

this was an initial exposure to the reagent it demonstrated that activation and expansion

of antigen-specific T cells is not required to elicit a differential response by Timp3-/-

animals in this model.

Loss of TIMP3 alters hematopoietic regeneration in peripheral blood

TIMP3 inhibits many metalloproteinases that target growth factors and receptors involved in hematopoiesis. We hypothesized that TIMP3 plays a role in normal hematopoietic development, and tested this by exposing wild-type and Timp3-/- FVB

mice to the myeloid-ablating drug 5-fluorouracil, also known as 5-FU, an anti-tumour

114 T3-/- T3-/- T3-/- T3-/-

wt wt wt wt

Figure 3. Timp3-/- animals have a stronger primary reaction to DNFB. Five days following an initial 25 µl of 0.5% DNFB, spread over 1.5 cm2, there appears a greater inflammatory response by Timp3-/- animals. Upper row: Timp3-/- mice display an erythematic (reddened) and scabbed surface. Lower row: wild-type mice show little or no redness or scabbing.

agent used for decades [313]. 5-FU functions as a pyrimidine analogue; when incorporated into the DNA of rapidly dividing cells it arrests DNA synthesis, causing the cells to undergo apoptosis. Hematopoietic stem cells divide very slowly, while terminally differentiated cells do not divide, therefore neither of these groups is affected by the 5-FU. The intermediate cells, however, since they are undergoing rapid division, are highly vulnerable. It is estimated that the hematopoietic system of adult humans produces over one hundred billion cells daily [314].

Animals administered a sub-lethal dose of 5-FU showed a gradual loss of red blood cells

(Fig. 4A). After 12 days RBC levels rise as newly developed erythrocytes repopulated the peripheral blood. The kinetics of RBC loss and recovery were the same for wild-type and TIMP3 deficient animals. On the other hand, there was an apparent difference in the kinetics of leukocyte repopulation between the two groups (Fig. 4B). Specifically, one week after 5-FU exposure WBCs in both genotypes begin to repopulate the periphery, in each case their numbers rising above normal levels before returning to the pre-injection state. By two weeks, however, it is apparent that the peak of WBC levels in the Timp3-/- mice is considerably lower than in the wild-type mice. At day 14 the number of circulating leukocytes in the blood of Timp3-/- mice is approximately half of what it is in the wild-type animals. There is also a difference in the rise of platelets during the second week after 5-FU exposure (Fig. 4C). In both the wild-type and the Timp3-/- animals the levels rise higher than normal, but again the rise is less pronounced in the Timp3-/- blood.

116 A 13 RBCs 11 wt T3-/- /mL

9 9 7 5 cells x 10 cells x 3 1 0246810121416

B 6 WBCs 5 /mL

7 4 3 2 cells x 10 cells x 1 0 0246810121416

40 platelets 32 /mL 8 24 16

cells x 10 cells x 8

0 0246810121416 days

Figure 4. Damping of hematopoietic kinetics in Timp3-/- mice compared to wild-type following 5-FU exposure. Analysis of peripheral blood. A) RBC levels. No difference between genotypes. B) WBC count. Significant difference in the number of nucleated cells in the periphery two weeks after 5-FU exposure. C) Significantly greater platelets in the wild-type animals after 8 days following 5-FU. For all panels solid lines represent wild-type animals, dashed lines represent Timp3-/- animals, n=5 for all wild-type data points, n=3 for all Timp3-/- data points. Significant differences were found between genotypes in panels B and C by two- way ANOVA (p<0.01).

The relatively low levels of WBCs and platelets found in the Timp3-/- blood in the second

week could be due to a slower rate of hematopoiesis in these mice. Alternatively, blood

cell production could be normal, but the Timp3-/- cells may have a delay in the migration

of cells from the bone marrow. Loss of TIMP3 in the first instance could be causing

increased metalloproteinase cleavage of growth factors or receptors responsible for

hematopoietic expansion; in the second instance, TIMP3 loss could be resulting in greater

metalloproteinases cleavage of chemokines or adhesion molecules involved in migration.

For example, megakaryocyte development requires the SCF/c-Kit pathway. TACE

cleaves the receptor c-Kit [225]; the loss of TIMP3 may lead to greater TACE activity

leading to less c-Kit on megakaryocyte precursors, reducing their development and

ultimately being responsible for fewer platelets in the periphery in the Timp3-/- mice,

compared to wild-type mice, as seen at day 8 after 5-FU.

Total bone marrow cell levels are equivalent or higher in Timp3-/- animals compared to

those in wild-type animals.

To investigate differences in the mitogenesis of hematopoietic precursor cells, animals were again administered the sub-lethal dose of 5-FU but this time we analyzed the bone marrow cells at various levels of development. Total cell numbers (minus the bone marrow RBCs) were assessed and then purified using an antibody affinity column to enrich for the earliest hematopoietic cells. Approximately 97% of the bone marrow cells were removed using six antibodies to markers of lineage commitment (see Methods for details). The cells that pass through the column, the so-called lineage negative cells (Lin-) lacking any of these markers, are higher up in the developmental hierarchy of hematopoiesis. Figure 5 shows the total bone marrow cell count, the Lin- cell count and

118

the ratios of Lin- to total cell count for Timp3-/- and wild-type animals following 5-FU

exposure (Fig. 5 A-C respectively).

No difference in Lin- level between wild-type and Timp3-/- animals

Panel A of Figure 5 shows the number of bone marrow cells harvested at each time-point

after 5-FU exposure. The numbers are remarkably similar between wild-type and Timp3-/- mice. The kinetics were virtually identical, bone marrow numbers decreased for the first six days and began to recover by nine days. There were slightly more Timp3-/- cells at

day nine than wild-type cells. The Lin- cells (Fig. 5B) also show little difference in

kinetics between the genotypes. Their numbers drop to an undetectable level by day 3,

destroyed by the 5-FU, and begin to rise again by day 6, again with higher numbers by

day 9 for the Timp3-/- animal. When the ratio of Lin- cells to total bone marrow cells is

calculated (Fig. 5C) there was no difference between the genotypes. The absence of a

difference in the ratio in Figure 5C suggests there is no obvious delay or acceleration in

the Timp3-/- mice from the Lin- cells to the more differentiated lineage specific cells.

To investigate whether more premature cells within the Lin- category differed between

Timp3-/- and wild-type animals, we stained the Lin- cells from Figure 5 to identify an

even more primitive subset of hematopoietic precursors. c-Kit and Sca-1 are markers

highly expressed on hematopoietic stem and progenitor cells [315]. c-Kit+, SCA-1+ cells from a population of Lin- cells are known as KSL cells. The Lin- cells were also stained

with the vital dye Hoechst 33342, used to detect the presence of an efflux pump active

119 A total BM WBC count after 5FU 60 50 wt 6 T3-/- 40 30

cells x 10 cells x 20 10 0 0369

B total # of Lin-ve cells after 5FU 2.5

6 2.0 1.5 1.0 cells x 10 cells x 0.5 0 0369

C Lin-ve cells/total BM WBC # after 5FU 12 10 8 6 4 % of cells 2 0 0369 days

Figure 5. 5FU-induced loss of bone marrow cells is similar for wild-type and Timp3-/- mice. For each animal bone marrow was harvested from 6 long bones. A) Total number of white blood cells harvested. B) Number of cells to pass through the lineage affinity column. C) Ratio of Lin- cells to all bone marrow WBCs, n=1 for all data-points.

in only the most primitive cells of the bone marrow. As cells progress from their stem

cell status to a more determined lineage they lose the power to discharge the dye [263]. It

has been shown that 90% of the most primitive cells of all, i.e. the hematopoietic stem

cells, are in the KSL fraction with an active efflux pump and that normally 25% of all of

the cells in that fraction are long-term repopulating hematopoietic stem cells [316]. The

Hoechst dye is detected in two channels of the fluorescence detector, blue and red, which

can be set as the vertical and horizontal axes of a flow cytometry dot plot. The majority

of KSL cells retain the dye, they appear in the middle of the plot and are known as the

main population or KSL-MP. The population of cells able to efflux the dye is relatively small, it appears shifted to the left and down, and is known as the side population or

KSL-SP. As cells mature and begin to retain the dye they migrate from the side population to the main population. In each of the dot plots of figure 6A the side population is in region P4, and the main population in region P5.

KSL-MP cell numbers double in Timp3-/- at day 9, compared to control mice

For each FACS analysis some cells are used for calibrating the machine and for

confirming the efficacy of the reagents. Comparing the total number of Lin- cells for

each data-point (Fig. 5B) to the total number of events recorded in each FACS we were

able to calculate the total number of KSL-SP and KSL-MP cells that would be found in

the pooled bone samples from each animal had all cells been recorded (Fig 6B). For both

the wild-type and Timp3-/- KSL-MP cells, their values drop 3 days after 5-FU exposure and begin to rise again by day 6, total numbers being very similar for days 0,3 and 6

121 A

Dec 11 Day 0 of 5FU-wt lin- H cKit Sca1 PI- Dec 11 Day 0 of 5FU-ko lin- H cKit Sca1 PI- 250 250 Hoechst BLUE-A (x1,000) Hoechst BLUE-A (x1,000) 50 100 150 200 50 100 150 200

50 100 150 200 250 50 100 150 200 250 Hoechst RED-A (x1,000) Hoechst RED-A (x1,000)

Dec 14 Day 3 of 5FU-wt lin- H cKit Sca1 PI- Dec 14 Day 3 of 5FU-ko lin- H cKit Sca1 PI- Hoechst BLUE-A (x1,000) Hoechst BLUE-A (x1,000) 50 100 150 200 250 50 100 150 200 250

50 100 150 200 250 50 100 150 200 250 Hoechst RED-A (x1,000) Hoechst RED-A (x1,000)

Dec 17 Day 6 of 5FU-wt lin- H cKit Sca1 PI- Dec 17 Day 6 of 5FU-ko lin- H cKit Sca1 PI- 150 200 250 150 200 250 100 100 Hoechst BLUE-A (x1,000) BLUE-A Hoechst Hoechst BLUE-A (x1,000) BLUE-A Hoechst 50 50

50 100 150 200 250 50 100 150 200 250 Hoechst RED-A (x1,000) Hoechst RED-A (x1,000)

Dec 20 Day 9 of 5FU-wt lin- H cKit Sca1 PI- Dec 20 Day 9 of 5FU-ko lin- H cKit Sca1 PI- 150 200 250 150 200 250 100 100 Hoechst BLUE-A (x1,000) BLUE-A Hoechst Hoechst BLUE-A (x1,000) 50 50

50 100 150 200 250 50 100 150 200 250 Hoechst RED-A (x1,000) Hoechst RED-A (x1,000)

B C

5 2 4 4 6 3 4 2 2 cells x 10 cells x cells x 10 cells x 1 0 0 0369 0369 days days

Figure 6. Timp3-/- and wild-type live KSL cells incubated with Hoechst 33342. A) Each row represents from top to bottom day 0,3,6 or 9 following 5-FU exposure. Wild-type animals are on the left, Timp3-/- animals on the right. B) Estimated total number of KSL-MP cells. C) Estimated total number of KSL-SP cells. For B and C solid line represents wild- type animals, dashed line represents Timp3-/- animals, n=1 for all data-points.

between the genotypes. By day 9 however, there is a remarkable rise in KSL-MP cells in

Timp3-/- as compared to wild-type cells. More than twice as many KLS-MP cells appear

in the Timp3-/- animal. Either the KSL cells divide more rapidly in the Timp3-/- mice, or

the loss of TIMP3 causes these cells to ‘hang back’, delaying their transition to the more mature c-Kit- Sca-1- state.

