The effects of chemotactic factors on the adhesiveness of rabbit neutrophil granulocytes

Copyright 0 1979by Academic Press, hc. All rights cf reproduction in any form reserved 0014-4827/79/090169-0~02.00/0

Experimental

THE EFFECTS ADHESIVENESS RONALD

Cell Research 122 (1979) 169-177

OF CHEMOTACTIC OF RABBIT

P. C. SMITH,’

FACTORS

NEUTROPHIL

JOHN M. LACKIE’

ON THE

GRANULOCUT

and PETER C. WILKINSON”

‘Department of Cell Biology, University of Glasgow, Glasgow G12 SQQ and ‘Department of Bacteriology and Immunology, University of Glasgow, Western Infirmary, Glasgow Gli 6NT, UK

SUMMARY A range of chemotactic factors has been shown to affect the adhesion of rabbit peritoneal neutrophil granulocytes to cultured endothelial cells and to serum-coated glass. At chemotactically optimal concentrations, cYs-casein,p-casein, alkali denatured human serum albumin (HSA) and several synthetic formyl-peptides reduced the number of adherent neutrophils after 30 min to around 50% of control values. These effects were still observed after neutrophils, but not endothelium or serum-coated glass had been exposed to chemotactic factors and washed before use in assays. Two non-chemotactic analogues, native HSA and a non-formyl-peptide were ineffective. The dose responses for adhesion after 30 min in the presence of Lu,-caseinand formyl-methionylleucyl-phenylalanine (FMLP) were found to be inversely related to those for migration towards these substances. After incubation for 60 min in high (10-8-10-7 M) concentrations of FMLP, neutrophil adhesion was found to be enhanced. Neutrophil aggregation was also affected by the presence of chemotactic factors in a similar time- and dose-dependent manner to the adhesion to substratum assays. Using FMLP, it was also shown that the timing of the adhesive changes depended on the concentration of chemotactic factor present.

During an acute inflammatory response, neutrophil granulocytes adhere to the endothelial cells which line the post-capillary venules. Since neutrophils do not normally adhere to blood vessel walls in large numbers, their localized adhesion in areas of inflammation suggests that a change in the strength of the neutrophil-endothehal interaction may occur. The cellular location of this change is not clear but evidence supports the involvement of both the endothelium and the neutrophils [ 11. Following adhesion to the endothelium, neutrophils migrate between the endothelial cells and towards the site of damage or infection. The direction of migration is probably influenced by diffusible chemotactic factors, although the evidence for this in

vivo, is largely circumstantial [2]. Adhesion is an essential prerequisite for cell movement, and the adhesiveness of the substratum may affect both the rate and direction of locomotion; this has been shown for fibroblasts [3, 4, 51 and is probably also t for neutrophils [6]. Thus, adhesion changes might be important during inflammation not only in the primary neutrophil-endothelial interaction, but also in the chemotacticahy guided migration of neutrophils. If chemotactic factors affect the adhesive properties of neutrophils then the two phases of a hesion and migration could be mediated by a common factor. Quantitative data on adhesion are very difficult and laborious to obtain in vivo. Accordingly, we have studied the effect on neutrophil adhesion of Exp Cell Rer 122 (197Yj

170

Smith, Lackie and Wilkinson

Table 1. Effect of chemotactic factors on the adhesion after 30 min at 37°C of neutrophil granulocytes to confluent endothelium and serum-coated glass % adhesion (compared to controls= 100%) k S.E.M. (n) Chemotactic factor a,-Casein p-Casein Alkali-denatured HSA Native HSA” f-tri-tyr f-met-leu-phe f-met-phe met-pheb

Cone Of)

Endothelium

Serum-coated glass

43i5 (6) 64+4 (i3j 57k8 (12)

625 3 (3)* 49+ 6 (6) 25f 6 (3)*

136i7 (14) 6327 (11)

143&10 (8)

