eosinophil interaction with antibody-...

J. Cell Sci. 42, 367-378 (1980) 367Printed in Great Britain © Company of Biologists Limited igSo

EOSINOPHIL INTERACTION WITH ANTIBODY-

COATED, NON-PHAGOCYTOSABLE SURFACES:

CHANGES IN CELL SURFACE PROTEINS

KAREEN J. I. THORNE, RHONDA C. OLIVERAND AUDREY M. GLAUERTStrangeways Research Laboratory, Worts Causeway, Cambridge, England, CBi 4.RN

SUMMARY

Plasma membrane changes during the interaction of human eosinophils with large, antibody-coated, non-phagocytosable surfaces have been investigated in a model system. Humanperipheral blood eosinophils were incubated with layers of agar into which tetanus toxoidand human anti-tetanus immunoglobulin, together with an eosinophil chemotactic factor(ECF), were incorporated. Changes in organization of the eosinophil plasma membraneproteins during interaction with the agar layer were detected by lactoperoxidase-catalysediodination with [125]iodide.

A protein of apparent mol. wt 55000 became newly accessible on the eosinophil surface asa specific consequence of interaction with antigen-antibody complexes in the agar layer. Thisprotein appeared in the early attachment phase of the interaction which preceded extracellulardegranulation. Cytochalasin D enhanced its appearance, while Mg2+-deficiency prevented it.A second newly accessible protein of apparent mol. wt 58000 was blocked when ECF waspresent and may therefore be a receptor for ECF. Other proteins of apparent mol. wt 68000and 46000 newly appeared at the surface of eosinophils even after incubation in suspension,apparently as a consequence of the rapid cycling of membrane components which occurs ineosinophils.

INTRODUCTION

The discovery that eosinophils appear to play a major role in host resistance toschistosomula of Schistosoma mansoni (Butterworth et al. 1975; Butterworth, 1977)has drawn attention to the importance of the eosinophil as a cytotoxic cell. Whenhuman peripheral blood eosinophils encounter antibody-coated schistosomula theyattach to and flatten down closely onto the schistosomulum surface in an apparentattempt to phagocytose it (Glauert & Butterworth, 1977; Glauert, Butterworth,Sturrock & Houba, 1978). This frustrated phagocytosis is followed by release of theeosinophil granule contents onto the surface of the schistosomulum and by subsequentdamage both to the surface tegument and to the underlying muscle layers (Glauertet al. 1978).

In order to investigate the mechanism of eosinophil attachment to and degranu-lation onto large non-phagocytosable surfaces we have developed a model system inwhich eosinophils interact with human antigen-antibody complexes in agar layers(Glauert, Oliver & Thorne, 1980). The antigen-antibody complex used is tetanustoxoid with human anti-tetanus antiserum. In addition the eosinophil chemotactic

368 K. J. I. Thome, R. C. Oliver and A. M. Glauert

factor, ala-gly-ser-glu, (ECF) (Kay, Stechschulte & Austen, 1971; Goetzl & Austen,1975) is incorporated into the agar layer. Eosinophils flatten down closely onto thislayer and adhere more intimately to it than do neutrophils (Glauert et al. 1980).Both cell types then degranulate, as detected by the extracellular release of collagenase,peroxidase and /?-glucuronidase (Glauert et al. 1980). The behaviour of eosinophils onthe agar layer therefore resembles their behaviour on the surface of the schistosomu-lum. This model is now being used to elucidate the biochemical aspects of theinteraction of eosinophils, in comparison with neutrophils, with non-phagocytosablesurfaces. In the present study we have looked for changes in organization of theplasma membrane proteins of the eosinophil during the interaction. The plasmamembrane proteins accessible on the cell surface have been detected by lactoper-oxidase-catalysed iodination with [125I]iodide. Increase or decrease in surfaceaccessibility has been related to individual stages in the interaction of the eosinophilwith the non-phagocytosable surface.

MATERIALS AND METHODS

Eosinophil and neutroph.il preparation

Eosinophils and neutrophils were prepared from normal human peripheral blood by amodification of the method of Vadas et al. (1979). The details have been described elsewhere(Thome, Glauert, Svvennsen & Franks, 1979) but a brief outline of the method is given here.Heparinized venous blood (20 ml) was depleted of erythrocytes by sedimentation with 0-35 %(w/v) methyl cellulose in phosphate-buffered saline. Granulocytes were prepared from theleucocyte-rich supernatant by centrifugation on a Ficoll/Hypaque gradient. These wereseparated into neutrophils and eosinophils on a 12-ml Metrizamide gradient, 20-25 % (w/v) inHanks buffered salts solution (HBSS) containing o-i % (w/v) gelatin and 15 /tg/ml DNAse I(Sigma Chemical Co., Kingston upon Thames, KT2 7BH). After centrifugation for 45 min at400 g the upper white band of neutrophils and the lower red band of eosinophils were collectedand the Metrizamide removed by washing with HBSS containing o-i % (w/v) gelatin.