As described in the thesis introduction (Chapter 1, Figure 8 and Table 2), several

hematopoietic growth factors are cleaved by metalloproteinases inhibited by TIMP3.

Reviewing these growth factors we can speculate about the likeliest candidates that, due

to the loss of TIMP3 inhibition, might increase KSL-MP levels. Neither Notch or c-Kit

are likely to be responsible for the increase in KSL-MP cells. Cleavage of Notch leads to

cell cycle arrest, not expansion and thus would likely reduce the number of cells passing

from the side population into the main population. Cleavage of c-Kit would, of course,

reduce the number of cells recognized as KSL. Remaining possibilities are shedding of

the M-CSF receptor c-fms by TACE [232]; shedding of IL-2Rα by MMP-9 [62]; and

shedding of the IL-15Rα by TACE [233]. These receptors are all associated with the

progression of hematopoietic cells to committed lineages. IL-15Rα, for example, is

involved in the transition of precursor cells to an early T-cell state [317]; the rise of KSL-

MP cells in the Timp3-/- cells could be due to an accumulation of cells that would

normally have been stimulated by IL-15 to progress towards a T cell lineage but instead

remain in the KSL state for lack of the appropriate trigger. Future experiments could

include stains for expression levels of candidate receptors such as the IL-15Rα, c-fms

and IL-2Rα to confirm whether they are at normal expression levels in the Timp3-/- mice.

123

Finally, the most primitive cells identifiable, the KSL-SP cells, began with similar cell

numbers between genotypes at day 0; and by day 9 in both genotypes cell numbers drop

to almost undetectable levels. However, there were quite different kinetics for the wild- type and Timp3-/- mice at days 3 and 6 after 5-FU exposure (Fig. 6C). These kinetics are difficult to interpret, but in any case they suggest a role for TIMP3 in the transition from

KSL-SP to KSL-MP. Future experiments will require more time-points to track KSL-SP activity. Larger sample numbers are also required. Nevertheless, the KSL-SP and KSL-

MP data indicate that the early stages of hematopoiesis were affected by the loss of

TIMP3. While this effect was obscured or compensated for as the cells continued to develop in the bone marrow—the number of Lin- and total bone marrow cells being the same between the genotypes—we saw a difference reappear when we examined peripheral blood post 5-FU (Fig. 5).

The results of this chapter raise many questions about the role of TIMP3 in regulating immune cells and inflammation. For example, in the DNFB model where primary contact caused increased skin inflammation, at what stage or at what location is the loss of TIMP3 significant to the phenotype? Is the challenged skin initially releasing more chemokines and cytokines, and subsequently directing more immune cells to the area?

Alternatively, is it that there is just more nitric oxide produced locally, causing

vasodilation and the failure of platelet function? Or can neutrophil metalloproteinases

more readily cause damage due to the lack of TIMP3 in the surrounding tissue? Or are monocytes recruited to the site of injury releasing more soluble TNF, triggering a stronger inflammatory response? Is the loss of TIMP3 significant in stromal cells or

124

circulating leukocytes? Answering these questions will depend on the development of

reliable reagents for in situ detection of TIMP3 and in situ detection of the activities of

the proteases TIMP3 inhibits. Without proper reagents, such as good antibodies to

murine TIMP3, it is difficult to characterize the TIMP3 microenvironment.

In the experiments of hematopoietic reconstitution we need to further identify the cells that expand more slowly in the blood of Timp3-/- mice, compared to wild-type animals.

We presently know that the level of nucleated blood cells in the wild-type mice are

almost double in number at two weeks after 5-FU treatment, compared to Timp3-/- mice.

Approximately three-quarters of the WBC in a normal mouse are lymphocytes [318],

suggesting lymphocytes are the most likely source of the difference, but another

possibility is that what appear to be WBCs are immature reticulocytes appearing in the

peripheral blood . A differential blood count at 14 days would allow us to distinguish

what cells are preferentially appearing in the periphery of the wild-type animals.

Knowing the type of cells that differ would provide clues as to what factors or what

process TIMP3 is involved in with hematopoietic expansion. Again it would be

instructive to know whether the effect of altered hematopoietic cell numbers is due to

TIMP3 in the stroma or to TIMP3 associated with hematopoietic cells.

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Chapter 6: CONCLUSION

Previously, research from our lab demonstrated a role for TIMP3 in tissue remodelling

under several conditions: mammary involution [244]; cardiac dilation in response to hypertension [279]; and increased growth of exogenous tumours in Timp3-/- hosts [319];

in each case the loss of TIMP3 led to the acceleration of ECM reshaping. For example,

Kassiri et al. showed that cardiac failure in the Timp3-/- animals was a product not only of

remodelling but also of dysregulated cytokine activity.

My PhD has focused on this second role of TIMP3. In collaboration with F. Mohammed,

for example, I demonstrated that lymphocyte infiltration found in the aged livers of

Timp3-/- mice was dependent on an intact TNF signalling pathway [294]. Similarly, in a

model of antigen-induced arthritis (AIA) I discovered a 2x increase in TNF levels in the

blood of Timp3-/- animals in response to killed bacteria, as compared to the response in wild-type mice [246]. Interestingly, previous research with the AIA model has shown that swelling and lymphocyte infiltration are reduced with neutralizing antibodies to

TNF, but that the matrix degradation in the joint is not [320]. The Timp3-/- mice showed

increased joint inflammation and cellular invasion in response to the antigen, but no

difference in cartilage cleavage. It would appear, therefore, in this model that inhibition

of cytokine shedding by TIMP3, and not the ability of TIMP3 to inhibit matrix

proteolysis, could entirely account for the difference from the control animals. In another

model of cardiac disease where aged Timp3-/- animals spontaneously developed

cardiomyopathy, I demonstrated that the excised Timp3-/- hearts also shed increased

amounts of TNF [321] the release of which is associated with ischemia-reperfusion injury

126

[322]. Each of these three studies to which I contributed illustrated the importance of

TIMP3 in regulating TNF.

Chapter 3 of this thesis revealed dysregulation not only of TNF in the Timp3-/- animals,

but also of TNF receptors p55 and p75, which are also targets of the sheddase TACE.

Furthermore, alterations in the TNF signalling pathway carried forward to IL-6

dysregulation, a cytokine not directly targeted by TACE proteolysis. Intriguingly,

although I showed in Chapter 3 that the increased LPS sensitivity in the Timp3-/- mice was dependent on TNF signalling, I could not confirm in Chapter 4 that intact TNF signalling was sufficient to drive the increased sensitivity to LPS seen in the Timp3-/- mice. A synthetic metalloprotease that only weakly targeted TACE and thus allowed for

TNF cleavage was still adequate for rescuing the Timp3-/- mice. As in the studies of

cardiac failure [279], it could be that TIMP3 functions in multiple roles to protect the

host, and that at least one other TACE-independent proteolytic act is responsible for the

increased sensitivity to LPS.

For example, recalling that the lethal stage of SIRS is the disseminated intravascular coagulation (DIC), promotion of DIC is likely to increase mortality. Tissue factor

pathway inhibitor (TFPI) is a powerful inhibitor of factor Xa and factor VIIa, proteins in

the clotting cascade. Depletion of TFPI leads to an increased sensitivity to endotoxin,

resulting in greater DIC [323]. TFPI is subject to proteolysis by several MMPs including

MMP-7, MMP-9, MMP-1 and MMP-12 [324] destroying the ability of TFPI to prevent

coagulation. Of interest, the loss of MMP-9 significantly reduces the sensitivity of mice

to LPS [325]. The loss of TIMP3 could augment animal sensitivity to LPS by the

127

increased activity of these MMPs against TFPI; i.e. no TIMP3 Æ more MMP activity Æ

less TFPI Æ more coagulation Æ more DIC Æ greater mortality. This is a hypothetical

contributor to the phenotypic response to LPS seen in the Timp3-/- mice; however, it

underscores that TIMP3 may be acting at several stages to regulate inflammation and that there are other significant contributors beyond the dysregulation of the TNF signalling pathway contributing to this phenotype.

Chapter 5 contains two models that show significant changes in the Timp3-/- mice. In the

first one, primary exposure to DNFB elicited a strong reaction in the skin of Timp3-/- mice (Chapter 5, Fig. 3). Although the work to dissect the underlying causes will be left to the future, I would like to discuss possible mechanisms that might be responsible for this local inflammation. On one hand, it could be a matter of ECM degradation, for example loss of TIMP3 from the endothelium allowing for increased attack on the basement membrane by the neutrophil collagenase MMP8. A second possibility is that

loss of TIMP3 leads to increased monocyte shedding of TNF into the local environment,

increasing nitric oxide production and promoting increased vasodilation. A third

alternative, described in a review paper jointly written by F. Mohammed, R. Khokha and

myself, is that the lack of TIMP3 could lead to greater trafficking of neutrophils to the

site of inflammation via a chemokine gradient [326]. In this scenario the chemokine KC,

which promotes endothelial migration of neutrophils [327], and is normally sequestered

through binding to the proteoglycan syndecan-1, is liberated when MMP-7 cleaves

syndecan-1 from the surface of cells [328]. Thus: the loss of TIMP3 could lead to more

MMP-7 activity, increasing syndecan-1 cleavage, releasing soluble KC, leading to chemo

attraction of neutrophils, resulting in greater local inflammation. These three possible

128

roles for TIMP3 in this model, protector of basement membrane, promoter of chemokine

sequestration, inhibitor of cytokine shedding, underscore the potential complexities of

TIMP3 activity, and also the challenges of confirming which is dominant in this model.

The second revealing model in Chapter 5, hematopoietic ablation using 5-FU, showed

both in the bone marrow and in the peripheral blood that TIMP3 participates in blood cell

development. This work was preliminary; more antibody stains of the KSL-MP cells in

the bone marrow are needed to identify what type of cells are in higher numbers in the

Timp3-/- bone marrow. Specifically identifying these cells will help point to which

growth factors, if any, are involved. In the peripheral blood, a differential white cell

count will identify which cells types are reduced, once those cells are identified their

numbers can be compared between periphery and bone marrow to see whether a change

in trafficking in the Timp3-/- mouse, or reductions in differentiation and mitogenesis that

lead to lower leukocyte levels in the periphery in week 2 post-5-FU treatment (Chapter 5

Fig. 4B).

To carry the LPS research forward, which has been the bulk of this thesis, when considering the work of Chapter 4 it would be extremely useful to develop an ex vivo liver model. We would apply techniques used for studying isolated perfused livers in the rat [329], some of which have already been adapted to the mouse in our lab. Briefly, the suprahepatic inferior vena cava and the portal vein are cannulated while the infrahepatic inferior vena cava and the adjacent hepatic artery are ligated. The liver is then kept at 37° and perfused with a buffer saturated with 95% O2 and 5% CO2. The rat model circulates

a 250 mL solution, as the mouse is considerably smaller we would likely circulate a 50-

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100 mL solution. At this volume, there would be no trouble detecting TNF, p55 or p75 using ELISA kits if the concentrations in the perfusate were comparable to serum levels, we would also be able to monitor ALT levels to have an ongoing record of the health of the liver. The advantage of this system is that a single organ would be isolated, as in the heart model. We would have a clean read-out of increased shedding in response to different conditions. One of the difficulties with the analysis of whole liver tissue in

Chapter 4 was our inability to distinguish between membrane-bound and solubilized cytokines and their receptors. Analysis of the kinetics of shedding would be straightforward in the ex vivo liver system as fluid samples could be removed directly from the perfusate continually. The animals used could be pre-treated to inactive Kupffer cells [330], they could be TACE conditional knockouts crossed with albumin-Cre transgenic mice, which produce animals lacking hepatic expression of TACE; there are numerous possibilities with this model. As TIMP3 is an insoluble ECM-binding protein it has been problematic studying its importance in tissue culture systems. This model would provide us with an intact extracellular matrix with which to study the loss of

TIMP3.

