Clin. exp. immunol. (1979) 38, 598-608.

Relations between Fc receptor function and locomotion in human lymphocytes J. M. SHIELD S & P. C. WILKINSON, Department of Bacteriology and Immunology, University of Glasgow (Western Infirmary), Glasgow, Scotland

(Accepted for publication 31 May 1979)

SUMMARY

The relationship between the surface binding sites on human lymphocytes for chemotactic factors and for the Fc fraction of IgG was investigated using both blood lymphocytes and established cultures of human lymphoblasts. Pretreatment of human blood lymphocytes with a variety of chemotactic factors inhibited Fc-rosette formation. This was true even of small formylated peptides, for example, formyl-methionyl-phenylalanine (chemotactic) inhibited Fc-rosetting but unformylated methionyl-phenylalanine (non-chemotactic) did not. Conversely pretreatment of lymphocytes with IgG inhibited their locomotor reactions to a variety of chemoattractants. Aggregated IgG was more inhibitory than non-aggregated IgG and the inhibition was mediated via the Fc piece. In a filter assay, native IgG was chemokinetic but not chemotactic for lymphocytes. Heat-aggregated IgG induced more locomotion of lymphocytes than native IgG, and was possibly chemotactic, but not unequivocally so. The possibility that chemotactic factors and the Fc portion of IgG compete for the same cell surface receptor was investigated by binding studies using cultured lymphoblasts. These studies suggested that the reciprocal inhibition could not be explained by competition for receptors. An alternative explanation was suggested by the finding that inhibition of locomotion by aggregated IgG was dependent on the presence of divalent cations at the time the IgG was added, and did not occur in the presence of the calcium ionophore A23187. Addition of aggregated IgG or chemotactic factors to lymphocytes thus may lead to a gated entry of calcium, and following closure of the calcium gate, the cells become relatively unresponsive to further stimulation.

INTRODUCTION Under defined conditions, lymphocytes show chemotactic and chemokinetic reactions to a variety of attractants (Russell et al., 1975; Wilkinson et al., 1976; Wilkinson et al., 1977; Ward et al., 1977) and it is probable that many such reactions are activated by binding of chemotactic factors to cell surface receptors, though receptors for chemotactic factors in lymphocytes have not yet been investigated. Lymphocytes of defined classes bear surface receptors for the Fc portion of immunoglobulin as do neutrophils and mononuclear phagocytes. Recently there have been suggestions that the Fc receptor may play a role in modulating leucocyte locomotion. Van Epps & Williams (1976) reported that neutrophils preincubated with polymeric IgA myeloma proteins (and to a lesser extent with polymerized IgG) became defective in their chemotactic responses to a variety of factors. Kay, Bumol & Douglas (1978) observed inhibition of chemotaxis in neutrophils which had bound antibody-coated erythrocytes (EA) and in the presence of IgG antibody or immune complexes. In a study from this laboratory, migration of guinea pig peritoneal macrophages towards denatured serum albumin was reduced in cells Correspondence: Dr P.C. Wilkinson, Department of Bacteriology and Immunology, University of Glasgow (Western Infirmary), Glasgow Gl1 6NT, Scotland. 0099-9104/79/1200-0598$02.00 (C 1979 Blackwell Scientific Publications

598

Lymphocyte locomotion and Fc receptors

599

which had first been coated with anti-sheep red cell antibody (Wilkinson, 1976). Thus it is possible that occupancy of the Fc receptor by immunoglobulin may diminish chemotactic reactions. This could be because Fc and the chemotactic factors both compete for the same receptor or because Fc binding inhibits chemotactic responses by some change in the cell which is independent of receptor binding. In the present work we have investigated the relationships between Fc binding and chemotaxis in human lymphocytes. The influence of pretreatment of lymphocytes with IgG on their subsequent migration in micropore filters towards various attractants was investigated. Likewise the effect of pretreatment of lymphocytes with chemotactic factors on their subsequent ability to form EA rosettes (Fc rosettes) was studied. The results presented will show both that cell-bound IgG reduced locomotor responses in lymphocytes and that pretreatment with chemotactic factors inhibited Fc rosetting. We therefore carried out direct binding studies using radiolabelled reagents to investigate whether these findings could be explained by competition between IgG and chemotactic factors for cell-surface binding sites or whether some other explanation was more likely.

