I. Differential Gene Expression in Human Peripheral Blood Monocytes and Alveolar Macrophages

I. DIFFERENTIAL GENE EXPRESSION IN HUMAN PERIPHERAL BLOOD MONOCYTES AND ALVEOLAR MACROPHAGES

II. MACROPHAGE COLONY-STIMULATING FACTOR IS IMPORTANT IN THE DEVELOPMENT OF PULMONARY FIBROSIS

DISSERTATION

Presented in Partial Fulfillment of the Requirements for the Degree

Doctor of Philosophy in the Graduate School of The Ohio State University

Judy Marcus Opalek, B.S.

∗ ∗ ∗ ∗ ∗

The Ohio State University

2004

Dissertation Committee: Approved by

Clay B. Marsh, M. D., Advisor

Anne VanBuskirk, Ph.D. Advisor W. James Waldman, Ph.D. Department of Pathology

ABSTRACT

Monocytes are precursors to tissue macrophages, and while numerous studies have examined monocyte differentiation in vitro, none have yet analyzed the gene expression profiles of native tissue macrophages compared to circulating monocytes. We aimed to generate a library of expression information detailing the distinction of these cell types. To this end, we performed a pilot study utilizing cDNA microarray technology. Our data indicates that several hundred genes are differentially regulated in peripheral blood monocytes and alveolar macrophages. These include genes involved in, or postulated to be involved in, cellular scavenging, intracellular signaling pathways, cellular survival and/or differentiation. Based on our observation that the chemokine receptor expression profiles of monocytes and alveolar macrophages differed in the gene array analysis, we confirmed these results by reverse transcriptase polymerase chain reaction, flow cytometry and functional analyses. Our data indicates that circulating monocytes express the chemokine receptors CCR1 and CCR2, and that monocytes functionally respond by migrating toward both MCP-1 and the macrophage inflammatory protein MIP-1α. In contrast, alveolar macrophages do not express CCR1 or CCR2, but do express the MIP-1α chemokine receptor

CCR5. Alveolar macrophages did not respond to MCP-1 but did respond to MIP-

ii 1α in a migration assay. The addition of an anti-CCR5 blocking antibody completely abrogated MIP-1α-induced migration in alveolar macrophages, but did not affect monocytes. These data may be helpful in understanding the regulated recruitment of inflammatory cells, and in distinguishing the regulatory pathways controlling the recruitment of circulating monocytes and native alveolar macrophages in areas of lung inflammation.

This data is relevant to human disease, as in pulmonary fibrosis high concentrations of MCP-1 are found in the lung lavage fluid from affected patients but not in normal volunteers. Pulmonary fibrosis is a serious lung disease characterized by progressive scarring of the lung tissue, eventually leading to hypoxemia and death of the affected patient. In human idiopathic pulmonary fibrosis, prognosis is worsened in patients with more monocytes and macrophages in their lungs. We used an animal model of bleomycin-induced pulmonary fibrosis to examine the role of Macrophage Colony-Stimulating Factor

(M-CSF) in the development of this disease. We chose this model because mice that are genetically deficient in M-CSF have decreased numbers of circulating monocytes and tissue macrophages, and provide a useful model for studying the role of these cells and this growth factor. We found that mice lacking M-CSF survived better, lost less weight and developed less fibrosis than their M-CSF normal littermates when treated systemically with weight-dependent doses of bleomycin. These data clearly implicate M-CSF-responsive monocytes and tissue macrophages in the development of pulmonary fibrosis.

iii

Dedicated to my father, Donald Richard Marcus.

ACKNOWLEDGMENTS

I would like to take this opportunity to thank the many people who helped

lead me in this direction, assisted me directly with this work or generally showed tremendous support for me in all of my endeavors (both scientifically and in life).

Thanks to Larry Wittle, for believing in me and pushing me to be better; to

Ping Lu for understanding and unselfish encouragement; and to Jim Waldman, for making sure I could stay in graduate school. I wouldn’t be here now if he weren’t so accommodating.

Of course, none of this work would have been possible without my advisor, Clay Marsh. Before my first day in his laboratory he invited me to the

2001 FASEB conference, and from that point there was no looking back. His

confidence and enthusiasm are contagious, and I am eternally grateful to him for

giving me so many wonderful opportunities.

Additionally, I would like to thank Cheryl Schneider for her phenomenal

support with my animals, Paul Stromberg for his histopathological expertise,

Naeem Ali for providing migration assay assistance, Ruth Berger and the folks at

Histotechniques for beautiful slides of my animal work, Pat Farmer for tirelessly

recruiting bronchoscopy volunteers, and Kurtis Yearsley for digital imaging.

Additionally, Susheela Tridandipani has been a wonderful friend and mentor,

v and I am ever so thankful. Also, I would like to thank my dissertation committee,

Neil Baker, Clay Marsh, Anne VanBuskirk, and Jim Waldman, for their support. I am indebted to my many labmates, both past and present, and especially to my good friends Mandy Zeigler and Chris Baran, who make this “work” fun.

Finally, and above all, I would like to thank my parents, Donald and Judy

Marcus, and my brother, Donny, who may not always understand what I do, but who always support me nonetheless. And of course, my husband and best friend, David, who can finally say that his wife has a real job. Without his unfailing support and friendship, I could never be what I am today.

VITA

May 14, 1975.…………………………………….. ……………….… Born- Adrian, MI 1997.…………………………………………….…….... Bachelor of Science, Biology Alma College Alma, Michigan

1997-1998………………………………………………….…..…Biomedical Sciences Wayne State University Detroit, Michigan

1998……………………………………………………………...…Research Associate The Department of Pathology Harper Hospital Detroit, Michigan

1998-present………………….………………………..Graduate Research Assistant The Department of Pathology The Ohio State University Columbus, Ohio

PUBLICATIONS

Research Publications

1. Wittle, L. W., Opalek, J. M., and Ruiter, T. C. (2000). Chromogranin A- Immunoreactive Cells in the Olfactory System of Anuran Amphibians. Gen. Comp. Endocrinol. 120(1):17-26.

2. Zeigler, M. M., Doseff, A. I., Galloway, M. F., Opalek, J. M., Nowicki, P. T., Zwier, J. L., Sen, C. K., and Marsh, C. B. (2003). Presentation of Nitric Oxide Regulates Monocyte Survival through Effects on Caspase-9 and Caspase-3 Activation. J. Biol. Chem. 278(15): 12894-902.

vii 3. Tridandapani, S., Wardrop, R., Baran, C., Wang, Y., Opalek, J. M., Caligiuri, M. A., and Marsh, C. B. (2003). Transforming Growth Factor-β 1 Suppresses Myeloid FcγR Function by Regulating the Expression and Function of the Common γ-Subunit. J.Immunol. 170(9): 4572-7.

FIELDS OF STUDY

Major Field: Pathology

viii

TABLE OF CONTENTS

Abstract………………………………………………………….……………….…..…..ii

Dedication…………………………………………………..……………….…………..iv

Acknowledgements………………………………………………..……….…….……..v

Vita………………………………………………………….……………………………vii

List of Figures……………………………………………………………….…………..xi

List of Abbreviations………………………………………………………………...... xiv

Chapters:

1. Introduction……………………………………...……………………….…………..1

2. Differential Gene Expression in Freshly Isolated Peripheral Blood Monocytes and Alveolar Macrophages

Introduction………………………………………………………….………….15 Results………………………………………………………………...………..17 Discussion………………………………………………………….…………..34 Materials and Methods…………………………………………...…………...38

3. Alveolar Macrophages Lack CCR2 Expression and do not Functionally Respond to Monocyte Chemoattractant Protein-1

Introduction………………………………………………………….………….42 Results………………………………………………………….……………....49 Discussion………………………………………………………..….………....62 Materials and Methods………………………………………….….…………66

ix 4. Mice Deficient in Macrophage Colony-Stimulating Factor are Protected from Bleomycin-Induced Pulmonary Fibrosis

Introduction………………………………………………………….………….69 Results……………………………………………………………….…………79 Discussion………………………………………………………..….…………91 Materials and Methods……………………………………………..…………94

5. Summary/Miscellaneous……………………………………………….………….99

List of References..………...……………………………………………….………..102

LIST OF FIGURES

Figure Title Page

1.1 Monocyte to Macrophage Differentiation……………………………..2

1.2 Monocytes and Macrophages in Immunity…………………………...4

2.1 Many Genes are Differentially Regulated in Peripheral Blood Monocytes and Alveolar Macrophages……………………………...18

2.2 Genes Most Increased in Alveolar Macrophages………………….20

2.3 Scavenger Receptors are Expressed More Highly in Alveolar Macrophages………………………………………………...21

2.4 Macrophage Receptor MARCO and Mannose Receptor are Expressed More Highly in Alveolar Macrophages…………….22

2.5 The Protein Tyrosine Kinase Axl is Expressed More Highly in Alveolar Macrophages……………………………………………...24

2.6 Genes Most Decreased in Alveolar Macrophages…………………25

2.7 Alveolar Macrophages Down-Regulate the Expression of MAP Kinase Pathway Genes…………………………………………27

2.8 Proposed M-CSF Signaling Pathway for Monocyte Survival……..29

2.9 Peripheral Blood Monocytes More Highly Express the PI3-K p100δ Subunit………………………………………………….31

2.10 Peripheral Blood Monocytes More Highly Express the Phosphatase and Tensin Homolog………………………………….32

2.11 Alveolar Macrophages More Highly Express the Survival Factor Akt……………………………………………………………….33

3.1 C-C Chemokine Receptor Expression on Monocytes and Monocyte-Derived Cells………………………………………………47

3.2 Alveolar Macrophages Express Less MCP-1 Receptor RNA than Peripheral Blood Monocytes.…………………………………..50

3.3 Peripheral Blood Monocytes, but not Alveolar Macrophages, Express CCR2 Surface Protein………………………………………52

3.4 MCP-1 Attracts Monocytes but not Alveolar Macrophages……….53

3.5 MIP-1α Attracts Monocytes and Alveolar Macrophages…………..55

3.6 Peripheral Blood Monocytes and Alveolar Macrophages Express Similar Levels of RNA for the MIP-1α Receptors, CCR1 and CCR5………...…………………………………………….56

3.7 Peripheral Blood Monocytes, but not Alveolar Macrophages, Express CCR1 Surface Protein………………………………………58

3.8 Alveolar Macrophages, but not Peripheral Blood Monocytes, Express CCR5 Surface Protein………………………………………59

3.9 CCR5 Blocking Antibodies Abrogate Alveolar Macrophage Migration Induced by MIP-1α……………….………………………..60

4.1 Human Idiopathic Pulmonary Fibrosis………………………………71

4.2 Pulmonary Fibrosis Induced by Intra-Tracheal Instillation of Bleomycin in the Mouse………………………………………………74

4.3 Pulmonary Fibrosis Induced by Intra-Peritoneal Administration of Bleomycin in the Mouse…………………………………………....75

4.4 Systemic Bleomycin Administration Protocol…………………...….78

4.5 M-CSF -/- Mice Exhibit a Survival Advantage vs. M-CSF Normal Mice After IP Bleomycin Treatment………………………...80

4.6 M-CSF -/- Mice Lose Less Weight than M-CSF Normal Mice After IP Bleomycin Treatment……..…………………………………82

xii 4.7 M-CSF -/- Mice Develop Less Fibrosis than M-CSF Normal Mice After IP Bleomycin Treatment (Trichrome Stain)……………84

4.8 M-CSF -/- Mice Develop Less Severe Fibrotic Disease than M-CSF Normal Mice After IP Bleomycin Treatment (Pathologists Assessment)…………...... …85

4.9 M-CSF -/- Mice Develop Less Sub-Pleural Fibrosis than M-CSF Normal Mice After IP Bleomycin Treatment...... 87

4.10 M-CSF -/- Mice Have Fewer CD68+ Mononuclear Cells than M-CSF Normal Mice After IP Bleomycin Treatment………………88

4.11 M-CSF Normal Mice Express More MCP-1 in Areas of Inflammation and Fibrosis than M-CSF Deficient Mice After IP Bleomycin Treatment…………………………………………………90

xiii

LIST OF ABBREVIATIONS

Word or Phrase Abbreviation

Monocyte(s) Mo Macrophage(s) MФ Alveolar Macrophage(s) AM Natural Killer Cell(s) NK Red Blood Cell(s) RBC Poly-Morpho-Nuclear Cell (Neutrophil) PMN Macrophage Colony-Stimulating Factor M-CSF Colony-Stimulating Factor 1 CSF-1 Macrophage Colony-Stimulating Factor Receptor M-CSF-R Protein Tyrosine Kinase PTK Phosphatidylinositol-3 Kinase PI-3K Granulocyte-Macrophage Colony Stimulating Factor GM-CSF Monocyte-Derived Macrophage(s) MDM Monocyte Chemo-attractant Protein 1 MCP-1 Macrophage Inflammatory Protein 1 MIP-1 C-C Chemokine Receptor CCR Transforming Growth Factor-β TGF-β Pulmonary Fibrosis PF Usual Interstitial Pneumonia UIP Desquamative Interstitial Pneumonia DIP Respiratory Bronchiolitis-Associated Interstitial Lung Disease RBILD Acute Interstitial Pneumonia AIP Non-Specific Interstitial Pneumonia NSIP Bronchoalveolar Lavage/ Fluid BAL/BALF Phosphatase and Tensin PTEN Thymus and Activation-Regulated Chemokine TARC Regulated on Activation, Normal T cell, Expressed and Secreted RANTES Peritoneal Macrophage(s) PM Intra-Tracheal IT Intra-Peritoneal IP Hematoxylin and Eosin H&E Idiopathic Pulmonary Fibrosis IPF Connective Tissue Growth Factor CTGF

xiv

CHAPTER 1

INTRODUCTION

The Mononuclear Phagocyte System: An Overview

The mononuclear phagocyte system is composed of immune cells whose

primary function is phagocytosis. Monocytes (Mo) and tissue macrophages (MФ) are the primary members, and they are derived from bone marrow precursors

(Figure 1.1). At the time of exit from the bone marrow, they circulate through the peripheral blood, and are known as monocytes. Monocytes are usually 10-15µm

in diameter, have a U-shaped nuclei, and a granular cytoplasm containing

phagocytic machinery including lysosomes and phagocytic vacuoles. In the

absence of growth factors, monocytes undergo caspase-dependent programmed

cell death, or apoptosis, after 24-72 hours1,2. It is widely believed that monocytes

are the immature precursor cells to macrophages. When stimulated, monocytes

can extravasate, differentiate and reside in the tissue, where they are capable of

surviving for several months as macrophages. Macrophages are found in all

tissues of the body. In different tissues, macrophages have different names,

including Kupffer cells in the liver, microglial cells in the central nervous system,

osteoclasts in bone, and alveolar macrophages (AM) in the lung. Additionally,

macrophages may become epithelioid in nature, or may fuse to form

Bone Marrow Blood Tissue (pro-monocyte) (monocyte) (macrophage)

Maturation and Differentiation

Figure 1.1. Monocyte-Macrophage Differentiation. Monocytes originate in the bone marrow as pro-monocytes or monoblasts, and are released into the circulation as incompletely differentiated monocytes. Upon activation, monocytes extravasate and enter the tissues, where they differentiate into macrophages and assume numerous forms, including microglial cells (central nervous system), Kupffer cells (liver), osteoclasts (bone) and alveolar macrophages (lung).

