Comprehensive Summaries of Uppsala Dissertationsfrom the Faculty of Medicine 1071

_____________________________ _____________________________

Granulocyte Adhesion to MatrixProteins and the Effect on theRelease of Granule Proteins

Development of a Simple Method and its Application in

Experimental and Clinical Studies

BY

XIAOYAN XU

ACTA UNIVERSITATIS UPSALIENSISUPPSALA 2001

Dissertation for the Degree of Doctor of Philosophy (Faculty of Medicine) in Clinical Chemistry presented atUppsala University in 2001

ABSTRACT

Xu, X. 2001. Granulocyte Adhesion to Matrix Proteins and the Effect on the Release of Granule Proteins.Development of a Simple Method and its Application in Experimental and Clinical Studies. Acta UniversitatisUpsaliensis. Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine 1071. 46 pp. Uppsala. ISBN 91-554-5112-8

Granulocyte adhesion and release of their granule proteins are key steps during selective accumulation of acertain cell to an inflammatory site. Eosinophils are specifically recruited to sites of allergic inflammation andparasitic infection, whereas neutrophil influx predominates in bacterial infection and rheumatoid arthritis.

A simple, reliable and convenient method was developed for the measurement of granulocyte adhesion andrelease of granule proteins by using the normal population of granulocytes. The design allows simultaneousquantitative assessment of eosinophil and neutrophil adhesion to proteins and degranulation.

Using this method, manganese ions (Mn2+) induced a higher level of eosinophil adhesion to fibronectin,fibrinogen and albumin as compared with neutrophils. PMA induced comparable levels of eosinophil andneutrophil adhesion. F-MLP stimulated a rapid, short-term adhesion of neutrophils to fibrinogen.

In the same conditions PMA alone stimulated a dose-dependent release of ECP from cells that adhered toboth fibronectin and fibrinogen. Meanwhile, Mn2+ amplified the release of ECP induced by PMA. Furthermore,release of ECP was shown to be associated with cell death.

PMA, in combination with Mn2+, induced a marked release of ∼ 80%of the intracellular content of lactoferrinand HNL in neutrophils. PMA or f-MLP alone induced 30-40% release of lactoferrin and HNL. A maximalrelease of MPO of 15-20% was obtained from neutrophils stimulated by PMA and Mn2+. Release of lactoferrinand HNL showed a significant negative relationship to the viability of cells.

Stimulated by PMA, eosinophils from pollen-atopic patients during early pollen season displayed a markedlyenhanced adhesion and release of ECP of eosinophils compared with eosinophils from the references. Primingwith IL-5 caused a significantly higher adhesion and release of ECP by eosinophils in response to PMA. GM-CSF priming enhanced eosinophil adhesion in response to PAF and PMA plus Mn2+, but did not enhance therelease of ECP.

In conclusion, the assay allows a simple quantification of eosinophil and neutrophil adhesion, as well asdegranulation by using the normal population of granulocytes. Cellular adhesion plays an important role in theregulation of both eosinophil and neutrophil degranulation, but adhesion and degranulation can be inducedseparately.

Key words: Eosinophils, neutrophils, adhesion, release of ECP, extracellular matrix proteins, granulocytes.

Xiaoyan Xu, Department of Medical Sciences, Clinical Chemistry, University Hospital, SE-751 85 Uppsala, Sweden

Xiaoyan Xu 2001

ISSN 0282-7476ISBN 91-554-5112-8

Printed in Sweden by Kopieringshuset AB, Uppsala 2001

To Zhurong, Yangzi and my parents

4

This thesis is based on the following papers, which are referred to by their

Roman numerals.

I. Xiaoyan Xu and Lena Håkansson

Simultaneous analysis of eosinophil and neutrophil adhesion to plasma and tissue

fibronectin, fibrinogen and albumin. J.Immunol.Methods 1999, 226, 93-104.

II. Xiaoyan Xu and Lena Håkansson

Regulation of the release of eosinophil cationic protein by eosinophil adhesion.

Clin.Exp.Allergy 2000, 30, 794-806

Ш. Xiaoyan Xu and Lena Håkansson

Degranulation of primary and secondary granules in adherent human neutrophils

Submitted for publication

IV. Xiaoyan Xu , L. Håkansson and G. Stålenheim

The capability of adhesion and release of ECP of eosinophils from pollen-atopic

patients and cytokine primed normal eosinophils

Manuscript

Reprints were made with the permission of the publisher.

5

CONTENTS

1. ABBREVIATIONS……………………………………………………………………..6

2. INTRODUCTION………………………………………………………………………7

General background…………………………………………………………………….8

Asthma and allergic diseases Eosinophil granulocytes Neutrophil granulocytes Extra cellular matrix proteins Adhesion Adhesion molecules

Degranulation Eosinophil priming Cytokines Cell death 3. AIMS OF THE STUDY………………………………………………………………20

4. MATERIAL AND METHODS………………………………………………………21

Isolation of granulocytes Purification of eosinophils Coating of wells Stimulation of cells Priming of eosinophils Adhesion assay Release assay Radioimmunoassays Flow cytometry Assessment of granulocyte death Inhibition of release and adhesion by monoclonal antibodies Statistical analysis

5. RESULTS AND DISCUSSION………………………………………………………..26

6. CONCLUSIONS………………………………………………………………………..34

7. ACKNOWLEDGEMENTS……………………………………………………………35

8. REFERENCES…………………………………………………………………………37

Error CorrectSummary, page 11, Line 18

Understood.. Understood.

Summary, page 23,Line11

Granulocytes 1×109

cells/mlGranulocytes 1×109 cells/L

Summary, page 28,Line 25

these actions (114). these cations (114).

Summary, page 29,Line 10

Eosinophils adhering tofibronectin

Eosinophils adhering tofibrinogen

Paper Ι, page 95,Measurement ofeosinophil adhesionLine 12 and 19

Containing 5% (w/v)CTAB

Containing 0.5% (w/v)CTAB

Paper ΙΙ , page 795,Degranulation ofeosinophils duringadhesion to proteinsLine 2

With PMA (1×10-9

µmol/L), f-MLP (1×10-7

µmol/L)

With PMA (1×10-9 mol/L),f-MLP (1×10-7 mol/L)

Paper ΙV, page 7Measurement ofeosinophil adhesionLine 5

Frozen at 20° C Frozen at -20° C

6

1. ABBREVIATIONS

BPI – bactericidal/permeability-increasing proteinCGRP – calcitonin gene-related peptideCTAB - N-cetyl-N,N,N-trimethyl-ammonium bromideECM – extracellular matrix proteinECP – eosinophil cationic proteinEDN – eosinophil-derived neurotoxinEPO – eosinophil peroxidaseERK – extracellular regulated kinase FDA – fluorescein diacetateFMCA – fluorometric microculture cytotoxicity assayf-MLP - formyl-methionyl-leucyl-phenylalanineGM-CSF – granulocyte-macrophage colony-stimulating factorHNL – human neutrophil lipocalinHSA - human serum albuminICAM – intercellular adhesion moleculeLTC – leukotriene CLTD – leukotriene DIL – interleukinmAb - monoclonal antibodyMadCAM – mucosal addressin cell adhesion moleculeMAPK – mitogen activated protein kinaseMBP – major basic proteinMEK – MAPK / ERK kinaseMFI - mean fluorescence intensityMPO – myeloperoxidaseNBS – new-born calf serumPAF – platelet-activating factorPI – propidium iodidePKC - protein kinase CPMA - phorbol-myristate-acetatePMN – polymorphonuclear leukocytesRIA - radioimmunoassaySTZ – serum-treated zymosanTNF – tumour necrosis factorVCAM-1 – vascular cell adhesion moleculeVLA – very late activation antigen

7

2. INTRODUCTION

Eosinophils are specifically recruited to sites of allergic inflammation and parasitic infection.