4.2x 1O-5 4.2x 1O-5

1.4x 10-5 1.4x10-5 10-g 10-s 10-S 10-S

46f4 (22) 43+3 (30) 84+6(13)**

6Ok 592 55+ 83i

5 (16) 5 (23) 4(33) 5 (13)*

n, no. of replicate coverslips. Using Student’s r-test, p (difference from controls=O)
several well defined chemotactic factors using several in vitro systems [7,8] to measure adhesion. Our results clearly show that exposure to chemotactic factors causes marked changes in the adhesion of neutrophils, and thus lend support to the suggestion that adhesive changes may affect neutrophil behaviour in vivo. MATERIALS

AND METHODS

Cells Endothelial cells were obtained from the aortas of freshly slaughtered pigs using a method based on that described by Booyse, Sedlak & Rafelson [9]. Cannulated aortas were washed with BSS to remove blood clots, ligated, drained and refilled with a solution of 0.05% collagenase (Sigma. tvne II) in BSS and incubated for 2%min at‘37’C. After decanting the enzyme solution, the aortas were gently rinsed with BSS to remove residual collagenase, massaged lightly along their length to loosen the sheets of endothelial cells, filled with growth medium and shaken several times. The resultant cell suspension was dispensed into plastic 25 cm2 tissue culture bottles. Growth medium was changed after 24 h and thereafter every 3 days. Confluent monolayers of polygonal cells were formed within 3-6 days depending on the initial plating density. Cells were sub-cultured using trypsin/EDTA as described by Edwards & Campbell [lo], except that the cells were shaken off the plastic bottles in growth medium, and this suspension was used for seeding fresh cultures. For use in adhesion assays, endothelial Exp Cell Res 122 (1979)

monolayers were grown on 13 mm diameter, detergent (Decon) washed glass coverslips (Chance Propper Ltd) in‘10 cmx 10 cm polystyrene sectored boxes (Sterilin Ltd). Cells were subcultured no more than 4 times. Rabbit peritoneal neutrophils were obtained and prepared for use as previously described, [S].

Media Growth medium for endothelium was Glasaow-modified Eagles Medium containing 10% fetal calf serum (FCS) (Gibco). The gas nhase was 5% CO,. 95 % air. Balanced Salt SoluzonA (BSS) contained -(per litre) 8 g NaCl, 0.4 g KCl, 0.2 g MgCl, 6H,O, 0.14 g CaCl,, 1 g glucose, 2.388 g HEPES (10 mM) with the pH adjusted to 7.4 with NaOH. All chemotactic factors, oc,-casein, p-casein (gifts from Dr D. G. Dalgleish, Hannah Dairy Research Institute. Avr, Scotland) alkali-denatured human serum albumin (HSA) [ 111;formyl-methionyl-phenylalanine (f-met-phe), formyl-methionyl-leucyl-phenylalanine (FMLP) [12], and formyl-tri-tyrosine (f-tri-tyr) r131 were obtained as nreviouslv described rll. 131 and stored at -20°C in-BSS. The concentrations re: quired for experiments were made up by serial dilutions from stocks using glass universal bottles and one glass pipette for each series.

Adhesion-to-substratum

assay

In principle, this assay is identical to that described by Walther, ijhman & Roseman [14]. Assays of neutrophi1 adhesion to coverslips with either confluent endothelial monolayers or serum coats as substratum were performed in “Linbro” plastic, multi-welled trays &low Laboratories, Irvine, UK) containing the test substance in BSS. A suspension of neutrophils (-lo6 cells/well) was added to-give a final volume of. 1 ml and the tray incubated at 37°C. The coverslips were then removed and non-adherent cells rinsed off by

Chemotactic factors and neutrophil adhesion

171

Table 2. &fect of pretreatment of neutrophil granulocytes, endothelium or serum coated glass on neutrophil adhesion after 30 min at 37°C % adhesion of neutrophils i S.E.M. (n) (compared to controls= 100%) Pretreatment with chemotactic factor of Endothelium, 30 min at 37°C