MATERIALS AND METHODS Reagents. Human IgG was purchased from Miles, Stoke Poges, England. This material gave only a single arc on immunoelectrophoresis against good polyspecific anti-human antisera and appeared highly purified on SDS-polyacrylamide gel electrophoresis. It was used either as an unaggregated preparation by dissolving in Gey's solution, pH 7-2, or was lightly aggregated by heating at 63°C for 20 min. IgG was digested with pepsin (Koch-Light, Colnbrook) in acetate buffer at pH 4 (370C, 18 hr; weight ratio of IgG: pepsin, 20: 1). A second sample of human IgG used in some of the studies was a gift from the Scottish Antibody Production Unit. Human serum albumin (HSA) was from Behringwerke, Marburg, Germany and was highly purified judged by immunoelectrophoresis and by SDS-poly-acrylamide gel electrophoresis. It was denatured by alkali treatment as detailed by Wilkinson & Allan (1978a). Conformational changes in the product were monitored by difference spectroscopy. Casein (Alkaliloslich) was from Merck, Darmstadt, Germany. Formyl-methionyl-leucyl-phenylalanine (f-Met-Leu-Phe) was from Miles. Methionyl-methionine, methionyl-phenylalanine, trityrosine and triphenylalanine were purchased from Sigma and formylated after Sheehan & Yang (1958). After formylation all of these peptides have been shown to attract human neutrophils (Wilkinson 1979). Fresh human plasma was activated by addition of Difco Escherichia coli 0127 :B8 lipopolysaccharide. The complement-derived peptide CAT 1.5.1. was a gift from Dr J.H. Wissler, Bad Nauheim, Germany. Its preparation and activity are described elsewhere (Wissler, 1972; Stecher & Sorkin, 1972). The calcium ionophore, A23187 was from Eli-Lilly, Windlesham. Cells. Lymphocytes were obtained from heparinized venous blood from normal donors by centrifugation on Ficoll-Triosil, Sp.g. 1.078 (Pharmacia, Uppsala, Sweden and Nyegaard, Oslo, Norway). The cells were washed in Gey's solution three times and resuspended at 4x 106 cells per ml for rosetting tests. Using the rosetting technique described below, 20 25% of the lymphocytes in normal human blood samples show EA rosettes. Lymphocytes fresh from peripheral blood do not show optimal locomotor or chemotactic responses and require to be cultured in vitro for 24-72 hr prior to the test. The mononuclear cell layer from the Ficoll gradient was therefore washed twice and resuspended in 15 ml Eagles medium containing 5% heat-inactivated foetal calf serum (MEM-FCS) (Gibco, Biocult, Glasgow, Scotland) and incubated for 24-48 hr at 37°C. More than 80% of the cells recovered after culture were lymphocytes. No mitogen or other activator was added to these cultures. A second group of human lymphocytes which show good locomotor and chemotactic reactions (Russell et al., 1975) which represent a more homogeneous population than blood lymphocytes are the established cultured B lymphoblast lines. Twelve such lines were obtained (courtesy of Dr P. Singer, Biochemistry Department, University of Glasgow) and screened for locomotor and chemotactic activity. One line, RPMI 1788, was selected on the criteria of good locomotor activity and responsiveness to chemotactic factors and maintained in culture at 37°C in RPMI 1640 medium (Gibco-Biocult, Glasgow, Scotland) supplemented with 20% FCS, 1% glutamine, penicillin (100 pg per ml) and streptomycin (100 pug per ml) throughout the whole period of the study. It was maintained at around 5 x 105 cells per ml and aliquots were taken from the bulk culture whenever required for testing, washed, tested for viability by trypan blue exclusion and used at an appropriate concentration (106 cells per ml for chemotaxis studies, 5 x 10' per ml for binding studies). The RPMI 1788 line was originally established from normal human peripheral blood lymphocytes, has a diploid karyotype, was reported to be free of herpes-like EB virus and is a strong IgM secretor (Fujioka & Gallo, 1971). The cells are variable in size, many being 20 pm or more in diameter, and tend to aggregate in culture. The aggregates are readily dispersed, especially after brief incubation in EDTA (10-I M). Fc rosette tests. The assay for Fc rosettes (EA rosettes) uses chicken erythrocytes coated with rabbit anti-chicken globulin. The preparation of the antibody and the erythrocytes, and the rosetting assay have been described fully elsewhere (Sandilands et al., 1975), and the tests were carried out following that method and as described by Wilkinson (1977). In these experiments, both monocyte and lymphocyte rosettes are seen. Monocyte rosettes are usually large and ragged and contain more than one monocyte. Lymphocytes form neat small rosettes with the lymphocyte easily distinguishable and

