Interaction between C3 and IL-2; inhibition of C3b binding to CR1 by IL-2

Immunology Letters, 21 (1989) 131-138 Elsevier IML 01216

Interaction between C3 and IL-2; inhibition of C3b binding to CR1 by IL-2 Istvfin Bartdk ~, A n n a Erdei 1, Athanasia Mouzaki 2, Hisao Osawa 2, J~inos Sz616si 3, Angelika Eigentler 4, Tibor Diamantstein 2, Manfred R Dierich 4 and Jfinos Gergely ~ IDepartment of Immunology, L. EOtvOs University, GOd, Hungary; 2Immunology Research Unit, Free University of Berlin, Berlin, F.R.G.; 3Department of Biophysics, Medical University, Debrecen, Hungary; 4Institute for Hygiene, Leopold-Franzens-University, Innsbruck, Austria (Received 15 October 1988; revision received 31 January 1989; accepted 1 February 1989)

1. Summary We previously reported [7] that C3 has a role in the enhancement of the IL-2 dependent proliferation of helper T cells. Because the IL-2R has a structural homology with the complement proteins, such as CR1 and CR2, we studied the possible ligand crossreactions on CR1 and IL-2-receptor, and the direct interaction between C3 and IL-2. While C3 has an enhancing effect on the IL-2 dependent proliferation of HT-2, a CRl-positive mouse T-cell line, the growth of the CTLL-16 line (CRl-negative) is not affected by C3. It has been proven that neither the insolubilised C3 nor the soluble C3b-like C3 react with the I b 2 binding epitope of the IL-2 receptor. However, using human RBC we have demonstrated that the binding of aggregated C3 to CR1 is inhibited by rlL2, in a dose-dependent manner. When RBC were in-

Key words." Complement receptor; IL-2 receptor; T cell; C3b; IL-2

Abbreviations." aggrC3, C3 aggregated by heating at 63 °C for 5 min; BSA, bovine serum albumin; C3, the third component of the complement system; C3a, C3b, iC3b, C3c and C3d, proteolytic fragments of C3; ConA, Concanavalin A; CR1, complement receptor type 1, specific for C3b; ELISA, enzyme linked immunosorbent assay; FITC, fluorescein isothiocyanate; HRPO, horse-radish peroxidase; IL-2R, receptor for interleukin 2; OPD, orthophenylene diamine; RBC, red blood cell(s); r i b 2 , recombinant interleukin 2

Correspondence to: Istvzin Bart6k, Department of Immunology, L. EOtv6s University, J~ivorka S.u. 14, H-2131 G6d, Hungary.

cubated with rlL-2 and FITC-labelled Fab-anti-CR1 simultaneously, there was no inhibition in the fluorescence intensity. As detected by ELISA, rlL-2 was bound to the same extent by insolubilised C3, C3b, and C3c, while C3d coat had lower binding capacity. The receptor-binding epitope of IL-2 is intact in the complex of complement proteins and rlL-2, as demonstrated by the binding of DMS1, a monoclonal antibody reacting with the receptor site of IL-2. It is strongly suggested that C3b may play a role in the growth of CR1 positive T cells. 2. Introduction The most important role of the complement system in induction and/or regulation of immune responses has been ascribed to the third component of complement (C3). Interaction of C3 and its proteolytic fragments - C3a, C3b, iC3b, C3c and C3d - with the corresponding receptors on lymphocytes and macrophages has been shown to modulate mitogen- and antigen-induced lymphocyte proliferation, lymphokine production, antibody formation, macrophage activation, the activity of natural killer cells and the generation of cytotoxic T cells [1-5]. It also has been demonstrated that C3 plays an important role in the immune response to T celldependent antigens [6]. However, the exact mechanism of this process has not yet been clarified. We have previously reported that C3b receptors (CR1) have a role in the enhancement of the IL-2dependent proliferation of helper T cells [7]. Recently it has been revealed that soluble and cell-

0165-2478 / 89 / $ 3.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)