2 multinucleated giant cells. Due to the widespread distribution of macrophages

throughout various tissues, one encompassing description of tissue

macrophages does not suffice. Generally, however, tissue macrophages are

larger than their monocyte precursors, have increased both the size and number

of phagocytic vacuoles and lysosomal granules, and are capable of large-scale

activation. Thus, macrophages are fully differentiated mononuclear phagocytes.

Monocytes and Macrophages in Immunity

Both monocytes and macrophages play a role in innate immunity by

phagocytosing foreign particles such as bacteria or other microbes, as well as

scavenging dead or damaged tissues (Figure 1.2, left). These cells can also

phagocytose complement-coated particles, and particles marked for clearance by

other mechanisms. The phagocytic vesicles and lysosomes found within the

cytoplasm of these cells secrete multiple enzymes and reactive molecules (such

as superoxide and H2O2), which serve to kill the microbes and control infections.

Monocytes and macrophages also produce cytokines and other growth factors

that can recruit and activate other inflammatory cells in areas where they are needed. Monocytes and macrophages play an important role in specific

immunity by acting as antigen-presenting cells (Figure 1.2, right). After phagocytosis, they are capable of displaying foreign antigens for recognition by antigen-specific T cells. These mononuclear phagocytes also express co-

stimulatory molecules, such as CD40, which help to activate T lymphocytes. In

INNATE IMMUNITY SPECIFIC IMMUNITY

Microbial Pathogens

Activated Mo/Mφ T Lymphocytes

Opsonized Particles IFN-γ

Figure 1.2. Monocytes and Tissue Macrophages are Important Effector Cells in both Innate and Specific Immunity. In innate immunity, these cells can directly phagocytose pathogens or opsonized particles. Additionally, these cells can present antigens to T lymphocytes, which in turn release cytokines that can further activate Mo/Mφ, such as IFN-γ, thus participating in specific immunity.

4 turn, these activated T lymphocytes secrete cytokines that further potentiate the phagocytic function of monocytes and macrophages, thus creating an escalating loop of effector cell activity.

Monocyte Differentiation

During hematopoiesis, pluripotent stem cells in the bone marrow proliferate and mature into peripheral blood cells. These pluripotent cells, by definition, have the capacity to differentiate into several cell types. This differentiation results in the formation of numerous cell lineages, including those of lymphoid descent; B lymphocytes, T lymphocytes and Natural Killer (NK) cells, as well as those of myeloid descent. Cells of the myeloid lineage include basophils, eosinophils and cells of the granulocyte-monocyte family. The granulocyte-monocyte family includes both neutrophils, or polymorphonuclear cells (PMN’s), and monocytes. Each of these cells plays an important role in immunity.

The differentiation of stem cells into mature cells of any lineage is a tightly regulated process dependent on the timing and presence of specific growth factors. c-Kit ligand, produced by bone marrow stromal cells, is crucial for enhancing stem cell responsiveness to other growth factors although it does not appear to induce differentiation on its own3-5. Numerous specific cytokines and growth factors play key roles in the development of differentiated hematopoietic cells. For example, interleukin-7 (IL-7) is believed to play a role in the development of T and B cells. In vitro, IL-7 rescues neglected T cells in culture6.

5 In vivo, IL-7 also plays an important role in lymphocyte homeostasis, as naïve T

cells adoptively transferred to IL-7 -/- mice disappear after approximately one

month7. Transgenic mice that over-express IL-7 generate markedly increased

numbers of B cell progenitors and peripheral T cells8, while mice deficient in IL-7

have significantly decreased numbers of T and B cells9,10.

The survival and differentiation of monocytes and macrophages is

dependent on the growth factor cytokine Macrophage Colony-Stimulating Factor

(M-CSF), and stimulation by M-CSF is restricted to mononuclear phagocytes11.

Retinoic acid can inhibit monocyte survival and monocyte to macrophage differentiation in vitro by decreasing M-CSF production via post-transcriptional

modifications. Interestingly, M-CSF production by fully differentiated monocyte-

derived macrophages is unaffected by retinoic acid treatment12.

The human M-CSF gene, colony-stimulating factor 1 (CSF-1), is located

on the short arm of chromosome 1, and the single gene product generates three

mature isoforms: a soluble glycoprotein, an extracellular matrix bound

proteoglycan and a cell-associated glycoprotein13. All of the gene products are

capable of maintaining CSF-dependent monocyte-like cell lines, but the cell-

associated form requires direct cell-to-cell contact14. While the physiologic half-

life of M-CSF is relatively short15, stable levels of M-CSF in the serum and other

tissues indicate persistent synthesis16,17. Bone marrow stromal cells can

synthesize M-CSF for local use18. Osteoblasts, fibroblasts and endometrial

epithelial cells can also produce M-CSF, however, the endothelial cells lining the

small blood vessels appear to be the primary source of circulating glycoprotein18.

6 Monocytic cells may also synthesize and secrete M-CSF in an autocrine or

paracrine manner in response to a number of stimuli, including Fcγ receptor

cross-linking19. Not surprisingly, mice lacking M-CSF have decreased numbers

of monocytes and macrophages. These mice arose spontaneously in the 1970’s

and provide an excellent model for examining the role of M-CSF and of

monocytes and macrophages under a variety of conditions. These mice will be

described in detail in Chapter 4.

In the absence of M-CSF or other stimulatory signals, monocytes undergo

programmed cell death, or apoptosis within 24-72 hours after entering the

peripheral circulation1,2. Apoptosis is a non-inflammatory, non-necrotic

mechanism for disposing of unneeded cells in the body.

The M-CSF receptor (M-CSF-R) is a receptor protein tyrosine kinase

(PTK) encoded by the proto-oncogene c-fms, located on human chromosome

520. Expression of the M-CSF-R is largely restricted to cells of the

monocyte/macrophage lineage21. This receptor belongs to a PTK family of

receptors that also includes c-kit, flk2/flt3 and EGF, PDGF, insulin and insulin-like

receptors15. The receptors in this family have intrinsic kinase activity and are

able to auto- and trans-phosphorylate tyrosine residues upon binding their

appropriate ligand. M-CSF binding to the M-CSF-R leads to downstream events

such as the phosphorylation and activation of phosphatidylinositol-3 kinase (PI-

3K)22 and Protein Kinase B, (also known as Akt)23, both of which are important mediators of multiple cellular processes including survival, proliferation and

7 differentiation. Much of the work from our laboratory focuses on the signal transduction pathways downstream of the M-CSF receptor and the regulation of monocyte survival19,23-25, however, these studies will not be detailed here.

Macrophage Differentiation

In vivo, monocytes differentiate into tissue macrophages, an intricate process involving activation of the monocyte, adhesion to the endothelium, extravasation and migration within the tissue.

Under normal conditions, monocytes, like all leukocytes, flow freely within the blood vessels, and do not interact with the endothelium. In non-inflammatory conditions, e.g. maintenance of normal monocyte/macrophage homeostasis, the differentiation of monocytes to tissue macrophages is not well understood. In fact, very little is known about this process, and questions remain as to whether or not certain subpopulations of monocytes are predestined for specific tissues, or whether any given monocyte can become any type of macrophage.

Nonetheless, the process is likely similar to that which is outlined below.

Near sites of inflammation, the endothelium becomes activated and up- regulates the expression of chemokines, proteoglycans, selectins and integrin ligands, which attract and tether monocytes. This tethering slows the flow of monocytes and results in tumbling or rolling along the endothelium, which activates the monocyte. Once activated, monocytes undergo cytoskeletal rearrangement and flatten against the endothelium, and monocyte integrins increase their affinity for ligands found on the surface of the endothelial cells.

8 The monocyte firmly adheres to the endothelial cell surface and slowly migrates

to an inter-endothelial cell junction where it transmigrates through the endothelial cell layer. Once through the endothelium, monocytes continue to migrate toward

sites of inflammation, most likely in response to chemotactic gradients, and

eventually settle and mature into tissue macrophages. These tissue

macrophages are generally larger than their monocyte precursors, and have enhanced phagocytic capabilities.

This differentiation can be modeled in vitro, by culturing monocytes with an appropriate stimulus. These stimuli can include M-CSF, Granulocyte-

Macrophage Colony Stimulating Factor (GM-CSF), or, simply, adhesion to a tissue culture plate, although slightly different phenotypic changes occur with each method. Some of these differences will be described in Chapter 2. These cells are referred to as monocyte-derived macrophages (MDM).

Recruiting Monocytes and Macrophages

In order to exert their effect in local areas, leukocytes must be recruited to the sites at which they are needed. This recruitment is largely mediated by locally produced chemokines that act via specific and selective receptors26,27.

Monocyte Chemoattractant Protein 1 (MCP-1, also known as CCL2) and

Macrophage Inflammatory Protein 1α (MIP-1α, also known as CCL3) are

believed to be the major chemokines involved in the recruitment of monocytes

and macrophages. Both are members of the low molecular weight C-C

chemokine superfamily, characterized by the presence of adjacent cysteine

9 residues in the amino terminus. This critical motif defines the selectivity of this family of chemokines to the recruitment of mononuclear cells. In contrast, the alternate C-X-C chemokine family may be more selective for recruiting neutrophils. The C-C chemokine genes are generally found on human

chromosome 1726, and these chemokines bind C-C chemokine receptors, or

CCR’s. These receptors have characteristic seven transmembrane domains,

and signal through heterotrimeric GTP-binding proteins. There are currently at

least 10 known C-C chemokine receptors28.

MCP-1 is but one of a family of 4 Monocyte Chemoattractant Proteins,

chemokines originally named for their ability to recruit monocytes. This family of

molecules also affects T lymphocytes and basophils26, but does not recruit

neutrophils. Among the four MCP’s, all of which share a pyroglutamate proline

NH2—terminal motif, MCP-1 appears the most potent monocyte chemoattractant.

MCP-1 selectively binds only the C-C chemokine receptor CCR2, while MCP-2, -

3 and –4 can bind not only CCR2, but also CCR1 (MCP-2 and –3) and CCR3

(MCP-3 and –4)26.

The presence of MCP-1, without any other stimulus, is sufficient to cause

monocyte accumulation but not activation29, and numerous studies have

demonstrated the importance of MCP-1 in recruiting monocytic cells. Maus et al

describe the recruitment of peripheral blood monocytes into the alveolar space of

mice treated intra-tracheally with recombinant MCP-1. These cells retain their monocyte-like phenotype for approximately 96 hours after recruitment30.

10 MIP-1α is another member of the C-C chemokine family that appears capable of recruiting both monocytes and tissue macrophages, although direct experimental data to confirm this is lacking. In a wound repair model, MIP-1α is critical for attracting tissue macrophages to wounding sites, as the recruitment of macrophages was blocked with anti-MIP-1α antibodies31. Interestingly, blocking

MIP-1α-induced macrophage recruitment also decreases angiogenic activity and

collagen production at the wound site31. MIP-1α selectively binds the C-C

chemokine receptors CCR1 and CCR5, both of which are increased during the in

vitro differentiation of monocytes to macrophages32. The distribution of these

chemokine receptors on native macrophages has not yet been explored.

For many years, chemokines were believed to be strictly chemotactic

molecules. Recent studies, however, suggest that both MCP-1 and MIP-1α may

also play a role in modulating cellular adhesion molecules33,34, and that MCP-1

may stimulate fibroblasts to release pro-fibrotic molecules such as collagen and

Transforming Growth Factor-β (TGF-β)35. Thus we postulate that these

chemokines may also be integral to the differentiation process, and in the

development of tissue fibrosis.

11 Monocytes and Macrophages in Disease

Monocytes and macrophages are important in the manifestation of numerous diseases, including: atherosclerosis36,37, inflammatory bowel syndrome38 and Alzheimer’s Disease39,40. In the lung, alveolar macrophages are

the resident immune cells, and are critical in Mycobacterium tuberculosis

infection, where they phagocytose and harbor live bacterium.

Our data and others’ suggest that monocytes and macrophages may also

play in important role in Pulmonary Fibrosis (PF). PF encompasses a family of

lung diseases characterized by progressive scarring of the lung parenchyma,

ultimately leading to the death of the patient. The average age of onset is

between 50 and 70 years, and the prevalence of pulmonary fibrosis is

approximately 13-20 cases per 100,000 population41. Patients typically present with progressive dyspnea, a non-productive cough, abnormal breath sounds

(“Velcro-like” crackles on lung examination), and pulmonary limitations. By the time of diagnosis, abnormalities are usually found on a chest X-ray, and in the most severe form of pulmonary fibrosis, the mean length of survival in the most severe forms is only two to five years41,42. To date, there exists no effective

treatment, and the cause of this devastating disease is largely unknown.

Recent changes in the nomenclature of the disease elucidate five distinct

histopathological conditions under the umbrella of Idiopathic Interstitial

Pneumonias: Idiopathic Pulmonary Fibrosis/Usual Interstitial Pneumonia (UIP);

Desquamative Interstitial Pneumonia (DIP); Respiratory Bronchiolitis-Associated

Interstitial Lung Disease (RBILD); Acute Interstitial Pneumonia (AIP); and Non-

12 Specific Interstitial Pneumonia (NSIP)41. To fit the scope of this dissertation, we will be focusing on pulmonary fibrosis in general terms, with specific deference to

IPF/UIP (Refer to Chapter 4).

There appear to be at least two ideologic camps regarding the role of

inflammation in pulmonary fibrosis. Some investigators believe that inflammation and fibrosis are separate and distinct entities, and that inflammation in the lung

does not progress to fibrosis. This outlook is largely based on evidence that

mice lacking the integrin αvβ6 are protected from bleomycin-induced pulmonary

fibrosis even though they do develop exaggerated inflammation in the lungs and

skin43,44. Others, however, argue that inflammation and fibrosis are a continuum,

wherein the early stages of the disease are characterized by inflammatory

infiltrates, and that these later become fibrotic lesions. For example, Bitterman,

et al found that alveolar macrophages spontaneously release fibroblast growth

factors in patients with fibrotic lung diseases45.

Regardless, patients with pulmonary fibrosis tend to have increased

numbers of monocytes and macrophages in their broncho-alveolar lavage (BAL)

fluid, and the presence of these cells in BAL fluid, or in lung biopsy samples, is

correlated with a poorer prognosis46. MCP-1 likely plays a role in recruiting these

cells to the alveolar space, as numerous studies have demonstrated an increase

in MCP-1 expression in pulmonary fibrosis. For example, while some cells in the

lung express MCP-1 in normal situations, the pulmonary epithelial cells of

patients with pulmonary fibrosis express MCP-1 at higher levels than epithelial

13 cells in non- fibrotic samples47. Furthermore, mice that lack the MCP-1 specific

receptor CCR2 are protected from developing fibrosis48.