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J. M. Shields & P. C. Wilkinson

centrally placed. Rosetting assays were performed using untreated cells as controls, or test cells which had been pre-incubated for 1 hr at room temperature with appropriate chemotactic factors, then washed three times in Gey's solution. Assays of locomotion and chemotaxis. These were performed using the method described earlier (Wilkinson, 1974) and cell migration was assayed by the leading front technique (Zigmond & Hirsch, 1973). Filters of 8 pm pore size (Sartorius, Gottingen, Germany) were used for cultured blood lymphocytes and 12 pm poresize filters (Sartorius) for the lymphoblastoid cell line. Chambers were incubated for 3 hr at 370C. The results reported are the means for five fields in each of two filters (+ s.e.m.). In tests using peptide factors, the cells were always suspended in Gey's solution+HSA (1 mg per ml). For studies of the inhibitory effect of IgG on locomotion, cells were preincubated for 30 min with appropriate IgG preparations used at 1 or 2 mg per ml, then washed twice and placed in the upper comportment of chemotaxis chambers. The distinction between chemokinesis and chemotaxis (Keller et al., 1977) was made using the checkerboard assay (Zigmond & Hirsch, 1973) in which a series of chambers was set up, each of which contained a different concentration of attractant above and below the filter, so that the absolute concentration and the gradient varied from chamber to chamber. In this way the distance migrated in various absolute concentrations of attractant with no gradient, as well as in various positive and negative gradients could be studied. A large number of such experiments were done and these are therefore presented in summary form only. The chemokinetic effect of any factor (studied under uniform concentration conditions) has been evaluated as a 'chemokinetic ratio': Distance migrated at maximum chemokinetic concentrations of factor Distance migrated in absence of factor. The overall chemotactic effect in any checkerboard assay was expressed as a 'chemotactic increment': Observed locomotion (pm)-calculated locomotion on basis of chemokinesis alone (p"m) 100 Calculated distance on basis of chemokinesis alone (pum) the 'calculated locomotion on the basis of chemokinesis alone' being derived from the calculations in the appendix of the paper by Zigmond & Hirsch (1973). A full description of the rationale of the 'chemotactic increment' is given by Wilkinson & Allan (1978b). Effect of divalent cations on inhibition of locomotion by IgG. Human blood lymphocytes in Gey's solution either with or without Ca2+ (10 -3 M) and Mg2 + (5 x 10-4 M) were preincubated with heat aggregated IgG (1 mg/ml) for 30 min. Further batches of cells in divalent-cation-containing medium were incubated with IgG either in the presence of the ionophore A23187 (5 x 10-9 M) or the ionophore was added 30 min after the IgG. The cells were then washed twice and tested against chemotactic factors in the usual way in Gey's solution containing divalent cations. Binding assays. IgG and HSA were iodinated with 125I (1 mCi per 40 mg protein) using the chloramine-T method of Hunter & Greenwood (1962). All binding assays were done using the RPMI 1788 cell line. In outline, a series of tubes was set up containing dilutions of labelled protein from 200 pg per ml to 5 jug per ml (final concentration after addition of cells). Cells were prepared from culture by centrifugation, resuspension and incubation in Gey's EDTA (EDTA 10-' M) for one hr at room temperature to disaggregate the cells, after which they were washed three times in Gey's solution. The cells (5 x 106 per tube) were then added to the tubes containing labelled protein to a final volume of 1 ml. After one hr the cells were centrifuged and washed several times with a 15 min interval between each wash. The amount of bound radioactivity was measured after the second, third, fourth and fifth washes. Equilibrium binding (i.e. before washing) was estimated by extrapolation of the linear plot (on a logarithmic scale) of bound protein after washes 2, 3, 4 and 5, back to wash zero. This procedure is based on that of Leslie & Cohen (1974) and is identical to that described in detail by Wilkinson & Allan (1978a). Binding was measured under the following conditions: (a) Binding of 125I native HSA to untreated cells. (b) Binding of 125I-alkali-denatured HSA to untreated cells. (c) Binding of '25I-alkali-denatured HSA to cells preincubated for one hour with unlabelled aggregated IgG (2 mg per ml) then washed. (d) Binding of 125I-heat-aggregated IgG to untreated cells. (e) Binding of 125I-heat-aggregated IgG to cells preincubated for one hour with unlabelled alkali-denatured HSA (2 mg per ml) then washed. (f) Binding of 1251-heat aggregated IgG to cells preincubated for one hour with unlabelled formyl-methionyl-phenylalanine (5 x 10-) then washed.

