Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine 951

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Mechanisms of Protein Mobiliation in Blood

BY

MAŁGORZATA KARAWAJCZYK

ACTA UNIVERSITATIS UPSALIENSIS UPPSALA 2000 Dissertation for the Degree of Doctor of Medical Science in Clinical Chemistry presented at Uppsala University in 2000

ABSTRACT

Karawajczyk, M. 2000. Mechanisms of granule protein mobilization in blood eosinophils. Acta Universitatis Upsaliensis. Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicin 951 p.60 Uppsala. ISBN 91-554-4799-6.

Serum levels of the granule proteins ECP, EPO and EPX, which are stored in the matrix of specific granules, were shown to correlate with the course of disease in disorders involving eosinophils. The concentrations of eosinophil proteins in serum are the result of their release in vivo, and also ex vivo during the sampling procedure. Generally, eosinophils release the contents of their specific granules in three ways, namely by exocytosis, piecemeal (PMD), or cytolysis. The manner of eosinophil protein release in blood eosinophils has not been determined. The aim of this work was to study the mechanisms of granule protein release from blood eosinophils in respect to protein subcellular localization and cell ultrastructure. In patients with bacterial infections, the serum level of ECP but not of EPO was increased, while in patients with viral infections both proteins remained within the range of healthy controls. When G-CSF, a cytokine involved in the response mechanism to bacterial but not viral infections, was administered to healthy subjects, it induced an increase in eosinophil count and a preferential increase in serum EPX and ECP in comparison to EPO. The PMD model consists of stepwise transportation of contents from the granules towards the plasma membrane. We observed that administration of G-CSF in healthy subjects and allergen exposure of allergic subjects during the birch pollen season caused ultrastructural changes in the eosinophil specific granules, such as loosening of the matrix, granule matrix lucency, and ragged losses of their core. Similar morphological alterations had previously been observed in eosinophils undergoing PMD. ECP, EPX and EPO were localized not only in the specific granules but also in extra- granular compartments, as shown both by immunoelectron microscopy and subcellular fractionations. An extragranular EPX compartment were present in healthy as well as in allergic and hypereosinophilic subjects, and there were no significant differences in its size between the groups. The extragranular compartments of ECP and EPO were increased in size in allergic subjects during the birch pollen season and were clearly separate from the extragranular compartment of EPX. Results of this investigation indicate differential mobilization of granule proteins in circulating eosinophils and point to piecemeal degranulation as its principal mechanism.

Key words: eosinophil granule proteins, ECP, EPX, EPO, serum levels, piecemeal degranulation, release, ultrastructure, subcellular fractionation

Malgorzata Karawajczyk, Department of Medical Sciences, Clinical Chemistry, University Hospital, 751 85 Uppsala, Sweden

© Malgorzata Karawajczyk

ISSN 0282-7476 ISBN 91-554-4799-6 Printed in Sweden by Uppsala Tryck&Medier, Uppsala 2000 Rodzicom

(to my parents) This thesis is based on the following papers, which will be referred to by their Roman numerals:

I. The differential release of eosinophil granule proteins. Studies on patients with acute bacterial and viral infections. Karawajczyk M., Pauksen K., Peterson C.G.B., Eklund E, Venge P. Clinical and Experimental Allergy, 1995; 25(8); 713 - 9

II. Administration of G-CSF to healthy subjects. The effects on eosinophil counts and mobilization of eosinophil granule proteins. Karawajczyk M., Höglund M., Ericsson J, Venge P. British Journal of Hematology, 1997: 96; 259-265

III. Piecemeal degranulation of peripheral blood eosinophils. A study of allergic subjects during and out of the pollen season. Karawajczyk M., Seveus L., Garcia R., Björnsson E., Peterson C.G.B., Roomans G..M. , Venge P. American Journal of Respiratory Cell and Molecular Biology. In press

IV. Subcellular localization of eosinophil protein X (EPX) in blood eosinophils. A novel extragranular compartment containing EPX in eosinophils. Karawajczyk M., Garcia R.C., Peterson C-G.B., Moberg L., Höglund M., Venge P. In manuscript

Reprints were made with the permission from the publisher. TABLE OF CONTENTS

Abbreviations 7

Introduction 8

Eosinophil 8 General information 8 Ultrastructure 8 Eosinophil granule proteins 10 Eosinophilopoiesis 12

Secretion of granule proteins 13 Exocytosis of granules 13 Piecemeal degranulation 14 Cytolysis 15

Serum levels of ECP, EPX and EPO 16

Granulocyte -colony stimulating factor (G-CSF) 17

Aim of the Investigations 19

Methods 20

ECP, EPX and EPO assays 20 Eosinophil isolation 20 Subcellular fractionations on density gradients 20 Separation of eosinophils of different densities 21 Conventional transmission electron microscopy (TEM) 21 Immunoelectron microscopy (IEM) 22 Marker protein asssays 22 Statistical analysis 23

Results and Discussion 24

Serum levels of ECP, EPX and EPO (papers I and II) 24 Bacterial and viral infections (paper I) 24 G-CSF administration in healthy subjects (paper II) 27

Piecemeal degranulation (papers II and III) 29 Ultrastructure of eosinophils after G-CSF administration (paper II) 29 Ultrastructure of eosinophils from allergic subjects during and out of the birch pollen season (paper III) 33 Subcellular localization of eosinophil granule proteins (papers III and IV) 37

General summary and conclusions 41

Acknowledgments 42

References 43 Abbreviations BSA - bovine serum albumin C5a - complement factor 5a CFU - colony-stimulating units CLC - Charcot-Leyden crystal protein ECP - eosinophil cationic protein EPO - EPX/EDN - eosinophil protein X / eosinophil derived neurotoxin ER - endoplasmic reticulum G-CSF- colony-stimulating factor GM-CSF granulocyte- colony stimulating factor GTP-y - guanosine-5´-O-(3-thiotriphosphate) ICAM-1 - intracellular adhesion molecule IEM - immunogold labeling for electron microscopy IL - interleukin INF-γ - interferon gamma LIF - leukemia inhibitory factor MBP - MPO - NSF - N-ethylmaleimide-sensitive factor PAF - platelet activating factor PBS - phosphate buffered saline pI - isoelectric point PKC - protein kinase C PMD - piecemeal degranulation RNase - ribonuclease RSV - respiratory syncytial virus SCF - stem cell factor SNARE - soluble NSF attachment receptor TEM - transmission electron microscopy TGF - transforming growth factor TNF - tumor necrosis factor WBC - white blood cell count α−γ−SNAP - soluble NSF attachment proteins alpha or gamma

7 INTRODUCTION

Eosinophil

General information

Eosinophil are produced in the bone marrow, where they constitute 3 % of the cells. After migration from the bone marrow they remain in circulation for an average of 18 hours and are then distributed to the tissues. Among the leukocytes in circulation, about 1 %- 8 % are eosinophils. Eosinophils are mainly tissue cells, and once out of the circulation they most probably do not re-enter it. Their life span in the tissues is not known, but it is considered to vary from a few days to even weeks. The largest storage sites of eosinophils in the body are the gastrointestinal tract, the skin, and the lungs.

Eosinophils are involved in numerous events, mainly through the release of preformed and newly formed mediators to the surroundings. They also possess some phagocytic properties, but these are much weaker than those of . Eosinophils are involved in processes related to parasitic infestation, allergy, cancer defense, wound healing, and other inflammatory diseases. Recently, the role of eosinophils in antiviral defense has been discussed. Eosinophils show both pro-inflammatory and tissue-destroying properties, as well as immunoregulatory functions. In spite of the amount of information available, the physiological and pathological roles of eosinophils are not yet completely understood.

Ultrastructure

A mature, healthy human eosinophil has an average diameter of 8 µm. It has a bilobated nucleus and its cytoplasm is filled with characteristic granules, which show strong affinity to the negatively charged dye eosin. To this affinity eosinophils owe their name, which was given by Paul Ehrlich, who first noticed this feature in a subpopulation of leukocytes in 1879 (1). Eosinophils contain several populations of granular structures, namely: specific (also called secondary) granules, primary granules, small granules, lipid bodies and a variety of vesicular structures. The most prominent are the specific granules. These are oval or round structures enclosed by a membrane. They consist of two compartments - a crystal core and a surrounding homogeneous matrix. Owing to their content of basic proteins, they show an affinity to eosin that gives the eosinophil its pink color under the light microscope. The crystal core consists mostly of major basic protein (MBP) (2), but other proteins are also present, such as interleukins IL-2, IL-4, IL-5, and granulocyte–macrophage colony-stimulating factor (GM-

8 Fig 1.

Specific granule Primary granule

vesicular structures

Lipid body

Receptors Fig.1. Schematic depiction of an eosinophil granulocyte, showing the major structures: the bilobated nucleus, specific granules, primary granule, lipid body, vesicular structures (single- or double- membrane - bound and tubovesicular structures), and surface receptors.