With these key players introduced, the bulk of this dissertation will focus

on the relationships between peripheral blood monocytes and alveolar

macrophages, the mechanisms by which each can be selectively recruited, and

the role of these cells in health and disease.

CHAPTER 2

DIFFERENTIAL GENE EXPRESSION IN FRESHLY ISOLATED PERIPHERAL BLOOD MONOCYTES AND ALVEOLAR MACROPHAGES

Introduction

Circulating monocytes are immature precursors to tissue macrophages.

Interestingly enough, while numerous investigators have examined monocyte

differentiation in culture, no studies have yet evaluated the gene expression

profiles of human monocytes versus native (in this case, alveolar) macrophages.

Previous studies have observed that peripheral blood monocytes cultured

with M-CSF and/or GM-CSF adopt a macrophage-like phenotype11,49,50, and some argue that GM-CSF-derived macrophages are most similar to native alveolar macrophages51, because alveolar macrophages are functionally

dependent on GM-CSF in a knockout mouse model52. After 7 days of

stimulation, both M-CSF and GM-CSF induce the expression of genes coding for apolipoproteins, cathepsins, and macrophage capping proteins, among others53.

Only GM-CSF alone induces macrophages to express the Monocyte-Derived

Chemokine (MDC), which plays a role in the recruitment of CCR4 positive lymphocytes53,54. GM-CSF-stimulated macrophages also maintain the

expression of the Transforming Growth Factor β gene in approximately the same

15 abundance as fresh monocytes, while stimulation with M-CSF reduces TGF-β

expression. In contrast to GM-CSF derived macrophages, M-CSF-stimulated

MDM express CD48, an activation-associated marker53.

Some studies have examined, in small scale, pathways and/or molecules

of interest and compared expression levels in monocytes and specific types of macrophages including MDM, pleural macrophages, peritoneal macrophages and alveolar macrophages. For example, monocytes cultured in the presence of

serum down-regulate CD14, and interestingly, AM express less CD14 than other types of macrophages55.

Understanding the differences between peripheral blood monocytes and

mature tissue macrophages is an important step in identifying molecular targets

specific to each cell type. These targets, in turn, can potentially be used to

generate therapeutic approaches for monocyte- or macrophage- specific disease

processes, such as monocytic leukemias, coronary artery disease, or M.

tuberculosis infection. We sought to examine, by large-scale microarray

analysis, the genetic expression profiles of freshly isolated human monocytes

and alveolar macrophages. We first performed a pilot study, examining both the

power and limitations inherent to microarray analysis.

16 Results

The enormous amount of data generated by microarray technology makes

in-depth analysis of each and every data point extremely cumbersome. Within

the scope of this dissertation, we have identified genes that are significantly

differentially expressed between fresh monocytes and alveolar macrophages,

and analyzed the expression of genes of interest to our laboratory.

Many genes are differentially regulated in peripheral blood

monocytes and alveolar macrophages

For each probe set, a comparison is made between samples derived from

freshly isolated peripheral blood monocytes and freshly harvested alveolar

macrophages. The comparison result is expressed qualitatively as increased,

marginally increased, decreased, marginally decreased, or unchanged. In every

comparison, the results are displayed using fresh monocytes as the control and

AM as the experimental sample (i.e. “increased” will always mean “higher in AM

compared to monocytes” and “decreased” will always mean “lower in AM

compared to monocytes” unless otherwise specified). To better illustrate the

enormity of the data generated, Figure 2.1 shows the raw number and

percentage of probe sets described as increased, marginally increased,

decreased, marginally decreased or unchanged across 2 donors of each cell

type. The vast majority of genes (~89%) are unchanged between these cells,

with approximately equal numbers of genes up- (~5.4%) and down- (~5.3%)

Comparison Result # of Probe Sets % of Total (p value) Increased 19 0.150 (p < 0.0025) Marginally Increased 680 5.386 (0.0025 < p < 0.003) Unchanged 11237 89.006 (0.003 < p < 0.997) Marginally Decreased 668 5.291 (0.997 < p < 0.9975) Decreased 21 0.166 (p > 0.9975)

Figure 2.1. Many Genes are Differentially Regulated in Peripheral Blood Monocytes and Alveolar Macrophages. The average of the p-values from each comparison between freshly isolated peripheral blood monocytes and alveolar macrophages was computed, and the same p-value rules applied to this average. P-values are shown in parentheses for each category.

18 regulated in alveolar macrophages. These percentages are deceptively small,

however, and we must remember that they represent hundreds of genes that are differentially expressed.

Although there are 699 probe sets demonstrating genes that are either

increased or marginally increased, providing a table of this number of genes

would prove unwieldy. The 24 genes with most increased expression in AM are

listed (in order of Probe Set ID) in Figure 2.2.

Macrophage receptors are up-regulated in alveolar macrophages

As expected, some of the RNA’s that are up-regulated in AM include

macrophage-specific receptors, such as Macrophage Scavenger Receptor-1

(U95Av2 probe set ID 39981_at), expressed at approximately 100-fold higher

levels in AM, and Macrophage Scavenger Receptor–2 (U95Av2 probe set ID

39982_r_at), expressed at approximately 10-fold higher levels in AM (Figure 2.3).

Additionally, the collagenous receptor MARCO (U95Av2 probe set ID 40331_at) and Macrophage Mannose Receptor (U95Av2 probe set ID 36908_at) are also expressed more highly in AM compared to peripheral blood monocytes (Figure

2.4). All of these receptors play a role in mediating the binding and internalization of macromolecules and microbial pathogens56,57. Moreover, scavenger receptor signaling has been implicated in M-CSF production by monocytes58, potentially providing another effector feedback loop for

monocyte/macrophage survival.

Probe Set Accession Number General Description ID 1278_at TIGR HG162-HT3165 Tyrosine Kinase Receptor Axl, Alt 311_s_at TIGR HG3044-HT3742 Fibronectin 31719_at NCBI X02761 Fibronectin Precursor 31720_s_at NCBI M10905 Cellular Fibronectin 32552_at NCBI X00129 Retinol Binding Protein (RBP) Placenta Copper Monamine Oxidase, 33756_at NCBI U39447 Vascular Adhesion Protein-1 36606_at NCBI X51405 Carboxypeptidase E 36657_at NCBI AA883870 Apolipoprotein C II 36908_at NCBI M93221 Macrophage Receptor (MRC1) Peroxisome Proliferator Activated 37104_at NCBI L40904 Receptor Lectin-like Oxidized Low Density 37233_at NCBI AF079167 Lipoprotein Receptor-1 37399_at NCBI D17793 Aldo-keto Reductase Family; 3α Hydroxy Steroid Dehyrogenase Type II 37684_at NCBI AB020687 Solute Carrier Family Member 38379_at NCBI X76534 Transmembrane Glycoprotein 38404_at NCBI M55153 Transglutaminase (Tgase) 38430_at NCBI AA128249 Fatty Acid Binding Protein 4 38433_at NCBI M76125 Tyrosine Kinase Receptor Axl 38796_at NCBI X03084 C1q Β-Chain of C’ System 39248_at NCBI N74607 Aquaporin 3 Macrophage Scavenger Receptor, 39981_at NCBI D13264 Type I 41209_at NCBI M15856 Lipoprotein Lipase ATP-Binding Cassette; Subfamily G 41362_at NCBI X91249 (WHITE) 41764_at NCBI AA976838 Apolipoprotein C-I 425_at NCBI X67325 Heat Shock Protein Hsp27

Figure 2.2. The 24 Genes Most Increased in Alveolar Macrophages Compared to Peripheral Blood Monocytes. The signal-log ratio was computed for each probe set and across each comparison, and the average signal-log ratio calculated. The 24 highest ratios are listed in order of Probe Set ID.

2500 2500

s n

e 2000 2000

a 1500 1500

i S

1000 1000

a r

e 500 500

v A

Mo AM Mo AM

Figure 2.3. Macrophage Scavenger Receptor I (39981_at), left, and II (39982_r_at), right, are Expressed at Higher Levels in Alveolar Macrophages than in Peripheral Blood Monocytes. Scavenger Receptor I is increased approximately 100-fold, and Receptor II approximately 10-fold. The average signal intensities and standard deviation over 2 donors of each cell type are shown.

80,000 80,000

e t 60,000 60,000

i 40,000 40,000

r 20,000 20,000

Mo AM Mo AM

Figure 2.4. Macrophage Receptor MARCO (40331_at), left, and Macrophage Mannose Receptor (36908_at), right, are Expressed at Higher Levels in Alveolar Macrophages than in Peripheral Blood Monocytes. MARCO is expressed at more than 60-fold higher levels in AM, and the mannose receptor at over 200-fold higher levels. The average signal intensity and standard deviation over 2 donors of each cell type are shown.

22 Alveolar macrophages up-regulate potential survival factors

Other genes of interest from Figure 2.2 that are highly up-regulated in

alveolar macrophages include the receptor tyrosine kinase Axl (U95Av2 probe

set ID 1278_at), the gene with the most increased expression in AM

(approximately 330-fold), and the low molecular weight heat shock protein Hsp

27 (U95Av2 probe set ID 425_at), also up-regulated well over 100-fold. Both of

these genes have been implicated in the survival of numerous cell types59-61, and this data allows us to postulate that they may also be important factors in the survival of tissue macrophages. The increased expression of Axl in alveolar macrophages was also confirmed by reverse transcriptase PCR (Figure 2.5).

Trafficking and adhesion molecules are down-regulated in alveolar macrophages compared to peripheral blood monocytes

Again, due to the large number (689) of probe sets that are decreased or marginally decreased in alveolar macrophages compared to monocytes, the 24 genes with most decreased expression in AM are listed in Figure 2.6. Many of the genes that are most decreased in AM code for molecules involved in cellular trafficking, such as a lymph node homing receptor (U95Av2 probe set ID 245_at) or proteoglycans (U95Av2 probe set ID’s 31682_s_at, 38111_at and

38112_g_at). Additionally, some chemokines and chemokine receptors, such as

Pro-Platelet Basic Protein (CXCL7) (U95Av2 probe set ID 39208_i_at) and V28

GAPDH

Axl

1.25

1.0

D P

A 0.75

i 0.5

a R 0.25

Mo AM

Figure 2.5. The Receptor Tyrosine Kinase Axl (1278_at) is Expressed at Higher Levels in Alveolar Macrophages than in Peripheral Blood Monocytes. Microarray Analysis indicated that Axl was the gene with the most increased expression in AM, with a 330-fold increase over monocyte expression. We used Reverse Transcriptase PCR to confirm these results (above). Bands are representative of 4 separate donors, and the densitometry graph shows average ratio of Axl to GAPDH control band intensity and standard deviation over the 4 donors.

Probe Set Accession Number General Description ID 1115_at NCBI M25897 Platelet Factor 4 (PF4) 245_at NCBI M25280 Lymph node homing receptor 31506_s_at NCBI L12691 Neutrophil peptide-3 Chondroitin Sulfate Proteoglycan 31682_s_at NCBI D32039 Versican (PG-MV3) 31687_f_at NCBI M25079 Sickle cell beta-globin 31793_at NCBI AL036554 Defensin α3 32052_at NCBI L48215 B-Hemoglobin (HBB) SWI/SNF Related, Matrix Associated, 32565_at NCBI U66619 Actin Dependent Regulator of Chromatin 33925_at NCBI X99076 Neurogranin, PKC Substrate Leukocyte Immunoglobulin-Like 35095_r_at NCBI AF025527 Receptor-4 (LIR-4) 36447_at NCBI S80990 Ficolin 36766_at NCBI X55988 Ribonuclease, RNAse A Family 37456_at NCBI AL022315 Galactoside-Binding Lectin 37701_at NCBI L13463 Regulator of G-Protein Signaling 37799_at NCBI X55284 Asialoglycoprotein Receptor 2 38052_at NCBI M14539 Factor XIII Subunit A1 Chondroitin Sulfate Proteoglycan 2 38111_at NCBI X15998 (Versican) Chondroitin Sulfate Proteoglycan 2 38112_g_at NCBI X15998 (Versican) S100 Calcium Binding Protein A12: 38879_at NCBI D83664 Calgranulin C Pro-Platelet Basic Protein; C-X-C Ligand 39208_I_at NCBI M54995 7 39837_s_at NCBI AC004877 Unknown Dioxin-Inducible Cytochrome P450 40071_at NCBI U03688 (CYP1B1) G Protein Coupled Receptor V28; C- 40646_at NCBI U20350 X3_C Receptor 1 SWI/SNF Related, Matrix Associated, 456_at NCBI U66619 Actin Dependent Regulator of Chromatin

Figure 2.6. The 24 Genes Most Decreased in Alveolar Macrophages Compared to Peripheral Blood Monocytes. The signal-log ratio was computed for each probe set and across each comparison, and the average signal-log ratio calculated. The 24 lowest (most negative) ratios are listed.

25 (U95Av2 probe set ID 40646_at), a receptor with considerable homology to

MCP-1 and MIP-1α receptors62, are also expressed at lower levels in AM

compared to monocytes (data not shown).

Members of the MAP kinase pathway are down-regulated in alveolar macrophages compared to peripheral blood monocytes

It is interesting to note that numerous members of the MAP kinase pathway are decreased in AM. For example, the MAP kinase Mek-1 (U95Av2 probe set ID 33009_at), transactivator Jun-B (U95Av2 probe set ID 2049_s_at) and proto-oncogene c-fos (U95Av2 probe set ID 2094_s_at) are expressed at much higher levels in monocytes. The difference between signal intensities in monocytes and AM remains approximately the same, between 5- and 10-fold.

Remarkably, the raw signal intensity increases by a factor of 10 between Mek-1 and Jun-B, and again by 10 between jun-B and c-fos (Figure 2.7). This data is surprisingly in contrast with other studies suggesting that jun and fos are up- regulated in monocytes exposed to M-CSF, and may play a role in the intracellular signaling of monocyte differentiation63,64. Other members of the

MAP kinase pathway that are expressed more highly in monocytes include: Ref,

Mek, and Erk (data not shown). Our laboratory has recently shown that Erk

activation may also play a role in M-CSF-induced monocyte survival25.

1,600 16,000

l l

a a

n n

y y

g g

i i t 1,200 t 12,000

i i

S S

s s

n n

e e

g g

t 800 t 8,000

a a

n n

r r

I I

e e

v 400 v 4,000

A A

Mo AM Mo AM

160,000

i t 120,000

t 80,000

v 40,000

Mo AM

Figure 2.7. Peripheral Blood Monocyte More Highly Express MAP-Kinase Pathway Genes Compared to AM. The MAP Kinase Mek-1 (33009_at), top left, transactivator Jun-B (2049_s_at), top right, and the proto-oncogene c-fos (2094_s_at), bottom, are expressed at approximately 5-10 fold lower levels in AM than in circulating monocytes. The average signal intensity and standard deviation over 2 donors of each cell type are shown. Note that scale increases by 10-fold between each graph.