Neutrophil influx, on the other hand, predominates in bacterial infection. This biological

selectivity suggests that granulocytes may respond differentially to inflammatory mediators

during the recruitment to inflammatory foci (1). For instance, secretory IgA and secretory

component preferentially activate human eosinophils (2). The selective migration of different

granulocyte types from the circulation into the tissue might partly be due to the demonstrated

increased surface expression and activation of adhesion molecules, such as CD11/CD18 and

VLA-4 (3;4). Thus, adhesion may be regarded as one of the first functional events in the

activation cascade, whereas degranulation of the granule proteins most likely represents a

final functional event in this activation cascade (5).

In the activation cascade, leukocytes encounter the extracellular matrix protein (ECM) that

underlies endothelia and epithelia and surrounds connective tissue cells. It has been

demonstrated that adherence to the ECM is important for the localisation of leukocytes to a

site of inflammation (6) and that a number of key functions of leukocytes, including adhesion,

cytokine production (7;8), cell migration and activation (9) and release of granule contents

(10), are significantly affected. Examples of this are that adhesion of eosinophils to

fibronectin attenuated activation and release of EDN (eosinophil-derived neurotoxin) (11) and

prolonged eosinophil survival (12). The ability of the ECM to convey information to the cell

is thought to involve β1 and β2 integrins (13). Therefore, it seems reasonable that adhesion

molecules play an important role in normal immune surveillance and in the pathogenesis of

tissue injury, including cell recruitment at the site of allergic inflammation. Earlier studies

have shown increased expression of ICAM-1, E-selectin and VCAM-1 on endothelium in

allergic asthma (14). Meanwhile, secretion of granule proteins from eosinophils and

neutrophils was increased in asthma and cystic fibrosis (15;16).

It has become evident that specialised CD4+ type 2 T-helper cells are important in the

pathogenesis of allergic inflammation (17). The Th2 cells produce cytokines (e.g., IL-3 IL-5

and GM-CSF) (18). These cytokines are important modulators or primers of eosinophil

function (e.g., degranulation, adhesion, survival and superoxide production) (19-21). It has

8

been reported that patients with asthma show altered surface expression of the adhesion

molecules on airway granulocytes compared with blood granulocytes (22).

Though the eosinophil is a central effector cell in allergic diseases, neutrophils are also found

in bronchial lavage from patients undergoing acute exacerbations that were not associated

with infection (23).

The aim of the present work was to develop a simple, reliable and convenient assay for the

simultaneous measurement of eosinophil and neutrophil adhesion and release. Most of the

studies in this field are based on separate experiments performed with either purified

eosinophils or neutrophils whereas these two types of cells coexist in the blood. The present

method may offer a useful tool for comparative studies on eosinophils and neutrophils from

different patient groups. The method should also be helpful in characterising the effect of

inflammatory mediators or pharmacological agents on the selective adhesion and release of

granule contents in different types of granulocyte. As an important priority, we wanted to

assay the function of eosinophils within the normal population of granulocytes, making it

possible to perform a large number of experiments during states approaching physiological

conditions. As a comparison, neutrophil adhesion and degranulation of granule proteins were

measured. To investigate whether priming in vivo affects adhesion and degranulation

eosinophils from atopic patients exposed to allergens were compared with granulocytes from

healthy individuals. Adhesion to extracellular matrix proteins (ECM) and release of ECP were

investigated. In an attempt to mimic the situation in vivo, IL-5 and GM-CSF were used to

prime normal eosinophils in vitro. We then investigated the effect of this procedure on

eosinophil adhesion and release after further stimulation by PMA, PAF and Mn2+.

GENERAL BACKGROUND

1. Asthma and allergic diseases

Asthma is a common disease in the industriliazed world, for instance affecting 4 to 10% of

the population in the United States (24). It is an inflammatory disease involving mast cells,

antigen-presenting cells, macrophages, eosinophils, neutrophils, Th2-lymphocytes and

epithelial cells.

9

Blood and local tissue eosinophilia are outstanding features of allergic diseases such as

asthma. The evidence for the contribution of the eosinophil and eosinophil-elaborated

mediators to the pathophysiology of these disorders is increasing (25). This observation is one

of the cornerstones of the current hypothesis suggesting a central role for eosinophil-derived

mediators in causing asthma and related allergic diseases. Accordingly, selective

accumulation of eosinophils in the airways in allergic diseases has attracted significant

attention as an important mechanism underlying the diseases. However, the exact role of

eosinophils in the pathophysiology of chronic allergic disease remains obscure. The

recruitment of eosinophils to inflamed tissues is a multi-stage process that entails eosinophil

priming (26), adhesion, migration, generated cytokines, degranulation and survival (4;27;28).

10

Figure 1. Schematic representation of a multi-step paradigm of eosinophil recruitment into an

inflammatory tissue site

Eosinophils: Biology and Role in Diseases

FlowRolling

Activation

Transmigration

Selectins/MucinsP-selectin/PSGL-1VLA-4/VCAM-1α4β7/MadCAM-1

Chemoattractants PAF, IL-5, ChemokinesIntegrins/Ig family

Integrins/Ig familyMac-1/ICAM-1VLA-4/VCAM-1

Interaction withmatrix proteins

CytokinesLipid mediatorsGranule proteinsNew receptors VLA-4/Fibronectin

VLA-6/Laminin

Inflammatory site

11

2. Eosinophil granulocytes

Since Paul Ehrhich’s discovery of eosinophils in 1879, eosinophils have been found to be

associated with helminth infection, bronchial asthma and other allergic diseases (29).

Eosinophils play a principal role during the late-phase reaction in allergic inflammation,

accounting for about 30% of the accumulated cells (30); in the blood; however, only 1-4% of

all leukocytes are eosinophils. The influx of eosinophils in the late-phase response has been

shown to contribute to the chronic inflammation of the bronchial mucosa that characterises

persistent asthma (31-33). On the other hand, eosinophils are normally not present in lung

tissue. However, eosinophils are primarily tissue-dwelling cells in the lamina propria of the

normal gut, where they appear as intact cells. In helminth and allergic diseases eosinophils

often undergo massive degranulation. Preformed and newly formed eosinophil-derived

inflammatory mediators including cationic proteins like ECP, EDN, MBP and EPO (34), lipid

mediators such as LTC4, LTD4 and PAF, and oxygen metabolites (35-37) are thought to cause

many of the symptoms observed in allergic disease (25). The fact that eosinophils accumulate

in large numbers despite being present in peripheral blood at much lower levels suggests that

a pathway exists that selectively permits their local influx and survival. The mechanisms

underlying the selective eosinophil accumulation at the sites of inflammation and the

activation and release of mediators are, however, still poorly understood.. A multi-step

process is thought to be involved (see below).

a. Increased eosinophilopoiesis and release from the bone marrow (38;39).

b. Adhesion to endothelium and matrix proteins via the adhesion receptor VLA-4 and

selective priming of eosinophil adhesion by cytokine IL-5 (4;40).

c. Regulation of eosinophil migration into the tissue by eosinophil chemoattractants,

particularly chemokines (41;42).

d. Maintenance of eosinophil numbers in the tissue by prolonged survival, i.e. inhibition of

apoptosis (43).