CSF) (3-5). Other enzymes such as , catalase and β-glucuronidase have also been reported to be localized in the crystal core (6-8). The matrix of the specific granules provides a store for a multitude of preformed eosinophil mediators, including eosinophil cationic protein (ECP), eosinophil protein X (EPX) and eosinophil peroxidase (EPO), which were the particular subjects of the present studies. Other biologically potent proteins such as IL-6 (9), tumor necrosis factor - α (ΤΝF−α) (10), RANTES (11), and bacterial/permeability- increasing protein (12) are also present in the matrix. The specific granules also house enzymes considered to be a hallmark of lysosomes, e.g., hexosaminidase, , acid hydrolases, , catalase, as shown in rat but partially also in human(7). For this reason the specific granules of eosinophils have been

9 thought to be a form of lysosome (13). The specific granule matrix also contains (14;15). The primary granules are considered to be precursors of the specific granules (16). They are spherical, membrane-bound structures with a homogeneous matrix, without a core. MBP is diffused in the primary granules and during their maturation it forms a crystal core characteristic of the specific granules. Primary granules are abundant in eosinophil progenitors but rare in mature cells, where they constitute 5% of all granules (17). Lipid bodies are round, membrane-devoid structures with a high affinity for osmium. They are the storage site of arachidonic acid (18) and of enzymes involved in lipid metabolism, such as cyclooxygenase and 5- and 15- lipoxyge- nase. The number of lipid bodies is increased in activated eosinophils (19-22). Small granules were identified in tissue eosinophils by Parmley et al. (23), and have been found to house arylsulphatase B, acid phosphatase (23;24), catalase (6), and ECP (25). The presence of a variety of vesicular structures is a characteristic feature of eosinophils (26). They are single-membrane and double-membrane-bound structures, round or elongated (tubovesicular) in shape, with a wide spectrum of electron density. Some of these structures show EPO activity in activated eosinophils (27). A subpopulation of vesicles contains cytochrome b558 and albumin (28), and another subpopulation transforming growth factor α (TGF- α) (29). Some of these vesicles probably belong to the endoplasmic reticulum (ER) system. Functionally, some of them may be secretory vesicles, some transporting vesicles and others endocytic vesicles.

Important elements of the eosinophil are the plasma membrane receptors, through which secretion is mediated or regulated. Among them are receptors for cytokines, e.g. IL-3, IL-5, GM-CSF, TNF-α, (30-33); immunoglobulins: IgG

(34), IgA (35), and IgE (36;37); and adhesion molecules: β1 and β2 integrins (38;39) , L-selectin (40) , intracellular adhesion molecule (ICAM-1) (41;42), and the counterligands for selectins E and P (43;44).

Eosinophil granule proteins

The present studies have been focused on three out of the plethora of proteins stored in the specific granules, namely ECP, EPX and EPO.

ECP – eosinophil cationic protein or RNase 3 (45), is a member of the ribonuclease A superfamily (46), which groups ribonucleases homologous to bovine pancreatic ribonuclease A. Structurally, ECP is a single-chain, zinc- containing polypeptide with a molecular mass ranging from 18 to 22 kDa, due to variations in glycosylation. A high content of arginine gives the molecule its characteristic basic pI of 10.8 (45;47).

10 The for both ECP and EPX are localized on 14 and originate from the duplication of a precursor (48-50). ECP and EPX amino acid sequences show 66 % homology. In spite of this homology, ECP ribonuclease activity is 100 times weaker than that of EPX (49). ECP possesses many activities other than that of ribonuclease. It is a potent cytotoxin, especially against brain tissue (51), and a helminthotoxin (52;53). ECP also posseses anti- bacterial(54) and antiviral (55) properties. The antiviral activity is related to its ribonuclease activity, but the postulated mechanism of its cytotoxicity is based on a perforin-like activity (56). ECP has been shown to activate factor XII of the coagulation cascade (57;58). ECP interacts with cells; for example it induces degranulation of mast cells (59), inhibits the constitutive synthesis of immunoglobulin by and proliferation of lymphoblastoid cell lines (60;61), and upregulates expression of receptors such as ICAM –1 (62) and insulin-like growth factor (63) on epithelial cells. ECP induces the production of proteoglycans by fibroblasts (64) and stimulates mucus secretion in airways (65). The mechanisms mediating the above-mentioned activities at the levels of cell recognition and signal transduction are still not completely clear. The properties cited suggest a role for eosinophils in the regulation of the immune response and also in the process of matrix remodeling, which is important in wound healing and . ECP has been shown to be localized in the matrix of specific granules and in the small granules of eosinophils (25). Eosinophils contain an average of 15 µg ECP/106 cells (66). Small amounts of ECP have been reported to be present in neutrophils (163 ng/106 cells) (67), monocytes (20 ng/106 cells) (68), and (77 ng/106 cells) (69).

EPX - eosinophil protein X (70), which is also known as eosinophil derived neurotoxin (EDN) (71) or RNase 2, is a basic protein (pI 8.9) with a molecular mass of 18 kDa. EPX belongs to the RNase superfamily A, (46;72). In addition to its ribonuclease activity (73;74), EPX possesses cytotoxic (75) and antiviral properties (76). The antiviral effect of EPX is dependent on its RNase activity (77). It has recently been proposed that EPX may be an antiviral agent directed especially against single-stranded viruses such as the respiratory syncytial virus (RSV) (78). EPX is a potent neurotoxin, as demonstrated by the Gordon phenomenon, but otherwise it has much lower cytotoxic activity than ECP (79). The neurotoxicity of EPX is dependent on its ribonuclease activity (80). In eosinophils, EPX has been reported to be stored in the matrix of specific granules, together with ECP and EPO (81). The average content of EPX in eosinophils is 10 µg/106 cells (70). EPX is produced and stored in small amounts (112 ng/106 cells) in neutrophils (67). Trace amounts of EPX have also been demonstrated in basophils (214 ng/106 cells) (69) and mononuclear cells (82).

11 EPO - eosinophil peroxidase (83) - belongs to a family of haloperoxidases. It is composed of two polypeptide chains with molecular weights of 50 and 15 kDa respectively, and contains a heme molecule. Similarly to other basic proteins, EPO contains large amounts of arginine and lysine, which result in its basic pI of 10.9 (84),(85). The amino acid sequence of EPO shows 70% homology to myeloperoxidase (MPO) (86;87). EPO is localized in the matrix of specific granules (25) and in small vesicular structures of activated eosinophils (27). The mean content of EPO in eosinophils has been estimated to be 15µg /106 cells (85). EPO has been to be endocytosed by basophils, mast cells and neutrophils (88;89). It has anti- bacterial properties (90) and acts as a helminthotoxin. This latter activity is enhanced by the presence of hydrogen peroxide and halides (91). EPO upregulates receptors on inflammatory cells (92) and induces degranulation of mast cells and eosinophils (93;94).

Eosinophilopoiesis

In adults, eosinophil development and maturation occur mostly in the bone marrow. Eosinophils originate from stem cells common to many lymphomyeloid lineages. Hematopoietic stem cells enter the differentiation program under the influence of IL-6, IL-11, IL-12, granulocyte-colony stimulating factor (G-CSF), stem cell factor (SCF), and leukemia inhibitory factor (LIF) (95;96). The commitment of multipotent cells to the eosinophil lineage is regulated by SCF, IL-3, IL-5 and GM-CSF (97), and possibly also eotaxin (98). Further maturation of eosinophils is mediated mainly by IL-5 and eotaxin (32;99;100). The mean duration of maturation of eosinophils is 3.5 days (101). The extent of replication in the progenitor compartment is increased in diseases involving eosinophils (101). Cytokines originating at the site of inflammation are responsible for the enhancement of hematopoiesis in disease, as it occurs, for example, in bronchial asthma (102;103). The specific granules of an eosinophil appear at the myelocyte stage as a result of the maturation of primary granules (16). The primary granules are recognizable at the promyelocyte stage. Granule proteins are synthesized in the rough ER, processed in the Golgi apparatus and transported to the granules (13;17). The synthesis of ECP, EPO and EPX diminishes in differentiated cells to almost nondetectable levels, as shown by a decrease in mRNA for these proteins in cultured eosinophils (104) and by the disappearance of EPO activity from the Golgi apparatus and ER (17). However, it was recently observed that eosinophils from healthy subjects expressed low levels of mRNA for ECP (68). The situation is different in the case of patients with hypereosinophilia. Their peripheral blood eosinophils contain mRNA for ECP and EPX, but only few subjects also express mRNA for EPO (104). Eosinophil progenitors have the capacity to secrete whole granules, as well as small vesicles, as shown by both

12 patch clamp and electron microscopy techniques (105;106). This release of eosinophil proteins was confirmed by the presence of higher concentrations of MBP and Charcot-Leyden crystal protein (CLC) in blood from bone marrow sinusoids than in peripheral blood (107). However, the importance of this phenomenon is obscure.