27 While this microarray data provides exciting insight on new molecules and

genes that could be of interest to our laboratory, it is also necessary to scrutinize

the data for new information about molecules with which we are already familiar.

Our laboratory has described a signaling pathway potentially involved in the survival of M-CSF stimulated monocytes, (Figure 2.8) wherein M-CSF binds the M-CSF-R, which in turn binds and activates the phosphatidylinositol-3 kinase

(PI3-K) to phosphorylate phosphitidylinositol-4,5 bisphosphate (PI(4,5)P2)

yielding phosphatidylinositol 3,4,5 triphosphate (PI(3,4,5)P3). PI(3,4,5)P3

induces the translocation of the serine/threonine kinase Akt to the cellular

membrane where Akt is activated. Akt activation plays an important role in

cellular survival. The phosphatase and tensin (PTEN) phosphatase regulates

this reaction, by de-phosphorylating PI(3,4,5)P3 and restoring PI(4,5)P2.

Accordingly, we sought to examine RNA expression of these important genes.

PI3-K p110δ and a PTEN homolog are down-regulated in alveolar

macrophages

To phosphorylate the phosphatidylinositol intermediates, PI3-kinase

functions through the p85 adapter subunit and the p110 catalytic subunit. The

p110δ subunit of PI3-K shares homology with other p110 subunits, and functions

in the binding of adaptor proteins, however, the δ subunit is selectively expressed

in leukocytes65, and RNA for PI-3K p110δ (U95Av2 probe set ID 32121_at) is

expressed at approximately 4-fold higher levels in circulating monocytes than in 28

M-CSF Receptor

p110 PI3K p85 Akt

PI(4,5)P2 PI(3,4,5)P3

PTEN P- Akt

Cellular Survival

Figure 2.8. Proposed MCSF signaling pathway for monocyte survival. M- CSF binding the M-CSF-R initiates a cascade of signaling events leading to cell survival.

29 AM (Figure 2.9). As well, the phosphatase and tensin (PTEN) homolog (U95Av2 probe set ID 31675_s_at) is increased, also by approximately 3 to 4-fold in monocytes compared to alveolar macrophages (Figure 2.10).

The survival factor Akt is up-regulated in alveolar macrophages

Both RNA and protein analysis indicate that the survival factor Akt

(U95Av2 probe set ID 2023_g_at), on the other hand, is expressed at approximately 2-fold higher levels in alveolar macrophages than in circulating monocytes (Figure 2.11a). Previous work in our laboratory has indicated than alveolar macrophages have more (total of active and inactive) Akt protein than peripheral blood monocytes when assessed by western blotting (Figure 2.11b).

1600

n 1200

i 800

e 400

Mo AM

Figure 2.9. RNA Expression of the p100δ Subunit of Phosphatidylinositol-3 Kinase (32121_at) is Decreased in Alveolar Macrophages. PI-3K is expressed at approximately a 3-fold higher level in circulating monocytes than in AM. The average signal intensity and standard deviation over 2 donors of each cell type are shown.

3200

n 2400

n 1600

r 800

Mo AM

Figure 2.10. RNA Expression of the Phosphatase and Tensin (PTEN) Homolog (31675_s_at) is Decreased in Alveolar Macrophages. The PTEN homolog is expressed at approximately a 3-fold higher level in circulating monocytes than in AM. The average signal intensity and standard deviation over 2 donors of each cell type are shown.

a.A. b. 200

t Monocytes AM

s Anti-

e t 150 Akt

S 100 Anti-

a β-actin

v 50

A Mo AM

Mo AM

Figure 2.11. Alveolar Macrophages Express both RNA and Protein for the Survival Factor Akt (2023_g_at) at Higher Levels than Peripheral Blood Monocytes. Microarray RNA signal intensity comparison is shown in (a), Immunoblotting with an anti-Akt antibody (upper panel, b), substantiates these data with protein analysis. Lysates were probed with anti-B actin for loading control (lower panel, b). The average signal intensity and standard deviation over 2 donors of each cell type are shown in (a), and the bands in (b) are representative of 3 separate donors.

33 Discussion

In addition to evaluating pre-existing hypotheses, microarray analysis is also a valuable tool for generating new hypotheses. Only recently did this type of high-throughput analysis become available and affordable. However, while microarray analysis is a powerful tool for identifying potential genes of interest, this technique measures only the expression of RNA transcripts. These RNA sequences must ultimately be translated into proteins in order to augment cellular function, and this translation may be differentially regulated as well. Finally, even the mere presence of such a protein does not in any way indicate whether the gene product is active or functioning, as many proteins must be post- translationally modified, such as by phosphorylation, to be activated. Even with these limitations recognized, microarray analysis is a powerful technique for gaining a broad-spectrum of gene expression information.

It is worthwhile to note that when comparing freshly isolated peripheral blood monocytes and alveolar macrophages, the gene expression profile is largely unchanged. Only approximately 10% of genes represented on the chip

(of a possible 10,000 full length genes represented on the U95Av2 chip) are differentially expressed between these cell types. Of the numerous phenotypic and biological differences between monocytes and macrophages, the most obvious are the difference in location and average lifespan of the cells.

Monocytes are circulating cells and typically survive for only a few days then undergo spontaneous cell death. Macrophages, however, are tissue-residing

34 cells and can survive for many months. Many of the genes that are differentially

regulated can be generally described as genes that play a role, or are postulated

to play a role in cellular trafficking, adhesion and/or survival.

Our laboratory is in the process of delineating the signaling cascade

regulating the survival of monocytes in the presence of M-CSF. This microarray

data suggests that while some critical subunits of the phosphatidylinositol-3

kinase and a phosphatase and tensin homolog are expressed more highly in

peripheral blood monocytes, the survival factor Akt is up-regulated in alveolar

macrophages. This data is consistent with evidence that PTEN and Akt levels

are inversely correlated66, and further bolsters our previous data indicating a

critical role for Akt in monocyte-macrophage survival and differentiation23,24.

Interestingly, the heat shock protein Hsp27 was among the genes with the most increased expression in AM versus fresh monocytes. Hsp 27 expression is induced by Activating Transcription Factor 3 (ATF3), and inhibits apoptosis in a neural cell model60. This protection from apoptosis might arise from either Hsp

27 binding to cytochrome c and inhibition of caspase activity, or through the

interaction between Hsp27 and Akt. The latter mechanism for Hsp 27 mediated

cell survival has been demonstrated in neutrophils67. Akt is studied extensively in

our laboratory, and is postulated as an important survival factor for monocytes,

downstream of the M-CSF receptor23. Additionally, in U937 cells, a monocyte-

like cell line, induction of heat shock proteins decreased cell death in response to

apoptosis-inducing agents59 and, in pro-myelocytic HL60 cells, differentiation

toward the macrophage lineage is accompanied by increases in both the quantity

35 and phosphorylation state of Hsp 2768. This evidence, together with our data

indicates a role for Hsp 27 as a survival factor in numerous cell types, and

specifically, in alveolar macrophages. We are currently further investigating the

role of Hsp27 in macrophage homeostasis.

The receptor tyrosine kinase Axl is also up-regulated in alveolar

macrophages compared to fresh monocytes. This molecule, which is structurally

similar to cellular adhesion molecules, is expressed in CD34+ bone marrow

stromal cells, and in peripheral monocytes69, however, macrophage expression

of Axl has not yet been defined. The ligand for Axl, growth arrest-specific gene-6

(Gas6), signals through the phosphatidylinositol-3 kinase/Akt pathway and

appears to be a survival factor for oligodendrocytes61. Interestingly, Axl

expression appears to correlate with adhesion in some lung carcinoma cell lines.

In adherent non-small cell lung cancer and bronchial epithelial cell lines Axl is

highly expressed, however in suspension-grown small cell lung cancer cells, it is

not70. These lines of evidence, taken with our observations of Axl expression

tissue residing alveolar macrophages but not peripheral blood monocytes, indicate that Axl might also play a role in the survival and differentiation of

monocytes and macrophages.

The apparent increased expression of Axl and Hsp27 is very intriguing.

We are currently looking at other methods to validate this data, including protein

analysis. Further investigation into the potential role of these molecules in

monocyte differentiation will be the focus of my first post-doctoral research

36 project. We will further define and test the hypotheses that Axl and/or Hsp 27

are involved in the differentiation of monocytes to macrophages by utilizing both

cultured monocyte-derived macrophages, and native alveolar macrophages.

Additionally, with the advent of new and enhanced Affymetrix microarray

expression analysis gene chips, and with improved protocols for sample

preparation, it is our goal to perform an authoritative monocyte versus alveolar

macrophage gene expression comparison, to potentiate the data gathered in this

pilot study. This study will ultimately utilize large numbers (n=10) of donor-

matched monocyte and macrophage samples, and should provide a library of

information regarding gene expression comparisons between these cell types.

The latest Affymetrix chips (named HGU133 Plus 2.0), contain over 47,000 probe

sets interrogating 38,500 known human genes, more than three times the

information garnered by our pilot study.

37 Materials and Methods

Cell Isolation

We recruited healthy volunteers, aged 25-42 (average age = 33y) to

donate 120cc of peripheral blood, and to undergo bronchoalveolar lavage (BAL)

after signing an IRB approved consent form.

Mononuclear cells were isolated by layering fresh blood diluted 1:1 with

sterile PBS (Biowhittaker, Walkersville, MD) over Histopaque (Sigma

Diagnostics, St. Louis, MO), followed by density gradient centrifugation.

Monocytes were negatively selected using a Monocyte Isolation Kit (Miltenyi

Biotech, Auburn, CA) containing monoclonal hapten-conjugated antibodies to

CD3, CD7, CD19, CD45RA, CD56 and IgE, which deplete T cells, NK cells, B

cells, dendritic cells and basophils. We modified the buffer to contain 2mM

EDTA and 0.5% HSA in PBS. The non-monocyte cells are indirectly

magnetically labeled with anti-hapten microbeads, and depleted by retaining

them on a magnetic column. Unlabeled monocytes are contained in the eluant.

The cellular content of the BAL fluid was washed 3x in RPMI media

(Cambrex, Walkersville, MD) prior to use.

RNA Extraction

Immediately after monocyte isolation or AM collection, cells were lysed

with TRIzol (Invitrogen, Carlsbad, CA) and total RNA was extracted following the

manufacturers protocol. RNA’s were quantitated using UV spectrophotometry on

a GeneQuant Pro (Amersham Biosciences, Piscataway, NJ). RNA’s were further

purified using a Qiagen RNeasy RNA cleanup protocol (Qiagen, Inc., Valencia,

38 CA) according to the manufacturer’s suggestion. At this point, RNA’s were again

quantitated and only those samples with 260/280 ratios above 1.8 were used to

synthesize cDNA.

Microarray Analysis

cDNA Synthesis: Double stranded cDNA’s were synthesized from 10µg total RNA with the Superscript Double Stranded cDNA Synthesis Kit (Invitrogen,

Carlsbad, CA). The first strand was synthesized at 42˚C and primed with an

HPLC purified T7(dT)24 primer (Sequence: 5’-

GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-T(24)-3’ GenSet

Corporation, San Diego, CA). The second strand was synthesized at 16˚C for 2 hours. cDNA synthesis was terminated by the addition of EDTA.

cDNA Clean-Up: cDNA’s were purified by phenol/chloroform extraction.

Briefly, an equal volume of phenol:chloroform:isoamyl alcohol (Ambion, Austin,

TX) was added to the synthesis reaction, vortexed then centrifuged. cDNA was precipitated in ethanol by the addition of NaOAc to a final concentration of 0.3M. cDNA’s were resuspended in DEPC-treated H2O for in vitro transcription.

In Vitro Transcription: cRNA was transcribed using a BioArray High Yield

RNA Transcript Labeling Kit (Enzo Diagnostics, Farmingdale, NY), according to the manufacturer’s protocol. cRNA’s were purified using a Qiagen RNeasy clean up protocol and quantitated by UV spectrophotometry, both as described.

Gene Chip Hybridization: cRNA’s were further prepared for hybridization and hybridized to Affymetrix U95Av2 oligonucleotide microarray chips in the

Microarray/Genetics Core Facility at the Dorothy M. Davis Heart and Lung

39 Institute at The Ohio State University, following the manufacturer’s protocols.

The Core Facility performs cRNA fragmentation, target hybridization, washing, staining, array scanning and basic data analysis. A test chip is used to assure the cRNA integrity and target labeling quality before the fragmented cRNA

targets are hybridized with the standard array. Control probe sets that

interrogate phage sequences such as BioB, BioC, BioD, and Cre are used as

hybridization controls to assure that the hybridization, washing, staining, and

scanning procedures are capable of detecting a broad linear range of labeled

cRNA with a high level of sensitivity. Five quality control criteria are evaluated:

(1) Ratios of 3’ signal to 5’ signal of two house keeping genes, beta-actin and

GAPDH; (2) Presence of hybridization controls BioB, BioC, BioD, and Cre; (3)

Scale factors between arrays; (4) Background intensity, and; (5) Percent of

genes present. The labeled nucleic acid targets are hybridized with standard

arrays when all quality control measures are acceptable.

Gene Chip Analysis: All gene chip data analysis was performed in the

Davis Heart & Lung Research Institute (DHLRI) Bioinformatics/ Computational

Biology (BCB) Core using Data Mining Tool (Affymetrix, Santa Clara, CA) and

Microarray Suite 5.0 (Affymetrix, Santa Clara, CA) software.

Reverse Transcriptase Polymerase Chain Reaction

Total RNA’s were treated with DNAse to ensure removal of genomic DNA in the RNA prep. Single-stranded cDNA was synthesized at 42°C from total RNA using the Superscript Single Stranded cDNA Synthesis Kit (Invitrogen, Carlsbad,

CA). Random hexamers, included in the kit, were used to as primers for the

40 cDNA synthesis reaction. sscDNA’s were subjected to multiplex PCR reactions with primers for Axl (Sequences: 5’ 5’-GGTGGCTGTGAAGACGATGA-3’ and 3’

5’-CTCAGATACTCCATGCCACT-3’) as described by Sun et al (Sun, 2003), and

GAPDH (R&D Systems, Minneapolis, MN). The PCR reaction consisted of 28 cycles at 94°C for 45s for denaturing, 55°C for 45s for annealing, and 72°C for

90s for extension. PCR products were separated on a 2% agarose gel stained

with ethidium bromide, then visualized and photographed under UV light using

QuantityOne software (Bio-Rad, Hercules, CA). The predicted band sizes were

320bp (Axl) and 576bp (GAPDH). Densitometry of the bands was performed using Bandleader Application Version 3.00 (Magnitec, Tel Aviv, Israel) and values are presented as a ratio of Axl band intensity to GAPDH band intensity.