The differentiation and proliferation of eosinophils are controlled by IL-3, GM-CSF and IL-5.

All these growth factors are also capable of priming eosinophils for enhanced biological

functions, such as increased mediator release and prolonged survival (3;20). The list of

stimuli that can elicit rapid activation of eosinophils includes antibodies, complement-derived

opsonins and anaphylatoxins, PAF, leukotrienes, formylated methionyl peptides and other

chemoattractants.

12

Table 1. Activation and Regulation of Eosinophil FunctionActivators & regulators Degranu

lation

Production

of lipid

mediators

Priming Chemotaxis/chemokinesis

Adhesion Survival Cytokineproduction

IgGIgEIgA

↑↑↑

↑↑

↑ ↑↑↑

C3aC5aZymosan

↑↑↑ ↑

↑↑ ↑

PAFLTB4

↑ ↑ ↑ ↑↑

↑ ↑

IL-3IL-4IL-5GM-CSFTNFα

↑↓↑↑↑

↑

↑↑

↑

↑↑

↑

↑↑

↑

↑↑↑

↑↑

IL-8RANTES ↑

↑↑ ↑

β2-integrinligandsFibronectinVCAM-1

↑ ↑

↑↑

↑

↑↑

↑

FMLPPMA

↑↑

↑ ↑ ↑↑

↑

3. Neutrophil granulocytes

Neutrophils are known to be involved in the defence of the body against bacteria, fungi and

virus. They are the first cell type to enter the lung following allergen challenge (44-46).

Unlike eosinophils, the influx of neutrophils is transient after allergen challenge. Furthermore,

high neutrophil counts are not only observed in bronchial lavage from patients undergoing

acute exacerbations that were not associated with infection, but are also present in high

numbers in the airways of patients with chronic severe asthma (47). Neutrophils are also

13

found to be another major participant in late-phase reactions. One of the mechanisms

underlying the attraction of neutrophils to the area of a type Ι reaction is probably by a

neutrophil chemotactic factor that is released from degranulating mast cells (30).

Neutrophils can produce a wide range of products, including lipids (LTB4,PAF), cytokines

(IL-8, TNFα), proteases (elastase, collagenase) and microbicidal products (lactoferrin, MPO)

(47). The granule proteins secreted by neutrophils are compartmentalised into at least three

distinct granule populations: primary, specific and tertiary granules. Primary granules contain

MPO (myeloperoxidase), elastase and BPI (bactericidal/permeability-increasing protein);

specific granules comprise lysozyme, lactoferrin and HNL (human neutrophil lipocalin); and

tertiary granules include gelatinase. Specific granules have been characterised as secretory

granules that are known to play an important role in the initiation of the inflammatory

response (48).

Specific granules and tertiary granules are associated with diapedesis and phagocytosis via

several mechanisms listed below (49):

a. Primary contact between endothelium and circulating neutrophils is established via

selectins and their ligands. The contact causes neutrophil to roll along the activated

endothelium.

b. Integration of the membrane of secretory vesicles and its associated CD11b/CD18

molecules into the cell membrane enhances the potential of the neutrophils for firm

adhesion to endothelium.

c. Exocytosis of gelatinase from tertiary granules may facilitate degradation of type IV

collagen in basement membranes.

d. Mobilisation of specific granules to the surface membrane may enhance the phagocytic

potential of the neutrophils by providing CD11b/CD18, i.e. the C3b-receptor.

4. Extracellular matrix proteins

The ECM is a relatively stable structural matrix that underlies epithelia and endothelia and

surrounds connective tissue cells. During adhesion to endothelium and subsequent

transendothelial migration, leukocytes encounter matrices and bind to components of the

ECM via integrins and other receptors on their surface (6). The ECM has been shown to

significantly affect a number of different leukocyte responses to inflammatory stimuli. For

instance, eosinophil degranulation and activation induced by PAF, C5a and IL-5 was

14

attenuated by fibronectin and laminin compared with fibrinogen (11). Furthermore, when

granulocytes become adherent to ECM proteins or endothelial cells, they are thought to be

primed to respond to additive stimuli (e.g., cytokines or chemoattractants) (50;51). Although

the function of cell-matrix interaction in inflammation remains ambiguous, some observations

indicate that fibronectin and collagen are found in increased amounts in the bronchoalveolar

lavage fluid or lung tissue of children with obstructive bronchitis (52). Recent evidence

suggests that diseases characterised by airway inflammation (e.g., asthma and chronic

obstructive lung disease) are also associated with increased ECM deposition (53).

The ECM participates in the following areas:

A. Attachment of cells

B. Tissue growth and repair

C. Proliferation and differentiation

D. Cell migration and activation

E. Cell survival/delay of apoptosis

F. Chemotaxis

G. Degranulation

5. Adhesion

Granulocyte adhesion during an inflammatory process involves a series of interactions as the

cells migrate into the tissue. First, there is adhesion to vascular endothelium followed by

interaction with an extracellular matrix and resident cells. Each of these steps involves a

distinct set of cell surface receptors. The first step, in which leukocytes tether and roll along

the vascular endothelium, is now recognised to be selectin-dependent, including E-selectin, L-

selectin and P-selectin (54). Following this, β2 and β1 integrin expressions on the surface of

eosinophils and neutrophils are rapidly increased (55;56), whereas ICAM-1 and VCAM-1 on

the surface of the endothelium are activated and increase their expression (14;57;58). This, in

turn, leads to firm adhesion between the leukocytes and endothelium. Finally, the cells reach

the site of inflammation and come in contact with ECM (e.g., fibronectin and laminin) via

integrins, which are cell surface receptors. Eosinophils and neutrophils express β2 integrins:

CD11a/CD18, CD11b/CD18 and CD11c/CD18. Although β2 integrins, especially

CD11b/CD18, play an essential role in the adherence of both cell types to albumin (59;60)

and fibrinogen (61), eosinophils also express several β1 integrins, particularly CD49d/CD29

15

(VLA-4), which neutrophils do not express (62-64). VLA-4, the α4β1 integrin and α4β7,

which mediate eosinophil adhesion to VCAM-1 and fibronectin (57;65), may account for the

selective accumulation of eosinophils to the site of allergic inflammation. However, VLA-4 is

also expressed by monocytes and subgroups of lymphocytes, implying that several pathways

are involved in the selective accumulation of eosinophils (66;67).