Serum-coated glass, 30 min at 37°C Neutrophi!s, 15 min at 37°C

Chemotactic factor (M)

Endothelium

f-met-phe, lo-” f-met-ieu-phe, 10m9 /3-casein, 4.2~ 10e5 f-met-phe, lo-” f-met-leu-phe, 1OF /3-casein, 4.2~ 10m5 f-met-phe, 10-j f-met-leu-phe, 1OF

94* 5 (15) 91tto (4) 94+17 (11)

53i 3 (7)*** 74i: 5 (4)*

Serum-coated glass

94-+5 (16) 105i4 (4) 102i2 (7) 5112 (lo)*** 71rir4 (4)**

II, no. of replicate coverslips. Using Student’s r-testp (difference from control=O) ***
dipping 5 times in BSS at 37°C. The number of adherent cells was then determined by either of two methods: (1) The coverslips were fixed overnight in buffered formalin, rinsed in HzO, stained in Giemsa (l/l0 in distilled H,O) for 30 min, rinsed and dehydrated in isopropyl alcohol, air-dried and mounted in DPX. Using a x2.5 objective and a 10x10 eyepiece grid, the cells in a total of 10 random fieldslcovershp along 2 diameters at right angles to each other were counted and the mean number/unit area calculated. (2) The radioactivity bound to the coverslips was counted in a Wilj 2001 gamma counter, having used .“‘Cr-labelled neutrophils.

RESULTS

Cell substrate adhesion Initial experiments were performed wit chemotactic factors present during the period of the adhesion assay (30 mm). The results (table I) show that a wide range of chemotactic factors used at chemotactically optimal concentrations reduced th of adherent neutrophils both to e monolayers and to serum-coated glass. Labelling of neutrophils Most of the factors tested reduced the numNeutrophils were suspended in 1 ml BSS (approx. 5 x ber of adherent cells to around 50 % of conlo7 cells) to which was added 100 &i Na, [j’Cr]O, (100-300 mCi/mg Cr) in 0.1 ml isotonic saline (The trol values obtained for cells in BSS alone. Radiochemical Centre, Amersham). After incubation Since in these assays, 2040% of the cells at 37°C for 30 min, the cells were washed 3 times in normalIy adhere, this corresponds to a BSS, filtered through plastic mesh (‘Nitex’) to remove clumps, and used immediately of lO--20% of the total number of added cells. There seemed no’ obvious relation beAggregation assay tween the potency of an active factor in Neutrophil aggregation assays were performed as pre- chemotaxis and its effect on adhesion viously described, [S], using a 37°C reciprocating shaker bath at 120 cycleslmin (stroke 4 cm) and elec- though for a particular ;factor, the effect on tronic particle counting on a Coulter Counter Model A adhesion did depend on the dose of factor with 100 pm orifice tube, threshold setting 050 and added. Also shown in table 1 are the effects aperture current switch setting 4. of two other compounds. The non-forMicropore filter assay mylated dipeptide, met-phe, which is only Assays of purified human blood neutrophil locomotion feebly chemotactic inhibited adhesiveness in filters were performed using micropore filter assays very slightly compared to f-met-phe; native as previously described [2], using the leading front method of measuring cell migration [15]. HSA, which is not chemotactic, actually in-

172

Smith, Lackie and Wilkinson

loo+l

80.