Western Blotting

Protein-matched whole cell lysates from negatively isolated peripheral

blood monocytes and alveolar macrophages were subjected to SDS-PAGE, then

transferred to a nitrocellulose membrane and probed with a 1/5000 dilution of

goat polyclonal antibody to Akt (Santa Cruz Biotechnology, Santa Cruz, CA) and

a 1/5000 dilution of mouse monoclonal antibody directed against B-actin (Sigma-

Aldrich Corp, St. Louis, MO). Bands were detected using ECL Reagent

(Amersham Biosciences, Piscataway, NJ) and exposing the membrane to X-ray

film.

CHAPTER 3

ALVEOLAR MACROPHAGES LACK CCR2 EXPRESSION AND DO NOT FUNCTIONALLY RESPOND TO MONOCYTE CHEMOATTRACTANT PROTEIN-1

Introduction

Monocytes and alveolar macrophages share many similarities in function, and both have been implicated in numerous physiologic and pathophysiologic conditions, though not necessarily in the same juncture. Because monocytes are precursors to tissue macrophages, they have been categorized together and inadvertently there is the assumption that they share many features. In reality, while these cells are related, they are independently regulated and have discrete functions. Characterizing the location, activity and responsiveness of each cell type will distinguish functional differences between monocytes and alveolar macrophages, and may provide cell-specific therapeutic targets. The ultimate goal is to understand the independent regulation of each cell type in lung inflammation.

One question that arises in distinguishing the two cell types revolves around the responses of peripheral monocytes and tissue macrophages to various chemokines. MCP-1 and MIP-1α are low molecular weight C-C

42 chemokines believed to play a role in the recruitment of mononuclear cells,

however, no studies have yet compared the effects of these molecules on

peripheral blood monocytes and alveolar macrophages.

MCP-1 and CCR2

The regulated recruitment of blood monocytes to an area of tissue

inflammation or injury is critical to host defense. Although several chemokines

can influence monocyte trafficking, MCP-1 appears to be critical. Mice deficient

in MCP-1 (known as JE in the mouse) demonstrate significantly decreased

recruitment of monocytes after thioglycollate challenge, and impaired monocyte

responses, including migration and induction of cytokine expression, to certain

opportunistic infections71. On the other hand, an excess of MCP-1 can also be

problematic. Transgenic mice over-expressing the MCP-1 gene have increased

numbers of mononuclear cells in affected organs72, are more susceptible to

encephalopathy induced by pertussis toxin73, and have exacerbated ischemic

brain injury in a stroke model74.

MCP-1 specifically binds only the cell surface receptor CCR2, and is

known to selectively attract mononuclear cells but not neutrophils26. Because

MCP-1 signals solely via its specific receptor, the significance of CCR2 cannot be minimized. In peripheral blood, CCR2 expression is largely limited to monocytes, however a small percentage of T lymphocytes are also CCR2 positive, both in humans and in mice75. CCR2 exists as two RNA splice-variants, CCR2A and

CCR2B. Both variants, which differ only in their carboxyl tails, bind MCP-1,

43 though CCR2B seems to be the predominant variant in both monocytes and in monocyte-like cell lines76,77. Mice lacking CCR2 develop normally and have no

overt hematopoietic or other phenotypic abnormalities78, however they do

demonstrate enhanced myeloid progenitor cell cycling and concomitant

apoptosis79. CCR2 deficient mice, like MCP-1 deficient mice, are unable to

recruit monocytes to sites of inflammation80, and additionally are unable to clear

some intracellular pathogens78.

MCP-1 and/or CCR2 signaling has been implicated in the genesis and

maintenance of many diseases, with increased MCP-1 expression in coronary

artery81 and autoimmune diseases82 and the observation that mice lacking CCR2

are protected from developing pulmonary fibrosis48. Thus, physiological regulation of the production, expression and effect of MCP-1, via CCR2, is critical for host homeostasis and suggests that MCP-1 and CCR2 have complex regulatory roles in immune function.

MIP-1α, CCR1 and CCR5

MIP-1α is another member of the C-C family of chemokines. Originally purified as a doublet containing both MIP-1α and MIP-1β, these molecules were

separately identified in 198883. Although MIP-1α tends to aggregate at high

concentrations, at physiological levels (less than 100ng/ml) it exists solely as a

monomer84. Under normal conditions, most hematopoietic cells synthesize and

secrete low levels of MIP-1α. Interestingly, MIP-1α secretion by monocytes is

increased during monocyte-endothelial interactions mediated by ICAM, and 44 some hypothesize that this enhancement may help to sustain the recruitment of

inflammatory cells84. Mice deficient in MIP-1α develop normally and have no

hematologic abnormalities, although they do experience decreased inflammatory

responses and inhibited viral clearance in some models85.

MIP-1α binds the C-C chemokine receptors CCR1 and CCR5. Some

literature suggests MIP-1α may also bind CCR4, but this receptor has more

recently been identified as specific only for Thymus and Activation-Regulated

Chemokine (TARC)86. Incidentally, CCR1 and CCR5 share 55% amino acid

homology87. CCR1 is expressed on monocytes, eosinophils, basophils and

activated T lymphocytes, and can also bind Regulated on Activation, Normal T

cell Expressed and Secreted (RANTES) and the monocyte chemotactic proteins

MCP–2 and MCP–327. CCR1 is rapidly internalized after exposure to its

ligand(s)88.

In contrast to CCR1, monocytes express little CCR5, while monocyte-

derived macrophages and NK cells have increased expression32,89. CCR5 plays

an important role in HIV infection, as it is required for infection by R5

(“macrophage trophic”) HIV strains87. Humans with a specific CCR5 deletion

mutation, CCR5-∆32, are almost wholly protected from infection by these strains90. Intriguingly, the frequency of this mutation in some populations has

been increasing rapidly, leading some investigators to suggest that positive

selection is at work84. CCR5 has been extensively studied in relation to HIV

infection, but no studies have yet examined CCR5 expression in alveolar

macrophages under normal conditions. 45 Altered expression of MIP-1α has also been implicated in numerous disease states, with increased expression in atherosclerotic lesions91, and some

adult T cell leukemias92. MIP-1a is also increased in rheumatoid arthritis93, and

in the BAL fluid of patients with pulmonary fibrosis94-96.

Chemokine Receptor Expression

Freshly isolated peripheral blood monocytes express a variety of

chemokine receptors, and expression of these receptors may be altered in

pathologic conditions like inflammation or disease (Figure 3.1). For instance,

normal blood monocytes express only CCR1 and CCR297. In rheumatoid

arthritis, however, circulating monocytes continue to express CCR1 and CCR2, but also express CCR3, CCR4 and CCR5. Curiously, monocytes in the synovial

fluid of rheumatoid arthritis patients down-regulate CCR1 and CCR2 but maintain

expression of CCR3 and CCR597. Even subpopulations of circulating monocytes

appear to differentially express these receptors, as CD14+ CD16+ monocytes

have been shown to express lower amounts of CCR2 and higher amounts of

CCR1 and CCR5 than CD14+ CD16- monocytes98,99.

Interestingly, circulating CD14+ CD16+ monocytes also exhibit features of

tissue macrophages100, and some investigators believe that the CD14+ CD16+ population of monocytes may be direct precursors to alveolar macrophages, since these macrophages are also CD14+ CD16+51. While chemokine receptor

expression on alveolar macrophages has not been characterized, other

CCR1 CCR2 CCR3 CCR4 CCR5 Peripheral Blood + 97,* + 97,* +/- 26 -* Monocytes Rheumatoid + 97 - 97 + 97 Arthritis PBM Rheumatoid Arthritis - 97 - 97 + 97,101 + 97,101 Synovial Fluid Monocytes CD14+ CD16+ + 98 - 98 +/- 98 Monocytes Peritoneal 103 + 102 + + 102 Macrophages (in mice) Monocyte- Derived + 32 - 32 + 32,89 Macrophages Alveolar -* -* +* Macrophages

Figure 3.1. A brief summary of C-C chemokine receptor expression in monocytes and monocyte derived cells. CC chemokine receptor expression on monocytic cells is fluid, and can change between disease states, cellular location or differentiation state. References are listed as superscripted numbers, and “*” indicates data shown in this dissertation.

47 populations of tissue macrophages possess a variety of CC chemokine

receptors. For example, human peritoneal macrophages (PM) express CCR1

and CCR5, and murine PM also express CCR2102,103.

Numerous studies have demonstrated that in vitro maturation of blood

monocytes to macrophages selectively changes the expression of specific

chemokine receptors. For instance, cultured monocytes begin to reduce

expression of CCR-2 in as little as 4 hours32, and continuing for up to 7 days,

when no CCR2 is detected104. In contrast, surface expression of CCR1 and

CCR5 increase within 24 hours and these changes in chemokine receptor expression correlate with altered chemotactic responses to MCP-1 and MIP-1α32.

Of note, some studies suggested that the loss of CCR2 expression is a result of

MCP-1 secretion by the differentiating monocytes75.

Understanding the regulation, recruitment and activation of monocytes and tissue macrophages will ultimately lead to a better understanding of the cellular processes for which these cells are responsible, and may also provide

molecular targets that are specific to one cell population, or sub-population.

These targets may eventually enable us to generate therapies specifically

intended for individual cell types, which may decrease nonspecific therapeutic

interactions and side effects. It is with this goal in mind we embarked upon

examining the chemokine receptor expression status and the resulting functional

effect in monocytes and alveolar macrophages.

48 Results

Our microarray analysis, detailed in Chapter 2, indicated that CCR2B might be differentially regulated between monocytes and alveolar macrophages, with AM expressing far less CCR2B RNA (refer to Figure 3.2a). This observation led us to hypothesize that alveolar macrophages would express both CCR2 RNA and the CCR2 surface protein at lower levels than freshly isolated peripheral blood monocytes, and that this lack of expression would prevent alveolar macrophages from functionally responding to MCP-1.

Freshly isolated human monocytes express more CCR2 RNA than alveolar macrophages

Previous studies found that CCR2 expression is reduced as monocytes undergo in vitro differentiation. Microarray expression analysis comparing freshly isolated peripheral blood monocytes and alveolar macrophages indicated that

CCR2B was differentially regulated between these two cell types (Figure 3.2a).

To verify gene array results, reverse transcriptase PCR was utilized, and confirmed that RNA for CCR2 is expressed at higher levels in monocytes (Figure

3.2b).

CCR2 surface protein is expressed on freshly isolated monocytes but not on alveolar macrophages

While differences in CCR2 RNA expression were intriguing, it was possible that RNA expression did not correlate with surface protein expression.

a. b.

Mo GAPDH AM CCR2

7500

H 1.5

5000 A 1.0

o .5

i 2500 t

Average Signal Intensity Signal Average Mo AM Mo AM

Figure 3.2. Alveolar Macrophages Express Less MCP-1R/CCR2 RNA than Peripheral Blood Monocytes. Microarray analysis indicated that AM express less MCP-1RB (39937_at) than PBM (a). Reverse transcriptase PCR for CCR2 (b) confirmed these results. Representative array scans (top panel, a) and the average signal intensity and standard deviation over 2 donors of each cell type (lower panel, a) are shown. In (b), bands are representative of 3 separate donors and densitometry graphs show the ratio of chemokine receptor to GAPDH control band intensity, averaged over the three donors.

50 Thus, we next sought to analyze surface expression of the chemokine receptor

CCR2 on human monocytes and AM. Using flow cytometric analysis, we found

that while freshly isolated peripheral blood monocytes expressed the chemokine

receptor CCR2 (Figure 3.3a), alveolar macrophages did not (Figure 3.3b).

AM are less responsive to MCP-1 than circulating monocytes in a migration assay

As surface expression of CCR2 was different between the cell types, we

sought to examine the functional result of these differences. Using an in vitro

migration assay, freshly isolated peripheral blood monocytes migrated toward

rhMCP-1 in a dose dependent manner (Figure 3.4 black bars). In contrast,

alveolar macrophages responded to rhMCP-1 only at the highest tested dose

(Figure 3.4 white bars). Even at this supraphysiologic dose, the average

increase in number of AM migrating in response to MCP-1 was only 2.3

cells/HPF. This lack of chemotaxis was not due to intrinsic defects in

macrophage chemotaxis as given an appropriate stimulus (see Figure 3.5), AM

responded normally in this assay. Thus monocytes, but not alveolar

macrophages, express the MCP-1 receptor CCR2 and are the primary

responders to this chemokine.

Figure 3.3. Peripheral Blood Monocytes, but not Alveolar Macrophages Express CCR2 Surface Protein. Monocytes (a) and AM (b) were isolated and subjected to flow cytometric staining for CCR2 (solid line) as detailed in materials and methods. IgG isotype control is shown (dashed line).

100 Mo AM 80

60 20 # cells migrating/HPF

0 1 10 100 Concentration of MCP-1 (in ng/ml)

Figure 3.4. MCP-1 Preferentially Attracts Freshly Isolated Peripheral Blood Monocytes Compared to AM in a Migration Assay. Monocytes (black bars) respond in a dose dependent manner to rhMCP-1. When compared to unstimulated cells, monocyte migration is significant to p< 0.01 at 1.0 ng/ml, and p< 0.001 at 10 and 100 ng/ml. AM chemotaxis (white bars) was only significant at 100ng (p< 0.01). The averages of 6 independent experiments and SEM are shown.

53 Peripheral blood monocytes and AM are responsive to MIP-1a in a migration assay

To clarify that alveolar macrophages recovered from the lungs of normal volunteers were functional, these cells were also assayed for chemotaxis toward

MIP-1α. Freshly isolated peripheral blood monocytes and alveolar macrophages

both responded, in a dose dependent fashion, to MIP-1α (Figure 3.5). In fact, peripheral blood monocytes responded more vigorously to lower concentrations of MIP-1α than to MCP-1. AM responses to MIP-1α peaked at 10ng/ml, however

at 100ng/ml MIP-1α, AM chemotaxis was still significantly different from non-

stimulated controls.

Freshly isolated human monocytes and alveolar macrophages

express similar levels of CCR1 and CCR5 RNA

After establishing that monocytes and alveolar macrophages both

responded to MIP-1α, we wanted to determine expression of the MIP-1α

receptors, CCR1 and CCR5 on each cell type. By reverse transcriptase PCR,

monocytes and alveolar macrophages shared similar levels of RNA expression

for both CCR1 (Figure 3.6, left panel) and CCR5 (Figure 3.6, right panel).

The MIP-1α receptors, CCR1 and CCR5 are differentially expressed

on peripheral blood monocytes and alveolar macrophages

We next sought to determine the surface protein expression of CCR1 and

CCR5 by flow cytometry. While freshly isolated peripheral blood monocytes 54

90 Mo AM

35 15 # cells migrating/HPF cells #

0 1 10 100 Concentration of MIP-1α (in ng/ml)

Figure 3.5. MIP-1α Attracts both Peripheral Blood Monocytes and AM. Monocytes (black bars) and AM (white bars) respond in a dose dependent manner in a chemotaxis assay. Compared to unstimulated cells, cellular migration is significant to p< 0.001at all MIP-1α concentrations tested for both cell types.

a. b. GAPDH GAPDH

CCR1 CCR5

H 1.5 H 1.5

D D

P P

A 1.0 A 1.0

G G

o o

t t

o .5 o .5

i i

t t

a a

R R

Mo AM Mo AM

Figure 3.6. Freshly Isolated Monocytes and Alveolar Macrophages Express Equivalent Amounts of CCR1 and CCR5 RNA. We performed multiplex reverse transcriptase PCR using primers for CCR1 and GAPDH (a) and CCR5 and GAPDH (b). Bands are representative of 3 separate donors. Densitometry graphs show average ratio of chemokine receptor to GAPDH control band intensity, averaged over the three donors.