Returning to the liver model used here in the thesis, i.e. in vivo changes following LPS exposure, one of the most significant findings that should continue to be investigated is the early activation of the JNK pathway found in the Timp3-/- mice (Chapter 4 Fig. 2).

This can be investigated in both directions of signalling i.e. tracing the upstream cause of the JNK activation and the downstream targets. Using TNF-/- /Timp3-/- or p55-/-/Timp3-/- mice would allow testing whether TNF signalling is responsible for the increase. An alternative being that the phosphorylation of SAPK is a direct result of LPS activation,

130

occurring before TNF is involved. Evidence of this would be an increase of ubiquitination of TRAF6, phosphorylation of ASK1 or TAK1 (see Introduction, Fig. 5).

JNK transcription targets could be analyzed using RT-PCR of liver tissue.

An important problem that should be addressed in any further studies with TIMP3 is the

lack of a reliable anti-TIMP3 antibody. Multiple attempts have been made and it would

appear TIMP3 has no strong, unique epitopes. A good antibody would allow greater

understanding of where TIMP3 is located in the microenvironment as it inhibits activity.

One way to address this problem would be to make a mouse expressing a modified

TIMP3 protein, one containing a strong epitope. For example, using FVB Timp3-/- embryos available in our lab, we could make transgenic animals expressing a functional

FLAG-tagged TIMP3 protein. The FLAG tag is an 8 peptide epitope for which there are excellent antibodies. The tag could be attached to the C-terminus of the protein, which is

unstructured and not enzymatically active [331]. Being so small the tag is unlikely to

interfere with the function of TIMP3. The transgene would insert randomly into the

genome; however, the expression of the transgene would be driven by the addition of the

TIMP3 promoter, so that TIMP3 would be normally expressed. Previous researchers

have already generated a transgenic mouse with a lacZ reporter gene driven by a human

TIMP3 promoter, a 1.2 kb 5’ UTR fragment the human TIMP3 gene [332]. Expression

of lacZ was temporally specific and tissue specific and reflected known expression

patterns of the gene, showing the proof-of-principle of the use of this fragment for

TIMP3 promotion.

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Overall, this work has shown the importance of TIMP3 in a number of models where cytokines and growth factors are active. I show that TIMP3 is involved in a hitherto unsuspected contribution to hematopoietic cell development. I also present some of the earliest evidence that TIMP3 plays a role in vivo in regulating TNF signalling, and does so at both a systemic and a local level. I have shown that TIMP3 acts as a key regulator of inflammation. Systemically and in every organ tested, i.e. heart, knees, skin and liver, my work illustrates the significant contribution TIMP3 makes in the normal animal to moderating the inflammatory response. As inflammation plays a role in so many disorders, this research is of great relevance to the study and understanding of human disease. It has been known for many years that certain tissues such as the eye are immunologically privileged from attack by lymphocytes. The presence of TIMP3 may provide a parallel mechanism in the innate immune system, binding to ECM where it is needed to limit inflammatory activity. This thesis provides the proof-of-principle that

TIMP3 is an important player in moderating the inflammatory response.

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REFERENCES

1. Arking, R., The Biology of Aging: Observations and Principles. 3rd ed. 2006, New York: Oxford University Press. 2. Jones, L.L., et al., NG2 is a major chondroitin sulfate proteoglycan produced after spinal cord injury and is expressed by macrophages and oligodendrocyte progenitors. J Neurosci, 2002. 22(7): p. 2792-803. 3. Fears, C.Y. and A. Woods, The role of syndecans in disease and wound healing. Matrix Biol, 2006. 25(7): p. 443-56. 4. Myllyharju, J. and K.I. Kivirikko, Collagens, modifying enzymes and their mutations in humans, flies and worms. Trends Genet, 2004. 20(1): p. 33-43. 5. Soderhall, C., et al., Variants in a novel epidermal collagen gene (COL29A1) are associated with atopic dermatitis. PLoS Biol, 2007. 5(9): p. e242. 6. Ayad, S., The extracellular matrix factsbook. 2nd ed. Factsbook series. 1998, San Diego, Calif.: Academic Press. x, 301 p. 7. Di Lullo, G.A., et al., Mapping the ligand-binding sites and disease-associated mutations on the most abundant protein in the human, type I collagen. J Biol Chem, 2002. 277(6): p. 4223-31. 8. Prockop, D.J. and K.I. Kivirikko, Collagens: molecular biology, diseases, and potentials for therapy. Annu Rev Biochem, 1995. 64: p. 403-34. 9. Khoshnoodi, J., V. Pedchenko, and B.G. Hudson, Mammalian collagen IV. Microsc Res Tech, 2008. 71(5): p. 357-70. 10. Kapyla, J., et al., The fibril-associated collagen IX provides a novel mechanism for cell adhesion to cartilaginous matrix. J Biol Chem, 2004. 279(49): p. 51677- 87. 11. Kreis, T. and R.E. Vale, Guidebook to the extracellular matrix and adhesion proteins. 1993, Oxford ; New York: Oxford University Press. xi, 176 p. 12. Hollyfield, J.G., M.E. Rayborn, and R. Tammi, Hyaluronan localization in tissues of the mouse posterior eye wall: absence in the interphotoreceptor matrix. Exp Eye Res, 1997. 65(5): p. 603-8. 13. Mow, V.C. and R. Huiskes, Basic orthopaedic biomechanics & mechano-biology. 3rd ed. 2005, Philadelphia, PA: Lippincott Williams & Wilkins. xvi, 720 p. 14. Laurent, T.C. and J.R. Fraser, Hyaluronan. Faseb J, 1992. 6(7): p. 2397-404. 15. Petitou, M., B. Casu, and U. Lindahl, 1976-1983, a critical period in the history of heparin: the discovery of the antithrombin binding site. Biochimie, 2003. 85(1- 2): p. 83-9. 16. Allen, H.J. and E.C. Kisailus, Glycoconjugates : composition, structure, and function. 1992, New York: Dekker. viii, 685 p. 17. Trowbridge, J.M. and R.L. Gallo, Dermatan sulfate: new functions from an old glycosaminoglycan. Glycobiology, 2002. 12(9): p. 117R-25R. 18. Funderburgh, J.L., Keratan sulfate: structure, biosynthesis, and function. Glycobiology, 2000. 10(10): p. 951-8.

133

19. Scott, J.E. and M. Haigh, Identification of specific binding sites for keratan sulphate proteoglycans and chondroitin-dermatan sulphate proteoglycans on collagen fibrils in cornea by the use of cupromeronic blue in 'critical-electrolyte- concentration' techniques. Biochem J, 1988. 253(2): p. 607-10. 20. Mecham, R.P., Laminin receptors. Annu Rev Cell Biol, 1991. 7: p. 71-91. 21. Dzamba, B.J., et al., Fibronectin binding site in type I collagen regulates fibronectin fibril formation. J Cell Biol, 1993. 121(5): p. 1165-72. 22. Lyon, M., et al., Elucidation of the structural features of heparan sulfate important for interaction with the Hep-2 domain of fibronectin. J Biol Chem, 2000. 275(7): p. 4599-606. 23. Zhou, A., et al., How vitronectin binds PAI-1 to modulate fibrinolysis and cell migration. Nat Struct Biol, 2003. 10(7): p. 541-4. 24. Esemuede, N., et al., The role of thrombospondin-1 in human disease. J Surg Res, 2004. 122(1): p. 135-42. 25. Hsia, H.C. and J.E. Schwarzbauer, Meet the tenascins: multifunctional and mysterious. J Biol Chem, 2005. 280(29): p. 26641-4. 26. Sadler, J.E., Biochemistry and genetics of von Willebrand factor. Annu Rev Biochem, 1998. 67: p. 395-424. 27. Federici, A.B., The factor VIII/von Willebrand factor complex: basic and clinical issues. Haematologica, 2003. 88(6): p. EREP02. 28. Handel, T.M., et al., Regulation of protein function by glycosaminoglycans--as exemplified by chemokines. Annual review of biochemistry, 2005. 74: p. 385-410. 29. Bernfield, M., et al., Functions of cell surface heparan sulfate proteoglycans. Annu Rev Biochem, 1999. 68: p. 729-77. 30. Yu, W.H., et al., TIMP-3 binds to sulfated glycosaminoglycans of the extracellular matrix. J Biol Chem, 2000. 275(40): p. 31226-32. 31. Yu, W.H. and J.F. Woessner, Jr., Heparan sulfate proteoglycans as extracellular docking molecules for matrilysin (matrix metalloproteinase 7). J Biol Chem, 2000. 275(6): p. 4183-91. 32. Lopez-Otin, C. Lab homepage [cited; http://www.uniovi.es/degradome/tables/table1.html]. 33. Suzuki, M., et al., Matrix metalloproteinase-3 releases active heparin-binding EGF-like growth factor by cleavage at a specific juxtamembrane site. J Biol Chem, 1997. 272(50): p. 31730-7. 34. McQuibban, G.A., et al., Matrix metalloproteinase activity inactivates the CXC chemokine stromal cell-derived factor-1. J Biol Chem, 2001. 276(47): p. 43503-8. 35. Schonbeck, U., F. Mach, and P. Libby, Generation of biologically active IL-1 beta by matrix metalloproteinases: a novel caspase-1-independent pathway of IL- 1 beta processing. J Immunol, 1998. 161(7): p. 3340-6. 36. Noe, V., et al., Release of an invasion promoter E-cadherin fragment by matrilysin and stromelysin-1. J Cell Sci, 2001. 114(Pt 1): p. 111-118. 37. Nuttall, R.K., et al., Expression analysis of the entire MMP and TIMP gene families during mouse tissue development. FEBS Lett, 2004. 563(1-3): p. 129-34. 38. Brinckerhoff, C.E. and L.M. Matrisian, Matrix metalloproteinases: a tail of a frog that became a prince. Nat Rev Mol Cell Biol, 2002. 3(3): p. 207-14.