6. Adhesion molecules

Multiple receptor systems mediate a variety of leukocyte adhesion capabilities. The cell

adhesion receptors are usually transmembrane glycoproteins that mediate binding interactions

at the extracellular surface and determine the specificity of cell-cell and cell-ECM

recognition. They include members of the integrin, cadherin, immunoglobulin, selectin and

proteoglycan superfamilies (68).

Since their initial discovery by Springer and others in the past decade (69), adhesion

molecules have been shown to have pivotal roles in normal immune surveillance and

function, as well as in the pathogenesis of tissue injury (70). The interaction of granulocytes

with ECM is mainly mediated by receptors of the integrin family (71). For example,

neutrophils are thought to bind to collagen via the β2-integrin CD11b/ CD18 and the β1-

integrins VLA-1 and VLA-2 and to laminin via β2-integrins and the β1 integrins VLA-5 and

VLA-6. Eosinophil adhesion to fibrinogen has been found to be mediated by β1 and β2

integrins and neutrophil adhesion to fibrinogen by the β2 integrin CD11b/CD18 and the β1

integrin VLA-5. Neutrophil adhesion to fibronectin is mediated by β2 integrins and by the β1

integrin VLA-5, whereas eosinophil adhesion to fibronectin is mediated by VLA-4 and α4β7

(12;72-75). In addition, recent evidence suggests that integrins, particularly the β2 integrin,

are capable of modulating the eosinophil degranulation response stimulated with various

physiologic secretagogues (76).

In contrast to the principal mechanism of modulation of the selectins and immunoglobulin

family adhesion receptors, which is accomplished through changing the number of expressed

receptors, integrin function is regulated by the conformational state (77).

16

Table 2. Matrix binding integrins expressed by leukocytes

Abbreviations: Coll-collagens, FN-fibronectin, VN-vitronectin, Fg-fibrinogen

LM-laminin, TS-thrombospondin, Alb-albumin

7. Degranulation

In 1879, Paul Ehrlich suggested that eosinophil granules were storage depots of materials that

were released into the environment when needed. The granule-derived proteins include EDN,

MBP, ECP and EPO. Accumulating evidence shows that the protein components of

eosinophil granules mediate the tissue damage (78) associated with allergic diseases. This

finding has focused an increasing interest on the regulation of the eosinophil degranulation

response.

The mechanism underlying the release of granule protein is poorly understood, but a wide

range of stimuli can induce human blood eosinophil release in vitro, particularly when

eosinophils are adherent. Accordingly, adhesion molecules may be involved in eosinophil

release (76). Several investigations have shown that antibodies to CD11b/CD18 inhibit

eosinophil release stimulated with different stimuli, such as the lipid mediator PAF and the

β 1-Subfamily α1β1 Coll, LM α2β1 Coll, LM α3β1 FN, Coll, LM α4β1 FN α5β1 FN α6β1 LM αvβ1 FN, VN

β2 -Subfamily αMβ2 (CD11b/CD18) Fg, Alb αXβ2 (CD11c/CD18) Fg αdβ2 (αd/CD18) VCAM-1, FN (?)

β3 -Subfamily αVβ3 VN, TS, Fg

β7-Subfamily α4β7 FN

Integrins Ligands*

17

cytokine GM-CSF (60). The finding of eosinophil granule proteins in diseased tissue or on the

surface of the parasite, where few or no intact eosinophils were seen, has suggested eosinophil

degranulation to be associated with cell death (79). From the results described above, it is

evident that the degranulation process can be affected by adhesion. Furthermore, in resting

neutrophils, 75% of Mac-1 is localised in gelatinase granules (tertiary granules) and specific

granules, 20% in secretory vesicles and 5% on the plasma membrane (80). It is also

biologically important that exocytosis of those compartments is regulated by different signals.

8. Eosinophil Priming

Priming of an eosinophil response is defined as an increased reaction to a particular stimulus

by an agent that, in itself, induces little or no response. It has previously been demonstrated

that eosinophils in the peripheral blood of patients with allergic disorders have a primed

phenotype that resembles eosinophils from normal donors primed in vitro with cytokines (18).

Priming factors for eosinophils include neuropeptides (CGRP, substance P) and PAF,

chemokines like RANTES, IL-8 and eotaxin, but the most essential proteins are the

eosinophilopoietic cytokines, of which GM-CSF is the most potent and IL-5 the most

eosinophil-specific (21;18; 26).

Despite the knowledge of the importance of priming of eosinophils, little is known about the

signals that mediate this priming response. Furthermore, over the years the distinction

between primed and activated eosinophils has been somewhat unclear. The shift in density of

eosinophils toward light density or hypodense eosinophils is a well known phenomenon,

which is related to such eosinophilic diseases as allergic asthma, allergic rhinitis and parasitic

diseases (81-83). The low density eosinophils are presumed to represent primed eosinophils,

but the literature is far from conclusive (84).

9. Cytokines

Cytokines are low-molecular-weight glycoproteins that, when released at sites of

inflammation, induce their biological effect by binding to cytokine cell surface receptors.

They regulate a number of cellular functions important to allergic inflammation. Some of

these functions include the immunoglobulin E (IgE) synthesis, expression of adhesion

molecules by endothelium and inflammatory cell function (85). Eosinophils have functional

18

receptors for IL-3, GM-CSF and IL-5 that can regulate cellular function (degranulation,

adhesion, survival, superoxide production, etc.) (19;20).

Recent kinetic data from patients undergoing antigen challenge showed that IL-5 levels

increased between days 2 and 7 post challenge, whereas GM-CSF peaked at day 2 and

remained elevated through to day 16 (86). Furthermore, recruitment of eosinophils is

associated with elevated IL-5 in the airways of asthma patients (87). Blood eosinophils from

allergic subjects have an enhanced ability to transmigrate across endothelial cells in vitro, a

process that involves in vivo priming by IL-5 and related cytokines (21).

IL-5, IL-3 and GM-CSF act on eosinophils at three levels (88):

• Stimulation of the production from bone marrow

• Stimulation of functional activity

• Prolongation of survival of eosinophils at the inflammatory site

10. Cell death

Cell death is the process that culminates with cessation of biological activity. In contrast to

cell quiescence or dormancy, which is also characterised by decreased rates of many

biological activities, the changes that accompany cell death are irreversible. It is generally

accepted that apoptosis and necrosis are two distinct, mutually exclusive, modes of cell death.

Apoptosis is an active and physiological mode of cell death, in which the cell itself designs

and executes the program of its own demise and subsequent body disposal (89). This evidence

points to the importance of apoptosis as a component of processes designed to limit and

resolve inflammation.

Eosinophil apoptosis, like neutrophil apoptosis, occurs constitutively in vivo and the cells are

recognised by macrophages by a specific process without discharge of their histotoxic

contents. The regulation of eosinophil survival and apoptosis seems to play a major role in

diseases, demonstrating increased numbers of circulating and tissue eosinophils such as

allergic reactions (43). The presence of eosinophils or their granule proteins, or both, was

observed in diseased tissues or on the parasite’s surface. Frequently, few or no intact

eosinophils are seen despite extensive deposition of the eosinophil granule major basic protein

(MBP) and ECP in diseased tissues (90). This observation suggests that either the eosinophils

degranulate and leave the tissue or degranulate and die. However, some studies show that cell

19

death is not always accompanied by the classical features of either apoptosis or necrosis but

had features of both (91-93). It has been reported that PMA-treated neutrophils show

morphological changes quite different from those of typical apoptosis or necrosis (94).