56 expressed surface CCR1 (Figure 3.7a), and current literature suggests CCR1 is

up-regulated during in vitro macrophage differentiation32, we were surprised to find that alveolar macrophages did not express CCR1 (Figure 3.7b).

On the other hand, while we found no CCR5 expression on peripheral

blood monocytes (Figure 3.8a), like in vitro differentiated MDM, alveolar

macrophages readily expressed CCR5 surface protein (Figure 3.8b).

Use of a CCR5 blocking antibody abrogates MIP-1a induced

chemotaxis in alveolar macrophages

Since peripheral blood monocytes and alveolar macrophages differentially

express the MIP-1α surface receptors CCR1 and CCR5, we wanted to

demonstrate the significance of this difference in our model. To verify that

surface expression of CCR1 and CCR5 predicted biological activity, we

examined the effect of anti-CCR5 blocking antibodies on MIP-1α-induced

migration. Consistent with a lack of CCR5 surface expression on monocytes, we

found that anti-CCR5 blocking antibodies did not reduce monocyte chemotaxis in

response to MIP-1α compared to the isotype control (Figure 3.9a). Overall, the

addition of any IgG slightly decreased monocyte responses to MIP-1α compared

to MIP-1α alone (data not shown), but there was no difference in response when

comparing CCR5 blocking antibody to the isotype control. In contrast, alveolar

macrophages were not significantly affected by the addition of any IgG compared

Figure 3.7. Peripheral Blood Monocytes, but not Alveolar Macrophages, Express CCR1 Surface Protein. Monocytes (a) and AM (b) were isolated and subjected to flow cytometric staining for CCR1 (solid line). IgG isotype control is shown (dashed line).

Figure 3.8. Alveolar Macrophages, but not Peripheral Blood Monocytes, Express CCR5 Surface Protein. Monocytes (a) and AM (b) were isolated and subjected to flow cytometric staining for CCR5 (solid line). IgG isotype control is shown (dashed line).

a. b. 80 16 IgG control * IgG control * Anti-CCR5 Anti-CCR5 * 60 12

40 8

20 4 # cells migrating/HPF cells # migrating/HPF cells #

0 1 10 100 0 1 10 100

Concentration of MIP-1α (in ng/ml)

Figure 3.9. Blocking antibodies to CCR5 decrease MIP-1α induced chemotaxis in AM, but not peripheral blood monocytes. The addition of 1µg/ml CCR5 blocking antibody does not significantly alter monocyte chemotaxis (a) at any concentration of MIP-1α when compared to the IgG control. In contrast, addition of CCR5 blocking antibodies alters MIP-1α induced alveolar macrophage chemotaxis (b) at every concentration (p<0.001 at 1, 10 and 100ng/ml). Results are averaged over 3 separate experiments, and the SEM is shown.

60 to MIP-1α alone (data not shown), whereas addition of anti-CCR5 blocking

antibodies specifically reduced the chemotaxis of AM (Figure 3.9b) suggesting

that AM utilize only CCR5 when responding to MIP-1α.

61 Discussion

This study evaluated the regulation and recruitment of monocytes and

alveolar macrophages to MCP-1 and MIP-1α. We found that alveolar

macrophages, which are derived from monocytes, do not express CCR2 and are unresponsive to MCP-1 as a chemotactic stimulus. Freshly isolated peripheral

blood monocytes express CCR2 and respond to MCP-1, both in our study and

according to previous literature104. These data suggest that in the regulation of lung inflammation, MCP-1 may be a monocyte-specific stimulus.

Monocyte-derived macrophages (MDM) are utilized in many laboratories as an easily acquired model to study the activity and responses of tissue macrophages. Indeed, our laboratory uses these cells to model numerous aspects of monocyte and macrophage biology. MDM appear to share many similarities with tissue macrophages, and decrease surface expression of CCR2 as they undergo in vitro differentiation. Additionally, MDM are unresponsive to

MCP-1. In the present study, we show that native alveolar macrophages also do

not express CCR2, and do not respond to MCP-1 in a migration assay. These

data clearly demonstrate further similarities shared by MDM and true tissue

macrophages.

Previous investigators have examined the effects of MCP-1 on monocytes

and macrophages without differentiating the two. Some papers use the term

“monocyte/macrophage” rather than identifying each cell separately105. For example, Lu et al were puzzled when they reported that mice genetically deficient in MCP-1 were indistinguishable from wild type mice in clearing M. tuberculosis

62 infection71. The authors speculated that this unexpected response might be due

to the retained ability of these mice to elicit a TH1 response. We would argue

that because alveolar, and perhaps other tissue macrophages lack CCR2, it is

not surprising that MCP-1 has little effect in this system.

In this study, we also confirm that both peripheral blood monocytes and

alveolar macrophages respond to MIP-1α, albeit through different receptors.

While monocytes express CCR1, alveolar macrophages express only CCR5. It

is worth mentioning that this expression appears to be regulated at a post-

transcriptional level, as both cell types express RNA for CCR1 and CCR5. More

work is needed to elucidate the mechanism of this regulation.

In raw numbers, many more monocytes than alveolar macrophages are

stimulated to migrate by MIP-1α, although responses for both are statistically

significant at all concentrations tested. The reason for the difference in raw

numbers of migrating cells is not clear. One possible explanation may lie in the

intrinsic properties of these cells; monocytes circulate through the peripheral

blood and are, by definition, mobile, whereas macrophages are tissue residing

cells, and may therefore be inherently less mobile than their monocyte

counterparts. Nevertheless, lung inflammation mediated by MIP-1α would

predictably involve both monocytes and alveolar macrophages.

Curiously, while previous studies have indicated that MDM up-regulate

both CCR1 and CCR5 during differentiation, our data indicates that AM respond

to MIP-1α solely through expression of CCR5, and do not express CCR1.

Blockade of CCR5 completely abrogates MIP-1α-induced chemotaxis in AM, 63 providing further evidence that these cells do not express another functional MIP-

1α receptor. Peripheral blood monocytes, on the other hand, appear to respond to MIP-1α solely via CCR1, as these cells do not express CCR5, and blockade of

CCR5 has no effect on MIP-1α-induced monocyte chemotaxis. Although blocking antibodies to CCR1 are not commercially available, we would hypothesize that CCR1 blockade would affect only the migration of peripheral blood monocytes to MIP-1α, and have no effect on alveolar macrophages. The differential expression of CCR1 and CCR5 between monocytes and AM alludes to differential regulation of the activity of monocytes and alveolar macrophages in areas of lung inflammation and injury. In contrast to our CCR2 data, this CCR1 and CCR5 data presents an interesting dichotomy between the more readily available MDM and true tissue macrophages.

Currently, it is difficult to separately identify newly recruited monocytes from resident macrophage cells in areas of lung inflammation and fibrosis. Our data may help to ease that burden. By extrapolating from our data, it is possible that examining the expression of C-C chemokine receptors may help delineate newly recruited monocytes from mature macrophages in the lung. Further analysis of the cellular content in the lungs of patients with inflammatory or fibrotic conditions will be necessary to test this hypothesis.

Understanding the preferential recruitment of inflammatory cells has important implications in lung pathology. Monocytes and macrophages are actively recruited into the lung in numerous pathological conditions, including pneumococcal pneumonia106 and Mycobacterium tuberculosis infection107. Our 64 data helps to clarify the cells responding to specific chemotactic stimuli, and in turn may help to specifically and differentially target monocytes and macrophages in the lung.

Additionally, our interests in MCP-1 and MIP-1α are piqued by the knowledge that both of these chemokines have been extensively implicated in

pulmonary fibrosis. The etiology and epidemiology of pulmonary fibrosis will be

discussed in detail in Chapter 4. Nonetheless, it is appropriate to note here that

increased levels of MCP-1 have been demonstrated both in human pulmonary

fibrosis 47 and in rodent models of fibrosis108. MCP-1 can directly stimulate

fibroblasts to produce TGF-β and synthesize collagen35, both of which are pro-

fibrotic molecules. Finally, and perhaps most importantly, previous studies have

demonstrated that mice deficient in CCR2 are protected from developing fibrosis

in a bleomycin model48. Our data helps to delineate that monocytes, but not resident alveolar macrophages are the cells most likely affected by these increases in MCP-1, and that these increases in MCP-1 serve to recruit circulating monocytes to the lung. Further research will be necessary to ascertain the role of those newly recruited monocytes in the development of pulmonary fibrosis.

65 Materials and Methods

Cell Isolation

Human monocytes and alveolar macrophages were isolated from healthy

normal, nonsmoking volunteers as detailed in Chapter 2.

Microarray Analysis

Gene expression analysis was performed on freshly isolated human monocytes and alveolar macrophages as detailed in Chapter 2.

Reverse Transcriptase PCR

Total RNA was extracted and cDNA’s synthesized as in Chapter 2.

Commerically available PCR primers for human CCR1, CCR2 and CCR5 (R&D

Systems, Minneapolis, MN) were utilized in a two-gene multiplex reaction with

GAPDH primers (R&D Systems, Minneapolis, MN) added as a loading control

(i.e. CCR1 and GAPDH, CCR2 and GAPDH, etc). The PCR reaction consisted

of 30 cycles at 94°C for 45s for denaturing, 55°C for 45s for annealing, and 72°C

for 45s for extension, according to the manufacturer’s protocol. The PCR

products were separated on a 2% agarose gel and stained with ethidium bromide

then photographed and analyzed as described in Chapter 2. PCR bands were

predicted at 201bp (CCR1), 406bp (CCR2), 459bp (CCR5) and/or 576bp

(GAPDH). Densitometric values are always presented as a ratio of chemokine

receptor band intensity to GAPDH band intensity.

66 Flow Cytometry

In preparation for flow cytometric analysis, freshly isolated peripheral

blood monocytes and alveolar macrophages were placed in a buffer solution

containing 100µg/ml human IgG (JacksonImmuno Research, West Grove, PA) in

sterile PBS, for 10 minutes to block nonspecific Fc receptor binding. All subsequent steps were carried out in this buffer. Primary antibodies (5ug/ml) to

CCR1, CCR2, CCR5 and an IgG2b isotype control (all from R&D Systems,

Minneapolis, MN) were incubated for 45 minutes on ice, followed by washing and

the addition of a tandem PE-Cy5 labeled goat F(ab’)2 anti-mouse IgG (H+L)

(alveolar macrophages) for 30 minutes on ice. After a final wash, cells were fixed with 10% buffered formalin (Fisher Scientific, Pittsburgh, PA). Cytometric analysis was performed using an FL3 long pass (>670nm wavelength) filter on a

BD FACSCalibur flow cytometer (BD Biosciences, San Jose, CA).

Migration Assays

A 48-well chemotaxis chamber (Neuroprobe, Rockville MD) was used for

all chemotaxis assays. Monocytes and all tested agents were cleared for endotoxin contamination by addition of Polymyxin B (Calbiochem, San Diego,

CA). MCP-1 (R&D Systems, Minneapolis, MN) or MIP-1α (R&D Systems,

Minneapolis, MN) were loaded into the bottom well at the appropriate concentrations, and 4.5 x 104 monocytes or AM were added to the upper

chamber. The chamber was incubated at 37˚C with 5% CO2 for 90 minutes.

Monocyte chemotaxis was measured on a 5-micron pore polycarbonate filter, and AM chemotaxis on an 8-micron pore polycarbonate filter (Osmonics, Inc. 67 Minnetonka, MN). The filters were removed, fixed and stained in Diff-Quik and viewed under high power (40X) lens. Three wells for each condition were counted under high power and reported as the number of cells/High Power Field

(HPF). At least five fields were counted per well and at least 15 were counted per condition. Results are reported as average number of cells per HPF for all of the fields viewed per condition. For experiments utilizing blocking antibodies,

1µg/ml antibody (R&D Systems, Minneapolis, MN) was added to the bottom well with the appropriate chemokine.

CHAPTER 4

MICE DEFICIENT IN MACROPHAGE COLONY-STIMULATING FACTOR ARE PROTECTED FROM BLEOMYCIN-INDUCED PULMONARY FIBROSIS

Introduction

The preceding portions of this dissertation focused on gene expression in monocytes and alveolar macrophages. In addition to these in vitro studies, we wanted to develop an in vivo model examining the importance of these cells in a model of human disease. Monocytes and alveolar macrophages have been implicated in a number of disease states; however, pulmonary fibrosis is of particular interest to our laboratory.

Pulmonary Fibrosis

Pulmonary Fibrosis is a devastating human disease characterized by chronic and progressive scarring of the lung parenchyma. PF manifests itself in numerous forms (briefly outlined in Chapter 1), however Idiopathic Pulmonary

Fibrosis (IPF) characterized by Usual Interstitial Pneumonia (UIP) is the most severe. The prevalence of IPF is approximately 13-20/100,000 in the United

States42. The average age of onset is between 50 and 70 years, and most patients present with a non-productive cough, progressive dyspnea, abnormal

69 breath sounds, and restricted lung volumes41. By the time of diagnosis, virtually

all patients have abnormalities visible upon X-ray109. No definitive explanation

exists, hence the idiopathic nomenclature, however IPF is correlated with

smoking110, environment111 and genetics112,113.

Histologically, IPF consists of areas of normal lung heterogeneously

interspersed with interstitial inflammation, fibrosis, and honeycomb changes

(Figure 4.1). It is important to note that human IPF primarily affects the

peripheral sub-pleural areas of the lung41. The fibrotic areas have increased

collagen content, and may include fibroblastic foci at the interface with normal

tissue. Honeycomb changes can occur and consist of cystic, fibrotic airspaces

lined with bronchiolar epithelium and filled with mucin41. Additionally, human IPF

is characterized by chronic interstitial inflammation with septal infiltrates and an

increase in type 2 pneumocytes41.

In animal studies, the development and progression of pulmonary fibrosis

frequently implicates TGF-β, a pro-fibrotic molecule that affects angiogenesis,

fibroblast activation, and the deposition of extracellular matrix41. PF is also thought to be a Th2-type disease and IFN-γ has been promoted as a therapy for the disease in humans. Unfortunately, while IFN-γ decreases the tissue effects

of TGF-β, IFN-γ is decreased in patients with pulmonary fibrosis, and has not

been proven as a consistently effective therapy114,115.

Figure 4.1. Human Idiopathic Pulmonary Fibrosis. Trichrome Stain. From ATS Slide Set “New Approaches to Managing Idiopathic Pulmonary Fibrosis (IPF)”. Note the characteristic heterogeneous appearance and diffuse sub- pleural distribution of fibrosis.