RESULTS Inhibition of Fc resetting in lymphocytes pretreated with chemotactic factors The experiments described in this paper began with the observation that pretreatment with chemotactic factors could inhibit Fc rosetting by human lymphocytes. Table 1 shows results obtained with a number of such factors. All chemotactic factors tested, i.e. casein, alkali-denatured HSA, the complementderived peptide CAT 1.5.1. with chemotactic and anaphylatoxic activity (Wissler, Stecher & Sorkin,

Lymphocyte locomotion and Fc receptors

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TABLE 1. Percentage of Fc rosettes in human blood mononuclear cell preparations preincubated with various attractants

Number of

Agent used to pretreat cells (a) Lymphocyte rosettes alone No agent Casein 2 mg per ml f Met-Phe 5x 10-5 M (b) Lymphocytes and monocytes* No agent Met-Phe 5x 10-5M fMet-Phe5xlO 5M fMet-Met 5x 105 M f-tri-Tyr 10-9 M f-tri-Phe 10-9 M HSA 14x 10-5 M Alkali-denatured HSA 1-4x 10-5 M Casein 2 mg per ml CAT 1.5.1. peptide Negative control: RBC not coated with antibody

experiments

Percent rosette-bearing cells (+ s.e.m. where more than one

experiment done)

1 1 1

18-5 10-5 11-5

6 2 2 3 2 1 5 4 5 1

24-0+2-6 25-2+1-5 12-5+1-5 11-0+3-8 9-6+3-1

5

0-75+ 006

14-6 19-2+2-8 13-0+ 3 0 130+ 3-0 12-0

* The majority of rosettes seen in all tests were lymphocyte rosettes and in no case was the reduction in resetting induced by chemotactic factors restricted to either lymphocyte or monocyte rosettes.

1972) and therefore presumptively related to C5a, and various formylated peptides all substantially reduced resetting. Formyl-methionyl-phenylalanine (f-Met-Phe) halved the number of rosettes while the unformylated analogue, methionyl-phenylalanine (Met-Phe) had no effect. Alkali-denatured HSA was more inhibitory than native HSA. However, HSA itself did cause some reduction in rosettes (HSA has no chemotactic activity but is chemokinetic for lymphocytes; Wilkinson et al., 1977). Most of these experiments were done using the Ficoll-Triosil interface layer from human blood which contains both monocytes and lymphocytes. The results at the top of Table 1 are for lymphocyte rosettes only, the remainder for combined lymphocyte and monocyte rosettes, both being reduced after treatment with chemotactic factors. The doses of all chemoattractants used were those at which the attractants cause maximal stimulation of locomotion. The effect on locomotion ofpretreatment of lymphocytes with IgG Following from the observation that chemotactic factors inhibit Fc resetting, the question whether cell-surface-bound IgG inhibits chemotactic and locomotor reactions in human blood lymphocytes was studied. In order to obtain motile blood lymphocyte populations, it is preferable to culture the cells in vitro briefly, and cells cultured for 24-72 hr in MEM-FCS were used. Table 2 shows the effects of preincubation for one hr with unaggregated and aggregated IgG on the locomotion of such cells in the presence of various attractants. It is evident that this preincubation reduced the response of the lymphocytes to the factors studied and that aggregated IgG had more effect than unaggregated IgG. In addition, aggregated IgG inhibited unstimulated locomotion of lymphocytes to Gey's solution whereas native IgG did not affect this locomotion. There was no change in cell viability after the treatment with IgG. The inhibitory effect of IgG on cell locomotion appeared to be mediated via the Fc portion of IgG since pepsin-digested immunoglobulin had little inhibitory effect. Similar results to those shown in Table 2 were obtained using the human B cell line RPMI 1788. Checkerboard assays were performed to study whether the IgG was inhibiting the directional response of the cells to chemotactic factors or was