20 t

o!0

,(gl

I

,$I

,o’

,08

,$

,&6

Fig. 1. Abscissa: molar cont. of FMLP; ordinate: mean Tr counts/sec/coverslip (adhesion). Distance into filter (pm) (migration). O-O, Migration I S.E.M.; A-A, adhesion to serum-coated glass i S.E.M.; W-W, adhesion to confluent endothelium kS.E.M. Effect of FMLP on adhesion after 30 min and migration through filters towards FMLP. For adhesion, each point is mean from 4 replicate coverslips. For migration, each point is mean of 5 measurements on each of duplicate filters.

creased adhesion in contrast to the reduction observed in the presence of denatured HSA. Since adhesion of neutrophils to both endothelium and serum-coated glass was inhibited, it appeared that the effect of chemotactic factors was on the neutrophils themselves. However, it is possible that the factors may interact with the endothelial cells and serum in some way to make these less adhesive. To determine which of these components of the adhesive interaction was being affected, experiments were performed in which each component was preexposed to chemotactic factor, washed, and then used in adhesion assays. The results (table 2) show that when neutrophils were exposed to chemotactic factors for short periods at 37°C (15 min), washed twice in BSS and then used in adhesion assays, the reduction in adhesion was still observed. This reduction was not so marked as in the Exp CellRes 122 (1979)

0’

I 0

I

0.2

04

0.6

0.8

I.0

Fig. 2. Abscissa:

casein cont. (mg ml-‘); ordinate: mean number of cells/microscope field (adhesion). Distance into filter, cLrn(migration). O-O, migration +S.E.M.; A-A, adhesion to serum-coated glass kS.E.M.; W--H, adhesion to confluent endothelium fS.E.M. Effect of cY,-caseinon adhesion after 30 minutes and migration through filters towards qcasein. For adhesion, each point is mean from 3 replicate coverslips. For migration, each point is mean of 5 measurements in each of duplicate filters.

presence of chemotactic factor but was still clearly evident. In another type of experiment, endothelial monolayers or serum coats were incubated in the presence of chemotactic factor for 30 min, washed twice and used as substrata in adhesion assays. Very little or no effect on neutrophi1 adhesion was observed (table 2). Any slight reduction may be due to residual chemotactic factor, especially since the compounds studied are active at very low concentrations. Thus it seems that the reduction in adhesion results from a change in the adhesive characteristics of the neutrophils and not of the endothelium or serum coats. Further it appears that exposure to and presumably binding of chemotactic factor to the neutrophil receptors is sufficien stimulus to induce this change. The con

Chemotactic factors and neutrophil adhesion

143

Table 3. Effect of chemotactic factors on the aggregation of neutrophil gra~~~Qcyte~ after 60 min at 37°C Chemotactic factor

Cont. Of)

% of controi (= 100%) particle number +S.E.M. (n)

a,-Casein &Casein f-met-phe f-met-leu-phe

4.2x 1O-5 4.2x 10-j 10-j 10-g 10-T

143.3k6.4 (24)p
n, total number of replicate assays. Using Student’s t-test, p (difference from controls =O), values as shown. Values greater than 100% indicate an inhibition of adhesion:

observed for c+casein, a high molecular weight chemotactic factor, whose dose re,o-” ,o-‘o lo-9 ,g8 ,o-7 sponses are of a different pattern. For this 0 factor, within the range studied, chemotaxis Fig. 3. Abscissa: molar cont. of FMLP; ordinate: reaches an optimum and remains high while mean no. of cells k S.E.M./microscope field. Effect of FMLP on adhesion to serum coated glass adhesion is reduced and remains low (fig. after 60 min at 37°C. Each point is the mean of ob2), thus it seems that chemotaxis and adservations on 4 replicate coverslips. hesion are very closely correlated, alth~~gb no causal relationship has been demontinued presence of chemotactic factor is un- strated. necessary. After 60 min incubation in the prese To clarify the effect that chemotactic fac- of FMLP, a further effect was observed, tors have on adhesion, the most potent fac- Fig. 3 shows a typical dose response curve, tor, FMLP was further studied. At this time, the high concentrations of The dose response curve of adhesion FMLP used (lo* M- 10T7 M) clearly inafter 30 min (fig. 1) to both serum coats and duced an increase in the number of adendothelium was very reproducible, adhe- herent cells. Low concentrations (IQ-lo sion reaching a minimum at lo-lo M - 10mgM lo-l1 M) still inhibited adhesion, though not FMLP and returning to control levels at so markedly as at 30 min. Examination of 1O+7M FMLP. The adhesive minimum is fixed and stained cells at high concentrasimilar to the chemotactic optimum and tions showed that many of the cells were comparison of these curves with a typical very spread and flattened on the substrachemotaxis dose response curve (fig. 1) tum. It is possible that the apparent inshows that there is a very close inverse crease in adhesiveness of the ~eutro~~i~s relationship between adhesion and chemo- may be a result of increased spreading and taxis, although adhesion declines at slightly resultant resistance to detachment during lower concentrations than those at which rinsing. We therefore studied ~e~t~op~~~agchemotaxis is apparent. A similar relation- gregation, since this sort of adhesion assay ship between chemotaxis and adhesion was largely eliminates any effect of cell spreadE.xp Cd Res 122 (IY7YI