131

membrane derived proteins which have the capacity to bind different fragments of C3 and C4, form a family based on the homology of their primary structure [8 - 10]. These molecules all contain an approximately 60 amino acid-long internal repeating unit with a characteristic framework of highly conserved residues. This structural homology is shared not only with complement proteins such as CR1, CR2, C2, factor B, factor H, C4b-binding protein, Clr and Cls, but also with the non-complement proteins; /32-glycoprotein I, the /3 subunit of clotting factor XlII and the receptor for IL-2 (IL-2R). In this paper we demonstrate that IL-2 inhibits binding of C3b to CR1. Moreover a ligand-ligand interaction between C3 and IL-2 is described, which may be responsible for the modulation of T cell growth by C3 and C3-derived fragments demonstrated earlier [7, 11, 12]. 3. Materials and methods

3.1. Reagents Concanavalin A (ConA), bovine serum albumin (BSA) and ortho-phenylene diamin (OPD) were purchased from Sigma (St. Louis, USA). Iodogen was from Pierce and Warriner (Chester, U.K.), and CNBr-activated Sepharose 4B from Pharmacia (Uppsala, Sweden). 3.2. Buffers The ligand-binding buffer used was phosphate buffered saline (pH 7.2) (PBS) containing 1070 w/v Triton X-100 and 2 mM phenylmethyl-sulfonylfluoride. 3.3. C3 and its derivatives C3 was prepared from human plasma by the purification method described by Hammer et al. [13]. C3b-like C3 was prepared by KBr-treatment as described by Janatova et al. [26]. C3 was cross-linked by glutaraldehyde according to the method of Papamichail and Pepys [14]. Heat-aggregation of C3 (1.8 mg/ml) was carried out by incubation at 63 °C for 5 min. C3b and C3c fragments were prepared according to established methods [15, 16]. C3d was generously 132

supplied by Dr. J. Lambris, Basel Institute for Immunology, Basel, Switzerland. The purity of each preparation was checked by SDS-polyacrylamide gel electrophoresis. Coupling of C3 to CNBr-activated Sepharose 4B beads was carried out according to the instructions of the manufacturer. 3.4. Radio-iodination Aggregated human C3 was radiolabelled with Na[125I] by the Iodogen method [27]. The specific activity of the preparation was 4×106cpm/t~g protein. 3.5. Antibodies Anti-C3c and anti-C3d antibodies were purchased from Behringwerke (ER.G.). Horse-radish peroxidase-(HRPO)-labelled anti-mouse lg and HRPO-labelled anti-rabbit Ig were from Nordic (Tilburg, The Netherlands). 3.6. Cells and IL-2-dependent cell lines ConA-blasts were obtained after culturing human peripheral blood leucocytes with the mitogen as described earlier [18]. Red blood cells (RBC) were separated from human blood drawn into heparin. CTLL-16, a cytotoxic mouse cell-line, was maintained in Click's medium (Biochrom KG, Berlin, ER.G.) supplemented with recombinant human IL-2 (50 U/ml) as described earlier [29]. The helper mouse cell-line HT-2 [30] was maintained in culture using conditioned medium derived from the culture supernatant of ConA-stimulated rat spleen cells. 3.7. C3-rnediated rosetting Zymosan-C3mo (Z-C3mo) was prepared using fresh mouse serum as described by Weir [191. Erythrocyte-C3Bhu (E-C3bhu) was prepared by incubation of RBC (100/~1; 1 × 108 cells/ml) with haemolytically active human C3 (30 ~1; 1 mg/ml) activated by trypsin (1 mg/ml; 20 #1) in the presence of the cells. For rosette formation, 25 ~1 of lymphocyte sus-

pension (5 x l06 cells/ml) were mixed with 25/A of the indicator cell suspension (1×108 particle/ml) followed by incubation at 37 °C for 30 min. Samples were put on ice for 2 h, then the percentage of mouse lymphocytes binding three or more Z-C3mo and EC3bhu was determined by light microscopy.

3.8. Cell proliferation assay Prior to the proliferation assay, cultured cells were washed into serum-free Click's medium and incubated for 30 rain at 37 °C. The cells were then plated at a concentration of 4xl03/well in 160/A Click's medium containing 10% FCS. Various dilutions of C3 and rlL-2 were added to the cells in 20 #1 of the medium. Cells were cultured for 24 h and pulsed with 0.5 izCi [3H]thymidine for the final 4 h. The results are shown as mean of triplicate samples.