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TABLE 3. Chemokinesis and chemotaxis of human lymphocytes to IgG and to formyl peptides

Chemotactic increments in:

Cells Cultured blood lymphocytes

Cell line RPMI 1788

Factor

Unaggregated IgG test I Aggregated IgG test I Unaggregated IgG test II Aggregated IgG test II f Met-Leu-Phet f Met-Leu-Phet f Met-Leu-Phet fMet-Leu-Phet f Met-Leu-Phet f Met-Phet

fMet-Phel Casein *

Chemokinetic ratio*

Positive gradients

1-48 1-74 3-5 2-3 1-51 1-04 1-22 1.1 1-25 1.1 1.1 1-44

-4-1% +7-2% +6-0% +28-0% 0

+8-7% -0-3% +5 0% -4.0% +5 0% +12-5% +12-9%

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1-0 = control locomotion in absence of an attractant.

t Tested over concentration range 10-7 M- 10-1

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$ Tested over concentration range 10-4 M- 10-7 M.

inhibiting locomotion itself. Cells cultured in the presence of unaggregated IgG then tested in a checkerboard assay against casein showed a mean chemotactic increment in positive gradients of + 33 % and in negative gradients of -11%, i.e. a clear chemotactic response, as much as that of control cells. A similar result was obtained after one hr preincubation with IgG. IgG pretreatment does not appear to abolish the ability of cells to respond by chemotaxis, but does reduce their ability to move a finding which would argue against an effect of the IgG on the receptors for chemotactic factors. Studies of chemokinetic and chemotactic activity of IgG and offormyl peptides for lymphocytes The effects of IgG as a lymphocyte attractant have not previously been studied and these were therefore tested using checkerboard assays. Likewise formyl peptides are known to be chemotactic for neutrophils and monocytes but their effects on lymphocyte locomotion have not been documented. A considerable number of checkerboard assays were performed and for reasons of economy of space, these are not presented in full but are given in summary form in Table 3. Two IgG preparations were tested. Both had a chemokinetic effect since the rate of lymphocyte locomotion increased as IgG concentration was raised. There was no evidence to suggest that unaggregated IgG had a chemotactic effect. However, aggregated IgG showed positive chemotactic increments in positive gradients, but no negative increments in negative gradients. A typical chemotactic factor should show a chemotactic effect on the increments in both positive and negative gradients, thus the experiments suggest but do not confirm a chemotactic effect of aggregated IgG for lymphocytes. In our hands aggregated IgG has chemotactic activity for monocytes (Wilkinson & Allan, 1979) and neutrophils (unpublished), judged by checkerboard assays. Table 4 also shows the chemotactic increments for f-Met-Phe and fe-Met-Leu-Phe using both blood lymphocytes and RPMI 1788 lymphoblasts. Neither peptide showed a marked chemokinetic effect on lymphocyte locomotion, but f-Met-Phe appeared to be chemotactic. However f-Met-Leu-Phe, which is strongly chemotactic for neutrophils showed little chemotactic activity for lymphocytes. The filter assay system used may not be ideal for seeing chemotaxis towards such peptides since lymphocytes require a three hr incubation and gradients of small molecules such as these peptides across a filter would probably flatten out during such a period (Zigmond, 1979).

Binding studies The experiments detailed above showed a reciprocal inhibitory activity of chemotactic factors and of Fc on lymphocyte locomotor and Fc binding functions. One possible explanation for this would be that

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chemotactic factors and IgG Fc share the same receptor. Alternatively, binding of either chemotactic factor or Ig, each to an independent site, might lower the cell's ability to move or to respond to further signals. We therefore attempted to measure receptor activity by direct binding studies rather than by a functional assay. For these studies the cultured B cell line 1788 was preferred to blood lymphocytes because of its homogeneity and because large numbers of cells (5 x 108) could be prepared for each test. In the first group of experiments, binding of '25I-alkali-denatured HSA (a chemotactic factor) was measured both in untreated lymphocytes and in lymphocytes pretreated with unlabelled heat-aggregated