Secretion of granule proteins

Eosinophils release the contents of their specific granules in three different ways: through exocytosis, by piecemeal degranulation, and by cytolysis.

Exocytosis of granules

In this phenomenon, secretory granules approach the plasma membrane and after fusion of the granule membrane with the plasma membrane the contents of the granule are discharged from the cell. Extrusion of whole granules of eosinophils has been observed both at the ultrastructural level (108-110) and by the patch clamp technique (111). Release of a group of granules, referred to as compound exocytosis, has also been observed (110;112). Here, granules fuse with each other and create a large sac, which after connection with the plasma membrane forms an extrusion channel. In this way the granules are released in a coordinated way in one direction. The occurrence of single or compound exocytosis has been shown to be regulated by GTPγ and Ca++. Low concentrations of GTP-γ favor single-granule exocytosis in streptolysin-O- permeabilized cells, whereas higher concentrations of GTP-γ induce compound exocytosis (113). The speed of pore expansion is determined by the Ca++ level and is dependent on protein kinase C (PKC) (114). In a permeabilized cell model, ATP has been shown to enhance the release elicited by GTP-γ and Ca++ (115). The occurrence of exocytosis has been documented by electron microscopy in many experimental models. During degranulation induced by immunoglobulin- coated beads, the presence of dense, membrane-free granule material (cores and also matrix) adjacent to the bottom of vacuoles evaginating into the surface of a stimulating bead has been observed (116). Exocytosis in streptolysin-O - permeabilized eosinophils stimulated with GTPγ in the presence of Ca++ has been also documented at an ultrastructural level (117). Further, extrusion of the contents of eosinophil granules has been observed on the surface of opsonized Schistosoma mansoni larvae (118). Although exocytosis is seen in many in vitro systems, its actual physiological relevance is under discussion. The target- oriented discharge of granule proteins seems to make sense in the case of anti- parasite defense, when cytotoxic proteins are to be released onto the surface of an invading parasite. However, exocytosis in vivo has only been observed in tissue eosinophils from patients with Crohn’s disease (109). This suggests that

13 this form of regulated secretion has only a minor physiological role. Exocytosis is a rapid process, however, and it is therefore difficult to capture events of such brief duration by means of conventional techniques (110;119).

Piecemeal degranulation

Piecemeal degranulation is another proposed mechanism for the release of eosinophil granule proteins. Accordingly to this hypothesis, the content of specific granules is gradually packed into small vesicles and transported to the plasma membrane, leaving the granules empty. The first morphological observations of ultrastructural changes in specific granules, such as swelling and vacuolization, were first described in patients with Hodgkin’s lymphoma in 1975 (120). Since then, other authors have observed similar alterations, such as ragged losses of matrix and core of different extents, ranging from slight changes to complete optical lucency of granules, in the ileum in patients with Crohn’s disease, in hypereosinophilia, and in gastroenteritis (121-123). However, at the early stage of these studies it was questioned whether the described alterations were not artifacts due to tissue preparations. In a study in 1992, peroxidase activity was shown to be associated with vesicular and tubovesicular structures in the vicinity of emptied granules (27). Recently, an abundance of eosinophils displaying morphological characteristics of piecemeal degranulation was shown in polyps and mucosa of the airways of asthmatic subjects (124). This mechanism allows selective release of individual proteins. Selective release of eosinophil mediators was indeed suggested by studies with in vitro stimulation: when IgE complexes were used to stimulate eosinophils from allergic subjects, secretion of EPO was observed, while IgG complexes induced ECP secretion (125). Recently, it was shown by confocal microscopy that stimulation of eosinophils with interferon-γ (INF-γ) results in the mobilization of RANTES but not of MBP from the specific granules (126). Moreover, the kinetics of mobilization of RANTES differed from that of another specific granule protein, β−hexosaminidase. In the experiments with coated beads mentioned above (116), the presence of specific granules showing varying degrees of lucency was observed in eosinophils in the close proximity of, but not attached to beads coated with albumin. In another experimental model, incubation of eosinophils with platelet activating factor (PAF) but not with complement factor 5a (C5a) resulted in the appearance of swollen granules with a lucent matrix (127;128).

The mechanism governing piecemeal degranulation has not yet been defined. On the basis of present data, I consider that this process might be mediated by a SNARE (soluble NSF attachment receptor) mechanism. In this model the proposed events are as follows: a trafficking vesicle carries on its surface a ligand (so called v-SNARE), which specifically recognizes target molecules (so

14 called t-SNARE) on the plasma membrane. The fusing and recycling processes are facilitated by binding of cytosolic proteins (α− γ− SNAP) to the SNARE complex . The SNARE mechanism was first shown to regulate the secretion of recycling secretory vesicles in neurons (129;130), but is now a more commonly found phenomenon. Recently, the presence of members of the SNARE group, namely VAMP-2, a v-SNARE, and syntaxin, a t-SNARE, were demonstrated in eosinophils (131). VAMP-2 was localized in the low-density region of subcellular fractionation gradients, which do not contain specific granules, suggesting that the SNARE complex is involved in vesicle-mediated release from eosinophils.

Cytolysis

Cytolysis is a process of cell destruction characterized by rupture of the plasma membrane, fragmentation of the nuclear membrane and chromatin disintegration. The mitochondria become swollen and specific granules with an intact membrane are extruded from the cell, often in the form of clusters. Eosinophils in different stages of disintegration, as well as clusters of intact specific granules, have been observed for example in patients with bullous pemphigoid (132), nasal polyps (133), asthma (134), or atopic dermatitis (135). These findings formed the basis of the hypothesis that cytolysis plays an important role in the pathology of eosinophil-related diseases (136). In in vitro systems, it was found that stimulation of secretion by immunoglobulin-coated beads (116) or by ionophore A (137) led to the death of the adhered eosinophils (as assessed by incorporation of propidium iodide) and that adherent cells showed ultrastructural characteristics of cytolysis. Piecemeal degranulation, exocytosis and cytolysis were shown to occur in the model of stimulation by opsonized beads (116). Cells showing ultrastructural characteristics of piecemeal degranulation or cytolysis have been observed in inflamed tissues (134). On the basis of these findings I hypothesize that there may be a continuum of events. It is proposed that piecemeal degranulation occurs when a cell is in close proximity to a bead, exocytosis occurs when a cell adheres to a bead, and finally that the over-stimulated cell undergoes cytolysis. A similar sequence of events may take place in inflamed tissues. Cytolysis may be the ultimate fate of stimulated cells, which in this way escape the normal death control program.

15 Serum levels of ECP, EPX and EPO

Measurements of the eosinophil count and of ECP, EPX, and to a lesser extent EPO concentrations in various body fluids have been widely used in evaluations of disease activity. Serum levels of eosinophil granule proteins are the result of active release by eosinophils during the sampling procedure added to the levels existing in vivo. EDTA plasma levels of eosinophil granule proteins represent in vivo levels. They are not significantly affected either by time, temperature, or the utensils used for sampling. Release of ECP and EPX into serum is an active process, it is time- and temperature-dependent and requires the presence of Ca++ and Mg++ (138-140). Characteristic of the sampling utensils such as the type of tubes and needles influence the release. For example, the presence of a separating gel such as in SST tubes increases the serum levels of eosinophil proteins (138;141). The use of needles with a tubing system elevates the serum levels of ECP and EPX in comparison to vacutainers (138), probably because passage through a tubing system upregulates CD11b/ CD18 receptors on the surface of granulocytes (142). The eosinophil surface receptors CD11b, and CD18, as well as others such as CD54 or CD49d, CD29 or alfa4beta7, have been shown to play a role in the release of ECP to serum (143). Thus, serum levels of eosinophil granule proteins reflect the state of activation of eosinophils in the circulation.