3.9. Ligand binding to ConA-blasts and RBCs 3.9.1. Assay for the competitive binding of C3b-

like C3 and rlL-2 to ConA-blasts 1 ×106 T-blasts in 100#1 binding buffer were incubated with 100 #1 buffer containing different amounts of C3b-like C3, ranging from 0.6/~g/ml to 10 #g/ml, 10 U/ml rIL-2 added in 100 #1, and AHT54, the monoclonal antibody reacting with the ligand-binding site of IL-2R (500-fold dilution of ascites fluid, 100/A) for 30 min at 22 °C. Then 12sIlabelled rIL-2 (13 000 cpm/10/A/tube) was added to the samples followed by a further 45 min incubation period. Cell-associated radioactivity was measured after centrifugation of the samples through an oil-phase.

3.10. Enzyme

linked

immunosorbent

assays

(ELISA) For the assays ELISA plates of Dynatech (Denmark) were used. 3.10.1. The interaction between C3 and rlL-2 was detected by indirect assay. Wells were coated with intact C3, C3b, C3c and C3d, alternatively (5 /~g protein/ml of PBS; 100/d/well), then five-fold dilutions of rlL-2 (ranging from 15 to 250 U/well) were measured into the wells (in 40/A PBS), in duplicates. To detect IL-2 molecules bound to C3 or its fragments, DMS1, a mouse monoclonal antibody reacting with the binding site of IL-2, was added to the samples (applying 1000-fold dilution of ascites), followed by incubation with HRPO-labelled antimouse Ig. The reaction was developed using OPD as substrate. The optical density of the samples was measured using Dynatech-reader. As a control, wells were coated with BSA (100 ~tg/ml in PBS; 100/A/well). Plates were saturated with 0.1% BSA in PBS. 3.10.2. IL-2Rs in the detergent-extract of ConA-blasts were detected as described earlier [20]. Cell equivalent IL-2R content of the samples was calculated using a standard curve of ConA-blast lysate with a computer program developed for ELISA [21]. 4. Results 4.1. Effect of C3 on the IL-2-dependent growth of

different mouse cell-Snes 3.9.2. Competitive binding of C3 and rlL-2 to

RBCs 25/~1 of human RBC (2x 107/ml) washed with PBS, were mixed with different amounts of rlL-2 in 25 #1 PBS (ranging from 2 - 1 2 5 0 U/ml) for 1.5 h at 4 °C rotation followed by an overnight incubation at 4 °C with [125I]aggrC3 (92000 cpm/25 #1 PBS). In the control sample 100-fold amount of cold aggrC3 replaced the rlL-2. The experiment was done in triplicate. Radioactivity associated with the cells was measured after pelleting cells through oil-gradient.

Using HT-2, a mouse helper T cell line, we could confirm our earlier results [7], showing that C3 exerts a significant enhancing effect on the IL-2 dependent growth of the mouse helper cell line (Fig. 1). To check whether the same phenomenon could be observed in the case of cytotoxic T cells, the CTLL16 line was tested. Cells were cultured in the presence o f a suboptimal dose of rIL-2 and various concentrations of aggrC3. As illustrated in Fig. 1 the C3preparation had no influence on the IL-2-dependent proliferation of the cytotoxic T-cells. Moreover, 133

TABLE 2 HT-2

Binding of IL-2R to C3-coupled Sepharose 4B beads.

15

ConA-blast extract adsorbed with a

IL-2R × 10 4 cell equivalent in the adsorbed supernatant

rlL-2-agarose b BSA-Seph. 4B C3-Seph. 4B

18 +- 3 32 +_2 36 ± 4

x u "~ 10

x~ ~//

CTLL" 16

al00-~l of ConA-blast lysate were mixed with 25 /zl of affinity support (respectively with rlL-2-agarose, C3-Seph. 4B and as controls agarose-bead, BSA-Seph. 4B). Mixtures were rotated overnight at 4°C. After incubation the amount of IL-2R in the supernatants was determined by ELISA as described in Section 3. bControl agarose beads did not adsorb IL-2R from the detergent extract of ConA-blasts.