71 Current therapies aimed at slowing the progression of PF have largely

been unsuccessful, even when combining corticosteroids, anti-fibrotic agents,

anti-oxidants, and other pharmaceuticals116-119. Hence, this relentless disease with no known cause also affords no effective treatment.

Bleomycin

Bleomycin is a complex of water-soluble peptides extracted from

Streptomyces verticillatus, originally touted as an anti-tumor antibiotic. While the

exact mechanism of effect is uncertain, the available evidence seems to indicate

that bleomycin inhibits DNA synthesis120. In the late 1960’s and 1970’s,

bleomycin garnered a reputation as a valuable treatment for some types of

cancer, including squamous cell carcinoma of the skin, cancers of the penis and

mycosis fungoides121. Unfortunately, interstitial pneumonitis and pulmonary

fibrosis were soon discovered as serious side effects of bleomycin, thus limiting

the efficacy of bleomycin as a cancer treatment122-124. The exact mechanism by

which bleomycin induces lung injury is unknown.

Animal studies evaluating the toxicologic and pathologic effects of bleomycin125,126 soon evolved into the use of bleomycin as an animal model of

pulmonary fibrosis, with various investigators illustrating differing susceptibilities

among mouse strains127,128, and between the sexes125,129.

Bleomycin can be administered locally to the lungs, by intra-tracheal

instillation, or systemically via intra-peritoneal or intravenous injection, or by use

of an implanted pump. The literature available suggests that most investigators

72 today use an intra-tracheal instillation method130-132. This method generally consists of one intra-tracheal administration of the drug followed by a monitoring period of approximately 2-4 weeks before analysis of lung pathology.

Mechanistically, this method is very simple, as it involves manipulating the animals only one time. The resulting pathology is characterized by early alveolar macrophage accumulation and increased septal cellularity133, followed by

widespread thickening of the septae, intra-alveolar fibrosis and dilation of the

brochioles and alveolar ducts130. The early stages of damage from intra-tracheal

instillation of bleomycin, including inflammatory cell infiltrates, increased collagen

synthesis and decreased lung compliance, moderately resemble human disease.

In later stages, however, intra-tracheal instillation of bleomycin results in focal

peri-bronchiolar inflammation and fibrosis (Figure 4.2), not the heterogeneous

sub-pleural lesions seen in human disease, and lesions created by IT instillation

of bleomycin do not restrict lung capacity134.

Some groups have administered bleomycin systemically126,135-137, a more time-consuming and labor-intensive endeavor. Systemic administration involves repeated injections over time126,137, or, alternatively, the implantation of an

osmotic mini-pump device135. Systemic administration of bleomycin results in the

appearance of focal sub-pleural lesions, patchy endothelial damage, diffuse

interstitial edema and an increase in macrophages135 (Figure 4.3). Later,

alveolar epithelial cells proliferate and are accompanied by the appearance of

extracellular debris in the alveoli. Over an extended period of time these

pathologic changes spread throughout extensive areas of the lung126. Hence, it

a. b.

Figure 4.2. Pulmonary Fibrosis Induced by Intra-Tracheal Instillation of Bleomycin in the mouse. Note the focal peri-bronchiolar fibrosis (a and c) with minimal pleural involvement (a and b).

a. b.

Figure 4.3. Pulmonary Fibrosis Induced by Intra-Peritoneal Injection of Bleomycin in the mouse. Note the predominantly sub-pleural distribution of inflammation and fibrosis (a and b), with little peri-bronchial involvement (c).

75 appears that the lesions resulting from systemic administration of bleomycin may

more closely mimic those seen in human pulmonary fibrosis than lesions

resulting from local bleomycin administration138.

M-CSF Deficient Mice

Mice lacking functional M-CSF arose spontaneously in 1970 at The

Jackson Laboratory139. Originally believed to be located on chromosome 12139,

this single base pair insertion is now known to be within the Csfm gene on mouse

chromosome 3140,141. Homozygous M-CSF deficient (M-CSF -/-) mice exhibit a

phenotype that includes shortened limbs, a dome-shaped head and teeth that do

not erupt. These skeletal abnormalities arise as a consequence of decreased

bone resorption by osteoclasts, and hence the mice are referred to as

‘osteopetrotic’139. M-CSF -/- mice also exhibit retarded growth after the 10th day of life, and have significantly decreased survival over time when compared to normal littermates139. These mice are completely lacking M-CSF, and have

severely decreased numbers of mononuclear phagocytes, including

monocytes142, peritoneal macrophages143, alveolar macrophages144, and osteoclasts139,142. For these reasons, these mice provide an excellent model for

examining the role of M-CSF and of monocytes/macrophages under a variety of

conditions. It should be noted, however, that deficiencies in alveolar

macrophage numbers appear to be corrected by 4 months of age, possibly by an

IL-3 mediated pathway145, and therefore all studies must be completed on young

M-CSF deficient mice.

Numerous studies demonstrate that monocytes and macrophages are

recruited to the lungs and may play a critical role in the development of

pulmonary fibrosis45,133,146, and thus we hypothesized that mice deficient in M-

CSF would be protected from bleomycin-induced pulmonary fibrosis compared to their M-CSF normal counterparts. We chose a systemic administration protocol for bleomycin treatment based on the observation that lesions induced by systemic bleomycin more closely resemble human disease (Figure 4.4).

0.035 U/gram Intra-peritoneal Injections Timeline: Bleomycin Bleomycin Bleomycin Bleomycin Bleomycin (weeks)

Birth Wean 1 2 3 4 Sacrifice Treatment Weeks

Harvest Tissues

Figure 4.4. Systemic Bleomycin Administration Protocol. After weaning, mice were weighed on a weekly basis and intra-peritoneally injected with weight- dependent doses (0.035U/g) of bleomycin twice per week for four weeks. Mice were sacrificed by CO2 asphyxiation 7 days after the last injection, and the heart, lungs, liver and skin harvested for analysis.

78 Results

To assess the role of M-CSF in the development of pulmonary fibrosis, we injected M-CSF deficient mice and their normal littermates with weight-dependent doses of IP bleomycin, monitored the mice throughout treatment and examined lung sections with histochemical and immunohistochemical stains.

M-CSF Deficient Mice Exhibit a Survival Advantage over M-CSF

Normal Mice when Treated With Systemic Bleomycin

We wanted to assess the survival outcome of M-CSF normal (+/+ and +/-) and M-CSF deficient (-/-) mice when treated with systemic bleomycin. We measured survival over 31 days of treatment and created Kaplan-Meier survival curves for each group of mice (Figure 4.5a and b). We performed a log rank test for differences between the curves, and attempted to compute the Cox proportional hazard ratio for Bleomycin treatment versus the vehicle control.

None of the M-CSF normal mice died within 31 days when treated with the vehicle control, while 32% (eight of twenty-five) M-CSF normal mice treated with bleomycin died within this time period. There is a statistically significant difference in the survival of M-CSF normal mice when treated with bleomycin versus the vehicle control; however this difference cannot be computed using the

Cox model because none of the vehicle treated mice died (Figure 4.5c). In essence, the statistical model creates a hazard ratio that is extremely large, on the order of 3.3 x 1030, indicating that the risk of dying is extraordinarily large

when comparing bleomycin treatment to vehicle control.

a. b. 1.00 1.00

0.75 0.75

0.50 0.50

0.25 0.25

0.00 Survivorship Probability 0.00 Survivorship Probability 0 10 20 30 0 10 20 30 Time in Days Time in Days c. Log Rank Test/Hazard Ratio Computation

Log Rank Test Cox Proportional 2 Group Treatment χ (1) P-value Hazard P-value M-CSF Normal Bleomycin 5.64 0.0176 ** ** M-CSF Normal Vehicle Control

M-CSF -/- Bleomycin 0.01 0.9137 0.87 0.914 M-CSF -/- Vehicle Control

Figure 4.5. M-CSF Deficient Mice Exhibit a Survival Advantage over M-CSF Normal Mice when Treated with Systemic Bleomycin. Kaplan-Meier survival curves are shown for M-CSF normal (a) and M-CSF deficient (b) mice treated with bleomycin (solid line) or vehicle control (dashed line). The log rank test and Cox Hazard Ratio computations indicate that while M-CSF normal mice have an exorbitantly increased risk of dying when treated with bleomycin (** indicates Cox Proportional hazard can only be approximated at 3.3 x 1030 with a p-value = 0, because none of the M-CSF normal mice treated with vehicle control died) M- CSF deficient mice are at no greater risk of dying when treated with bleomycin or the vehicle control (c).

80 In contrast, M-CSF deficient mice exhibited no difference in survival when comparing bleomycin and vehicle control treatment. Twenty-two percent (two of nine) M-CSF deficient mice died during treatment with bleomycin, and 25% (one of four) during treatment with the vehicle control. Using the Cox model, the hazard ratio for M-CSF deficient mice treated with bleomycin compared to vehicle control is 0.87, which indicates these mice are at 0.87 times the risk of dying than those treated with the vehicle control. This difference, however is not statistically significant, with a P value of 0.91 and a 95% confidence interval (0.08

- 9.69) that includes 1, indicating non-significance. Thus, while M-CSF normal mice die at a significantly higher rate when treated with bleomycin compared to vehicle control, M-CSF deficient mice show no change in survival when treated with either bleomycin or the vehicle control.

M-CSF Deficient Mice Lose Less Weight than M-CSF Normal Mice

After Systemic Bleomycin Treatment

While survival indicates the ultimate outcome of bleomycin treatment, we also wanted to assess the relative health of the animals throughout the treatment regimen. We tracked the weight of the animals on a weekly basis and used a linear regression model to examine changes in weight over time (Figure 4.6a).

We found that M-CSF normal mice lost weight throughout the treatment regimen.

In contrast, M-CSF deficient mice gained weight at the beginning of treatment, plateaued in the middle and began to lose weight at the end of the treatment protocol. The difference between the groups is statistically significant

a. Bleomycin: Deficient vs Normal

6 6

3 3

0 0

-3 3 Weight change in grams in Weight change

-6 6 23 45

Time in Weeks

Observed Deficient Observed Normal Fitted Deficient Fitted Normal

b. XTGEE Analysis

Source | SS df MS Number of obs = 125 ------F( 2, 122) = 7.78 Model | 27.8396025 2 13.9198013 Prob > F = 0.0007 Residual | 218.252398 122 1.78895408 R-squared = 0.1131 ------Adj R-squared = 0.0986 Total | 246.092 124 1.98461291 Root MSE = 1.3375

delta | Coef. Std. Err. t P>|t| [95% Conf. Interval]

time | -.242875 .1093249 -2.22 0.028 -.4592945 -.0264554 group | -.8927799 .2689418 -3.32 0.001 -1.425177 -.3603828 _cons | .8857756 .4406217 2.01 0.047 .0135211 1.75803

Figure 4.6. M-CSF Deficient Mice Lose Less Weight than M-CSF Normal Mice when Treated with Intra-peritoneal Bleomycin. A linear regression model (a) shows no significant interaction between the two groups as evidenced by the parallel lines. XTGEE Analysis shows that there is a statistically significant negative slope for weight change over time for both groups, and that there is a statistically significant difference between the two groups (p = 0.001), with M-CSF normal mice losing weight throughout the treatment regimen and M- CSF deficient mice gaining weight the first week, plateauing during the middle weeks and losing weight at the end of the protocol.

82 (p = 0.001) (Figure 4.6b). Both M-CSF deficient and M-CSF normal mice gain weight throughout the regimen when treated with the vehicle control (data not shown). Hence, although they begin to lose weight at the end of the protocol, M-

CSF deficient animals are better able to maintain their weight when treated with bleomycin than M-CSF normal mice, as these mice lose weight throughout the treatment regimen.

M-CSF Deficient Mice Exhibit Less Lung Fibrosis than M-CSF Normal

Mice After Systemic Bleomycin Treatment

We used several parameters to assess the development of pulmonary fibrosis in M-CSF normal and M-CSF deficient mice treated with systemic bleomycin. Masson’s Trichrome staining revealed M-CSF normal mice (Figure

4.7a) developed more pulmonary fibrosis than M-CSF deficient mice (Figure

4.7b).

Additionally, we used the expertise of a board certified veterinary pathologist to group the animals according to severity of disease. All samples were examined in a blinded manner. By assessing the presence and severity of fibrotic lesions and inflammatory infiltrates, including macrophages, lymphocytes, plasma cells and PMN’s, we were able to group the animals into three categories. Figure 4.8 shows the percentage of animals from each treatment group that were categorized as having either no overt pathological changes, mild fibrotic changes, or marked fibrosis.

a. b.

Figure 4.7. M-CSF deficient mice demonstrate less fibrosis after Bleomycin treatment than M-CSF normal mice. M-CSF Normal (a) or M-CSF Deficient (b) mice were treated with IP Bleomycin or vehicle control (inset) and stained with Masson’s Trichrome.

100 M-CSF -/- or normal, vehicle M-CSF normal bleo M-CSF -/- bleo

o 50

No Mild Marked Fibrosis Fibrosis Fibrosis

Figure 4.8. M-CSF Deficient Mice Develop Less Severe Lung Fibrosis than M-CSF Normal Mice after Systemic Bleomycin Treatment. In a blinded fashion, an independent pathologist assessed lung histology in M-CSF normal and M-CSF deficient mice treated with bleomycin or vehicle control, and categorized the severity of disease as no fibrosis (normal), mildly fibrotic or markedly fibrotic.

85 Finally, because systemic administration of bleomycin causes a distinct pattern of subpleural fibrosis, we determined the amount of subpleural involvement as a quantifiable measure of the severity of fibrosis. We found that

M-CSF deficient mice had, on average, only half as much pleural involvement as

M-CSF normal mice (Figure 4.9).

M-CSF Deficient Mice Have Fewer CD68+ Cells in the Lung than M-

CSF Normal Mice After Systemic Bleomycin Treatment

Since M-CSF deficient mice are known to have fewer monocytes and tissue macrophages, we wanted to look at the presence and activation of these cells in the lungs of mice treated with bleomycin. Macrosialin is the mouse

homolog to human CD68, a heavily glycosylated transmembrane protein

expressed almost exclusively in mononuclear phagocytes147. The exact function

of macrosialin/CD68 is unknown, however, it may act as a scavenger receptor,

and is up-regulated on activated macrophages148.

We used CD68 to identify macrophages in the lungs of mice treated with

bleomycin. Not surprisingly, we found that M-CSF normal mice (Figure 4.10a)

had more CD68+ cells than their M-CSF deficient counterparts (Figure 4.10b)

after bleomycin treatment, and these differences were statistically significant

(Figure 4.10c). Incidentally, CD68 counts were very low (approximately 0.2–0.3 cells/mm2) in both M-CSF normal and M-CSF deficient mice treated with the

vehicle control (data not shown).