The elevation of serum levels of eosinophil granule proteins in the exacerbation of diseases is believed to be due to an increase in eosinophil susceptibility to stimulation, a phenomenon called priming. This occurs, for example, in allergic individuals during the pollen season. Eosinophil activation in this situation disease is mediated by Th2 clones of lymphocytes (144). Indeed, in vitro studies have shown that pre-incubation with IL-5, GM-CSF or IL-3 enhances the release of granule proteins by eosinophils stimulated by serum-opsonized particles (which acts through activation of the CD11/CD18 complex (145)), or by IgG- and IgA-opsonized particles (36)(146). Also, eosinophils obtained from allergic subjects during the birch pollen season release more ECP and EPX upon in vitro stimulation (147). In conclusion, the mechanism proposed for the elevation of serum levels of eosinophil granule proteins during pathological events is priming by cytokines produced during the immune response. One of the proposed mechanisms of the priming of secretion involves eosinophil surface receptors. It has been shown that GM-CSF, IL-5, IL-3, TNF-α and PAF increase the affinity and expression of CD11b/CD18 (148;149). Priming may also affect the release by altering signal transduction pathways of degranulation (150;151). We have to bear in mind that the stimulation of release comprises two elements – the cell and the stimulus. In the whole-blood model, the triggering factors are components of the plasma (140) and the walls of the sampling utensils, as

16 discussed above. The suggested ligands for the integrin receptors may be fibrinogen or factors belonging to the complement system. We may speculate that different diseases may alter the concentrations of triggering factors in the plasma or serum. Another factor that influences the levels of ECP, EPX and EPO in plasma and serum is protein turnover. Our knowledge of these processes is far from complete. The half-life of ECP in the circulation is 65 minutes (152). Proteins can be removed from the circulation in the kidneys. Large amounts of EPX have indeed been found in the urine of healthy subjects (153). In asthmatic children, urinary EPX but not ECP has been shown to reflect the course of the disease (154). These data suggest a differential mechanism of kidney clearance of ECP and EPX. ECP might be removed by other cells present in the circulation. It can be taken up by neutrophils (155) and can bind to α2−macroglobulin (156). Complexes of α2−macroglobulin with other proteins are taken up by (157). More work remains to be done to elucidate these turnover processes.

Elevation of serum levels of ECP, EPX and EPO has been shown on many occasions during the course of pathological events. Early reports on serum ECP levels in subjects with bronchial asthma and on the influence of treatment with steroids and β−adrenergic drugs date from 1978 (158;159). Increased serum levels were later observed in patients with allergic diseases (160), acute myocardial infarction (161), rheumatoid arthritis (162), atopic dermatitis (163;164), ulcerative colitis (165), and parasite infestations (166), and after an allergen challenge test (167). Since then, measurements of serum levels of eosinophil granule proteins have been widely used for the diagnosis of diseases and monitoring of their course (168;169). Although considerable research has been performed, much remains to be done before the relationship between systemic events such as changes in the size and activity of the circulating eosinophil pool and local inflammatory events is fully understand. The physiological role of the proteins released into the circulation is not completely known. It remains to be elucidated whether they are part of antiviral or antibacterial defense mechanisms, as suggested by their bactericidal and ribonuclease activities, or whether they have an immunoregulatory role, as indicated by their capacity to stimulate immunoregulatory cells.

Granulocyte colony-stimulating factor (G-CSF)

G-CSF is a 20 kD glycoprotein which shows different degrees of glycosylation (170), (171), (172). Its gene is localized on chromosome 17, in close proximity to the gene encoding IL-6 (173). G-CSF is produced by stromal cells, endothelial cells, fibroblasts, and monocyte–macrophages (174), (175),(176;177). Its production is stimulated by IL-1, TNF-α, INF-γ and

17 bacterial lipopolysaccarides (LPS) (178;179). G-CSF acts through a single, high-affinity receptor with a molecular mass of 140 kD. This receptor is present on cells from the neutrophil lineage (180), but also in small amounts on promonocytes, some leukemic cells (181), placenta and trophoblastic cells (182), and endothelial cells (183;184). G-CSF is a growth, differentiation and activating factor for neutrophils and their precursors (185). Its primary role seems to be in maintaining normal neutrophil levels and regulating the acute response to bacterial infections (186;187).The latter is supported by the type of factors stimulating G-CSF synthesis, which are mentioned above. G-CSF has also been shown to exert activities against cells other than neutrophils. For instance it increases the number of differentiating stem cells (188) and also affects the proliferation and migration of endothelial cells (183). Recently, an activating effect of G-CSF on TNF-α and GM-CSF production by monocytes was reported (189;190). Two recombinant forms of G-CSF are available on the market. One is an E.coli- produced, nonglycolysated form, Filgrastim (191), and the other is a glycolysated form called Lenograstim, produced in Chinese hamster ovary cells (192). There are no significant differences between the biological actions of these two forms of G-CSF (193). Administration of G-CSF to humans causes an increase in circulating neutrophils (194) and white cell progenitors (195). Although G-CSF is considered to be specific for the neutrophil lineage, an increase in monocytes (189) and eosinophils (196) has been observed after its administration. The reported mechanisms underlying the increase in the number of circulating neutrophils are: a) enhanced mobilization from the bone marrow storage pool; b) an increase in the proliferation rate of neutrophil progenitors, and c) shortening of the myelocyte – to blood transition time to 1 to 2 days (188;197;198). Administration of G-CSF to humans also increases the secretion of IL-1, soluble TNF receptors and IL-6, IL-8 and IL-10, and reduces the release of TNF-α, GM-CSF and INF-γ from monocytes (199;200).

18 AIM OF THE INVESTIGATIONS

A correlation between the state of eosinophil activation and serum levels of the granule proteins ECP, EPX and EPO has been demonstrated on in a number of studies. However, the roles of these granule proteins in the circulation and the mechanisms regulating their release are not sufficiently known. The general aim of this investigation was to elucidate the way in which the eosinophil granule proteins ECP, EPX and EPO are mobilized in the blood under different circumstances. The specific aims were:

• to compare the serum profiles of ECP, EPX and EPO in subjects with acute viral and bacterial infections; • to determine the influence of G-CSF administration on the eosinophil count and serum levels of ECP, EPO and EPX; • to study the influence of changes in the cytokine panel on the ultrastructure of blood eosinophils in two different situations: a) during administration of G-CSF to healthy subjects, and b) during and out of the birch pollen season in allergic subjects; • to investigate the localization of eosinophil granule proteins in blood eosinophils from allergic subjects during and out of the birch pollen season and from healthy subjects, both by immunoelectron microscopy and by subcellular fractionation on density gradients.

19 METHODS

ECP, EPX and EPO assays

Blood sampling for serum isolation was performed in a standardized manner. Blood was taken with SST Vacutainers, turned upside down 5 times, left at room temperature for 1 h and then centrifuged at 1300 x g for 10 min. Serum was carefully collected and frozen at –20oC or –70oC until the assays were performed. The ECP and EPX contents in serum samples or in gradient fractions were measured by specific radioimmunoassays (Pharmacia & Upjohn Diagnostic, Uppsala, Sweden) (152). EPO was measured by a specific fluoro-immunoassay, as previously described (201).

Eosinophil isolation

Eosinophils were separated by negative immunomagnetic selection as described by Hanzel (202). Mononuclear cells were separated from red cells and granulocytes by centrifugation on 67 % Percoll (Pharmacia-Biotech, Uppsala, Sweden). Red cells were lysed with water. The remaining granulocytes were incubated with magnetic beads coated with antibody against CD16, a receptor that is expressed on neutrophils. As CD16 was also found to be expressed on eosinophils to some extent, the antibody used to coat the beads was tested and found to bind to the eosinophils in very small amounts (Lena Håkansson –to be published). After incubation, the cell suspension was loaded on a separation column, which was then placed in a strong magnetic field. Neutrophils coupled to magnetic beads via antibody were captured by the strong magnetic field in the column. Non-retained cells were eluted with buffer and consisted of >98 % eosinophils. Purified eosinophils were used immediately after purification.

Subcellular fractionations on density gradients

By this method organelles can be separated from each other by virtue of differences in their equilibrium density in sucrose gradients. Purified eosinophils were disrupted by sonication. Sonication was chosen as the method of cell disruption after comparison with nitrogen cavitation and repeated passage through a needle. The lowest energy that causes disruption of almost all cells was used. After sonication, the postnuclear supernatant was loaded on the top of a sucrose density gradient. During the course of this work we used two types of gradients. Gradient I was partially continuous and consisted of a lower section made of layers of 60%, 55 %, 50%, 46% and 43% sucrose that were left at room temperature for 3 h before being overlaid with layers of 34 %, 32 %, 30%, 25 % and 20% sucrose. Postnuclear supernatants were loaded on the top of such

20 gradients within 10 min of addition the upper layer. Gradient II was totally continuous and consisted of layers of 60%, 55 %, 50%, 46%, 42%, 38%, 34% and 20% sucrose that were left to diffuse for 3 h at room temperature before sample loading. After overnight centrifugation at 84,000 x g (rav) in a Beckman ultracentrifuge (SW28.1 rotor) at 4oC, 40 fractions of 425 µl each were collected from the top of the gradients by upward displacement with 60% sucrose. Samples were stored at -20oC until analyzed for marker enzyme activities and contents of eosinophil proteins. The subcellular fractionation method used gave reproducible results. The distribution of organelles in the gradient was estimated by assaying marker proteins for different organelles and by analyzing the organelle morphology by electron microscopy as described further below.