Attachment to agar layers

Thin layers of agar, containing IO~5M ala-gly-ser-glu tetrapeptide, an eosinophil chemotacticfactor (ECF) (Uniscience Ltd., Cambridge CB5 8BA), and tetanus toxoid (Lister Institute,Elstree, Herts., WD6 3AY) were prepared on glass coverslips as described by Glauert et al.(1980). Immediately before use the agar layers were treated with 10 times diluted human anti-tetanus immunoglobulin (Lister Institute) for 45 min at 37 °C. Excess immunoglobulin wasthen removed and the layers washed thoroughly with HBSS. Purified eosinophils (70-100%)or neutrophils (90-100%) in HBSS, containing o-i % (w/v) bovine serum albumin (BSA) oro-i % (w/v) gelatin were then allowed to settle onto and attach to the agar layers, supportedon glass coverslips in Falcon 3008 multi-cell tissue culture plates. After incubation for 15-60 min at 37 °C, the HBSS containing the unattached cells was removed and the layers werewashed once with HBSS, containing BSA or gelatin. The number of cells adhering to thelayers was estimated by counting the number of unattached cells present in the removed HBSSand the wash. After 15 min about 20 % of the added eosinophils were attached. The unattachedcells were then removed from the washes by centrifugation and the residual supernatant wasassayed for released granule enzymes.

[125I]Iodination of cell surface proteins

Eosinophils and neutrophils were pretreated with non-radioactive potassium iodide to blockpre-existing accessible tyrosine residues in the resting cells. The cells were incubated in

Eosinophil surface proteins 369

suspension in 1 ml HBSS with 20 /tg/ml lactoperoxidase (Sigma Chemical Co.), 004 units ofType V glucose oxidase (Sigma Chemical Co.), and o-i mil potassium iodide for 10 min atroom temperature. They were then washed 3 times with HBSS containing o-i % gelatin. Thecells were attached to agar layers on glass coverslips as described above. After removal of theunattached cells the cell monolayer was treated with 1 ml HBSS with 20 /tg/ml lactoperoxidase,0-04 units of glucose oxidase and about 100 /tCi L125I]sodium iodide for 10 min at roomtemperature. The iodinating solution was then removed and the cells on agar were washed 3times with HBSS containing o-i % gelatin. The 125I-labelled cells were solubilized with 0-2 mlof digestion mixture (4 % (w/v) sodium dodecyl sulphate (SDS), 20 % (v/v) mercaptoethanoland 0-09% (w/v) bromphenol blue in aqueous solution) at 100 °C for 1 min. The digest wasthen separated by polyacrylamide gel electrophoresis on rods of 75 % polyacrylamide ino-i % SDS and o-i M Tris/bicine, pH 83. After electrophoresis the gels were sliced intoi-mm-long segments and counted in a Packard PGD auto gamma counter. The gels werecalibrated with the following standard proteins: phosphorylase a (mol. wt iooooo), transferrin(mol. wt 78000), bovine serum albumin (mol. wt 68000), ovalbumin (mol. wt 42000), carbonicanhydrase (mol. wt 29000), soya bean trypsin inhibitor (mol. wt 21000) and cytochrome c(mol. wt 13000).

Electron microscopy

Eosinophils and neutrophils were incubated in 0-2 ml of HBSS and gelatin on agar layers onAraldite instead of on glass coverslips. The cells were then fixed in situ by the addition of20 n\ of 25 % glutaraldehyde and incubation was continued at 37 °C for 30 min. The glutar-aldehyde was then removed and the cells washed with o-i M cacodylate buffer pH 7-2 contain-ing 3 mM CaCL. The fixed cells on the agar layers were then processed for electron microscopyas described earlier (Glauert et al. 1980).

Enzyme assays

Peroxidase (E.C. 1.11.1.7) was assayed by the method of Migler & DeChatelet (1978)using p-phenylenediamine as hydrogen donor. /?-glucuronidase (E.C. 3.2.1.31) was assayedemploying phenolphthalein /?-D-glucuronide as substrate. The enzyme was incubated at 37 °Cfor 2 h with 1 mM phenolphthalein /?-D-glucuronide and o-i M sodium acetate buffer, pH 4-5.The reaction was stopped by the addition of 2 vol. of 1 M sodium carbonate and the amount offree phenolphthalein determined from its optical density at 555 nm.