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IgG. Fig. la shows that the binding of '25I-alkali-denatured HSA was moderately reduced in the IgG-treated cells in comparison with controls. The results for two experiments were plotted as Scatchard plots to give an average intrinsic association constant (Ka) and number of binding sites per cell (n). The Ka for alkali-denatured HSA was 2-7 x 105 litres per mole for one experiment (Fig. lb) and 2-5 x 10' litres per mole for the other, and n was 2 0 x 107 in the first experiment and 9 0 x 106 in the second. After aggregated IgG treatment (experiment 2), Ka (alk-den HSA) was 2-7 x 105 (control untreated cells 2-5 x 105) and n 77 x 106 (control 90 x 106). Aggregated IgG pretreatment therefore did not alter the affinity of chemotactic factor binding, but caused a small reduction in the number of binding sites. A similar result was obtained in a second experiment. These values for '25I-alkali-denatured HSA-binding by lymphoblasts may be compared with those obtained earlier using rabbit neutrophils (Wilkinson & Allan, 1978a) (Ka 1 82 x 106 litres per mole, n 1 24 x 106). The number of binding sites for '25I-denatured HSA per lymphoblast was higher (they are much larger cells) but the affinity was lower than for neutrophils. Binding of 125I-aggregated IgG to RPMI 1788 lymphoblasts was measured using untreated cells, cells pretreated with unlabelled alkali-denatured HSA and cells pretreated with the unlabelled peptide, f-Met-Phe. In the absence of chemotactic factors aggregated IgG bound to lymphoblasts with a Ka of 1-26 x 106 and n = 1-98 x 106, i.e. with a higher affinity but to less binding sites than denatured HSA. Treatment with either denatured HSA or with f-Met-Phe did not reduce the number of binding sites for aggregated IgG (denatured HSA-treated cells, n = 2 57 x 106; f-Met-Phe treated cells, n = 1-91 x 106), nor did it reduce the affinity, rather it increased it (Ka for 1251I IgG using denatured HSA-treated cells = 2-4 x 106 litres per mole; using f-Met-Phe-treated cells 2 x 106 litres per mole: compare control untreated cells 1 26 x 106 litres per mole). Thus the binding studies failed to show that these chemotactic factors blocked binding of aggregated IgG to the surface of lymphoblasts.

Role of divalent cations Table 4 shows the effect of divalent cations and the divalent cation ionophore A23 187 on the inhibition (deactivation) by aggregated IgG of locomotion of lymphocytes to attractants. In the experiment shown, aggregated IgG pretreatment in the presence of divalent cations did not inhibit unstimulated locomotion, but did inhibit locomotion to casein and, less markedly, to denatured HSA. There was very little inhibition of locomotion to these agents if the preincubation was carried out in divalent cation-free medium. Addition of divalent cations 30 min after the IgG did not result in deactivation of locomotion, suggesting that at that time, the cells had become unresponsive to divalent cations. Furthermore, if the divalent cation inophore A23187 was added to cells preincubated with IgG (in Ca2 +, Mg2 +-rich medium), deactivation of locomotion was not seen. The ionophore had to be present when the IgG was added. If it were added 30 min after the IgG, it did not prevent the locomotion, inhibiting effect of the IgG. These results suggest that locomotion-inhibition by IgG requires the presence of divalent cations at the time the IgG is added, and that addition of these cations after the IgG does not lead to inhibition. The data in Table 4 refer to Ca2 + and Mg2 + together, but when they were tested separately Ca2 + but not Mg2 + was found to be essential for the inhibitory effect of IgG (not shown). These findings are consistent with the possibility that deactivation of locomotion by IgG depends on a gated entry of Ca2 + on addition of the IgG. Once the calcium gate is closed, the cells become unresponsive to further addition of Ca2 . The ionophore A23187 would prevent inhibition by providing an alternative non-gated channel for calcium, thus allowing the cell to remain responsive to chemoattractants added later. DISCUSSION The studies described above showed that pretreatment of human lymphocytes with several chemotactic factors inhibited Fc rosetting. Conversely membrane-bound immunoglobulin reduced the attraction of lymphocytes to chemotactic factors-though not by inhibiting their capacity to migrate directionally. The hypothesis we originally favoured to explain these findings was that chemotactic factors shared the same cell-surface receptor that mediated binding of antibody-coated red cells, i.e. the Fc receptor and