Separation of eosinophils of different densities

Eosinophils of different densities were separated by Percoll gradient centrifugation according to the method of Gärtner (203), with modifications. Two milliliters of a suspension of eosinophils (98 % pure) were loaded on a Percoll gradient consisting of successively underlaid layers of freshly made Percoll solutions in PBS of the following densities (g/mL): 1.075, 1.078, 1.086, 1.092, 1.100 and 1.111, respectively. Percoll solution densities were measured with a densitometer (Paar, Graz,Austria). The gradient was centrifuged at 600 x g for 1h at room temperature. The cells, which are present as bands at the interfaces between Percoll layers, were collected with a Pasteur pipette, washed twice with phosphate buffered saline (PBS), and subjected to subcellular fractionation as described above.

Conventional transmission electron microscopy (TEM)

For conventional morphological examinations, cells or organelles from one of the fractionation experiments were fixed with 2.5% glutaraldehyde and 0.5% paraformaldehyde (final concentrations) in cacodylate buffer (pH 7.4) for 2 h on ice. Cells or organelles were washed in cacodylate buffer and post-fixed with osmium tetroxide. Samples were dehydrated stepwise with ethanol (50%, 75 %, 90%, 95%, and absolute ethanol twice) and embedded in Spur’s resin (Agar Scientific Ltd., Standstill, England) or Agar 100. Samples were sectioned with an Ultrotome V (LKB Bromma, Sweden) provided with a diamond knife (Diatome, Switzerland), and stained with uranyl acetate and lead citrate. This fixation and embedding procedure gives good membrane contrast, but the preservation of protein antigenicity is usually poor.

21 Immunoelectron microscopy (IEM)

An immunogold labeling procedure was carried out on cells fixed with 4% paraformaldehyde plus 0.5 % glutaraldehyde in cacodylate buffer on ice, for 2 h. Fixed cells were washed in Ringer’s buffer, dehydrated in ethanol (50%, 75%, 90%, 95%, and absolute ethanol twice) and embedded in Lowicryl KM4 (Agar Aids). Polymerization was performed by UV light (360 nm). Dehydration, embedding and polymerization were carried out at –20oC. Sections 50 nm thick were cut with an Ultrotome V provided with a diamond knife. They were mounted on Formvar (Merck, Darmstad, Germany)-coated golden grids and counterstained with uranyl acetate in methanol. This technique preserves the protein antigenicity, but membrane contrast is poorer than with epoxy resins. The location of antigens by immunolabeling is shown by the presence of gold particles, which have high electron density and are clearly seen under an electron microscope. The antigen of the specimens is detected by a specific primary antibody. The primary antibody is then visualized by a secondary antibody coupled to a gold particle. Briefly, sections were first incubated in 0.1 M phosphate buffer (pH 7.4)-0.15 M glycine followed by incubation with 1 % bovine serum albumin (BSA) in 0.1 M phosphate buffer (pH 7.4) in order to block unspecific binding. They were then incubated with primary antibody (mouse monoclonal antibody) at room temperature for 1 h, rinsed with 0.2% BSA in phosphate buffer, and incubated with secondary antibody (goat anti-mouse IgG) conjugated to 10 nm gold (British BioCell International, Cardiff, UK). The sections were finally washed and fixed with 3% glutaraldehyde in 0.1 M phosphate buffer. As controls we used samples that were incubated with secondary antibody only, or samples where the primary antibody was replaced by a nonrelevant antihuman murine IgG (Dakopats, Glostrup, Denmark). The sections were counterstained with uranyl acetate in methanol. Samples were analyzed in a Hitachi 7100 (Tokyo, Japan) transmission electron microscope.

Marker protein asssays

The following enzymes were used as organelle markers: β-hexosaminidase (specific granules and lysosomes), α-mannosidase II (Golgi apparatus and lysosomes) (204), and alkaline phosphodiesterase I (plasma membranes). The activity of the enzymes were estimated as described by Storrie and Madden (205). Basically, the organelles were incubated in the appropriate buffer with an enzyme substrate. The reaction product was chromogenic or luminescent upon excitation with a defined wavelength. Absorbances were then measured in a spectrophotometer and luminescence in a fluoro-spectrometer. Hexosaminidase is widely considered to be a marker of lysosomes in a number of cell types. In eosinophils it is also present in the specific granules and can be

22 released upon stimulation. In our gradients, we have observed that some alkaline phosphodiesterase and α-mannosidase II are present in the specific granule compartment. The presence of markers for the Golgi apparatus and plasma membranes associated with the specific granules (storage compartment) could be interpreted as the consequence of trafficking between compartments. Albumin was used as a marker for a subpopulation of vesicles containing CD11b receptor (28). The concentration of albumin was measured by a radioimmunoassay using I125-labeled human serum albumin and polyclonal rabbit antibodies against human albumin. Albumin-antibody complexes were separated from unbound protein using the decanting suspension nb3 (Pharmacia & Upjohn Diagnostics, Uppsala, Sweden). Determinations were performed in duplicate (CV<10%). Total protein content was determined with a commercial kit (Biorad, Hercules, California).

Statistical analysis

To evaluate the statistical significance of observed changes, non-parametric tests were used, namely the Mann –Whitney U test, Wilcoxon’s matched pair test for paired samples, Fisher’s exact test, and Spearman rank correlation. The program STATISTICA (Statsoft, Tulsa, OK, USA) was used for the analyses. P values < 0.05 were considered significant.

23 RESULTS AND DISCUSSION

Serum levels of ECP, EPX and EPO (papers I and II)

Bacterial and viral infections (paper I) As described in detail in the Introduction, serum levels of ECP, EPX and EPO reflect the number and activity of blood eosinophils. Many studies have therefore been focused on the possible correlation between the course of inflammatory diseases and the activity of circulating eosinophils. Studies on stimulated releases in vitro have suggested that eosinophil granule proteins are selectively secreted depending on the type of stimuli. As the serum levels of ECP, EPO and EPX are mostly the results of active release during the sampling procedure, one aim of the current studies was to determine whether the effects of differential mobilization of ECP and EPO could be observed in serum. Previous in vitro results (125) had indicated that stimulation via IgG receptors promoted ECP release, while stimulation via IgE receptors promoted EPO release. Bacterial infections usually result in an increase in IgG levels as well as in complement activation (206). On the other hand, previous work in our department (207) had shown that ECP levels were increased in patients with bacterial infections. In study I we therefore compared the serum levels of ECP and EPO in patients with acute bacterial infections. It has been suggested that viral infections are related to the development of asthma or allergy (208-210). In addition, direct interactions between viruses and eosinophils have been described, for example eosinophils have been found to ingest viruses (211). For this reason we also estimated serum levels of ECP and EPO in patients with viral infections.

The serum levels of ECP were significantly higher in the group with bacterial infections then in those with viral infections or in healthy subjects, while the serum EPO remained within the normal range in both infection groups. Serum ECP correlated with serum EPO in the group of normal subjects and in the group with viral infections, but not in the group of subjects with bacterial infections. In the latter group, the ECP and MPO levels were correlated.

We later we decided to complement our observations on EPX in cooperation with Dr. Shengyun Xu and Dr. Karlis Pauksen. We compared serum levels of ECP and EPX in similar groups of patients with bacterial or viral infections. The study comprised 27 patients with bacterial infections, 26 patients with viral infections and 20 healthy controls. The median serum level of EPX was significantly higher in the patients with bacterial infections (38 µg/L) than in those with viral infections (27.3 µg/L) (p= 0.0003) and in the healthy

24 Fig.2

Fig.2 Serum levels of EPX and ECP in the patients with bacterial and viral infections. EPX, open boxes; ECP, filled boxes. The EPX serum levels are significantly higher in patients with bacterial infections than in those with viral infections and the healthy group. The difference between the viral and healthy groups is also significant as estimated by the Mann-Whitney U test. Data depicted as median values and 25-75% quartiles. The numbers of patients in the bacterial, viral and healthy groups were 27, 26 and 20 respectively.

individuals (20.3µg/L) (p=0.0004; Fig 2), as estimated by the Mann-Whitney test. The difference in EPX between subjects with viral infections and healthy controls was also significant (p=0.009). The differences in serum ECP between the three groups showed the same pattern as those found in the first study, with values of 20.2, 12.6 and 10.5 µg/L, respectively (Fig. 2). There was a correlation between ECP and EPX in patients with bacterial infections ( r = 0.68 , p<0.05, Fig .3), but not in those with viral infections or in healthy subjects. The median eosinophil count was 45 x 106/L in the bacterial infection group, 30 x 106/L in the viral infection group, and 130 x 106/L in the group of healthy subjects.