174

Smith, Lackie and Wilkinson

O

0

10

20

30

40

50

60

time (min); ordinate: mean particle no. x 1OW’f S.E.M. O-O, BSS alone; A-A, 1OWM FMLP; W--m, lo-’ M FMLP. Time course of neutrophil aggregation in the presence of FMLP. Each mean is the mean from 3 replicate assays (where omitted, error bars fall within symbol)

Fig. 5. Abscissa:

molar cont. of FMLP; ordinate: m/m,, + S.E.M. where n is the mean total number of particles at time t. A-A, t, 30 min; O-O, t, 60 min; nt,-10G cells. Effect of FMLP on neutrophil aggregation. Each point is the mean from 4 replicate assays.

Fig. 4. Abscissa:

occur in the neutrophils themselves. The caseins inhibited aggregation at 60 min, unlike the peptides which at chemotactic concentrations enhanced it. This may reflect a fundamental difference in the mechanism of action of the caseins, or it may be that Cell aggregation at the concentrations used, stimulation of Since the extent of cell aggregation is the adhesion had not occurred (cf low concenresultant of adhesion and separation, trations of FMLP). For technical reasons, changes in either of these processes may af- higher concentrations of casein were not fedt the final degree of adhesion observed. studied. Our data do not distinguish between these The aggregation dose response curve for possibilities but do indicate that a change in FMLP (fig. 4) was determined and showed one or both of these processes occurs, pro- that adhesiveness was reduced by low conducing a net change in the observed degree centrations at 30 min and increased by high of adhesion. concentrations at 60 min, essentially the The results of cell aggregation assays same result as was obtained using adhesion(table 3) show that all the factors tested to-substratum assays. induced adhesive changes in the neutroBy following the time course of decrease phils. The effects of these factors were in particle number during an aggregation qualitatively the same as in adhesion-to- assay, it should be possible to estimate the substratum assays. This confirms the ear- time at tYhich the neutrophils become more lier conclusion that the adhesive changes adhesive. Fig. 5 shows that for FMLP, the ing. A further advantage of aggregation assays is that studies can be made at much shorter time intervals after beginning the experiment since no time is necessary to allow cell settling.

Exp Cd/ Res 122 (1979)

Chemotactic factors and neutrophil adhesion timing of changes in adhesion depends on the concentration present, Thus in 1O-7M FMLP the neutrophils were more adhesive from time 0 min (as judged by the slope of the time course) than controls. In lop9 M FMLP the cells aggregated more slowly than controls in BSS during the first 15 min, and are thus assumed to be less adhesive; after I5 min aggregation occurred more rapidly and the cells are thus judged to be more adhesive than controls. These times are crude estimates based on a qualitative examination of the time course. An exact estimate of the times is not possible since they varied from experiment to experiment. The overall pattern however, was always the same, i.e. that neutrophils became more adhesive earlier in high concentrations than controls in BSS alone. 10eg M FMLP was the lowest concentration which reliably induced an increase in adhesiveness. In IO-“’ M, throughout the time courses performed (up to 1 h), the neutrophils remained less adhesive than controls. Thus, in general, the results of aggregation assays confirm the conclusions from the adhesion-to-substratum assays, namely that chemotactic factors induce a series of time and concentration dependent changes in the adhesiveness of neutrophils, starting with an inhibition followed by a marked stimulation at high concentrations. DISCUSSION The results here indicate a close inverse relation between the adhesiveness of neutrophils in the presence of a chemotactic factor (when measured after 30 min exposure) and the distance migrated through filters in standard chemotaxis assays in response to that factor. Comparison of these two sets of data is complicated by the presence of a gradient across the filter (though 12-791810