Lymphocyte locomotion and Fc receptors

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some at least of the chemotactic receptors were identical or at least overlapping. In favour of this hypothesis was the finding that aggregated IgG itself could enhance lymphocyte locomotion (Table 3). In the binding studies described above, bound aggregated IgG partially reduced uptake of alkalidenatured HSA but the reverse was not shown. This result could be explained if, as was shown, there were more receptors for denatured HSA than for IgG, but the affinity of aggregated IgG was higher than that of denatured HSA. Then aggregated IgG would block some, but not all, of the HSA receptors and would not be displaced by denatured HSA. HSA would block the IgG receptors but would be readily displaced from them by aggregated IgG. However, the results of such studies are too tentative to give strong support to such a hypothesis and it must be emphasized that these cell-surface binding data at best present an average result for what may be very heterogeneous binding. For instance, we have not investigated separated IgG subtypes. Moreover, a number of unrelated chemotactic factors including small formyl dipeptides inhibited rosetting (Table 1) and it would be surprising if the Fc receptor were blocked by such small molecules. Indeed, the binding studies provided no evidence that formyl-methionyl-phenylalanine could block IgG binding to the lymphocyte surface. This makes the receptorblocking hypothesis unlikely to provide a full explanation of the findings and some other explanation has to be sought. The inhibition of lymphocyte locomotion by aggregated IgG was shown to depend on the presence of divalent cations at the time the IgG was added. If IgG was added in divalent-cation-free medium and divalent cations added later, no inhibition was seen. This is consistent with the possibility that aggregated IgG induces a brief gated entry of calcium into the cell, as is seen in histamine release by IgE-coated mast cells on addition of antigen (Foreman & Mongar, 1973; Foreman & Garland, 1974). Once the calcium gate is closed, the cell becomes unresponsive to further stimulation or to the addition of further calcium. This possibility is supported by the ability of A23187, present when the IgG was added, to prevent inhibition, since A23187 would provide an alternative non-gated channel for divalent cations. Thus we feel that our results on reciprocal inhibition of lymphocyte functions are better explained by induction of a state of relative unresponsiveness in the cells following closure of a calcium gate than by competition between IgG and chemotactic factors for cell-surface receptors. Another possibility to be considered is that IgG inhibits lymphocyte locomotion by increasing the adhesion of the cells to substrata as has been shown for neutrophils (Keller et al., 1979). These results suggest that caution should be exercised in interpreting results in which extrinsic agents can be shown to inhibit receptor-mediated functions, such as resetting. Such results have often been interpreted as showing blocking of receptors, without quantitative binding studies having been done. Our results show that there are other interpretations of such results than direct competition for receptors and that the ability of lymphocytes to show Fc rosettes can be inhibited under conditions where the number and affinity of Fc receptors on the cell surface is little changed. This work was supported by the Medical Research Council.

REFERENCES FOREMAN, J.C. & GARLAND, L.G. (1974) Desensitization in KELLER, H.U., BARANDUN, S., KISTLER, P. & PLOEM, J. the process of histamine secretion induced by antigen (1979) Locomotion and adhesion of neutrophil granuloand dextran. J. Physiol. (Lond.), 239, 381. cytes: effects of albumin, fibrinogen and gamma globulins FOREMAN, J.C. & MONGAR, J.L. (1973) The interaction of studied by reflexion-contrast microscopy. Exp. Cell. calcium and strontium with phosphatidyl serine in the Res. (In press.) anaphylactic secretion of histamine. 3. Physiol. (Lond.), KELLER, H.U., WILKINSON, P.C., ABERCROMsIE, M., 230, 493. BECKER, E.L., HIRSCH, J.G., MILLER, M.E., RAMSEY, FuJIo"A, S. & GALLO, R.C. (1971) Aminoacyl transfer W.S. & ZIGMOND, S.H. (1977) A proposal for the definiRNA profiles in human myeloma cells. Blood, 38, 246. tion of terms related to locomotion of leucocytes and other HUNTER, W.H. & GREENWOOD, F.C. (1962) Preparation cells. Clin. exp. Immunol. 27, 377. of iodine-131 labelled human growth hormone of high LESLIE, R.G.O. & COHEN, S. (1974) Cytophilic activity of specific activity. Nature, 194, 495. IgG 2 from sera of immunized guinea pigs. Immunology, KAY, N.E., BUMOL, T.F. & DOUGLAS, D.S. (1978) Effect 27, 577. of phagocytosis and Fc receptor occupancy on com- RUSSELL, R.J., WILKINSON, P.C., SLEss, F. & PARROTT, plement dependent neutrophil chemotaxis. J. Lab. D.M.V. (1975) Chemotaxis of lymphoblasts. Nature, 256, Glin. Med. 91, 850. 646.