25 Fig.3

Fig 3 Correlation between serum levels of EPX and ECP in the patients with bacterial infections(n=27), as estimated by Spearman correlation. (r= 0.62, p= 0.0006).

In this study (paper I) we interpreted the fact that ECP was increased during bacterial but not viral infections as due to the activation of eosinophils by a specific set of cytokines that were active only during the former but not the latter infection. The discrepancy between the ECP and EPO levels in the bacterial infection group, and especially the lack of correlation between ECP and EPO, were considered to indicate differential mobilization of these two proteins. The correlation between serum ECP and MPO could be due to parallel activation of neutrophils and eosinophils during bacterial infections.

Recent data have shown that ECP and EPX are also present in small amounts in neutrophils (67) and that ECP can be released from neutrophils upon stimulation in vitro (155). We cannot therefore rule out the possibility that in the case of

26 bacterial infections, where the number and activity of neutrophils were elevated, the increases in ECP and EPX were due to release from activated neutrophils. The fact that ECP is stored in the same granules as MPO (155) speaks in favor of this interpretation.

Whatever the reason for the increase in ECP in bacterial infections, it would seem important to keep in mind that bacterial infections cause elevation of ECP levels, as this can be misleading in cases where allergic diseases are complicated by infections. EPO seems to be more specific for eosinophils and hence may be more useful in monitoring allergic disease (212).

G-CSF administration in healthy subjects (paper II) It has been found that patients with bacterial but not with viral infections have elevated serum levels of G-CSF (213). As we had hypothesized that difference in cytokine patterns between patients with viral and bacterial infections could be the reason for the differences in the serum profiles of ECP, we considered it of interest to investigate the influence of administration of G-CSF in healthy subjects on the number of circulating eosinophils and serum levels of ECP, EPO and EPX.

In this paper we reported for the first time the influence of G-CSF administration on peripheral blood eosinophils. Healthy subjects received G- CSF subcutaneously a dose of 7.5 (n=8) or 10 (n=6) µg/kg body weight, daily for six consecutive days. The eosinophil count was already increased significantly 24 h after the first dose of G-CSF, from 0.22 to 0.37 x109/L, and reached a maximum of 0.61x109/L on day 5. Interestingly, the eosinophil count started to decrease on day 6 in spite of the continuing G-CSF administration, in contrast to that of neutrophils. In correspondence with the eosinophil count, increases in the serum levels of ECP (r=0.63), EPX (r=0.67) and EPO (r=0.61) were observed. The baseline serum value of EPO was 9 µg/L, of ECP 12 µg/L, and of EPX 28 µg/L. There were no increases either in the serum granule protein levels or in the eosinophil count during the first 3 h after G-CSF injection, although an increase in the serum level of G-CSF was observed (data not published). On the second day, a significant increase in the serum concentrations of all three proteins was observed, with maintenance of the same relative order as for the baseline levels. A maximum was reached on day 4 or 5 (EPO 20 µg/L, ECP 62 µg/L and EPX 88 µg/L). The changes in the ratio between serum granule protein level and eosinophil count differed between the different proteins. The EPX /eosinophil count ratio increased on day 2 and remained elevated until the end of the study, even after discontinuation of G-CSF. The corresponding ratio for ECP, after an increase on day 2, gradually declined to the initial value at the end of the study period. The

27 amount of EPO released by eosinophils diminished slightly throughout the G- CSF administration.

Administration of G-CSF in healthy subjects has been found to induce an increase in the cytokines GM-CSF (190) and TNF-α (189). As GM-CSF, which is an eosinophil-activating cytokine, was already markedly elevated 24 h after the injection of G-CSF, it is difficult for us to determine whether the changes observed in eosinophils are due to the direct action of G-CSF or occur indirectly through the production of eosinophil-specific cytokines.

The increase in the eosinophil count in the blood could be due to an increased production of these cells in the bone marrow (188) or to enhanced mobilization from the marginal pool, as was found for neutrophils (197). G-CSF administration also increases the number of circulating colony-forming units (CFU cells) committed to the eosinophil lineage in humans (195), possibly as a result of increased mobilization of cell progenitors and/or of an increase in their number in the bone marrow. The mechanisms regulating the eosinophil count seem to differ from those of the neutrophils, as suggested by the different time courses of the reduction in their number that we found in the present study.

The increases in the serum levels of EPX, ECP and EPO are related both to an increase in the number of eosinophils and to changes in eosinophil reactivity, as suggested by the differences in the protein/ eosinophil count ratios between the different granule proteins. These differences might be interpreted as follows: the propensity to release EPX increases and then remains unchanged; ECP increases at the beginning and then declines with time; and EPO remains unchanged or even diminishes.

In the G-CSF model, preferential mobilization of EPX and ECP was observed in comparison to that of EPO. However, for example in asthma, the elevation of serum EPO was much more pronounced than that of EPC (214). These observations suggest that different mechanisms regulate the release of the different proteins.

As discussed above, neutrophils contain small amounts of ECP and EPX, and we therefore have to consider the possibility that some ECP and EPX in serum originates from neutrophils, particularly in view of the fact that the neutrophil count increased significantly in the G-CSF model. However, there was no correlation either between the neutrophil count and the ECP and EPX levels or between the MPO level and the level of ECP or EPX (data not published). This suggests that in the G-CSF model eosinophils, but not neutrophils, are the main

28 source of granule proteins. The difference in the correlation between MPO and ECP in bacterial infections as compared with that in the G-CSF model seems interesting with respect to the discussion concerning the source of ECP. The reason may lie in the different degrees of neutrophil activation in the two cases. G-CSF was found to increase the number of circulating neutrophils, but not to prime all neutrophil functions. For example G-CSF administration reduces neutrophil chemotactic activity and neither enhances the respiratory burst (194) nor improves the antibacterial response (215). The single cytokine model has both advantages and disadvantages. The advantages are that the number of involved factors are reduced to a single one at least at the beginning of the experiment, and the possibility of observing the effects from the beginning of the cytokine action. Natural infections, on the other hand, evoke a complicated and multifactorial response, and patients go to the physician when the symptoms are fully developed, which means that several days have already elapsed since the time of bacterial invasion. One disadvantage of the single cytokine model is that some physiological events may be missing. For example during a natural infection granulocytes are attracted to the site of infection and hence disappear from the circulation. Thus caution is required in making direct extrapolations between the model situation and real events such as bacterial infection.

In conclusion, the serum profiles of ECP, EPX and EPO differ from each other depending on the situation at hand. This may be due to differences in the regulation of the release mechanisms of each protein.

Piecemeal degranulation (papers II and III)

Ultrastructure of eosinophils after G-CSF administration (paper II)

The model of piecemeal degranulation has been proposed as a mechanism of eosinophil granule protein release on the basis of ultrastructural studies (27). This mechanism constitutes the basis for the selective release of eosinophil mediators. According to this model, the content of specific granules is packed into small vesicles and gradually transported to the plasma membrane, leaving the specific granules progressively empty. In EM preparations, the specific granules show gradual changes in their structure - from a diminished optical density that gives the impression of a loosening matrix structure, through ragged losses, towards complete lucency. The crystal cores show similar changes - from ragged losses to complete disappearance. The presence of peroxidase activity in vesicular structures in the vicinity of dissolving granules has also been demonstrated. These ultrastructural alterations were observed in eosinophils derived from cord blood cells in culture with IL-5(27). Similar changes, e.g.,

29 gradual dissolution of granule contents, have been observed under many other conditions, as reviewed in the Introduction.

As described above, we observed differential increases in the serum levels of ECP, EPX and EPO during the administration of G-CSF, suggesting independent mobilization of each protein into the serum. A further aim of study II was therefore to determine whether the administration of G-CSF caused changes in the ultrastructure of eosinophils.

Eosinophils obtained before G-CSF administration displayed the typical ultrastructure of a normal eosinophil. Most specific granules (on average 17/cell) had a homogenous matrix and a clear crystal core. Few granules in a cross-section (on average 5 per cell) showed ragged losses of the matrix. As early as 24 hours after G-CSF administration, the number of specific granules with ultrastructural changes had increased to 23 per cell (range 6 to 31). The ragged losses of the matrix were similar to those described as piecemeal degranulation by Dvorak (27). An additional ultrastructural examination was performed on eosinophils from a bone marrow donor (unpublished data). A 60-year-old woman without any history of allergic symptoms received G-CSF for 5 consecutive days. Her serum ECP level was 20 µg/L and the eosinophil count 0.390 x109/L (6.3% of WBC) before the administration of G-CSF. The eosinophils were fixed in a mixture of buffered glutaraldehyde with osmium tetroxide and embedded in Agar 100. An illustrative example of an eosinophil after 5 days of G-CSF administration is shown in Figure 4. Apart from the alterations in the ultrastructure of specific granules that are described above, the formation of vesicles was observed on the borderline of specific granules (Fig. 4b). The fate of emptied granules is not known. In some cross-sections we observed structures that resembled membranes encapsulating the remnant (a ‘ghost ‘) of a granule (Fig.5). These structures could have arisen to digest old, used granules (creation of autolysosomes).