134

39. Overall, C.M. and O. Kleifeld, Tumour microenvironment - opinion: validating matrix metalloproteinases as drug targets and anti-targets for cancer therapy. Nat Rev Cancer, 2006. 6(3): p. 227-39. 40. Garnero, P., et al., The collagenolytic activity of cathepsin K is unique among mammalian proteinases. J Biol Chem, 1998. 273(48): p. 32347-52. 41. Clendeninn, N.J. and K. Appelt, eds. Matrix Metalloproteinase Inhibitors in Cancer Therapy. 1st ed. Cancer Drug Discovery and Development, ed. B.A. Teicher. 2001, Humana Press: Totowa. 42. Lauer-Fields, J.L., D. Juska, and G.B. Fields, Matrix metalloproteinases and collagen catabolism. Biopolymers, 2002. 66(1): p. 19-32. 43. Morgunova, E., et al., Structure of human pro-matrix metalloproteinase-2: activation mechanism revealed. Science, 1999. 284(5420): p. 1667-70. 44. Nagase, H., R. Visse, and G. Murphy, Structure and function of matrix metalloproteinases and TIMPs. Cardiovasc Res, 2006. 69(3): p. 562-73. 45. Yang, M. and M. Kurkinen, Cloning and characterization of a novel matrix metalloproteinase (MMP), CMMP, from chicken embryo fibroblasts. CMMP, Xenopus XMMP, and human MMP19 have a conserved unique cysteine in the catalytic domain. J Biol Chem, 1998. 273(28): p. 17893-900. 46. Chin, J.R., G. Murphy, and Z. Werb, Stromelysin, a connective tissue-degrading metalloendopeptidase secreted by stimulated rabbit synovial fibroblasts in parallel with collagenase. Biosynthesis, isolation, characterization, and substrates. J Biol Chem, 1985. 260(22): p. 12367-76. 47. Vu, T.H. and Z. Werb, Matrix metalloproteinases: effectors of development and normal physiology. Genes Dev, 2000. 14(17): p. 2123-33. 48. Miralles, F., et al., TGF-beta plays a key role in morphogenesis of the pancreatic islets of Langerhans by controlling the activity of the matrix metalloproteinase MMP-2. J Cell Biol, 1998. 143(3): p. 827-36. 49. Lelongt, B., et al., Matrix metalloproteinases MMP2 and MMP9 are produced in early stages of kidney morphogenesis but only MMP9 is required for renal organogenesis in vitro. J Cell Biol, 1997. 136(6): p. 1363-73. 50. Schnaper, H.W., et al., Type IV collagenase(s) and TIMPs modulate endothelial cell morphogenesis in vitro. J Cell Physiol, 1993. 156(2): p. 235-46. 51. Gill, S.E. and W.C. Parks, Metalloproteinases and their inhibitors: regulators of wound healing. Int J Biochem Cell Biol, 2008. 40(6-7): p. 1334-47. 52. McGuire, J.K., Q. Li, and W.C. Parks, Matrilysin (matrix metalloproteinase-7) mediates E-cadherin ectodomain shedding in injured lung epithelium. Am J Pathol, 2003. 162(6): p. 1831-43. 53. Atkinson, J.J., et al., Membrane type 1 matrix metalloproteinase is necessary for distal airway epithelial repair and keratinocyte growth factor receptor expression after acute injury. Am J Physiol Lung Cell Mol Physiol, 2007. 293(3): p. L600- 10. 54. Hallmann, R., et al., Expression and function of laminins in the embryonic and mature vasculature. Physiol Rev, 2005. 85(3): p. 979-1000. 55. Banks, R.E., et al., Release of the angiogenic cytokine vascular endothelial growth factor (VEGF) from platelets: significance for VEGF measurements and cancer biology. Br J Cancer, 1998. 77(6): p. 956-64.

135

56. Xu, J., et al., Proteolytic exposure of a cryptic site within collagen type IV is required for angiogenesis and tumor growth in vivo. J Cell Biol, 2001. 154(5): p. 1069-79. 57. Nisato, R.E., et al., Dissecting the role of matrix metalloproteinases (MMP) and integrin alpha(v)beta3 in angiogenesis in vitro: absence of hemopexin C domain bioactivity, but membrane-Type 1-MMP and alpha(v)beta3 are critical. Cancer Res, 2005. 65(20): p. 9377-87. 58. Kalluri, R., Basement membranes: structure, assembly and role in tumour angiogenesis. Nat Rev Cancer, 2003. 3(6): p. 422-33. 59. Tholozan, F.M., et al., FGF-2 release from the lens capsule by MMP-2 maintains lens epithelial cell viability. Mol Biol Cell, 2007. 18(11): p. 4222-31. 60. Yu, W.H., et al., CD44 anchors the assembly of matrilysin/MMP-7 with heparin- binding epidermal growth factor precursor and ErbB4 and regulates female reproductive organ remodeling. Genes Dev, 2002. 16(3): p. 307-23. 61. Ito, A., et al., Degradation of interleukin 1beta by matrix metalloproteinases. J Biol Chem, 1996. 271(25): p. 14657-60. 62. Sheu, B.C., et al., A novel role of metalloproteinase in cancer-mediated immunosuppression. Cancer Res, 2001. 61(1): p. 237-42. 63. Martin, D.C., et al., Insulin-like growth factor II signaling in neoplastic proliferation is blocked by transgenic expression of the metalloproteinase inhibitor TIMP-1. J Cell Biol, 1999. 146(4): p. 881-92. 64. Westermarck, J. and V.M. Kahari, Regulation of matrix metalloproteinase expression in tumor invasion. Faseb J, 1999. 13(8): p. 781-92. 65. Gettins, P., A.H. Beth, and L.W. Cunningham, Proximity of thiol esters and bait region in human alpha 2-macroglobulin: paramagnetic mapping. Biochemistry, 1988. 27(8): p. 2905-11. 66. Chu, C.T., et al., Alpha 2-macroglobulin: a sensor for proteolysis. Ann N Y Acad Sci, 1994. 737: p. 291-307. 67. Rundhaug, J.E., Matrix metalloproteinases and angiogenesis. J Cell Mol Med, 2005. 9(2): p. 267-85. 68. Bond, J.S. and R.J. Beynon, The astacin family of metalloendopeptidases. Protein Sci, 1995. 4(7): p. 1247-61. 69. Hege, T. and U. Baumann, The conserved methionine residue of the metzincins: a site-directed mutagenesis study. J Mol Biol, 2001. 314(2): p. 181-6. 70. Stocker, W., et al., The metzincins--topological and sequential relations between the astacins, adamalysins, serralysins, and matrixins (collagenases) define a superfamily of zinc-peptidases. Protein Sci, 1995. 4(5): p. 823-40. 71. Tan, K., et al., Crystal structure of the TSP-1 type 1 repeats: a novel layered fold and its biological implication. J Cell Biol, 2002. 159(2): p. 373-82. 72. Puente, X.S., et al., Human and mouse proteases: a comparative genomic approach. Nat Rev Genet, 2003. 4(7): p. 544-58. 73. Llamazares, M., et al., Identification and characterization of ADAMTS-20 defines a novel subfamily of metalloproteinases-disintegrins with multiple thrombospondin-1 repeats and a unique GON domain. J Biol Chem, 2003. 278(15): p. 13382-9.

136

74. Rodriguez-Manzaneque, J.C., et al., ADAMTS1 cleaves aggrecan at multiple sites and is differentially inhibited by metalloproteinase inhibitors. Biochem Biophys Res Commun, 2002. 293(1): p. 501-8. 75. Informatics, M.G., 2007. 76. Blobel, C.P., Functional and biochemical characterization of ADAMs and their predicted role in protein ectodomain shedding. Inflamm Res, 2002. 51(2): p. 83- 4. 77. Blobel, C.P., et al., A potential fusion peptide and an integrin ligand domain in a protein active in sperm-egg fusion. Nature, 1992. 356(6366): p. 248-52. 78. Cho, C., Mammalian ADAMs with Testis-Specific or -Predominant Expression. Proteases in Biology and Disease: The ADAM Family of Proteases, ed. Hooper, Lendeckel. Vol. 4. 2005, Dordrecht: Springer. 79. White, Bridges, ; ,DeSimone, ; ,Tomczuk, ; ,Wolfsberg, Introduction to the ADAM Family, in The ADAM family of Proteases, Hooper, Lendeckel, Editor. 2005, Springer: Dordrecht. p. 344. 80. Preece, G., G. Murphy, and A. Ager, Metalloproteinase-mediated regulation of L- selectin levels on leucocytes. J Biol Chem, 1996. 271(20): p. 11634-40. 81. Yan, Y., K. Shirakabe, and Z. Werb, The metalloprotease Kuzbanian (ADAM10) mediates the transactivation of EGF receptor by G protein-coupled receptors. J Cell Biol, 2002. 158(2): p. 221-6. 82. Black, R.A., et al., A metalloproteinase disintegrin that releases tumour-necrosis factor-alpha from cells. Nature, 1997. 385(6618): p. 729-733. 83. Moss, M.L., et al., Cloning of a disintegrin metalloproteinase that processes precursor tumour-necrosis factor-alpha. Nature, 1997. 385(6618): p. 733-736. 84. Dinarello, C.A., Biologic basis for interleukin-1 in disease. Blood, 1996. 87(6): p. 2095-147. 85. Reddy, P., et al., Functional analysis of the domain structure of tumor necrosis factor-alpha converting enzyme. J Biol.Chem.2000 May 12.;275.(19.):14608.-14., 2000. 275(19): p. 14608-14614. 86. Colotta, F., et al., Interleukin-1 type II receptor: a decoy target for IL-1 that is regulated by IL-4. Science, 1993. 261(5120): p. 472-5. 87. Gabay, C. and I. Kushner, Acute-phase proteins and other systemic responses to inflammation. N Engl J Med, 1999. 340(6): p. 448-54. 88. Althoff, K., et al., Shedding of interleukin-6 receptor and tumor necrosis factor alpha. Contribution of the stalk sequence to the cleavage pattern of transmembrane proteins. Eur.J Biochem.2000 May;267.(9.):2624.-31., 2000. 267(9): p. 2624-2631. 89. Peschon, J.J., et al., An essential role for ectodomain shedding in mammalian development. Science, 1998. 282(5392): p. 1281-1284. 90. Mann, G.B., et al., Mice with a null mutation of the TGF alpha gene have abnormal skin architecture, wavy hair, and curly whiskers and often develop corneal inflammation. Cell, 1993. 73(2): p. 249-61. 91. Jackson, L.F., et al., Defective valvulogenesis in HB-EGF and TACE-null mice is associated with aberrant BMP signaling. Embo J, 2003. 22(11): p. 2704-16. 92. Yamazaki, S., et al., Mice with defects in HB-EGF ectodomain shedding show severe developmental abnormalities. J Cell Biol, 2003. 163(3): p. 469-75.

137

93. Boutet, P., et al., Cutting edge: The metalloproteinase ADAM17/TNF-alpha- converting enzyme regulates proteolytic shedding of the MHC class I-related chain B protein. J Immunol, 2009. 182(1): p. 49-53. 94. Doedens, J.R. and R.A. Black, Stimulation-induced down-regulation of tumor necrosis factor-alpha converting enzyme. J Biol.Chem.2000 May 12.;275.(19.):14598.-607., 2000. 275(19): p. 14598-14607. 95. Doedens, J.R., R.M. Mahimkar, and R.A. Black, TACE/ADAM-17 enzymatic activity is increased in response to cellular stimulation. Biochem Biophys Res Commun, 2003. 308(2): p. 331-8. 96. Leco, K.J., et al., Tissue inhibitor of metalloproteinases-3 (TIMP-3) is an extracellular matrix-associated protein with a distinctive pattern of expression in mouse cells and tissues. J Biol Chem, 1994. 269(12): p. 9352-60. 97. Meyer-Bertenrath, J.G., 150 Years of croton oil research. Experientia, 1969. 25(1): p. 1-5. 98. Van Duuren, B.L. and A. Sivak, Tumor-promoting agents from Croton tiglium L. and their mode of action. Cancer Res, 1968. 28(11): p. 2349-56. 99. Mizuguchi, J., et al., Protein kinase C activation blocks anti-IgM-mediated signaling BAL17 B lymphoma cells. J Immunol, 1987. 139(4): p. 1054-9. 100. Ono, Y., et al., Phorbol ester binding to protein kinase C requires a cysteine-rich zinc-finger-like sequence. Proc Natl Acad Sci U S A, 1989. 86(13): p. 4868-71. 101. Diaz-Rodriguez, E., et al., Extracellular signal-regulated kinase phosphorylates tumor necrosis factor alpha-converting enzyme at threonine 735: a potential role in regulated shedding. Mol Biol Cell, 2002. 13(6): p. 2031-44. 102. Zhang, Q., et al., Phosphorylation of TNF-alpha converting enzyme by gastrin- releasing peptide induces amphiregulin release and EGF receptor activation. Proc Natl Acad Sci U S A, 2006. 103(18): p. 6901-6. 103. Herrlich, A., et al., Ectodomain cleavage of the EGF ligands HB-EGF, neuregulin1-beta, and TGF-alpha is specifically triggered by different stimuli and involves different PKC isoenzymes. Faseb J, 2008. 22(12): p. 4281-95. 104. Sithu, S.D., et al., Membrane-type 1-matrix metalloproteinase regulates intracellular adhesion molecule-1 (ICAM-1)-mediated monocyte transmigration. J Biol Chem, 2007. 282(34): p. 25010-9. 105. Van Lint, P. and C. Libert, Matrix metalloproteinase-8: cleavage can be decisive. Cytokine Growth Factor Rev, 2006. 17(4): p. 217-23. 106. Hedrich, H.J. and G.R. Bullock, The Laboratory Mouse. 2004: Academic Press. 107. Thomas, A.H., E.R. Edelman, and C.M. Stultz, Collagen fragments modulate innate immunity. Exp Biol Med (Maywood), 2007. 232(3): p. 406-11. 108. Schulz, B., et al., ADAM10 regulates endothelial permeability and T-Cell transmigration by proteolysis of vascular endothelial cadherin. Circ Res, 2008. 102(10): p. 1192-201. 109. Carmeliet, P., et al., Targeted deficiency or cytosolic truncation of the VE- cadherin gene in mice impairs VEGF-mediated endothelial survival and angiogenesis. Cell, 1999. 98(2): p. 147-57. 110. Aye, M.T., Erythroid colony formation in cultures of human marrow: effect of leukocyte conditioned medium. J Cell Physiol, 1977. 91(1): p. 69-77. 111. Golde, D.W., et al., Production of erythroid-potentiating activity by a human T- lymphoblast cell line. Proc Natl Acad Sci U S A, 1980. 77(1): p. 593-6.