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WILKINSON, P.C. & ALLAN, R.B. (1979) The locomotor behaviour of human blood monocytes in chemotactic and chemokinetic environments and the role of the substratum in monocyte locomotion. Functional Aspects of Mononuclear Phagocytes (ed. by R. van Furth). Martinus Nijhoff, The Hague. (In press.) WILKINSON, P.C., PARROTT, D.M.V., RUSSELL, R.J. & SLESS, F. (1977) Antigen-induced locomotor responses in lymphocytes. ]. Exp. Med. 145, 1158. 5. Exp. Med. 144, 1227. WARD, P.A., UNANUE, E.R., GORALNICK, S.J. & SCHREINER, WILKINSON, P.C., ROBERTS, J.A., RUSSELL, R.J. & McLOUGHLIN, M. (1976) Chemotaxis of mitogen-activated G.F. (1977) Chemotaxis of rat lymphocytes. 5. Immunol. human lymphocytes and the effect of membrane-active 119, 416. enzymes. Clin. exp. Immunol. 25, 280. WILKINSON, P.C. (1974) Chemotaxis and Inflammation, WISSLER, J.H. (1972) Chemistry and biology of the anaphyp. 33. Churchill-Livingstone, Edinburgh. latoxin-related serum peptide system. I. Purification, WILKINSON, P.C. (1976) Cellular and molecular aspects of crystallization and properties of classical anaphylatoxin chemotaxis of macrophages and monocytes. Immunofrom rat serum. Eur. J. Immunol. 2, 73. biology of the macrophage (ed. D.S. Nelson), pp. 349-365. WISSLER, J.H., STECHER, V.J. & SORKIN, E. (1972) Chemistry Academic Press, New York. and biology of the anaphylatoxin-related peptide system. WILKINSON, P.C. (1977) Action of sphingomyelinase C and III. Evaluation of leucotactic activity as a property of a other lipid-specific agents as inhibitors of Fc binding and new peptide system with classical anaphylatoxin and locomotion in human leucocytes. Immunology, 33, 407. cocytotaxin as components. Eur. I. Immunol. 2, 90. WILKINSON, P.C. (1979) Synthetic peptide chemotactic efficacy and inhibitory activity, and susceptibility of the ZIGMOND, S.H. (1979) Gradients of chemotactic factors in various assay systems. Functional aspects of Mononuclear cellular response to enzymes and bacterial toxins. Phagocytes (ed. R. van Furth). Martinus Nijhoff, The Immunology, 36, 579. Hague. (In press.) WILKINSON, P.C. & ALLAN, R.B. (1978a) Binding of protein chemotactic factors to the surface of neutrophil leucocytes ZIGMOND, S.H. & HIRSCH, J.G. (1973) Leucocyte locomotion and chemotaxis. New methods for evaluation and and its modification with lipid specific bacterial toxins. demonstration of cell-derived chemotactic factor. 7. Mol. Cell. Biochem. 20, 25. Exp. Med. 137, 387. WILKINSON, P.C. & ALLAN, R.B. (1978b) Chemotaxis of neutrophil leucocytes towards substratum-bound protein attractants. Exp. Cell. Res. 117, 403.

SANDILANDS, G.P., GRAY, K., COONEY, A., BROWNING, J.D. & ANDERSON, J.R. (1975) Formation of auto-rosettes by peripheral blood lymphocytes. Clin. exp. Immunol. 22, 493. SHEEHAN, J.C. & YANG, D.D.H. (1958) The use of N-formylamino acids in peptide synthesis. 3. Am. Chem. Soc. 80, 1154. VAN Epps, D.E. & WILLIAMS, R. (1976) Suppression of leucocyte chemotaxis by IgA myeloma components.

Relations between Fc receptor function and locomotion in human lymphocytes.

Clin. exp. immunol. (1979) 38, 598-608. Relations between Fc receptor function and locomotion in human lymphocytes J. M. SHIELD S & P. C. WILKINSON,...
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