4E -

,-<

5

i x\ \\

4.2. C3-mediated rosetting of the helper (HT-2) and

cytotoxic (CTLL-16) cells

r IL-2

~11

medium

[~

rlL-2*C3(4Otug/mO

[~/'/JJ r l L - 2 + C 3 ( 2 0 / U g l m l )

Fig. 1, Effect of aggregated C3 on the proliferation of the IL-2dependent cell-lines CTLL-16 and HT-2. Cells were cultured at the density of 4× 103 cells/well, rIL-2 (1 U/ml) and aggregated human C3 were added to the cells at the initiation of the culture in 20/xl medium. [3H]thymidine was added at 20 h and cells were harvested 4 h later. Samples were set up in triplicates. Means _+ SD of one representative experiment are shown.

To test whether there is a difference in the C3 bind-

Z0

44c~

44-

4-

o x

15

E

neither non-aggregated C3 nor C3 polymerized by glutaraldehyde affected the growth of the CTLL-16 line (data not shown).

N i ,,,,,,,,, 10 - imHirl

I IN "6 5

TABLE 1 Rosetting of CTLL-16 and HT-2 ceils with C3 fragment carrying particles. Cells

CTLL-16 HT-2

°7o of rosettes formed with Z-C3mo

E-C3bhu

5+_2 32 +_3

7+_ 1 29 _+2

25 /xl suspension of the CTLL-16 and HT-2 lines (5× 106 cells/ml) were mixed with 25 /~1 of the indicator cell suspension ( I x 108 cells/ml), respectively. The percentage of rosetteforming cells was evaluated after incubating the samples on ice for 2 h. Means +- SD of three experiments.

134

E

butler

"cold " rlL-2

AHT 54

~uglml C3"b - [ike C3

Fig. 2. Binding of [125I]rlL-2 to human T-blasts is not inhibited by C3b-like C3. 106 T cell blasts in 100/xl buffer were incubated with different amounts of C3b-like C3, cold IL-2, anti-lL-2R antibody AHT-54, and buffer, respectively. After incubation for 30 min at 22 °C radiolabelled rlL-2 was added to the samples followed by 45 min incubation period. Cell-associated radioactivity was measured after centrifugation of the samples through an oilgradient. One representative experiment is shown.

tein/1 ml bead) was incubated with the IL-2R containing cell-extract of ConA-blasts. The unbound material was then tested for the presence of IL-2R by ELISA. As a positive control, agarose beads bearing rlL-2 were used for adsorption. Immobilised C3 did not bind IL-2R while rlL2 coupled to beads significantly decreased the IL-2R content of the detergent extract of ConA-blasts (Table 2).

20

% x

g15

I

ff 10 //

+

/t

35

% //

preincul~a'on of tesl cells with:

'~:old" PBS oggr-C3

4.3.2. In another set of experiments a competitive binding assay was performed using intact ConA-blasts, C3b-like C3 and radioiodinated rlL-2. As shown in Fig. 2, C3b-like C3 does not react with the IL-2 binding epitope of the receptor on intact cells at the concentrations tested. 4.4. Inhibition of ligand-binding to CR1 by rlL-2

unit ~ IL - 2

ing capacity of the HT-2 and CTLL-16 lines, C3mediated rosetting of these cells was tested. Helper cells of the HT-2 line formed rosettes with Z-C3mo as well as E-C3bhu, while the cytotoxic cells of the CTLL-16 line failed to do so (Table 1). This finding is in agreement with our earlier results proving that the ST1 helper line expresses CR1 [71.

In order to investigate further whether the homology between C3-binding structures and IL-2R has any effect on their ligand-binding capacity, the possible influence of rlL-2 on the interaction between C3 and CR1 was tested. For this experiment, human RBC were used, since they carry the CR1 but lack the IL-2R. Cells were incubated with a constant amount of ]25I-labelled heat-aggregated human C3, and cell-bound radioactivity was measured. As a control the inhibitory capacity of a 100-fold amount of 'cold' aggregated C3 (not radiolabelled) was assayed. The binding of C3 was inhibited by rIL-2, in a dose-dependent manner (Fig. 3). The binding of Fab-anti-CR1 to RBC was not inhibited by rIL-2 as FACS analysis revealed (data not shown), demonstrating that there was no direct binding of IL-2 to the binding site of CRI.

4.3. No interaction between IL-2R and C3

4.5. Interaction between C3 and IL-2

Based on the recently found homology between C3-binding molecules and the IL-2R [8, 9] we assumed that this structural similarity might play a role in the process observed by us [7] (see sections 4.1. and 4.2.). Two series of experiments were performed to clarify whether C3 interacts directly with the receptor for IL-2.