20

AIMS OF THE STUDY

There were two aims of this investigation. The first was to develop a reliable, convenient and

specific method for the simultaneous analysis of eosinophil adhesion and release of ECP by

granulocytes that adhered to ECM. The second aim was to apply the method in experimental

and clinical studies. This would extend our knowledge of selective accumulation of different

types of granulocyte into the site of various inflammatory reactions. The specific aims of this

study were the following:

1. To compare the adhesion of eosinophils and neutrophils to fibronectin, fibrinogen and

albumin.

2. To study the relationship between eosinophil adhesion and release of ECP from

granulocytes that adhered to ECM.

3. To investigate the relationship between the release of ECP and eosinophil death.

4. To compare eosinophil adhesion and ECP release from granulocytes and purified

eosinophils that adhered to ECM.

5. To compare degranulation from primary and secondary granules of neutrophils that

adhered to ECM.

6. To compare the eosinophil priming in vivo and in vitro on eosinophil adhesion and

simultaneous release of ECP.

21

4. MATERIAL AND METHODS

Figure 2. Summary of the method used in the present studies

Whole blood

Normal population granulocytes or further purified eosinophils

The cells in coated 96 wells

Adherent cells

Radioimmunoassay of ECP and MPO

Relative % of adherenteosinophils and neutrophils

Percoll gradient (67%)

Priming (GM-CSF, IL-5)Incubated with / without stimuli(PMA, PAF, f-mlp, Mn2+,partilces)

Non-tissue culturetreated and ECM

Flowcytometry

Ab

Cell death

Supernatant

CTABadded

Supernatant

Relative % of released ECP and MPO

22

Isolation of granulocytes (papers I- IV)

Granulocytes were isolated from peripheral blood of non-allergic, normal volunteers or atopic

patients by Percoll (Pharmacia & Upjohn Diagnostic AB, Uppsala, Sweden) gradient

centrifugation as previously described (95;96). In a 50 mL albumin-coated tube ~18 mL

heparinized blood was mixed with an equal volume of PBS, and 15 mL Percoll (67%) was

applied at the bottom of the tube. After centrifugation at 1000 x g for 30 minutes at room

temperature, the mononuclear leukocytes on top of the Percoll gradient and the Percoll

gradient were removed. The remaining cell suspension in the bottom of the tube contained

granulocytes and erythrocytes. The erythrocytes were lysed by the addition of 40 mL of

NH4Cl (155 mmol/L) and incubated on ice for 15 minutes. The purified granulocytes (≥99%)

were washed twice with the assay buffer plus 1% (w/v) HSA and then suspended in the same

buffer. The cell concentration was 1 × 109 cells/L. The eosinophil content in the purified

granulocytes was 6% ± 2% (SD)

Purification of eosinophils (papers I, II)

Granulocytes, isolated as above, were incubated with anti-CD16-labelled magnetic beads in

PBS plus 2% NBS for one hour at 4° C (10 × 106 cells: 15 µl particles: 30 µl PBS + 2% NBS).

Resuspended cells (PBS + 2% NBS) were loaded onto the separation column positioned in a

strong magnetic field. The neutrophils bound to the anti-CD16 labelled magnetic beads

remained in the column while the eosinophils were eluted. The pure eosinophils (≥99%) were

diluted to a concentration corresponding to that in the granulocyte suspension from which

they were isolated.

Coating of wells (papers I-IV)

Ninety-six well, flat-bottom, non-tissue culture treated, high protein binding plates (Greiner

Labortechnik, Germany) were coated overnight at 37°C with 50 µg/ml fibronectin (both

plasma and tissue form) or albumin, or with 25 µg/ml fibrinogen (all matrix proteins were

from Sigma Chemical Co. St. Louis, Mo) in coating buffer, 100 µl/well. Before use, the wells

were washed twice in the assay buffer supplemented with 1% HSA. Finally, one more

washing was done with the same buffer in the absence or presence of Mn2+ (5 mmol/L).

23

Stimulation of cells (papers I-IV)

The stimulating agents tested were PMA, PAF, f-MLP, Mn2+ and serum-opsonized Sephadex

G-15 particles. The serum-opsonized Sephadex G-15 particles were prepared as described

previously (15). Suspensions containing 1 × 109 granulocytes/L and PMA, PAF, f-MLP or

Mn2+ at the concentrations of 10-9 mol/L, 10-7 mol/L, 10-7 mol/L and 5 mmol/L, respectively,

were added to the wells. For the particle stimulation, one volume of washed serum-opsonized

Sephadex G-15 particles was mixed with two volumes of granulocytes, with or without Mn2+

(5 mmol/L). The final concentration of cells was 1 × 109cells/L and the final concentration of

the serum-opsonized Sephadex G-15 particles was 27.8 g/L.

Priming of granulocytes (papers IV)

Granulocytes 1×109 cells/ml from normal donors were primed with IL-5 or GM-CSF for 20

minutes at 37°C in 5% CO2 in a humidified atmosphere. IL-5 was used at a concentration of

10-12 mol/L. GM-CSF was used at a concentration of 10-14 mol/L. The selected concentration

were found to be optimal for priming in preliminary experiments.

Adhesion assays (papers I- IV)

After incubation at 37° C in a humidified atmosphere containing 5% CO2 for the tested time,

the non-adherent cells were removed by three repeated washes with a room temperature assay

buffer. Adhering cells were then solubilized by the addition of a 100µl CTAB detergent

solution (5 g CTAB/L, 9 g NaCl/L). The supernatants of the extracts were harvested and

centrifuged at 600 × g for 10 minutes. The supernatants were collected again and frozen at

-20°C until assayed for the content of ECP (eosinophil marker) and MPO (neutrophil marker).

The total intracellular ECP and MPO contents extracted from granulocytes or from purified

eosinophils, solubilized with CTAB detergent solution, were measured in parallel. Results

from the adhesion experiments were expressed as the percentage of the total amount (100%)

of ECP and MPO in the respective population of eosinophils and neutrophils, which

corresponds to the relative amount of eosinophils and neutrophils.

24

Release assay (paper II- IV)

The samples for determination of the release of granule contents were collected from adhesion

experiments. After incubation, the supernatants were transferred to tubes before washing

away the non-adherent cells. These tubes were centrifuged at 600 × g for 10 minutes at room

temperature. The supernatants were carefully collected and mixed with an equal volume of

CTAB detergent solution and frozen at -20°C until assayed for ECP and MPO by

radioimmunoassay. The release of ECP was calculated according to the same principle as the

calculation of the relative amount of adherent eosinophils.

Radioimmunoassay (papers I- IV)

The ECP and MPO concentrations of the supernatants were analysed by a double antibody

competition radioimmunoassay(RIA) as described earlier (Pharmacia & Upjohn Diagnostics

AB, Uppsala, Sweden) (97;98).