138

112. Docherty, A.J., et al., Sequence of human tissue inhibitor of metalloproteinases and its identity to erythroid-potentiating activity. Nature, 1985. 318(6041): p. 66- 9. 113. Stetler-Stevenson, W.G., N. Bersch, and D.W. Golde, Tissue inhibitor of metalloproteinase-2 (TIMP-2) has erythroid-potentiating activity. FEBS Lett, 1992. 296(2): p. 231-4. 114. Kinoshita, T., et al., TIMP-2 promotes activation of progelatinase A by membrane-type 1 matrix metalloproteinase immobilized on agarose beads. J Biol Chem, 1998. 273(26): p. 16098-103. 115. Baker, A.H., et al., Divergent effects of tissue inhibitor of metalloproteinase-1, -2, or -3 overexpression on rat vascular smooth muscle cell invasion, proliferation, and death in vitro. TIMP-3 promotes apoptosis. J Clin Invest, 1998. 101(6): p. 1478-87. 116. Bond, M., et al., Localization of the death domain of tissue inhibitor of metalloproteinase-3 to the N terminus. Metalloproteinase inhibition is associated with proapoptotic activity. J Biol Chem, 2000. 275(52): p. 41358-63. 117. Qi, J.H., et al., A novel function for tissue inhibitor of metalloproteinases-3 (TIMP3): inhibition of angiogenesis by blockage of VEGF binding to VEGF receptor-2. Nat Med, 2003. 9(4): p. 407-15. 118. Lee, M.H., et al., Full-length and N-TIMP-3 display equal inhibitory activities toward TNF-alpha convertase. Biochem Biophys Res Commun, 2001. 280(3): p. 945-50. 119. Lee, M.H., M. Rapti, and G. Murphy, Total conversion of tissue inhibitor of metalloproteinase (TIMP) for specific metalloproteinase targeting: fine-tuning TIMP-4 for optimal inhibition of tumor necrosis factor-{alpha}-converting enzyme. J Biol Chem, 2005. 280(16): p. 15967-75. 120. Bode, W., et al., Insights into MMP-TIMP interactions. Ann N Y Acad Sci, 1999. 878: p. 73-91. 121. Bode, W. and K. Maskos, Structural basis of the matrix metalloproteinases and their physiological inhibitors, the tissue inhibitors of metalloproteinases. Biol Chem, 2003. 384(6): p. 863-72. 122. Wisniewska, M., et al., Structural determinants of the ADAM inhibition by TIMP- 3: crystal structure of the TACE-N-TIMP-3 complex. J Mol Biol, 2008. 381(5): p. 1307-19. 123. Willenbrock, F., et al., The activity of the tissue inhibitors of metalloproteinases is regulated by C-terminal domain interactions: a kinetic analysis of the inhibition of gelatinase A. Biochemistry, 1993. 32(16): p. 4330-7. 124. Langton, K.P., M.D. Barker, and N. McKie, Localization of the functional domains of human tissue inhibitor of metalloproteinases-3 and the effects of a Sorsby's fundus dystrophy mutation. J Biol Chem, 1998. 273(27): p. 16778-81. 125. Lee, M.H., et al., Tailoring tissue inhibitor of metalloproteinases-3 to overcome the weakening effects of the cysteine-rich domains of tumour necrosis factor- alpha converting enzyme. Biochem J, 2003. 371(Pt 2): p. 369-76. 126. Lee, M.-H., private communication. 2007 127. Lee, M.H., et al., Mapping and characterization of the functional epitopes of tissue inhibitor of metalloproteinases (TIMP)-3 using TIMP-1 as the scaffold: a new frontier in TIMP engineering. Protein Sci, 2002. 11(10): p. 2493-503.

139

128. Pulukuri, S.M., et al., Epigenetic inactivation of the tissue inhibitor of metalloproteinase-2 (TIMP-2) gene in human prostate tumors. Oncogene, 2007. 26(36): p. 5229-37. 129. Galm, O., et al., Inactivation of the tissue inhibitor of metalloproteinases-2 gene by promoter hypermethylation in lymphoid malignancies. Oncogene, 2005. 24(30): p. 4799-805. 130. Ivanova, T., et al., Frequent hypermethylation of 5' flanking region of TIMP-2 gene in cervical cancer. Int J Cancer, 2004. 108(6): p. 882-6. 131. Gu, P., et al., Frequent loss of TIMP-3 expression in progression of esophageal and gastric adenocarcinomas. Neoplasia, 2008. 10(6): p. 563-72. 132. Wang, Y., et al., Multiple gene methylation of nonsmall cell lung cancers evaluated with 3-dimensional microarray. Cancer, 2008. 112(6): p. 1325-36. 133. Hoque, M.O., et al., Tissue inhibitor of metalloproteinases-3 promoter methylation is an independent prognostic factor for bladder cancer. J Urol, 2008. 179(2): p. 743-7. 134. Yuan, Y., et al., Frequent epigenetic inactivation of spleen tyrosine kinase gene in human hepatocellular carcinoma. Clin Cancer Res, 2006. 12(22): p. 6687-95. 135. Lui, E.L., et al., DNA hypermethylation of TIMP3 gene in invasive breast ductal carcinoma. Biomed Pharmacother, 2005. 59 Suppl 2: p. S363-5. 136. Jeong, D.H., et al., Promoter methylation of p16, DAPK, CDH1, and TIMP-3 genes in cervical cancer: correlation with clinicopathologic characteristics. Int J Gynecol Cancer, 2006. 16(3): p. 1234-40. 137. Peng, D.F., et al., DNA methylation of multiple tumor-related genes in association with overexpression of DNA methyltransferase 1 (DNMT1) during multistage carcinogenesis of the pancreas. Carcinogenesis, 2006. 27(6): p. 1160-8. 138. Brueckl, W.M., et al., Alterations in the tissue inhibitor of metalloproteinase-3 (TIMP-3) are found frequently in human colorectal tumours displaying either microsatellite stability (MSS) or instability (MSI). Cancer Lett, 2005. 223(1): p. 137-42. 139. Dulaimi, E., et al., Promoter hypermethylation profile of kidney cancer. Clin Cancer Res, 2004. 10(12 Pt 1): p. 3972-9. 140. Kang, G.H., et al., CpG island methylation in premalignant stages of gastric carcinoma. Cancer Res, 2001. 61(7): p. 2847-51. 141. Nakamura, M., et al., Frequent LOH on 22q12.3 and TIMP-3 inactivation occur in the progression to secondary glioblastomas. Lab Invest, 2005. 85(2): p. 165- 75. 142. Gonzalez-Gomez, P., et al., Promoter methylation status of multiple genes in brain metastases of solid tumors. Int J Mol Med, 2004. 13(1): p. 93-8. 143. Saunders, W.B., et al., Coregulation of vascular tube stabilization by endothelial cell TIMP-2 and pericyte TIMP-3. J Cell Biol, 2006. 175(1): p. 179-91. 144. Will, H., et al., The soluble catalytic domain of membrane type 1 matrix metalloproteinase cleaves the propeptide of progelatinase A and initiates autoproteolytic activation. Regulation by TIMP-2 and TIMP-3. J Biol Chem, 1996. 271(29): p. 17119-23. 145. Amour, A., et al., The in vitro activity of ADAM-10 is inhibited by TIMP-1 and TIMP-3. FEBS Lett, 2000. 473(3): p. 275-9.

140

146. Amour, A., et al., TNF-alpha converting enzyme (TACE) is inhibited by TIMP-3. FEBS Lett., 1998. 435(1): p. 39-44. 147. Loechel, F., et al., ADAM 12-S cleaves IGFBP-3 and IGFBP-5 and is inhibited by TIMP-3. Biochem Biophys Res Commun, 2000. 278(3): p. 511-5. 148. Yu, H. and T. Rohan, Role of the insulin-like growth factor family in cancer development and progression. J Natl Cancer Inst, 2000. 92(18): p. 1472-89. 149. Coussens, L.M. and Z. Werb, Inflammation and cancer. Nature, 2002. 420(6917): p. 860-7. 150. Li, W.Q. and M. Zafarullah, Oncostatin M up-regulates tissue inhibitor of metalloproteinases-3 gene expression in articular chondrocytes via de novo transcription, protein synthesis, and tyrosine kinase- and mitogen-activated protein kinase-dependent mechanisms. J Immunol, 1998. 161(9): p. 5000-7. 151. Gatsios, P., et al., Oncostatin M differentially regulates tissue inhibitors of metalloproteinases TIMP-1 and TIMP-3 gene expression in human synovial lining cells. Eur J Biochem, 1996. 241(1): p. 56-63. 152. Bugno, M., et al., Reprogramming of TIMP-1 and TIMP-3 expression profiles in brain microvascular endothelial cells and astrocytes in response to proinflammatory cytokines. FEBS Lett, 1999. 448(1): p. 9-14. 153. Meira, L.B., et al., DNA damage induced by chronic inflammation contributes to colon carcinogenesis in mice. J Clin Invest, 2008. 118(7): p. 2516-25. 154. Karin, M. and F.R. Greten, NF-kappaB: linking inflammation and immunity to cancer development and progression. Nat Rev Immunol, 2005. 5(10): p. 749-59. 155. Tillett, W. and T.J. Francis, Serological reactions in pneumonia with a non- protein somatic fraction of pneumococcus. J Exp Med, 1930. 52(4): p. 561-571. 156. Avery, O.T. and C.M. MacLeod, The occurence during acute infections of a protein not normally present in the blood: III. immunological properties of the C- reactive protein and its differentiation from normal blood proteins. The Journal of Experimental Medicine, 1941. 73: p. 191-200. 157. Bone, R.C., et al., Definitions for sepsis and organ failure and guidelines for the use of innovative therapies in sepsis. The ACCP/SCCM Consensus Conference Committee. American College of Chest Physicians/Society of Critical Care Medicine. Chest, 1992. 101(6): p. 1644-55. 158. Sharma, S. and A. Kumar, Septic shock, multiple organ failure, and acute respiratory distress syndrome. Curr Opin Pulm Med, 2003. 9(3): p. 199-209. 159. Aird, W.C., The role of the endothelium in severe sepsis and multiple organ dysfunction syndrome. Blood, 2003. 101(10): p. 3765-77. 160. Fry, D.E., Multiple Organ Dysfunction Syndrome, in Surgery : Basic Science and Clinical Evidence, J. Norton, et al., Editors. 2008, Springer Science-Business Media, LLC. 161. Rietschel, E.T., et al., Bacterial endotoxin: molecular relationships of structure to activity and function. Faseb J, 1994. 8(2): p. 217-25. 162. Raetz, C.R. and C. Whitfield, Lipopolysaccharide endotoxins. Annu Rev Biochem, 2002. 71: p. 635-700. 163. Medzhitov, R. and C. Janeway, Jr., Innate immunity. N Engl J Med, 2000. 343(5): p. 338-44.