RESULTS

Attachment of eosinophils and neutrophils to agar layers

Eosinophils and neutrophils differed markedly in the nature of their attachment toagar layers containing the chemotactic factor (ECF) and antigen-antibody complexes(Figs. 1, 2). After 30 min eosinophils had flattened down closely onto the surface ofthe agar layer while neutrophils made many contacts of limited area with interveningregions where the cell was not in contact with the agar. As already described (Glauertet al. 1980) attachment was usually complete in 30 min and this was followed bydegranulation. Release of granule enzymes from neutrophils commenced as soon asthe cells were added to the agar layers, while release of eosinophil enzymes began tobe detectable only after a lag of about 1 h.

Changes in plasma membrane proteins during interaction with agar layers

The surface proteins of human eosinophils which were accessible to lactoperoxidase-catalysed iodination proved to be more numerous than those previously observed on

Eosinophil surface proteins

3

1 2f - 1o

B

-

-

10 20

7 3

30 40 5

4

X£

jf 2

1

3

2

1

10

10

10

20 30 40 50

20 30 40 50

20 30 40 50 60

I

100 68 42 29 21 13Mol. wtX 10'3

Fig. 3. Lactoperoxidase-catalysed iodination of newly accessible cell-surface proteins.After iodination the proteins were separated by SDS-PAGE in 7*5 % polyacrylamide.The gels were sliced into i-mm segments and the radioactivity of each slice plottedagainst the distance along the gel. A, B, C, cells on agar layers containing antigen-antibody complex and ECF. A, eosinophils incubated for 15 min; B, eosinophilsincubated for 60 min; c, neutrophils incubated for 15 min; D, erythrocytes incubatedin suspension for 15 min.

rabbit peritoneal exudate neutrophils (Thome, Oliver & Lackie, 1977 a). In order,therefore, to detect changes in the organization of membrane proteins pre-existingaccessible tyrosine residues on the cell surface were first iodinated with unlabelledsodium iodide; the cells were then allowed to interact with the agar layers, unattachedcells were removed, and the attached cells were iodinated with [125I]iodide for detection

Figs. 1, 2. Electron micrographs of eosinophils and neutrophils adhering to agarlayers containing io"° M ECF and antigen-antibody complexes. Bar lines, 1 /im.

Fig. 1. After 60 min incubation an eosinophil has made close parallel contact withthe agar surface (ag).

Fig. 2. After 30 min incubation a neutrophil is only loosely attached to the agarsurface (ag) by broad pseudopodia.


of new groups on the cell surface. The cells retained their biological activity as judgedby their ability to release peroxidase during a subsequent 2-h incubation with theagar layers (30% released from untreated cells; 36% from iodinated cells). They alsoretained their ability to phagocytose Trypanosoma dionisii (Thorne et al. 1979). Afteriodination the radioactive proteins were separated by polyacrylamide gel electro-phoresis in SDS (Fig. 3). The results are expressed as radioactivity/io° cells attached

50

Fig. 4. Lactoperoxidase-catalysed iodination of newly accessible cell surface proteins.(Experimental procedure as Fig. 3.) Effect of ECF and antigen-antibody complexes.Eosinophils incubated for 15 min (A) in suspension for 15 min with ECF and antigen-antibody complex, (B) on agar layers containing antigen-antibody complex, and (c) onagar layers containing ECF.

to the agar layers. All experiments were performed at least twice, and the experimentshown in Fig. 3A was performed 6 times, with consistent results.

After eosinophils had interacted with antigen-antibody complexes on agar layersfor 15 min, 3 new proteins appeared at the cell surface (Fig. 3 A). These proteinswere designated 1 (apparent mol. wt 68000), 3 (apparent mol. wt 55000) and 4(apparent mol. wt 46000). The considerable amount of radioactive material moving


at the front is believed to be lipid. The appearance of the new proteins at the surfaceof the eosinophil appeared to represent an early response to the antibody-coatedagar layer, since if the cells were left for 60 min on agar very little new protein wasdetected (Fig. 3 u). Neutrophil labelling was much less than the labelling of eosinophils(Fig. 3 c). To eliminate the possibility that the new proteins detected on the eosinophilsurface were from contaminating erythrocytes the labelling pattern of erythrocyteswas determined (Fig. 3 D). It was found to be quite different from that of eosinophils.Nor could the labelling be attributed to the bovine serum albumin in the mediumsince the same results were obtained when albumin was replaced with gelatin. Indifferent experiments the radioactivity of the main peak 1 varied from 3 to 9 x io4

cpm/106 cells attached.