Flow cytometry (paper II)

To observe the relationship between the release of granule contents and cell death

granulocytes were stimulated with different stimuli in plasma fibronectin or fibrinogen-coated

wells, as above. After incubation, optimally titrated FITC-labelled mAbs CD9 and propidium

iodide were added and afterwards plates were incubated for another 30 minutes at 4° C. The

EDTA-solution was added to detach the adherent cells (details are described in paper II). An

EPICS XL flow cytometer (Coulter Corporation, Miami, FL) was employed to analyse the

cells. Eosinophils were identified as CD9-positive cells (99). The relative amount of

eosinophils and neutrophils stained with propidium iodide was measured.

Assessment of granulocyte death (papers II- Ш)

The trypan blue exclusion assay was applied to assess the amount of dead granulocytes. Fifty

µl PMN suspension with different stimuli were incubated in plasma fibronectin- or

fibrinogen-coated wells for 90 minutes. After aspiration of the supernatant, 50 µl trypan blue

solution was added for 5 minutes and then aspirated. Next, 100 µl of PBS was added to each

25

well and the relative number of cells stained with trypan blue was evaluated blindly with an

inverted microscope.

Inhibition of release and adhesion by monoclonal antibodies (paper II)

The inhibitory effect of mAbs against CD18 and CD49d was tested by co-incubation of

granulocytes or purified eosinophils with antibodies (final concentration 10 µl/ml) and

activating agents for 90 minutes.

Patients (paper IV)

Eleven birch pollen-atopic patients with asthma (seven men and four women), with a mean

age of 39 years (range, 20 to 51 years), were selected for the study. The patients were

considered atopic because of positive skin tests to birch allergen. None of the patients were

smokers and all patients had a positive methacholine test. Blood was collected during early

pollen season from March to April. The range of eosinophil counts was 60 - 520 x 106/L

(median 210 x 106/L).

References (paper IV)

The reference group consisted of 9 healthy, non-allergic, laboratory employees and normal

blood donors matched for age and sex with the patients. The range of eosinophil counts was

60 – 160 x 106/L (median 135 x 106/L).

Statistical analysis (papers I- IV)

Statistical analyses were performed using Student’s paired t test, one-way ANOVA, Mann-

Whitney U test, and Wilcoxon’s paired test. Statistically significant differences were assumed

when p< 0.05. All calculations were done using CSS Statistica (Statsoft Inc., Tulsa, OK).

26

5. RESULTS AND DISCUSSION

Development of a method for the simultaneous analysis of eosinophil adhesion and ECP

release (I,II)

The measurement of ECP and MPO has been used in earlier research to quantitatively

estimate eosinophil and neutrophil involvement (97;98). In the present study, the number of

adherent eosinophils and neutrophils was quantified indirectly by expressing the amount of

eosinophil cationic protein (ECP) and myeloperoxidase (MPO) extracted from the adherent

fraction of cells in percentage of the total amount of ECP and MPO extracted from the

granulocytes added to the wells. Degranulation was quantified in the same way.

The cellular content of ECP has been estimated to be 10 µg/106 eosinophils (100). The normal

level of eosinophils in peripheral blood is about 1-4% of all leukocytes. The RIA measures

ECP within the range of 2-200 µg/L. In all experiments, the concentration of 1 x

109granulocytes/L was used. Thus, although there was only 1% of eosinophil adhesion or

release, the concentration of ECP in supernatants was enough for measurement by the RIA.

The adhesion of purified eosinophils was compared with that of eosinophils in the normal

granulocyte population. At a short incubation time (15 minutes), eosinophils in the

granulocyte population showed a significantly higher basal adhesion to both fibronectin and

fibrinogen. The results indicate that the dominating presence of neutrophils might induce a

short time enhancement of the spontaneous adhesion of eosinophils. After a prolonged

incubation period (90 minutes), purified eosinophils evidenced a higher adhesion to

fibronectin induced by Mn2+ and to fibrinogen induced by PMA as compared with eosinophils

in the granulocyte population. Comparison of the release of ECP from granulocytes and

purified eosinophils adhered to fibronectin and fibrinogen showed a higher release of ECP

from purified eosinophils than that from eosinophils when induced by serum-treated

Sephadex G-15 particles. The overall difference between isolated eosinophils and eosinophils

in the normal granulocyte population was minor. The results indicate that the pattern of

responses of isolated eosinophils versus eosinophils in the granulocyte population to the

different stimuli was similar. In conclusion, the method allows the measurement of eosinophil

adhesion and degranulation by using granulocytes.

27

The effects of PMA, f-MLP and Mn2+ on eosinophil and neutrophil adhesion (I)

Depending on both the stimulus and the protein that the cells reacted to, induction of adhesion

by the activation of integrins revealed different patterns of eosinophil and neutrophil adhesion

to tissue and plasma fibronectin, albumin and fibrinogen. When the incubation time was 105

minutes, Mn2+ preferentially enhanced eosinophil adhesion compared with neutrophil

adhesion in the presence of these four proteins. Furthermore, background adhesion of

eosinophils to albumin was higher as compared with the other three proteins. These data are

compatible with the previous finding that approximately 50% of the eosinophils adhered to

albumin-Sepharose or albumin-coated plastic surfaces, as compared with only 25-30% of the

isolated neutrophils (101). The level of eosinophil adhesion, however, is lower in our study

than the level reported in the above study. The difference might be due to the use of

eosinophils purified to varying degrees and by slightly different procedures. Furthermore,

tissue fibronectin supported a greater eosinophil and neutrophil adhesion than plasma

fibronectin. This may be explained by the fact that tissue fibronectin, in comparison with

plasma fibronectin, contains approximately 50% more of the CS-1 cell binding region

recognised by the integrin α4β1 (102;103). The number of RGD cell binding domains

recognised by the integrin α5β1 (104) in tissue fibronectin is not higher than in plasma

fibronectin, however (27). This observation means that there are probably other structural

differences between plasma and tissue fibronectin to explain this phenomenon.

It has been demonstrated that PKC is important for eosinophil activation. For example, the

PKC activator PMA induces a variety of eosinophil functions, such as adhesion (105),

respiratory burst (106) and degranulation (107). In the present studies, PMA induced a similar

pattern of adhesion of both eosinophils and neutrophils to the four tested proteins. The results

are in agreement with studies that have shown that human neutrophils and eosinophils from

the peripheral circulation had similar average levels of activable PKC (108) and that PMA

removed the environmental restriction on eosinophil respiratory burst (109). This is probably

because PMA induces a direct stimulation of PKC of the cells, which would allow that the

receptor mechanism is bypassed (110).