141

164. Beutler, B. and A. Poltorak, The sole gateway to endotoxin response: how LPS was identified as Tlr4, and its role in innate immunity. Drug Metab Dispos, 2001. 29(4 Pt 2): p. 474-8. 165. Thompson, P.A., et al., Lipopolysaccharide (LPS)-binding protein inhibits responses to cell-bound LPS. J Biol Chem, 2003. 278(31): p. 28367-71. 166. Pugin, J., et al., Lipopolysaccharide activation of human endothelial and epithelial cells is mediated by lipopolysaccharide-binding protein and soluble CD14. Proc Natl Acad Sci U S A, 1993. 90(7): p. 2744-8. 167. Wright, S.D., CD14 and innate recognition of bacteria. J Immunol, 1995. 155(1): p. 6-8. 168. Shimazu, R., et al., MD-2, a molecule that confers lipopolysaccharide responsiveness on Toll-like receptor 4. J Exp Med, 1999. 189(11): p. 1777-82. 169. Sandor, F. and M. Buc, Toll-like receptors. II. Distribution and pathways involved in TLR signalling. Folia Biol (Praha), 2005. 51(6): p. 188-97. 170. Matsuzawa, A., et al., ROS-dependent activation of the TRAF6-ASK1-p38 pathway is selectively required for TLR4-mediated innate immunity. Nat Immunol, 2005. 6(6): p. 587-92. 171. Vassalli, P., The pathophysiology of tumor necrosis factors. Annu Rev Immunol, 1992. 10: p. 411-52. 172. Kinkhabwala, M., et al., A novel addition to the T cell repertory. Cell surface expression of tumor necrosis factor/cachectin by activated normal human T cells. J Exp Med, 1990. 171(3): p. 941-6. 173. Lopez-Cepero, M., et al., Soluble and membrane-bound TNF-alpha are involved in the cytotoxic activity of B cells from tumor-bearing mice against tumor targets. J Immunol, 1994. 152(7): p. 3333-41. 174. Remick, Friedland, ed. Cytokines in Health and Disease. Second Edition, Revised and Expanded ed. 1997. 175. Freudenberg, M.A., D. Keppler, and C. Galanos, Requirement for lipopolysaccharide-responsive macrophages in galactosamine-induced sensitization to endotoxin. Infect Immun, 1986. 51(3): p. 891-5. 176. Freudenberg, M.A. and C. Galanos, Tumor necrosis factor alpha mediates lethal activity of killed gram-negative and gram-positive bacteria in D-galactosamine- treated mice. Infect Immun, 1991. 59(6): p. 2110-5. 177. Wiktor-Jedrzejczak, W., et al., Colony-stimulating factor 1-dependent resident macrophages play a regulatory role in fighting Escherichia coli fecal peritonitis. Infect Immun, 1996. 64(5): p. 1577-81. 178. Gelman, A.E., et al., Toll-like receptor ligands directly promote activated CD4+ T cell survival. J Immunol, 2004. 172(10): p. 6065-73. 179. Wilson, R.K., et al., Structure, organization and polymorphism of murine and human T-cell receptor alpha and beta chain gene families. Immunol Rev, 1988. 101: p. 149-72. 180. Miethke, T., et al., T cell-mediated lethal shock triggered in mice by the superantigen staphylococcal enterotoxin B: critical role of tumor necrosis factor. J Exp Med, 1992. 175(1): p. 91-8. 181. White, J., et al., The V beta-specific superantigen staphylococcal enterotoxin B: stimulation of mature T cells and clonal deletion in neonatal mice. Cell, 1989. 56(1): p. 27-35.

142

182. Jue, D.M., et al., Processing of newly synthesized cachectin/tumor necrosis factor in endotoxin-stimulated macrophages. Biochemistry, 1990. 29(36): p. 8371-7. 183. Granell, S., et al., Circulating TNF-alpha and its soluble receptors during experimental acute pancreatitis. Cytokine, 2004. 25(4): p. 187-91. 184. Erickson, S.L., et al., Decreased sensitivity to tumour-necrosis factor but normal T-cell development in TNF receptor-2-deficient mice. Nature, 1994. 372(6506): p. 560-3. 185. Tartaglia, L.A., et al., The two different receptors for tumor necrosis factor mediate distinct cellular responses. Proc Natl Acad Sci U S A, 1991. 88(20): p. 9292-6. 186. Grell, M., et al., The transmembrane form of tumor necrosis factor is the prime activating ligand of the 80 kDa tumor necrosis factor receptor. Cell, 1995. 83(5): p. 793-802. 187. Wajant, H., K. Pfizenmaier, and P. Scheurich, Tumor necrosis factor signaling. Cell Death Differ, 2003. 10(1): p. 45-65. 188. Beutler, B.A., I.W. Milsark, and A. Cerami, Cachectin/tumor necrosis factor: production, distribution, and metabolic fate in vivo. J Immunol, 1985. 135(6): p. 3972-7. 189. Hoeck, J. and M. Woisetschlager, STAT6 mediates eotaxin-1 expression in IL-4 or TNF-alpha-induced fibroblasts. J Immunol, 2001. 166(7): p. 4507-15. 190. Nakayama, T., et al., Inducible expression of a CC chemokine liver- and activation-regulated chemokine (LARC)/macrophage inflammatory protein (MIP)-3 alpha/CCL20 by epidermal keratinocytes and its role in atopic dermatitis. Int Immunol, 2001. 13(1): p. 95-103. 191. Massey, K.D., et al., Cardiac myocytes release leukocyte-stimulating factors. Am J Physiol, 1995. 269(3 Pt 2): p. H980-7. 192. Hwang, C.S., et al., Adipocyte differentiation and leptin expression. Annu Rev Cell Dev Biol, 1997. 13: p. 231-59. 193. Rui, L., et al., Insulin/IGF-1 and TNF-alpha stimulate phosphorylation of IRS-1 at inhibitory Ser307 via distinct pathways. J Clin Invest, 2001. 107(2): p. 181-9. 194. Bullo-Bonet, M., et al., Tumour necrosis factor, a key role in obesity? FEBS Lett, 1999. 451(3): p. 215-9. 195. Aguirre, V., et al., The c-Jun NH(2)-terminal kinase promotes insulin resistance during association with insulin receptor substrate-1 and phosphorylation of Ser(307). J Biol Chem, 2000. 275(12): p. 9047-54. 196. Sharma, R. and S.D. Anker, Cytokines, apoptosis and cachexia: the potential for TNF antagonism. Int J Cardiol, 2002. 85(1): p. 161-71. 197. Beutler, B., et al., Purification of cachectin, a lipoprotein lipase-suppressing hormone secreted by endotoxin-induced RAW 264.7 cells. J Exp Med, 1985. 161(5): p. 984-95. 198. Argiles, J.M., et al., Cancer cachexia: the molecular mechanisms. Int J Biochem Cell Biol, 2003. 35(4): p. 405-9. 199. Masaki, T., et al., Tumor necrosis factor-alpha regulates in vivo expression of the rat UCP family differentially. Biochim Biophys Acta, 1999. 1436(3): p. 585-92. 200. Richards, C.D., Interleukin 6, in Cytokines, A. Mire-Sluis and R. Thorpe, Editors. 1998, Academic Press.

143

201. Klir, J.J., et al., Role of hypothalamic interleukin-6 and tumor necrosis factor- alpha in LPS fever in rat. Am J Physiol, 1993. 265(3 Pt 2): p. R512-7. 202. Castell, J.V., et al., Interleukin-6 is the major regulator of acute phase protein synthesis in adult human hepatocytes. FEBS Lett, 1989. 242(2): p. 237-9. 203. Mold, C., B. Rodic-Polic, and T.W. Du Clos, Protection from Streptococcus pneumoniae infection by C-reactive protein and natural antibody requires complement but not Fc gamma receptors. J Immunol, 2002. 168(12): p. 6375-81. 204. Turner, M.W., The role of mannose-binding lectin in health and disease. Mol Immunol, 2003. 40(7): p. 423-9. 205. Taga, T. and T. Kishimoto, Gp130 and the interleukin-6 family of cytokines. Annu Rev Immunol, 1997. 15: p. 797-819. 206. Heinrich, P.C., et al., Interleukin-6-type cytokine signalling through the gp130/Jak/STAT pathway. Biochem J, 1998. 334 ( Pt 2): p. 297-314. 207. Skiniotis, G., et al., Signaling conformations of the tall cytokine receptor gp130 when in complex with IL-6 and IL-6 receptor. Nat Struct Mol Biol, 2005. 12(6): p. 545-51. 208. Imada, K. and W.J. Leonard, The Jak-STAT pathway. Mol Immunol, 2000. 37(1- 2): p. 1-11. 209. Akira, S., et al., Molecular cloning of APRF, a novel IFN-stimulated gene factor 3 p91-related transcription factor involved in the gp130-mediated signaling pathway. Cell, 1994. 77(1): p. 63-71. 210. Chomarat, P., et al., IL-6 switches the differentiation of monocytes from dendritic cells to macrophages. Nat Immunol, 2000. 1(6): p. 510-4. 211. Ryffel, B., et al., Pathology induced by interleukin-6. Toxicol Lett, 1992. 64-65 Spec No: p. 311-9. 212. Barton, B.E. and J.V. Jackson, Protective role of interleukin 6 in the lipopolysaccharide-galactosamine septic shock model. Infect Immun, 1993. 61(4): p. 1496-9. 213. Xing, Z., et al., IL-6 is an antiinflammatory cytokine required for controlling local or systemic acute inflammatory responses. J Clin Invest, 1998. 101(2): p. 311-20. 214. El-Ani, D. and R. Zimlichman, TNFalpha stimulated ATP-sensitive potassium channels and attenuated deoxyglucose and Ca uptake of H9c2 cardiomyocytes. Ann N Y Acad Sci, 2003. 1010: p. 716-20. 215. Binder, C., et al., Induction of inducible nitric oxide synthase is an essential part of tumor necrosis factor-alpha-induced apoptosis in MCF-7 and other epithelial tumor cells. Lab Invest, 1999. 79(12): p. 1703-12. 216. Nathan, C. and Q.W. Xie, Nitric oxide synthases: roles, tolls, and controls. Cell, 1994. 78(6): p. 915-8. 217. Lloyd-Jones, D.M. and K.D. Bloch, The vascular biology of nitric oxide and its role in atherogenesis. Annu Rev Med, 1996. 47: p. 365-75. 218. Hollenberg, S.M., M. Guglielmi, and J.E. Parrillo, Discordance between microvascular permeability and leukocyte dynamics in septic iNOS-deficient mice. Crit Care, 2007. 11(6): p. R125. 219. Laubach, V.E., et al., Mice lacking inducible nitric oxide synthase are not resistant to lipopolysaccharide-induced death. Proc Natl Acad Sci U S A, 1995. 92(23): p. 10688-92.