Vehicle Control 50 Bleomycin

10 % pleural involvement

M-CSF M-CSF Normal -/-

Figure 4.9. M-CSF Deficient Mice Develop Less Sub-pleural Lung Fibrosis than M-CSF Normal Mice After IP Bleomycin Treatment. The percentage of the sub-pleura with fibrotic involvement was calculated for each mouse. The average and SEM for each treatment group are shown.

a. b.

c. 8 2 6

cells/mm 4 +

CD68 2

M-CSF M-CSF Normal -/-

Figure 4.10. M-CSF Deficient Mice Have Fewer CD68+ Mononuclear Cells in the Lung than M-CSF Normal Mice After Systemic Bleomycin Treatment. M-CSF Normal (a) and M-CSF Deficient (b) mice were treated with IP bleomycin or vehicle control (inset) and lung sections were stained for CD68/macrosialin. We averaged the number of CD68+ cells/mm2 counted using a random 1mm2 grid over three mice for each group (c). There is a statistically significant difference in CD68+ staining cells between M-CSF normal and M-CSF deficient mice (p < 0.05).

M-CSF Deficient Mice Express Less MCP-1 than M-CSF Normal Mice after Systemic Bleomycin Treatment

Because M-CSF normal and M-CSF deficient mice had different numbers of CD68+ cells, we wondered if M-CSF deficiency had any effect on recruiting these cells to the lung. Because of our interest in the role of MCP-1 in recruiting monocytes (Chapter 3), and previous literature describing a potential role for

MCP-1 in fibrosis48,94,95 , we stained lung sections from some of the animals using antibodies to MCP-1. Our preliminary evidence shows that M-CSF deficient mice appear to have decreased expression of MCP-1 in the lung compared to M-CSF normal mice, particularly in areas of fibrosis (Figure 4.11).

a. b.

Figure 4.11 When Treated with IP Bleomycin, M-CSF Normal Mice Express more MCP-1 in Areas of Inflammation and Fibrosis Compared to M-CSF Deficient Mice. M-CSF Normal (left) and M-CSF Deficient (right) mice were treated with IP bleomycin and lung sections were stained for MCP-1.

90 Discussion

Monocytes and tissue macrophages have been extensively but indirectly

implicated in the pathogenesis of pulmonary fibrosis. This study was designed to

evaluate the role of the monocyte and macrophage specific growth factor M-CSF

in the development of fibrosis in an animal model.

Our data comparing systemic bleomycin treatment in M-CSF normal and

M-CSF deficient mice demonstrate that mice lacking functional M-CSF are

protected from bleomycin-induced pulmonary fibrosis. M-CSF deficient mice

survive better, maintain their weight and develop less fibrosis than their M-CSF normal counterparts. This protection may be afforded strictly by the absence of growth factor, however it is reasonable to also hypothesize that protection may be due to the presence of decreased numbers of monocytes and tissue macrophages resulting from the growth factor deficiency. One possible mechanism to test this hypothesis would be to deplete these mononuclear cells in animals with a full contingent of M-CSF, and assess their response to bleomycin treatment. Alternatively, at least one study has shown that M-CSF deficient mice regain normal numbers of alveolar macrophages by four months of

age145, and thus, completing a similar study utilizing older M-CSF deficient mice

could feasibly answer this important question. Regardless, these data demonstrate a role for M-CSF and, potentially, mononuclear cells in the development of pulmonary fibrosis induced by systemic bleomycin.

91 We used a systemic model of bleomycin administration based on the observation that this method produces lesions similar to those found in human disease. The contrast between the more common intra-tracheal method and systemic administration are startling (Refer to Figures 4.02 and 4.03). Systemic administration of bleomycin, while more time consuming, results in heterogeneous fibrosis in the lung and includes sub-pleural inflammation, reminiscent of human pulmonary fibrosis. Importantly, the route of bleomycin administration appears critical in determining the pathological consequences of lung response, as systemic bleomycin injection is different than IT challenge in the murine model. Additionally, bleomycin lung toxicity remains an important clinical consideration in the treatment of some cancers, as these patients are treated systemically.

It is quite likely the route of bleomycin administration plays a role in the development of disease, as evidenced by the dissimilar patterns of fibrosis induced by each method. Thus, it would be most interesting to examine whether the route of bleomycin administration in M-CSF deficient mice has an effect, and in fact we have already begun studies to evaluate this hypothesis.

With regard to our systemic bleomycin model, the molecular mechanism behind protection from fibrosis in M-CSF deficient mice is unclear; however we are investigating several possibilities. Our preliminary investigations into the mechanism of protection suggest that expression of the monocyte chemoattractant protein MCP-1 may be decreased in M-CSF deficient animals, which provides an attractive hypothesis. MCP-1 has been shown to directly stimulate

92 collagen synthesis and TGF-β expression35, both of which are directly involved in

the development of fibrosis44,149,150. Additionally, this hypothesis would be in line

with Moore’s study demonstrating that mice lacking the MCP-1 receptor are

protected from developing pulmonary fibrosis48, potentially via this same

mechanism. Interestingly, as well, our laboratory has evidence that bleomycin

stimulated monocytes are themselves able to secrete TGF-β, thus providing

another potential feedback loop that could be disrupted by M-CSF deficiency

(unpublished observations).

We anticipate further examination and quantification of MCP-1 expression,

as well as analysis of TGF-β expression and collagen synthesis, perhaps via

Connective Tissue Growth Factor (CTGF), will help to clarify the mechanism of

protection afforded by M-CSF deficiency.

Although clearly more work remains, the data presented in this chapter

provide valuable insight into the role of M-CSF, monocytes, and macrophages in

the pathogenesis of this pulmonary fibrosis. Understanding these factors may

ultimately belie more effective treatment options for this devastating and

incurable disease. and significance of M-CSF, and of monocytes and macrophages in the development of pulmonary fibrosis may help to identify cellular or molecular therapeutic targets that may help patients with this severe lung disease.

93 Materials and Methods

Animals

Heterozygous B6C3Fe-a/a-Csfmop mice were obtained from The Jackson

Laboratory (Bar Harbor, ME). All mice were maintained and bred under sterile

conditions inside the Davis Heart and Lung Institute vivarium at The Ohio State

University. All phenotypically normal mice were fed a standard laboratory LM-

485 mouse/rat sterilizable diet (Harlan Teklad 7912, Madison, WI) ad libitum.

Osteopetrotic M-CSF deficient mice were fed a powdered diet (Harlan Teklad

7913, Madison, WI) mixed with water, also ad libitum. All animal studies were performed following ILACUC standards, under the advice of a licensed veterinarian and with the approval of The Ohio State University animal protocol number 00A0108.

Intra-Tracheal Bleomycin

For IT instillation of bleomycin, animals were anesthetized with inhaled

IsoSol isofluorane (Abbott Laboratories, North Chicago, IL) and the trachea

surgically exposed. A 30µl volume containing 0.025U bleomycin (Faulding

Pharmaceutical Company, Paramus, NJ) in sterile water was injected into the

trachea with a 28 gauge insulin syringe. The incision was closed with a single

suture and the animals monitored until regaining consciousness. Animals were weighed on a weekly basis and sacrificed 21 days after the procedure.

94 Intra-Peritoneal Bleomycin

Mice were weighed on a weekly basis and injected IP twice per week for 4

weeks with a weight-dependent (0.035U/g) dose of bleomycin (GensiaSicor

Pharmaceuticals, Irvine, CA). Age and sex-matched mice were injected with

150µl sterile LAL Reagent water (Cambrex, East Rutherford, NJ) as a control.

Mice were monitored on a daily basis for signs of stress or ill health, and

sacrificed one week after the last injection.

Tissue Procurement and Processing

All animals were humanely sacrificed by CO2 asphyxiation, and the

tongue, trachea, heart and lungs removed en block. The lungs were insufflated

with PBS at 18cm pressure, and the lobes of the right lung were removed and

snap-frozen in liquid nitrogen for later RNA and/or protein analysis. The left lung

was tied off to maintain insufflation pressure and placed, still en block with the

heart, in 10% buffered formalin. Liver and skin samples were also fixed in

formalin. After 24 hours fixation, the samples were processed immediately or transferred to PBS until processing, to prevent over-fixation. These formalin-

fixed specimens were processed, embedded in paraffin, blocked and cut in 4µm sections for histological analysis.

Histochemistry and Immunohistochemistry

Tissue sections were processed and stained with H&E and Masson’s

Trichrome by Histotechniques, Ltd (Powell, Ohio), and for CD68 and MCP-1 by

the Histology Core Facility at The Ohio State University, according to standard

protocols, briefly; 95 Hematoxylin and Eosin

Tissue sections were deparaffinized and hydrated, then placed in hematoxylin for 8 minutes, rinsed and differentiated for 3-10 seconds in 0.5% acid alcohol. The alcohol was rinsed and the sections stained with ammonia water for 8-10 seconds, then washed with water. Slides were then counterstained in eosin for 20 seconds and rinsed in 95% alcohol then dehydrated and cleared in xylene for mounting.

Masson’s Trichrome

Tissue sections were deparaffinized, hydrated and placed in Bouins fixative for 1 hour at 56°C then cooled, washed and placed in Weigert’s Iron

Hematoxylin for 10 minutes. After washing again, they were then placed in

Biebrich Scarlet-Acid Fuscin for 5 minutes, rinsed and placed in phosphotungstic-

phosphomolybdic acid for 10-15 minutes then in aniline blue for 5 minutes,

followed by a wash. Finally, slides were placed in 1% aqueous acetic acid for 3-

5 minutes, dehydrated and cleared for mounting.

CD68 Staining

Tissue sections were deparaffinized, hydrated and rinsed in TBS-Tween,

followed by quenching endogenous peroxidase activity in 3% hydrogen peroxide

in methanol for 15 minutes. Sections were pre-treated for 15 minutes with

Proteinase K (Dako, Carpinteria, CA) and blocked with Avidin D and Biotin

solutions (Vector Laboratories, Burlingame, CA) for 20 minutes each. Sections

were then protein blocked with 5% rabbit serum for 30 minutes. A 1:400 dilution

of rat anti-mouse CD68 (Serotec, Raleigh, NC) was added overnight, followed by

96 addition of a 1:400 dilution of Biotinylated Rabbit anti-Rat (mouse-adsorbed) for

30 minutes. Staining was achieved using HRP-labelled Streptavidin for 30

minutes followed by DAB Substrate Solution (Vector Laboratories, Burlingame,

CA). Sections were counterstained with Hematoxylin and blued with ammonia

water, then dehydrated, cleared and mounted.

MCP-1 Staining

Tissue sections were deparaffinized and hydrated then quenched for 15

minutes in 3% hydrogen peroxide in methanol to block endogenous peroxidase then blocked for endogenous biotin in avidin-biotin blocking solution (Vector

Laboratories, Burlingame, CA). Tissue sections were then blocked for protein by the addition of 5% rabbit serum for 1 hour. A 1:400 dilution of goat anti-mouse

MCP-1 (Santa Cruz Biotechnology, Santa Cruz, CA) was added overnight, followed by addition of a 1:400 dilution of biotinylated rabbit anti-goat (Vector

Laboratories, Burlingame, CA) for 30 minutes. Staining was achieved using

Vectastain Elite (Vector Laboratories, Burlingame, CA) for 30 minutes followed by DAB Substrate Solution (Vector Laboratories, Burlingame, CA) for 1 minute.

Sections were counterstained with Hematoxylin then dehydrated, cleared and mounted.

Expert Pathologists Assessment

We asked an independent expert veterinary pathologist to view H&E and

Trichome stained slides containing mouse lung sections, and group the animals

according to the severity of pathologic changes exhibited. In a blinded manner,

he separated the slides into 3 groups based on the presence and severity of

97 fibrotic lesions and the presence and severity of inflammatory infiltrate, including numbers of macrophages, lymphocytes, plasma cells, and PMN’s. Using these criteria, he independently separated the slides into three groups; 1. no fibrosis,

2. mild fibrosis, and 3. marked fibrosis.

Statistical Analyses

Statistical analyses examining survival were computed using Kaplan-

Meier curves and the log-rank test to compare the curves. We also attempted to

calculate the Cox Proportional Hazard ratio. To analyze changes in mouse

weight we utilized the Cross-Sectional Generalized Estimating Equation

(XTGEE). All calculations were performed using Stata software, Version 8 (Stata

Corporation, College Station, TX).

CHAPTER 5

SUMMARY/MISCELLANEOUS

Summary and Future Directions

This dissertation describes three distinct projects, each concentrating on a different aspect of monocyte or macrophage biology. We began with microarray analysis of the genetic expression profiles of monocytes and alveolar macrophages. The data garnered from this large-scale investigation led to studies focusing on the recruitment of mononuclear cells using an in vitro model of cellular migration. Concurrently, we also designed an in vivo analysis of the role of M-CSF in bleomycin-induced pulmonary fibrosis.

In summary, we have begun to delineate the genetic components that distinguish circulating peripheral blood monocytes from alveolar macrophages, including genes involved in survival, adhesion and trafficking. Additionally, we have examined the expression of receptors for two important chemotactic molecules, MCP-1 and MIP-1α. Our finding that alveolar macrophages lack

CCR2 expression and are unresponsive to MCP-1 has important implications in the area of pulmonary inflammation. Likewise, differential expression of the MIP-

1α receptors CCR1 and CCR5 on monocytes and alveolar macrophages point to

99 other potential targets in lung inflammation. Finally, our in vivo model of pulmonary fibrosis clearly implicates M-CSF, and perhaps monocytes and/or macrophages themselves in the development of this disease.

Future Studies

The completion of this dissertation in no way implies that work in these areas is complete. Our microarray analysis, as it stands, could provide a career’s worth of interesting projects to pursue, and we will specifically and aggressively pursue our keen interest in the role of Hsp27 and Axl in monocyte to macrophage differentiation in the coming months.

With respect to the pulmonary fibrosis study, we have tissue sections from other organs, including the heart, liver and skin of the study animals that will be evaluated for systemic pathological changes in response to bleomycin treatment.

Additionally, we have flash-frozen lung specimens from these animals, and are planning extensive protein and gene expression studies to further elucidate the mechanism of protection from pulmonary fibrosis imparted by M-CSF deficiency.

In total, however, these projects help to provide understanding and insight into the independent regulation and recruitment of peripheral blood monocytes and alveolar macrophages, and have identified a potential role for these cells in pulmonary fibrosis.

100 Collaborative Studies

In addition to the studies carried out in Clay Marsh’s laboratory and described in this dissertation, we are also involved in numerous collaborative efforts, and many of these studies are now coming to fruition. For example, in collaboration with Dr. Haifeng Wu (Department of Pathology) we have developed a protocol examining the proteomic expression profiles of freshly isolated peripheral blood monocytes and alveolar macrophages using 2D gel electrophoresis. Not only does this work fit nicely with our gene expression analyses in these cells, but it also may provide validation of the RNA data. An abstract describing this work will be presented at the 2004 American Thoracic

Society meeting, and a manuscript is in preparation for publication.

101

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