175

consideration of the neutrophils chemokinetic response is simpler) and by the fact that filter assays are performed for various times, usually longer than 1 h. owever, the close correlation does lend support to the suggestion that adhesive changes have a role in chemotaxis. These adhesive changes are net changes as detected by the assays used. Changes in the degree of cell detachment (as opposed to cell adhesion) have been implicated in the decreased aggregation of em cells in the presence of tumour cells [16]. Macrophage migration inhibition has been reported to be correlated with decrease cell detachment [17]. It must be borne in mind therefore that as the term ‘adhesion’ in this discussion is defined operationally and is not intended to descri processes occurring, that celP se~~~at~o~ changes may also be involved in the phenomena described. If, as has been suggested for fibroblast locomotion [4], the rate of leucocyte locomotion depends on the degree of adhesion, then the inhibition of adhesion caused by chemotactically optimal concentrations of these factors may bring the strength of adhesion into a range optimal for locomotion. Many chemotactic factors also stimulate chemokinesis (random locomotion) and it seems plausible that a change in adhesiveness could account for this. At high concentrations of chemotactic factor chemoki~esi§ is often reduced. The return to control values of adhesion observed here in t ence of higher concentrations of tactic factor could lead to a decrease in the rate of locomotion, thus reducing the distance moved into filters. A similar explanation could account for the observations of Dierich, Wilhelmi & Till [18], who found that neutrophils well into filters with surface-bou

176

Smith. Lackie and Wilkinson

and that high concentrations of fluid phase casein (>3 mg ml-‘) depressed the migration from the maximum observed in lower concentrations. The second effect of chemotactic factors on neutrophil adhesion is a dose- and timedependent stimulation of adhesiveness. This effect was most marked at the high concentrations of factor used (lops- lo-: M FMLP). In the presence of cytochalasin B (CB), such concentrations also stimulate enzyme release from neutrophils [19], a process which Lackie [8] showed to be correlated with increased neutrophil adhesiveness. Chemotactic factor-induced increases in adhesiveness might then result from increased secretion. Increased adhesiveness under these circumstances may be appropriate if it helps to trap the neutrophils at the focus of infection where enzyme release is desirable. This increase in adhesion at high concentrations may also be relevant to the process of chemotaxis. Carter [3] and Harris [5] have shown that moving cells tend to accumulate on more adhesive substrata, and such a mechanism has been suggested to explain the action of the chemotactic factor, casein [18]. If, instead of a gradient of substratum adhesiveness, there exists a gradient of cellular adhesiveness (as our results indicate), then the end result should be the same, i.e. that cells accumulate where they are most adhesive. It is not clear whether any of the adhesive changes observed are relevant to events occurring during margination of neutrophils in vivo. At high concentrations of FMLP, cells became more adhesive almost immediately after exposure whereas at optimally chemotactic concentrations, the cells became less adhesive initially and then either remain so or become more adhesive only after some time. As concentrations of Exp Cc// Rej 122 i/575/