Eosinophils incubated in suspension for 15 min with ECF and antigen-antibodycomplexes produced only proteins 1 and 4 (Fig. 4A). These proteins of mol. wt68000 and 46000 appeared under all conditions tested, in the presence and absenceof antigen-antibody complexes and in the presence and absence of ECF.

If eosinophils were incubated with agar layers in the absence of ECF or in theabsence of antigen-antibody complexes some cells attached but the pattern of labellingof these cells was altered. In the absence of ECF an additional protein, 2 (apparentmol. wt 58000) could be detected (Fig. 413). This was not seen when ECF waspresent. In the absence of antigen-antibody complexes very little protein 3 wasfound (Fig. 4c).

Effect of inhibitors

Inhibitors of adherence and of degranulation were tested for their effect on themodification of the eosinophil surface proteins. The influence of these agents on theinteraction with antibody-coated agar layers, containing ECF, is summarized inTable 1. Little effect on the total number of cells attached was observed, although thenature of the attachment could be affected. For example, some cells may adhere muchmore intimately than others (see Glauert et al. 1980). Since extracellular release of thegranule enzyme peroxidase is believed to be consequent upon attachment to the agarlayer, the amount of peroxidase found in the medium after 2 h incubation is given bothas a percentage of the total cell content and as a percentage of that in the attached cells.

Cytochalasins inhibit both close adherence of cells to glass or plastic surfaces(Weiss, 1972) and phagocytosis (see review by Allison, 1973), but enhance extracellulardegranulation in the presence of aggregated immunoglobulin (Zurier, Hoffstein &Weissmann, 1973). Cytochalasin D was used in the present experiments in preferenceto cytochalasin B since it affects microfilaments without also inhibiting hexosetransport (Miranda, Godman, Deitch & Tanenbaum, 1974; Miranda, Godman &Tanenbaum, 1974). Attachment in the presence of cytochalasin D (Fig. 5 c) enhancedthe appearance of protein 3 and also revealed protein 2, which had previously beendetected only in the absence of ECF (Fig. 4). Cytochalasin D increased the amountof enzyme released on agar layers nearly 3-fold.

cAMP inhibits degranulation in neutrophils (Ignarro, Paddock & George, 1974),mast cells (Kaliner & Austen, 1974) and platelets (Maclntyre et al. 1977). In our


Table i. Effect of inhibitors on attachment of eosinophils to antibody-coated layersand on release of peroxidase

Agent

Cytochalasin D,io/tg/ml

005 mM dibutyryl

cAMP + o-os niMtheophylline

Ca2+-free + o-1 mMEGTA

Mg2+-free+ 1 m^iEDTA

Mg2+- and Ca2+-free + 1 mM EDTA

Attachment1

+ Agent,0//o

55

35

65

43

2 4

Eosinophils were incubated for 2 hand absence of the listed agents.

Control,%

69

4 2

5 i

5°

28

with agar

Total>1

+ Agent,%±S.D.

35 ±1°

I - 2 ± 0 ' 3

7-5 + 2-9

4 1 ± 1 3

0

Peroxidase release

cells

Control,%±S.D.

l6 + 2

5'2 ±2-4

5-1 ±2-9

49 ±05

2-6±o-s

Attached cells1

+ Agent,%

64

3

1 1

9'5

0

layers containing antigen-antibody and

Control,0//o

23

13

1 0

1 0

9

ECF, in the

Ratio

2 8

0 2 5

I - I

1 0

0

presence

agar model system dibutyryl cAMP and theophylline reduced peroxidase releasefrom adherent eosinophils by three quarters (Table 1), but had no effect on theappearance of new proteins on the eosinophil surface (Fig. 5 B), suggesting that theirappearance precedes and is not a consequence of degranulation.

Ca2+-depletion with EGTA in a Mg2+-containing medium not only had little effecton eosinophil attachment and degranulation (Table 1) but also had no detectableeffect on the appearance of new surface proteins (Fig. 6B). Mg2+-depletion alonealso had little effect, but elimination of both cations inhibited peroxidase releasecompletely (Table 1). However, it was the absence of Mg2+ which prevented theappearance of protein 3 (Fig. 6c).

DISCUSSION

The technique used in the present paper for observing changes in cell surfaceproteins is lactoperoxidase-catalysed iodination with [125I]iodide and hydrogenperoxide. Changes in platelet membrane proteins occurring during lipopolysaccharide-induced aggregation have been detected by a double-labelling method (Thorne,Oliver, Maclntyre & Gordon, 19776) where resting platelets were labelled with[125I]iodide and stimulated platelets were labelled with [131I]iodide and the ratio ofisotope in each membrane protein determined. This method worked well for rabbitplatelets since relatively few proteins on the control platelet surface were accessibleto iodination. Human peripheral blood eosinophils and neutrophils, however, had


too many proteins on the cell surface for clear separation and quantitation. For thisreason the cells were pretreated instead with unlabelled iodide to block the exposedtyrosine residues.