Fig.4 a) Eosinophil from a subject after 5 days of G-CSF administration. Cells were fixed in osmium tetroxide with glutaraldehyde and embedded in Agar 100. Matrix of some specific granules show decreased electron density. Some specific granules present ragged losses of matrix. Chains of vesicles are seen on the granule membrane (arrows) of altered specific granules. There are vesicular structures in the cytoplasm (thin arrows). b) Cell fragment showing specific granules with vesicles on the granule membrane.

30 Fig.4 a

Fig.4 b

31 Fig.5 Eosinophil obtained from a bone marrow donor after 5 days of G-CSF administration. Cells were fixed in osmium tetroxide with glutaraldehyde and embedded in Agar 100. Membranes are seen to wrap around remnants of granules (arrows). Specific granules show the ragged loses of matrix (arrowheads).

32 It was concluded that administration of G-CSF caused alterations in the ultrastructure of blood eosinophils similar to those reported to occur during the piecemeal degranulation. Since an increase in the serum and plasma levels of ECP and EPX was observed at the same time, we suggest that ultrastructural alterations are related to enhanced release of granule proteins. This implies that piecemeal degranulation is the main mechanism of granule protein secretion in the blood. The formation of vesicles observed on day 5 indicates a vesicular transport of the granular content. The observed changes could be due to a direct action of G-CSF or be mediated by a secondary set of cytokines.

Ultrastructure of eosinophils from allergic subjects during and out of the birch pollen season (paper III) We decided to continue our investigations on a natural model of eosinophil activation, namely pollen allergy. According to the current theory, exposure of an allergic subject to the allergen to which he/she is sensitive, causes a cascade of events from the production of cytokines by Th2 clones of lymphocytes to the activation of eosinophils (216-218). Indications of blood eosinophil activation during the birch pollen season, such as an increase in the eosinophil counts and in the serum levels of ECP (219), EPO (212) and EPX (147), have been reported. Activation of Th2 cells during the pollen season has been also reported (220;221). Since the influence of IL-5 on eosinophil production, activation and accumulation has been well documented in different models (222;223), activation of blood eosinophils during the pollen season was thought to be primarily due to the priming effect of IL-5 and other cytokines originating from T helper 2 (Th2) lymphocytes.

The first aim of study III was to investigate the ultrastructure of eosinophils during and out of the birch pollen season.

Three mildly allergic subjects, not treated with steroids or immunotherapy, entered the study. Their serum ECP levels were 12, 17 and 18 g/mL, and EPO 18, 25 and 36 µg/L. In these subjects the seasonal changes in the ultrastructure of eosinophil specific granules was observed. Out of the season, the majority of the eosinophil specific granules had the appearance of normal, non-activated granules, i.e., a homogeneous matrix and a clear crystal core, similar to those observed in healthy subjects. Only some specific granules showed ragged losses of matrix structure. In the allergic subjects during the birch pollen season, we noted an increase in the number of eosinophils, which exhibited specific granules with a variety of alterations such as ragged losses of their core and/or loosening of their matrix structure. These alterations varied in extent, from very minor changes to complete emptying of the matrix compartment or

33 disappearance of the core (Fig. 6). The co-existence of activated and non- activated eosinophils in the same specimen is exemplified in Figure 7.

Fig.6 An eosinophil from an allergic subject during a pollen season was immunolabeled for ECP by monoclonal antibodies. The cells were fixed in paraformaldehyde and embedded in Lowicryl KM4. Many specific granules show alterations of ultrastructure- from losses of matrix and core ( arrow ) to complete lucency of matrix. Labeling for ECP (10 nm gold particles) is observed in the matrix of unaltered specific granules (arrowhead) and in the cytoplasm both in the vicinity of granules and in the granule-free area (asterix). Some labeling is associated with the plasma membranes (empty arrows).

34 Fig. 7 Eosinophils from a mild allergic subject without medication during the pollen season. Purified eosinophils were fixed with 4 % paraformaldehyde and embedded in Lowicryl KM4. Eosinophils with most specific granules showing normal ultrastructure (arrow) are in the vicinity of cells showing most of the granules altered (arrowhead). Between them is cell with few granules showing alterations, while the rest of the cells display a normal structure.

We carefully considered the possibility of artifacts having been caused by the fixation, embedding or sectioning procedures used. We excluded them, however, for various reasons. Firstly we observed granule structures at different stages of emptying – from loosening of the matrix and core to more advanced stages up to complete emptying – often all within the same cell. Secondly, we observed cells containing both affected and unaffected granules adjacent in the same section (Fig. 7). In addition, eosinophils both from healthy individuals and

35 from allergic subjects out of the pollen season showed a clear predominance of cells containing unaffected specific granules, even though the fixation and embedding procedure was the same as in the case of cells showing alterations. Further, cells other than eosinophils present in the specimens, such as neutrophils, did not show any alterations in the structure of their granules either during or after the season. Results of other authors also support the exclusion of artifacts. For example, the appearance of lucency in specific granules was observed in cells stimulated with PAF but not with C5a, and not with buffer (127;128). For all these reasons I consider that differences observed in the ultrastructure of specific granules actually reflect their state of activation.

The seasonal appearance of the changes in the eosinophil ultrastructure indicates that they were caused by events following allergen exposure. As similar alterations in culture experiments have been found to be caused by IL-5, it seems plausible that IL-5 and possibly also GM-CSF (as suggested by G-CSF experiments) are a direct cause of ultrastructural changes also in the pollen season (27;224) .

The site of eosinophil degranulation could not be determined in the present studies. Eosinophils degranulate in the circulation, as indicated for example by the presence of granule proteins in plasma (data not shown), but they also degranulate in the bone marrow, as shown by others (105-107). The function of proteins released to the circulation in allergic events seems to be obscure, especially in the view of the fact that the eosinophil is regarded as a tissue cell. Other groups have reported the presence of eosinophils showing characteristics of piecemeal degranulation in tissues, for example polyps or airways mucosa (124), suggesting that this process is an important form of eosinophil degranulation in situ. Regarding the role of circulating eosinophil proteins, in cases of infection EPX or ECP may be considered to be antiviral or antibacterial factors (77). In allergy it may be speculated that the role of eosinophil granule proteins is related to their propensity to regulate the function of other cells from the . Another possibility is that activation of eosinophil degranulation is due to some kind of inadequate response, which makes them pathogenic.

In conclusion, the administration of G-CSF and allergen exposure induced ultrastructural features characteristic of piecemeal degranulation in blood eosinophils, suggesting an active release of eosinophil proteins in the blood. The changes in cytokine profiles during the studied events are presumably the cause of the observed alterations.

36 Subcellular localization of eosinophil granule proteins (papers III and IV)

The next question addressed was whether piecemeal degranulation might be the mechanism of release of ECP, EPX and EPO in the blood. Previously, these three granule proteins have been shown to be localized in the matrix of specific granules by immunogold labeling in nonstimulated eosinophils (25;225). In eosinophils undergoing piecemeal degranulation, EPO was found to be present in vesicular structures (27).

The aim of studies III and IV was therefore to investigate the localization of ECP, EPX and EPO in blood eosinophils from allergic subjects during and out of the pollen season and in healthy and hypereosinophilic subjects, by immunogold labeling for electron microscopy and by subcellular fractionations on density gradients.

In conformity with previous findings (225), immunogold labeling showed that most of the labeling for ECP and EPX was localized in the matrix of the specific granules. When specific granules displayed a normal structure, e.g., a clear crystal core and a homogeneous matrix, labeling for ECP and EPX was associated with the matrix. In the altered granules with a partially disintegrated core, the labeling for ECP was also seen to be associated with the core remnants, and in the granules without a visible core the labeling was scattered over the whole granule cross-section. In many cross-sections of eosinophils, labeling for ECP and EPX was also observed beyond the specific granules. Some labeling for ECP and EPX was found to be associated with the plasma membrane, but most of it was observed in the cytoplasm, both in the vicinity of granules and in the central part of the cell devoid of specific granules.