144

220. Wei, X.Q., et al., Altered immune responses in mice lacking inducible nitric oxide synthase. Nature, 1995. 375(6530): p. 408-11. 221. Salkowski, C.A., et al., Regulation of inducible nitric oxide synthase messenger RNA expression and nitric oxide production by lipopolysaccharide in vivo: the roles of macrophages, endogenous IFN-gamma, and TNF receptor-1-mediated signaling. J Immunol, 1997. 158(2): p. 905-12. 222. Hoffbrand, A.V., P.A.H. Moss, and J.E. Pettit, Essential Haematology. 5th ed. 2006, Malden. 223. Iscove, N.N. and K. Nawa, Hematopoietic stem cells expand during serial transplantation in vivo without apparent exhaustion. Curr Biol, 1997. 7(10): p. 805-8. 224. Heissig, B., et al., Recruitment of stem and progenitor cells from the bone marrow niche requires MMP-9 mediated release of kit-ligand. Cell, 2002. 109(5): p. 625- 37. 225. Cruz, A.C., et al., Tumor necrosis factor-alpha-converting enzyme controls surface expression of c-Kit and survival of embryonic stem cell-derived mast cells. J Biol Chem, 2004. 279(7): p. 5612-20. 226. LaVoie, M.J. and D.J. Selkoe, The Notch ligands, Jagged and Delta, are sequentially processed by alpha-secretase and presenilin/gamma-secretase and release signaling fragments. J Biol Chem, 2003. 278(36): p. 34427-37. 227. Brou, C., et al., A novel proteolytic cleavage involved in Notch signaling: the role of the disintegrin-metalloprotease TACE. Mol Cell, 2000. 5(2): p. 207-16. 228. Six, E., et al., The Notch ligand Delta1 is sequentially cleaved by an ADAM protease and gamma-secretase. Proc Natl Acad Sci U S A, 2003. 100(13): p. 7638-43. 229. Dyczynska, E., et al., Proteolytic processing of delta-like 1 by ADAM proteases. J Biol Chem, 2007. 282(1): p. 436-44. 230. Chklovskaia, E., et al., Cell-surface trafficking and release of flt3 ligand from T lymphocytes is induced by common cytokine receptor gamma-chain signaling and inhibited by cyclosporin A. Blood, 2001. 97(4): p. 1027-34. 231. Horiuchi, K., et al., Cell surface colony-stimulating factor 1 can be cleaved by TNF-alpha converting enzyme or endocytosed in a clathrin-dependent manner. J Immunol, 2007. 179(10): p. 6715-24. 232. Rovida, E., et al., TNF-alpha-converting enzyme cleaves the macrophage colony- stimulating factor receptor in macrophages undergoing activation. J Immunol, 2001. 166(3): p. 1583-9. 233. Budagian, V., et al., Natural soluble interleukin-15Ralpha is generated by cleavage that involves the tumor necrosis factor-alpha-converting enzyme (TACE/ADAM17). J Biol Chem, 2004. 279(39): p. 40368-75. 234. Duncan, A.W., et al., Integration of Notch and Wnt signaling in hematopoietic stem cell maintenance. Nat Immunol, 2005. 6(3): p. 314-22. 235. Pruijt, J.F., R. Willemze, and W.E. Fibbe, Mechanisms underlying hematopoietic stem cell mobilization induced by the CXC chemokine interleukin-8. Curr Opin Hematol, 1999. 6(3): p. 152-8. 236. Masure, S., et al., Production and characterization of recombinant active mouse gelatinase B from eukaryotic cells and in vivo effects after intravenous administration. Eur J Biochem, 1997. 244(1): p. 21-30.

145

237. Nie, Y., Y.C. Han, and Y.R. Zou, CXCR4 is required for the quiescence of primitive hematopoietic cells. J Exp Med, 2008. 205(4): p. 777-83. 238. Iscove, N.N., et al., Colony formation by normal and leukemic human marrow cells in culture: effect of conditioned medium from human leukocytes. Blood, 1971. 37(1): p. 1-5. 239. Chesler, L., et al., Metalloproteinase inhibition and erythroid potentiation are independent activities of tissue inhibitor of metalloproteinases-1. Blood, 1995. 86(12): p. 4506-15. 240. Niskanen, E., et al., In vivo effect of human erythroid-potentiating activity on hematopoiesis in mice. Blood, 1988. 72(2): p. 806-10. 241. Zins, K., et al., Colon cancer cell-derived tumor necrosis factor-alpha mediates the tumor growth-promoting response in macrophages by up-regulating the colony-stimulating factor-1 pathway. Cancer Res, 2007. 67(3): p. 1038-45. 242. Lotem, J. and L. Sachs, Cytokine control of developmental programs in normal hematopoiesis and leukemia. Oncogene, 2002. 21(21): p. 3284-94. 243. Mohler, K.M., et al., Protection against a lethal dose of endotoxin by an inhibitor of tumour necrosis factor processing. Nature, 1994. 370(6486): p. 218-20. 244. Fata, J.E., et al., Accelerated apoptosis in the Timp-3-deficient mammary gland. J Clin Invest, 2001. 108(6): p. 831-41. 245. Pfeffer, K., et al., Mice deficient for the 55 kd tumor necrosis factor receptor are resistant to endotoxic shock, yet succumb to L. monocytogenes infection. Cell, 1993. 73(3): p. 457-467. 246. Mahmoodi, M., et al., Lack of tissue inhibitor of metalloproteinases-3 results in an enhanced inflammatory response in antigen-induced arthritis. Am J Pathol, 2005. 166(6): p. 1733-40. 247. Jin, G., et al., A continuous fluorimetric assay for tumor necrosis factor-alpha converting enzyme. Anal Biochem, 2002. 302(2): p. 269-75. 248. Price, A., et al., Marked inhibition of tumor growth in a malignant glioma tumor model by a novel synthetic matrix metalloproteinase inhibitor AG3340. Clin Cancer Res, 1999. 5(4): p. 845-54. 249. Peterson, J.T., et al., Matrix metalloproteinase inhibition attenuates left ventricular remodeling and dysfunction in a rat model of progressive heart failure. Circulation, 2001. 103(18): p. 2303-9. 250. Semma, M. and S. Sagami, Induction of suppressor T cells to DNFB contact sensitivity by application of sensitizer through Langerhans cell-deficient skin. Arch Dermatol Res, 1981. 271(3): p. 361-4. 251. Leist, T.P., et al., Functional analysis of T lymphocyte subsets in antiviral host defense. J Immunol, 1987. 138(7): p. 2278-81. 252. Garza, K.M., et al., Role of antigen-presenting cells in mediating tolerance and autoimmunity. J Exp Med, 2000. 191(11): p. 2021-7. 253. Berg-Brown, N.N., et al., PKCtheta signals activation versus tolerance in vivo. J Exp Med, 2004. 199(6): p. 743-52. 254. Okada, S., et al., In vivo and in vitro stem cell function of c-kit- and Sca-1-positive murine hematopoietic cells. Blood, 1992. 80(12): p. 3044-50. 255. Jones, N.H., et al., Isolation of complementary DNA clones encoding the human lymphocyte glycoprotein T1/Leu-1. Nature, 1986. 323(6086): p. 346-9.

146

256. Mundt, C., et al., Loss of precursor B cell expansion but not allelic exclusion in VpreB1/VpreB2 double-deficient mice. J Exp Med, 2001. 193(4): p. 435-45. 257. Azzam, H.S., et al., CD5 expression is developmentally regulated by T cell receptor (TCR) signals and TCR avidity. The Journal of experimental medicine, 1998. 188(12): p. 2301-11. 258. Paige, C.J., H. Skarvall, and H. Sauter, Differentiation of murine B cell precursors in agar culture. II. Response of precursor-enriched populations to growth stimuli and demonstration that the clonable pre-B cell assay is limiting for the B cell precursor. J Immunol, 1985. 134(6): p. 3699-704. 259. Muto, S., V. Vetvicka, and G.D. Ross, CR3 (CD11b/CD18) expressed by cytotoxic T cells and natural killer cells is upregulated in a manner similar to neutrophil CR3 following stimulation with various activating agents. J Clin Immunol, 1993. 13(3): p. 175-84. 260. Hestdal, K., et al., Characterization and regulation of RB6-8C5 antigen expression on murine bone marrow cells. J Immunol, 1991. 147(1): p. 22-8. 261. Hirsch, S. and S. Gordon, Polymorphic expression of a neutrophil differentiation antigen revealed by monoclonal antibody 7/4. Immunogenetics, 1983. 18(3): p. 229-39. 262. Kina, T., et al., The monoclonal antibody TER-119 recognizes a molecule associated with glycophorin A and specifically marks the late stages of murine erythroid lineage. Br J Haematol, 2000. 109(2): p. 280-7. 263. Goodell, M.A., et al., Isolation and functional properties of murine hematopoietic stem cells that are replicating in vivo. J Exp Med, 1996. 183(4): p. 1797-806. 264. Baker, S.J. and E.P. Reddy, Modulation of life and death by the TNF receptor superfamily. Oncogene, 1998. 17(25): p. 3261-3270. 265. Reimold, A.M., TNFalpha as therapeutic target: new drugs, more applications. Curr Drug Targets Inflamm Allergy, 2002. 1(4): p. 377-92. 266. Diwan, A., et al., Targeted overexpression of noncleavable and secreted forms of tumor necrosis factor provokes disparate cardiac phenotypes. Circulation, 2004. 109(2): p. 262-8. 267. Georgopoulos, S., D. Plows, and G. Kollias, Transmembrane TNF is sufficient to induce localized tissue toxicity and chronic inflammatory arthritis in transgenic mice. J Inflamm, 1996. 46(2): p. 86-97. 268. Ruuls, S.R., et al., Membrane-bound TNF supports secondary lymphoid organ structure but is subservient to secreted TNF in driving autoimmune inflammation. Immunity, 2001. 15(4): p. 533-43. 269. Xanthoulea, S., et al., Tumor necrosis factor (TNF) receptor shedding controls thresholds of innate immune activation that balance opposing TNF functions in infectious and inflammatory diseases. J Exp Med, 2004. 200(3): p. 367-76. 270. Borland, G., G. Murphy, and A. Ager, Tissue inhibitor of metalloproteinases-3 inhibits shedding of L-selectin from leukocytes. J Biol Chem, 1999. 274(5): p. 2810-5. 271. Garton, K.J., et al., Tumor necrosis factor-alpha-converting enzyme (ADAM17) mediates the cleavage and shedding of fractalkine (CX3CL1). J Biol Chem, 2001. 276(41): p. 37993-8001. 272. Munoz, J.J. and W.A. Sewell, Effect of pertussigen on inflammation caused by Freund adjuvant. Infect Immun, 1984. 44(3): p. 637-41.