28

It has been reported that chemoattractants differentially regulate the avidity of β1 and β2

integrins in eosinophils. For example, f-MLP stimulated the expression of an activation-

specific Mac-1 epitope defined by the CBRM1/5 mAB on the surface of eosinophils, but did

not upregulate the 9EG7 neoepitope, which is related to the β1 subunit (111). On the other

hand, neutrophils mobilise Mac-1 to a greater extent when responding to receptor-dependent

factors related to bacterial infections, although eosinophils had a larger total pool of Mac-1

than neutrophils (112). In accordance with these results, the present study showed that f-MLP

induced a very early and short-term increment of the adhesion of neutrophils to fibrinogen

and albumin but not to fibronectin. A lower f-MLP induced adhesion of eosinophils to

fibrinogen was also observed. Moreover, PMA amplified the effect of Mn2+ on the adhesion of

both eosinophils and neutrophils to these four proteins suggesting that Mn2+ and PMA exert

synergistic effects on granulocyte adhesion. This event is in contrast to Mn2+ in combination

with f-MLP which had no synergetic effect on granulocyte adhesion.

The effect of eosinophil adhesion on the release of ECP (II)

Evidence suggests that ligation of the β2 integrins triggers activation and degranulation of

human eosinophils (76) and that ECM can attenuate activation and degranulation of

stimulated eosinophils (11). These findings clearly indicate that cellular adhesion is important

for cellular degranulation. Our findings extend the knowledge that adhesion molecules are not

simply involved in cell-cell or cell-matrix interactions but may also modulate selective cell

functions (72). Manganese ions (0.5-15mmol/L), which are known to activate integrins

directly (113), did not induce any ECP release, regardless of the degree of adhesion and to

which protein the cells adhered. The results could be explained by the results of the study

performed in cell suspensions. That study demonstrated that Mn2+ had dual effects: it

stimulated the ECP release in the absence of Mg2+ and Ca2+ and it inhibited the release in the

presence of these actions (114). In our system, the buffer contains Mg2+ and Ca2+. PMA alone

induced a dose-dependent eosinophil degranulation, independent of whether the eosinophils

were adhering to fibronectin or fibrinogen. The implication of these results is that PMA

removes the influence of ECM on eosinophil degranulation, which has been demonstrated to

attenuate activation and degranulation of eosinophils stimulated by PAF (11). An intriguing

finding was that PMA at the concentration of 10-9 mol/L did not induce any significant release

of ECP, but this concentration of PMA in combination with Mn2+ (5 mmol/L) dramatically

29

enhanced the release of ECP. Furthermore, antibody inhibition studies showed that the release

of ECP induced by PMA and Mn2+ in combination was dependent on β2 integrins on

granulocytes that adhered to fibronectin and fibrinogen, whereas the adhesion of eosinophils

to fibronectin could be inhibited by both anti-β1 (α4) and anti-β2 (CD18) monoclonal

antibodies. These results indicate that adhesion and degranulation can be regulated by

different signalling pathways. The anti-CD18 inhibited adhesion of eosinophils to fibronectin,

previously considered β1 dependent, may be explained by the newly discovered αdβ2 integrin

(115). This integrin was recently described to function as an alternative ligand for VCAM-1

and, in analogy with VLA-4, might also function as a ligand to fibronectin (57).

A small but significant release of ECP was found when eosinophils adhering to fibronectin

and HSA, but not to fibronectin, were stimulated by f-MLP. Earlier investigations have

demonstrated that eosinophils adhere to fibronectin via β1 and β7 integrins, to fibrinogen via

β1 and β2 integrins and to HSA via β2 integrins (61;75;111;116-119). Consequently, the

difference in f-MLP induced release of ECP could plausibly be explained by a differential

regulation of β1 and β2 integrins (111;112). In another study it was found that when adherent

to laminin- and fibronectin-coated wells, degranulation of eosinophils (induced by PAF and

C5a) was reduced by about 50% compared with cells adherent to non-coated, HSA-blocked

wells (11). Furthermore, Mn2+ suppressed f-MLP induced eosinophil degranulation.

When induced by serum-opsonized Sephadex particles, extracellular release of approximately

12-14% of ECP from eosinophils occurred in the presence of both plasma fibronectin and

fibrinogen. In the presence of the serum-opsonized particles adhesion to protein-coated wells

was decreased. This preferential adherence to the particles could be explained by sterical

hindrance in the sense that the particles prevent contact between eosinophils and

fibronectin/fibrinogen. Another probable mechanism is the competition between C3b particles

and fibronectin/fibrinogen in the binding to eosinophils. In a study on the release induced by

serum-opsonized Sephadex particles, almost all eosinophils were found to adhere to the

particles (101). Furthermore, ECP release appears to be associated with cell death when it is

induced by PMA plus Mn2+ or serum-opsonized Sephadex particles. One earlier study has

demonstrated that if stimulated by protein-coated beads, eosinophil degranulation is

accompanied by cellular injury and necrosis (79). Further investigations are needed to

confirm the kind of cell death in our study, i.e. apoptosis or necrosis.

30

Comparison of purified eosinophils and eosinophils in the normal population of granulocytes

regarding the release of ECP in general demonstrated the same pattern of responses. The only

difference was that serum-opsonized Sephadex particles stimulated a slightly, but

significantly, higher release of ECP from purified eosinophils than eosinophils in the normal

population of granulocytes.

The effect of neutrophil adhesion on degranulation of primary and secondary granules

(Ш)

Neutrophils are the first cell type to enter the lung after allergen challenge and another major

participant in late phase reactions (30). It has also been shown that neutrophils help eosinophil

migration across lung epithelium (120). Furthermore, neutrophil release granule constituents,

lipid-derived mediators, and reactive oxygen intermediates (ROI) which are involved in

killing of pathogens and tissue damage (121).

In the present study, we found that PMA, in combination with Mn2+, induced a marked release

of ∼ 80% of the intracellular content of lactoferrin and HNL after 60-75 minutes of incubation,

regardless to which protein the cells were associated. Under the same conditions, a maximal

release of MPO of 15-20% was obtained after a 90-minute stimulation of neutrophils by PMA

and Mn2+. This finding is agreement with a report describing the hierarchy of the mobilisation

of granule populations. This hierarchy leads to a higher release of lactoferrin and HNL from

the secondary granules as compared with MPO from the primary granules (48). The

biological relevance of the observed lag-phase between primary and specific granule

degranulation could be to facilitate cell adhesion and migration to inflammatory sites.

Adhesion molecules are considered as stores in specific granules (80) and exocytosis of

gelatinase from tertiary granules may facilitate degradation of type IV collagen in basement

membranes. Depending on the protein that the cells were plated on, PMA or f-MLP alone

induced 30-40% release of lactoferrin and HNL after 60 minutes of incubation; fibrinogen

and HSA were observed to support a higher release of lactoferrin than fibronectin. The

difference can likely be explained by differences in signalling from β2 and β1 integrins.

According to recent findings, adhesion to albumin is mediated by CD11b/CD18 (122),

whereas adhesion to fibronectin is mediated by VLA-5 and possibly αdβ2 (115;123). Lastly,

31

adhesion to fibrinogen is mediated by both CD11b/CD18 and VLA-5 (61;74;75). It is also

known that neutrophils from patients with leukocyte adhesion deficiency (LAD) lacking

CD18, are unable to spread on thrombospondin, and also fail to release lactoferrin in response

to f-MLP (124).