It seems that there is less extracellular labeling for ECP in cells in which most of the specific granules show the normal ultrastructure than in cells with many affected granules. However, we did not perform any exact quantitative estimation in this model. To estimate the size of the extragranular compartment more quantitatively, we examined the localization of eosinophil granule proteins by subcellular fractionation on density gradients. Most of the cell protein content was present in fractions of high density (1.2 –1.3 g/mL) containing specific granules. ECP, EPX and EPO were colocalized in this specific granule region of the gradient (Fig.8). Nevertheless, considerable amounts of EPX, ECP and EPO were also found in fractions of low density (from 1.05 to 1.17 g/mL), which contain markers for cytosol, plasma membranes, albumin-containing vesicles and the Golgi

37 apparatus. The subcellular distributions of ECP, EPX and EPO differed in the low-density region (Fig.8) EPX was found to be present in the light region in almost all examined subjects: healthy individuals, allergic subjects during and out of the pollen season, and hypereosinophilic subjects. This low-density peak of EPX was localized mainly in fractions with a density of 1.05 to 1.15 g/mL, overlapping partially with markers for cytosol, albumin-containing vesicles and plasma membranes.

220

200

180

160

140

120

100

80

60 protein content(µg/L) 40

20

0 1,00 1,05 1,10 1,15 1,20 1,25 1,30 density of sucrose (g/mL)

Fig.8 Subcellular distributions of ECP (open triangles), EPX(closed squares), and EPO(closed circles) in an allergic subject during the pollen season. Data are depicted as percentage of gradient content. ECP, EPX and EPO are co-localized in the high–density region of the gradient (fractions with a density of 1.2 to 1.3 g/ml), which contains the specific granules. Significant amounts of EPX are present in the low-density region of the gradient (from 1.05 to 1.15 g/mL). The buoyant densities of the extragranular ECP and EPO are 1.10 to 1.17 g/mL, values which are higher than the buoyant density of EPX. The EPX peak is separate from the ECP and EPO peaks.

In contrast, the low-density peaks of ECP and EPO were clearly pronounced in the allergic subjects during the pollen season. These two granule proteins were

38 completely co-localized in fractions with densities of 1.11 to 1.17g/mL, overlapping with markers for plasma membranes. The ECP and EPO peaks were separate from the EPX low-density peak. In the healthy individuals and in the allergic subjects out of the pollen season the amounts of ECP and EPO in the low-density region were significantly smaller.

The presence of ECP, EPX and EPO not only in the specific granules but also in an extragranular compartment was shown both by immunoelectron microscopy (IEM) and subcellular fractionations. EPX and ECP were partially associated with the plasma membranes, as shown by IEM and confirmed by their co- localization with markers for plasma membranes in subcellular fractionations. However, the bulk of the extragranular labeling for ECP and EPX was observed in the cytosol and at present it is difficult to draw a conclusion as to the exact nature of this compartment. The results of Dvorak suggest that the proteins are localized within the vesicular structures. The results of the present subcellular fractionations, e.g., the findings that EPO, ECP did not co-localize and EXP only partially co-localized with markers for cytosol, and the fact that the two proteins ECP and EPX, of almost identical molecular weight, sedimented differentially, seems to exclude the possibility of transport as free proteins. On the other hand, we were not able to distinguish the membrane structures around ECP or EPX labeling in the extragranular compartment in our micrographs.

There are some possible explanations for these seemingly controversial results, as discussed below. Still further work is required to elucidate the problem of how eosinophil granule proteins are transported from the granules to the plasma membranes. The method of fixation in paraformaldehyde and embedding in Lowicryl KM4 allows for good antigen preservation, but the membrane contrast is too poor, in comparison with fixation in glutaraldehyde and embedding in Agar 100, to allow visualization of transporting vesicles. Thus one explanation for the apparently contradictory results may be that for technical reasons we did not visualize the structures containing ECP, EPX and EPO at immunoelectron microscopy. Another possible explanation is that eosinophil granule proteins are not membrane-bound but are complexed to large molecules, and this alters their buoyant density in gradients compared to other cytosolic proteins. It may be speculated that the large complexes might be proteoglycans, which build up the matrix (14;15). In the cases of ECP and EPX they could be large complexes of cytosolic proteins, as occurs for other ribonucleases with an intracellular function (226).

Further, the possible roles of ECP and EPX in the cytoplasmic compartment need to be considered. Both ECP and EPX, and also EPO, are eosinophil secretory products. They are found in an extragranular compartment in various

39 proportions according to the physiological state of the cell. If this extragranular compartment is part of a secretory pathway, it must be involved in the piecemeal degranulation mechanism. The lack of co-localization of ECP and EPX in this compartment in the subcellular fractionations may reflect differences in the regulation of their mobilization. Another possibility is that EPX, which has much stronger ribonuclease activity then ECP, also plays an intracellular role in maintaining the homeostasis of cellular RNA, as do other ribonucleases (226).

To conclude, EPX, ECP and EPO are present not only in specific granules but also in the extragranular compartment. The amounts of ECP and EPO in this compartment change seasonally in allergic subjects. ECP and EPX compartments seem to be separate, as judged by their different buoyant densities. Localization of ECP, EPX and EPO to the extragranular compartment suggests the existence of a mechanism of protein translocation through the cytoplasm to the plasma membranes.

40 GENERAL SUMMARY AND CONCLUSIONS

This thesis describes the mobilization of eosinophil granule proteins in blood eosinophils.

Preferential mobilization of EPX and ECP in comparison with that of EPO was observed both in bacterial infections and during administration of G-CSF in healthy subjects. The differences were more clearly seen when the serum levels of the proteins were correlated with the cellular content of each protein or with the eosinophil count. During G-CSF administration and during allergen exposure in the pollen season, an increase in the number of specific granules showing ultrastructural alterations of various degrees, e.g., ragged losses of cores and /or matrix, was observed. The presence of ECP, EPO and EPX in an extragranular compartment of eosinophils was found both by EM and subcellular fractionations. The EPX- containing compartment was separate from that of ECP and EPO, as shown by their different buoyant densities. The amounts of ECP and EPO in the extra- granular compartment increased in allergic subjects during the season, while the EPX extragranular compartment did not exhibit seasonal changes.

In conclusion, the differences in the serum profiles of ECP, EPX and EPO are most probably due to their differential release from blood eosinophils. The simultaneous occurrence of both ultrastructural characteristics of piecemeal degranulation and an elevation of the serum levels of ECP, EPX and EPO suggests that piecemeal degranulation is the principal mechanism of their release in the circulation. The presence of EPX, ECP and EPO in an extragranular localization supports the hypothesis of secretion via a cytoplasmic compartment. The different buoyant densities of EPX and ECP and the difference in the size of their extragranular compartments during the pollen season support the hypothesis of a selective mechanism regulating the intracellular transport of different eosinophil granule proteins.

41 ACKNOWLEDGMENTS

This work was carried out at the Department of Medical Sciences, Clinical Chemistry, of the University Hospital, Uppsala.

I am very grateful to all those who contributed in various ways to the accomplishment of this thesis. I would particularly like to thank:

Professor Per Venge, my supervisor, for introducing me to the world of eosinophils, for giving me the opportunity to perform my studies, for his enthusiasm and optimism and for being confident in my ideas; Dr. Rodolfo Garcia, for sharing his knowledge, for his support and patience and for many interesting discussions; Associate Professor Lena Douhan-Håkansson, for always finding time to help me and answer my questions; Dr. Lahja Seveus, for introducing me to the field of electron microscopy; Dr. Christer Peterson, for providing me with antibodies and for discussions;

All co-authors, for their contribution to the articles;

All blood donors, without whom this thesis would not have been possible;

Lena Moberg, for all the laborious experiments she performed and for the fun of working together; Charlotte Woschnagg, Mia Lampinen, Jonas Byström and Shengyuan Xu, for sharing the room, the lab, the knowledge, the eosinophils (even their own), hopes and frustrations of being a PhD student, and a beer from time to time; Kristina Sandström, for sharing the lab bank and for discussions during the incubations; Xiaoyen Xu, for her help in purifying eosinophils; Agneta Trulson, for advice and support; Lixin Liu for being a good colleague in the lab; and Kerstin Lindblad, Ulla Britta Jansson and Ing Britt Person for their help during daily life in the lab; Gunilla Strömstedt - for always having a smile and for patient guidance through the labyrinth of all official forms and regulations;

All colleagues at the Department, for help, chats and smiles and for creating a pleasant atmosphere;

Anders Ahlander, for his excellent technical assistance with electron microscopy techniques, for his willingness to try something new and for all our chats; Marianne Ljungkvist and Frank Bitkowski, for their help with pictures;

42 Joanna Kufel, for understanding and support; Soheir Beshara and George Metry, for constant encouragement; All my friends from Poland and from Uppsala, for your friendship;

My parents Janina and Arkadiusz for their love and caring; Ron, for his patient support and love.

Malgorzata

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