147

273. Engelberts, I., et al., The interrelation between TNF, IL-6, and PAF secretion induced by LPS in an in vivo and in vitro murine model. Lymphokine Cytokine Res, 1991. 10(1-2): p. 127-31. 274. Ghezzi, P., et al., Lps induces IL-6 in the brain and in serum largely through TNF production. Cytokine, 2000. 12(8): p. 1205-10. 275. Beutler, B., I.W. Milsark, and A.C. Cerami, Passive immunization against cachectin/tumor necrosis factor protects mice from lethal effect of endotoxin. Science, 1985. 229(4716): p. 869-71. 276. Rothe, J., et al., Mice lacking the tumour necrosis factor receptor 1 are resistant to TNF-mediated toxicity but highly susceptible to infection by Listeria monocytogenes. Nature, 1993. 364(6440): p. 798-802. 277. Peschon, J.J., et al., TNF receptor-deficient mice reveal divergent roles for p55 and p75 in several models of inflammation. J Immunol, 1998. 160(2): p. 943-52. 278. Koskivirta, I., et al., Tissue inhibitor of metalloproteinases 4 (TIMP4) is involved in inflammatory processes of human cardiovascular pathology. Histochem Cell Biol, 2006. 126(3): p. 335-42. 279. Kassiri, Z., et al., Combination of tumor necrosis factor-alpha ablation and matrix metalloproteinase inhibition prevents heart failure after pressure overload in tissue inhibitor of metalloproteinase-3 knock-out mice. Circ Res, 2005. 97(4): p. 380-90. 280. Beutler, B. and A. Poltorak, Sepsis and evolution of the innate immune response. Crit Care Med, 2001. 29(7 Suppl): p. S2-6; discussion S6-7. 281. Godenschwege, T.A., et al., Inflated wings, tissue autolysis and early death in tissue inhibitor of metalloproteinases mutants of Drosophila. Eur J Cell Biol, 2000. 79(7): p. 495-501. 282. Parks, W.C., C.L. Wilson, and Y.S. Lopez-Boado, Matrix metalloproteinases as modulators of inflammation and innate immunity. Nat Rev Immunol, 2004. 4(8): p. 617-29. 283. Sessler, C.N., J.C. Perry, and K.L. Varney, Management of severe sepsis and septic shock. Current opinion in critical care, 2004. 10(5): p. 354-63. 284. Iimuro, Y., et al., Blockade of liver macrophages by gadolinium chloride reduces lethality in endotoxemic rats--analysis of mechanisms of lethality in endotoxemia. J Leukoc Biol, 1994. 55(6): p. 723-8. 285. Kumins, N.H., et al., Partial hepatectomy reduces the endotoxin-induced peak circulating level of tumor necrosis factor in rats. Shock, 1996. 5(5): p. 385-8. 286. Moshage, H., Cytokines and the hepatic acute phase response. J Pathol, 1997. 181(3): p. 257-66. 287. Casey, L.C., R.A. Balk, and R.C. Bone, Plasma cytokine and endotoxin levels correlate with survival in patients with the sepsis syndrome. Ann Intern Med, 1993. 119(8): p. 771-8. 288. Ge, Y., et al., Relationship of tissue and cellular interleukin-1 and lipopolysaccharide after endotoxemia and bacteremia. J Infect Dis, 1997. 176(5): p. 1313-21. 289. Agarwal, M.K., M. Parant, and F. Parant, Role of spleen in endotoxin poisoning and reticuloendothelial function. Br J Exp Pathol, 1972. 53(5): p. 485-91. 290. Karanfilian, R.G., et al., Effect of age and splenectomy in murine endotoxemia. Adv Shock Res, 1983. 9: p. 125-32.

148

291. Huston, J.M., et al., Splenectomy inactivates the cholinergic antiinflammatory pathway during lethal endotoxemia and polymicrobial sepsis. J Exp Med, 2006. 203(7): p. 1623-8. 292. Ogle, C.K., et al., Heterogeneity of Kupffer cells and splenic, alveolar, and peritoneal macrophages for the production of TNF, IL-1, and IL-6. Inflammation, 1994. 18(5): p. 511-23. 293. O'Neill, P.J., et al., Role of Kupffer cells in interleukin-6 release following trauma-hemorrhage and resuscitation. Shock, 1994. 1(1): p. 43-7. 294. Mohammed, F.F., et al., Abnormal TNF activity in Timp3-/- mice leads to chronic hepatic inflammation and failure of liver regeneration. Nat Genet, 2004. 36(9): p. 969-77. 295. Castell, J.V., et al., Plasma clearance, organ distribution and target cells of interleukin-6/hepatocyte-stimulating factor in the rat. Eur J Biochem, 1988. 177(2): p. 357-61. 296. Ripperger, J., et al., Isolation of two interleukin-6 response element binding proteins from acute phase rat livers. Ann N Y Acad Sci, 1995. 762: p. 252-60; discussion 260-1. 297. Hemmann, U., et al., Differential activation of acute phase response factor/Stat3 and Stat1 via the cytoplasmic domain of the interleukin 6 signal transducer gp130. II. Src homology SH2 domains define the specificity of stat factor activation. J Biol Chem, 1996. 271(22): p. 12999-3007. 298. Schumann, R.R., et al., The lipopolysaccharide-binding protein is a secretory class 1 acute-phase protein whose gene is transcriptionally activated by APRF/STAT/3 and other cytokine-inducible nuclear proteins. Mol Cell Biol, 1996. 16(7): p. 3490-503. 299. Negoro, S., et al., Activation of signal transducer and activator of transcription 3 protects cardiomyocytes from hypoxia/reoxygenation-induced oxidative stress through the upregulation of manganese superoxide dismutase. Circulation, 2001. 104(9): p. 979-81. 300. Davis, R.J., Signal transduction by the JNK group of MAP kinases. Cell, 2000. 103(2): p. 239-52. 301. Schiff, E.R., M.F. Sorrell, and W.C. Maddrey, Schiff's Diseases of the Liver. Vol. 2. 2007, Philadelphia: Lippincott Williams and Wilkins. 302. Hokeness, K.L., et al., Monocyte chemoattractant protein-1 and CCR2 interactions are required for IFN-alpha/beta-induced inflammatory responses and antiviral defense in liver. J Immunol, 2005. 174(3): p. 1549-56. 303. Bogdan, C., Nitric oxide and the immune response. Nat Immunol, 2001. 2(10): p. 907-16. 304. Frangogiannis, N.G., et al., Resident cardiac mast cells degranulate and release preformed TNF-alpha, initiating the cytokine cascade in experimental canine myocardial ischemia/reperfusion. Circulation, 1998. 98(7): p. 699-710. 305. DeFranco, A.L., R.M. Locksley, and M. Robertson, Immunity : the immune response in infectious and inflammatory disease. Primers in biology. 2007, London Sunderland, MA: New Science Press ; Sinauer Associates. xxx, 387 p.

149

306. Sallusto, F., et al., Rapid and coordinated switch in chemokine receptor expression during dendritic cell maturation. Eur J Immunol, 1998. 28(9): p. 2760- 9. 307. Bazan, J.F., et al., A new class of membrane-bound chemokine with a CX3C motif. Nature, 1997. 385(6617): p. 640-4. 308. Matias-Roman, S., et al., Membrane type 1-matrix metalloproteinase is involved in migration of human monocytes and is regulated through their interaction with fibronectin or endothelium. Blood, 2005. 105(10): p. 3956-64. 309. Van Den Steen, P.E., et al., Gelatinase B/MMP-9 and neutrophil collagenase/MMP-8 process the chemokines human GCP-2/CXCL6, ENA- 78/CXCL5 and mouse GCP-2/LIX and modulate their physiological activities. Eur J Biochem, 2003. 270(18): p. 3739-49. 310. Bour, H., et al., Major histocompatibility complex class I-restricted CD8+ T cells and class II-restricted CD4+ T cells, respectively, mediate and regulate contact sensitivity to dinitrofluorobenzene. Eur J Immunol, 1995. 25(11): p. 3006-10. 311. Farrar, J.J., et al., Thymoma production of T cell growth factor (Interleukin 2). J Immunol, 1980. 125(6): p. 2555-8. 312. Schwarz, K., et al., The use of LCMV-specific T cell hybridomas for the quantitative analysis of MHC class I restricted antigen presentation. J Immunol Methods, 2000. 237(1-2): p. 199-202. 313. Shirasaka, T., Development history and concept of an oral anticancer agent S-1 (TS-1): its clinical usefulness and future vistas. Jpn J Clin Oncol, 2009. 39(1): p. 2-15. 314. Vaziri, H., et al., Evidence for a mitotic clock in human hematopoietic stem cells: loss of telomeric DNA with age. Proc Natl Acad Sci U S A, 1994. 91(21): p. 9857- 60. 315. Ema, H., et al., Isolation of murine hematopoietic stem cells and progenitor cells. Curr Protoc Immunol, 2005. Chapter 22: p. Unit 22B 1. 316. Benveniste, P., et al., Hematopoietic stem cells engraft in mice with absolute efficiency. Nat Immunol, 2003. 4(7): p. 708-13. 317. Huang, Z., G.K. Ha, and J.M. Petitto, IL-15 and IL-15R alpha gene deletion: effects on T lymphocyte trafficking and the microglial and neuronal responses to facial nerve axotomy. Neurosci Lett, 2007. 417(2): p. 160-4. 318. Swiniarski, H., et al., A CTL assay requiring only 150 microliter of mouse blood. J Immunol Methods, 2000. 233(1-2): p. 1-11. 319. Cruz-Munoz, W., I. Kim, and R. Khokha, TIMP-3 deficiency in the host, but not in the tumor, enhances tumor growth and angiogenesis. Oncogene, 2006. 25(4): p. 650-5. 320. Lewthwaite, J., et al., Role of TNF alpha in the induction of antigen induced arthritis in the rabbit and the anti-arthritic effect of species specific TNF alpha neutralising monoclonal antibodies. Ann Rheum Dis, 1995. 54(5): p. 366-74. 321. Fedak, P.W., et al., TIMP-3 deficiency leads to dilated cardiomyopathy. Circulation, 2004. 110(16): p. 2401-9. 322. Wan, S. and A.P. Yim, Cytokines in myocardial injury: impact on cardiac surgical approach. Eur J Cardiothorac Surg, 1999. 16 Suppl 1: p. S107-11. 323. Broze, G.J., Jr., Tissue factor pathway inhibitor and the revised theory of coagulation. Annu Rev Med, 1995. 46: p. 103-12.

150

324. Belaaouaj, A.A., et al., Matrix metalloproteinases cleave tissue factor pathway inhibitor. Effects on coagulation. J Biol Chem, 2000. 275(35): p. 27123-8. 325. Dubois, B., et al., Gelatinase B deficiency protects against endotoxin shock. Eur J Immunol, 2002. 32(8): p. 2163-71. 326. Mohammed, F.F., D.S. Smookler, and R. Khokha, Metalloproteinases, inflammation, and rheumatoid arthritis. Ann Rheum Dis, 2003. 62 Suppl 2: p. ii43-7. 327. Zhang, X.W., et al., CXC chemokines, MIP-2 and KC, induce P-selectin- dependent neutrophil rolling and extravascular migration in vivo. Br J Pharmacol, 2001. 133(3): p. 413-21. 328. Li, Q., et al., Matrilysin shedding of syndecan-1 regulates chemokine mobilization and transepithelial efflux of neutrophils in acute lung injury. Cell, 2002. 111(5): p. 635-46. 329. Imamura, H., A. Brault, and P.M. Huet, Effects of extended cold preservation and transplantation on the rat liver microcirculation. Hepatology, 1997. 25(3): p. 664-71. 330. Urata, K., et al., Role of Kupffer cells in the survival after rat liver transplantation with long portal vein clamping times. Transpl Int, 2000. 13(6): p. 420-7. 331. Brew, K., D. Dinakarpandian, and H. Nagase, Tissue inhibitors of metalloproteinases: evolution, structure and function. Biochim Biophys Acta, 2000. 1477(1-2): p. 267-83. 332. Zeng, Y., et al., Temporal and spatial regulation of gene expression mediated by the promoter for the human tissue inhibitor of metalloproteinases-3 (TIMP-3)- encoding gene. Dev Dyn, 1998. 211(3): p. 228-37.

151