Adhesion and release of lactoferrin and HNL were quantitatively related when induced by

PMA and PMA plus Mn2+, but not when they were induced by f-MLP. This pattern may result

because f-MLP is more of a chemoattractant and preferentially induces migration, whereas

PMA directly activates proteinkinase C, which would by-pass the early steps in physiological

ligand-receptor activation of PMN (125). Earlier study has reported that concanavalin A (Con

A) at low doses is a potent stimulus for adhesion and does not induce any O2- production,

whereas at high doses it stimulate both adhesion and O2- production (126). The mean release

of lactoferrin and HNL induced by different stimuli showed a significant negative relationship

to the viability of cells. Thus, adhesion is important for the regulation of degranulation, but

adhesion and degranulation are not necessarily quantitatively related. The results also indicate

that neutrophil responses have various transductional pathways that may depend on the type

of stimulus.

The effect of priming in vivo or in vitro on eosinophil adhesion and release of ECP (IV)

Granulocytes obtained from pollen-atopic patients during early pollen season were stimulated

by PMA, Mn2+ and serum-opsonized particles. Afterwards, adhesion and release of ECP from

eosinophils were measured. In an attempt to mimic the situation in vivo, IL-5 and GM-CSF

were used to prime normal eosinophils in vitro and the effect on eosinophil adhesion and

release further stimulated by PMA, PAF and Mn2+ was investigated.

When stimulated by PMA, eosinophils from pollen-atopic patients during early pollen season

exhibited greater adhesion to fibronectin and fibrinogen as compared with eosinophils from

the reference group. In addition, eosinophils from pollen-atopic patients during early pollen

season displayed greater release of ECP than eosinophils from the reference group. The other

stimuli did not induce this kind of differential response. These findings indicate that

eosinophils from atopic patients during early pollen season already possess a higher PKC

activity. This view concurs with the finding that low-density eosinophils from allergic

asthmatic patients possess higher PKC activity than high-density eosinophils (108). The

32

finding of a higher proportion of low-density blood eosinophils was among the earliest

evidence suggesting a primed eosinophil phenotype in allergic disease was (21). It has also

been shown that there is an increase in the expression of the PKC ζ isoform in eosinophils

after allergen challenge (127). The evidence from all these studies suggests that protein kinase

C (PKC) is involved in eosinophil priming in vivo.

The priming is likely to be caused by IL-3, IL-5 and GM-CSF in the peripheral blood, where

IL-5 is especially important in the recruitment and activation of eosinophils. It has been

demonstrated that IL-5 mRNA and protein levels were elevated in lung tissue and

bronchoalveolar lavage fluid from symptomatic asthma patients (18;128). Priming with IL-5

caused a significantly higher adhesion to fibrinogen and simultaneous release of ECP in

response to PMA at 90 minutes. In the same conditions simulation with PMA plus Mn2+ for

90 minutes enhanced the adhesion to fibrinogen. These differences between eosinophils

primed in vivo versus in vitro can be explained by varying effects of short- versus long-term

priming (129) and that eosinophils from the patients have been primed in vivo by

combinations of cytokines. It has been found that allergen challenge induces the late-phase

nasal responses and the local expression of Th2-type cytokines, IL-4 and IL-5 (130;131), as

well as proinflammatory cytokines such as IL-6 (132).

GM-CSF, which shares a common β chain of the receptor with IL-5, was observed to prime

eosinophil adhesion to fibronectin and fibrinogen in response to PAF or PMA plus Mn2+,

whereas IL-5 did not prime this kind of response. The results indicate that IL-5 and GM-CSF

can signal through cytokine-specific pathways not dependent on the common β chain.

Furthermore, priming with GM-CSF only slightly affected the release of ECP. IL-5, but not

GM-CSF, was found to prime eosinophil generation of O2- and intracellular calcium

movement (133); however, an increased release of ECP by eosinophils upon short-term (20

minutes) priming with GM-CSF and stimulation with PAF has also been reported (134) . It is

likely that different experimental conditions are responsible for these apparently conflicting

results.

In conclusion, eosinophils from pollen-atopic patients primed in vivo and normal eosinophils

primed in vitro displayed similar, although not identical, patterns of upregulation of

33

eosinophil adhesion and release. The fact that the responses to PMA were primed both in vivo

and in vitro suggests that PKC activity is involved in priming of eosinophils.

34

6. CONCLUSIONS1. Normal population granulocytes can be used to measure eosinophil adhesion and

degranulation.

2. Mn2+ preferentially enhanced eosinophil adhesion compared with neutrophil adhesion,

though it did not induce any release of ECP.

3. PMA stimulated a dose-dependent eosinophil adhesion and degranulation, regardless to

which protein the eosinophils adhered. Mn2+ amplified the effects of PMA on eosinophil

adhesion and degranulation.

4. When stimulated by f-MLP, eosinophil adhesion and degranulation were affected by the

matrix proteins to which eosinophils adhered. Co-stimulation with f-MLP and Mn2+

exhibited different effects on eosinophil adhesion and degranulation.

5. When induced by PMA and Mn2+, eosinophil degranulation was β2 integrin-dependent,

regardless of whether the eosinophils adhered to fibronectin or fibrinogen; adhesion to

fibrinogen was β2 integrin-dependent and adhesion to fibronectin was β1 and β2 integrin-

dependent.

6. Adhesion modulates neutrophil degranulation, but it is not always quantitatively related or

related in time.

7. PKC is involved in the priming of eosinophils.

35

7. ACKNOWLEDGEMENTSI would like to express my sincere gratitude to everyone who contributed to this work and in

particular to the following persons:

Associate Professor Lena Håkansson, for being my tutor and supervisor, for introducing me to

the field of granulocyte adhesion, for giving me the privilege of sharing her plentiful, unique

knowledge in cellular adhesion science and flow cytometer, for her patience in reading and

revising my manuscripts and for her concern about my daily life.

Professor Per Venge, head of the Department of Clinical Chemistry, my supervisor, for

sharing his vast knowledge of science and for giving me a chance to work and study in a

friendly and inspiring atmosphere which he has worked hard to create.

Gunilla Strömstedt and Barbro Bjurhäll, for offering superb secretarial assistance.

Agneta Trulson, my former roommate, for friendship and kindness.

Kerstin Lindblad, Ing-Britt Persson, Lena Moberg and Ulla-Britt Jönsson, for their excellent

work in interpreting Swedish into English and narrating Swedish culture and customs.

Charlotte Woschnagg, Jonas Byström, Kristina Sandström, Malgorzata Karawajczyk and

Maria Lampinen, for skilful technical assistance and friendship.

Lixin Liu, Linshu Zhao and Shengyuan Xu, my Chinese colleagues, for their friendship and

reminding me of my homeland.

All the staff at the Department of Clinical Chemistry for their warm friendship.

Yangzi Liu, my dear daughter, for creating a rich and colourful life for me outside the

laboratory during these years in Sweden.

Zhurong Liu, my husband, for his love, understanding and continuous support.

36

My parents, sisters and brother, for their expectations, encouragement and taking care of my

daughter in times of need.

This work was supported by grants from the following sources:

The Medical Faculty of the University of Uppsala

The National Association against Asthma and Allergy

The Swedish Foundation for Health Care and Allergy Research

37

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