Human eosinophils in suspension in medium appeared to be in a dynamic stateand to be constantly bringing more of 2 proteins of apparent mol. wt 68000 (protein 1)

12

1012

8O

X

E

12

10

20 30 40 50

20 30mm

40 50

Fig. 5. Lactoperoxidase-catalysed iodination of newly accessible cell surface proteins.(Experimental procedure as Fig. 3.) Effect of cAMP and cytochalasin D. Eosinophilsincubated for 15 min on agar layers containing ECF and antigen-antibody complexes.A, no inhibitors; B, 0-05 mM theophylline; C, io/tg/ml cytochalasin D.

and 46000 (protein 4) to the surface. In this they differed from rabbit peritonealneutrophils, in which a new protein of mol. wt 150000 was detectable at the cellsurface only when the rate of membrane cycling was accelerated by phagocytosisfollowed by exocytosis (Thorne et al. 1977a). Eosinophils brought new proteins tothe surface even when they were not presented with a phagocytosable particle.Sanderson & Thomas (1978) showed by time-lapse photomicrography that ratperitoneal eosinophils show rapid membrane movement. The newly accessible


protein on the human eosinophil surface of mol. wt 68000 (protein 1) is unlikely tobe serum albumin from the medium since it was also detected when the experimentswere performed in gelatin.

An additional newly accessible protein of mol. wt 55000 (protein 3) appeared to bea specific consequence of interaction of the eosinophil with an antibody-coated, non-phagocytosable surface and may therefore be associated with, or be part of, the Fc

12

Fig. 6. Lactoperoxidase-catalysed iodination of newly accessible cell surface proteins.(Experimental procedure as Fig. 3.) Eosinophils incubated for 15 min with agarlayers containing ECF and antigen-antibody complexes. A, complete system; B,Caa+-free medium containing o-i mM EGTA; C, Mga+-free medium containing1 mM EDTA.

receptor in the eosinophil membrane. The position of protein 3 in the membranemay be partly controlled by links with intracellular microfilaments, since its appearanceat the cell surface is enhanced by cytochalasin D. The protein appears to be involvedin an early antibody- and Mg2+-dependent event preceding degranulation.

It is of interest that incubation in the presence of cytochalasin modifies themembrane changes induced in eosinophils during interaction with agar layers, while


cAMP has no effect, since both agents inhibit the killing of schistosomula by eosino-phils (David et al. 1977). Presumably cytochalasin interferes with the killing ofschistosomula by modifying the attachment of the eosinophil, while cAMP inhibitsby preventing the subsequent release of the toxic basic protein from the eosinophilgranules (Butterworth et al. 1979).

If the chemotactic factor (ECF) was omitted from the agar model system a secondnew protein of apparent mol. wt 58000 (protein 2) was exposed during interactionwith the antibody-coated agar layers. It is tempting to postulate that this might be areceptor for ECF and that in the complete system it is covered by ECF. Boswell,Austen & Goetzl (1976) have evidence that the eosinophil-reeeptor for the tetrapeptidecontains a hydrophobic domain which recognizes the N-terminal valine or alanine,a hydrogen-bonding domain which recognizes serine, and an ionic domain whichrecognizes the C-terminal glutamate.

The newly accessible proteins on the cell surface of the eosinophil may be dividedinto 2 groups. The first group of proteins, 1 and 4, seems to be constantly reappearingproteins on the eosinophil surface and to appear even on the cell in suspension.These proteins may be brought to the surface by the rapid cycling of membranecomponents resulting from endocytosis followed by exocytosis, of which the eosinophilis capable. The second group of proteins, 2 and 3, appears on the eosinophil surfaceas a consequence of its interaction with a non-phagocytosable antigen-antibodycomplex, and seems therefore to result from rearrangement of pre-existing plasmamembrane proteins. Protein 2 appears to be associated with the receptor for thechemotactic factor (ECF), while the appearance of the protein 3 is an early event inthe attachment of the eosinophil to non-phagocytosable antigen-antibody complexes,and appears to be involved in the initial antibody-dependent interaction whichprecedes extracellular secretion of lysosomal granule enzymes.

We thank Mr R. A. Parker for technical assistance, and The Wellcome Trust for financialsupport and for providing the Philips 201C electron microscope.

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(Received 2 August 1979)

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