chemotactic factor near endothelial cells in vivo are likely to be low due to the proximity of the bloodstream which should act as a sink and since high concentrations are likely at the source, it is difficult to imagine how the effects of chemotactic factors on neutrophil adhesion that we observe could be related to margination. The effects of chemotactic factors on neutrophil aggregation have also been studied by O’Flaherty, Kreutzer & Ward [20], who reported an immediate, transient increase in aggregation of rabbit neutrophils. Their assay system is radically different from the one employed here so that comparison of results is difficult. We, however, found no evidence of a transient increase in aggregation and on the contrary, observed an inhibition at some of the times and concentrations studied by O’Flaherty et al. The way in which chemotactic factors induce adhesive changes in neutrophils is not known. Gallin, Durocher & Kaplan [21] reported that chemotactic factors induced a reduction in net surface negative change (after exposure for 1 h). Such a reduction may well make the cells more adhesive since the repulsion between negatively charged surfaces would be reduced. A large increase in the amount of lactoperoxidasecatalysed iodination of surface protein has been shown to be associated with endotoxin-induced neutrophil aggregation [22]. It is possible that FMLP-induced aggregation is similarly associated with such a change. The close correlation between adhesive changes and chemotaxis has, of course, another interpretation. The adhesive changes may be a consequence of locomotory activity, the formation of ruffled membrane and pseudopodia, associated with the chemotactic stimulus. Such changes in cell surface topography may alter the overall

adhesiveness of the cell surface. If this is so1 then changes in adhesion will be an integral part of any chemotactic response. An understanding of the effect of altering leucocyte adhesiveness may contribute to the elucidation of the cellular processes occurring during chemotaxis. We wish to thank the veterinarv and other workers at the Glasgow abattoir for their generous assistance in the supply of aortas. R. P. C. Smith was supported by a research studentship from the MRC.

KEFERENCES I Grant. L, The inflammatory process (ed B W Zweifach, L Grant & R T McCluskey) 2nd edn, vol. 2, pp. 205-244. Academic Press, New York and London (1973). 2. Wilkinson, P C, dhemotaxis and inflammation, p. 169. Churchill Livingstone, Edinburgh and London ( 1974). 3. Carter. S B, Nature 208 (196.5)1183. 4. Gail, M H & Boone, C W. Exp cell res 70 (1972) 33. 5. Harris. A, Exp cell res 77 (1973) 285. 6. Lackie, J M. In preparation.

7. Lackie. J M & De Bono, D, Microvasc res !3 (1974) 107. 8. Lackie, J M, Inflammation 3 (1977) 1. 9. Booyse, F M, Sedlak, B J & Rafelson, M E. Thrombos diathes haemorrh (Stuttg) 34 (1975) 815. 10. Edwards, J G & Campbell, J A. J cell sci 8 (1971) 53. 11. Wilkinson. P C. Nature 251 (1974) 58. 12. Schiffmann. E. Corcoran, B A h Wahi, S M, Pro:: natl acad sci US 72 (1975) 1059. 13. Wilkinson. P C, Immunology 36 (1979) 579. 14. Walther. B T, Ohman. R & Roseman. S, Proc nztl acad sci US 71 (1973) 1569. 15. Zigmond. S H & Hirsch. J G. J exp med 137 ( 1373) 387. 16. Maslow. D E & Weiss, L, J cell sci 29 I 19785271. 17. Weiss, L & Glaves. D, J immunol 115t.1975)136-7. 18. Dierich, M P, Wilhelmi. D & Till. 6. Nature 170 I 1977)35 1. 19. Showell, H J. Freer, R J, Zigmond. S H, Schlffmann, E. Aswanikumar, S. Corcoran. B 8r Becker, E L, J exp med 143 i 1976’11154. 20. O’Flaherty, J T, Kreutzer, D L & Ward. P li. J immunol 119(1978) 232. 21. Gallin, J I, Durocher. J R & Kaplan. A P, ! clir: invest 55 (1975) 967. 22. Thorne, K J I. Oliver. R C & Lackie, J. J czll sci 27 (1977) 213. Keceived December 18. 1978 Revised version received February 10. 1974 Accepted February 22. 1979