The results described in the previous sub-section raised the possibility of the interaction between the two ligands, C3 and IL-2. The binding of rlL-2 to the third component of complement was tested by an ELISA, using human C3 for coating the plates, rlL-2 was added to wells in five-fold dilutions. The binding of DMSI, a monoclonal antibody reacting with the receptor binding site of IL-2, was then used to evaluate the amount of bound IL-2. The results, shown in Fig. 4, clearly demonstrate that rlL-2 reacts with

Fig. 3. IL-2 mediated inhibition of the binding of [1251]aggrC3 to human RBC. Human RBC (2× 107/ml; 25/zl) were mixed with rlL-2 in 25 #I PBS and [125I]aggrC3 (92300 cpm in 25/zl PBS) overnight at 4 °C. Cells were separated by centrifugation through an oil-phase and cell-associated radioactivity was measured in a gamma-counter. The experiment was done in duplicates. Means _+ SD of one representative experiment are shown.

4.3.1.

C3 coupled to Sepharose 4B-beads (1 mg pro-

135

OD

0.6

Q2

..........

........

" ~ L ~ . ~ < ~ c3c

..................

......... o ................

o BSA

i

i

i

i

i

250

125

62

31

15

U

rlL-2

Fig. 4. B i n d i n g of rIL-2 to C3, C3b, C3c a n d C3d. E L I S A - p l a t e s were c o a t e d with C3 ( ), C3b ( . . . . . ), C3c ( - ) a n d C3d ( - . • ) (5 t~g/ml, 100 #l/well), t h e n i n c u b a t e d with serial dilut i o n s o f rIL-2. The reaction was developed using DMS-1, a m o n o c l o n a l a n t i b o d y to IL-2, H R P O - l a b e l l e d a n t i - m o u s e Ig a n d O P D as substrate. The results o f one representative e x p e r i m e n t are shown. BSA ( o • • - o ) was used as a control.

C3 in a dose-dependent manner. As a control, BSAcoated wells were also incubated with rIL-2, but no binding occurred in this case (Fig. 4). In order to identify the region of the C3 molecule which is involved in the interaction with IL-2, C3b, C3c and C3d fragments were used to coat the ELISA plates. As is shown in Fig. 4, C3b and C3c but not C3d bind IL-2 to the same extent as the intact, whole protein. From these results we conclude that I ~ 2 binds to intact C3, C3b and C3c, and this interaction does not affect the receptor binding site(s) of IL-2. Moreover, the epitopes of C3 recognized by anti-C3c and antiC3d antibodies are not identical with the growth factor binding sites (data not shown). 5. Discussion

In our previous [7] and present papers it is shown that the proliferation of helper T-cells can be affected by aggregated C3 or C3b-like C3 in contrast to the cells of the CTLL-16 line (Fig. 1). An explanation for this discrepancy between the response of the two T cell subsets can be that helper T cells carry higher amounts of receptors for C3b compared to the cyto136

toxic cells (Table 1). These data agree with the results of Wilson et al. [22] showing that 65% of human T cells possessing CR1 are OKT4-positive, while the percentage among the OKT8-positive cells is only 17%. Consequently the enhancing effect of aggregated C3 on the IL-2-dependent proliferation of helper T-cells could be ascribed to the triggering of the cells through clustering of CR1, a phenomenon described in another system by Schlessinger [28]. However, our findings reported here suggest yet another mechanism. We demonstrated that IL-2 reacts directly and dose-dependently with C3b (Fig. 4). The interaction between the two ligands does not interfere with the epitopes recognised by antibodies against the C3c and C3d regions of C3 (data not shown), demonstrating that not all epitopes are covered by IL-2. Moreover, the receptor binding site of IL-2 is not blocked by the interaction, since the DMS-1 monoclonal antibody which neutralizes the action of I ~ 2 on the cells does react with C3-bound IL-2 (Fig. 4). According to our experiments (not shown) 60 U of rIL-2 saturate solid-phase C3 adhering to the ELISA plate from 100/A solution containing 5 ~g C3. Under culture conditions the proliferationenhancing effect of C3 was observed only in the presence of suboptimal IL-2 concentration (i.e., 6 U/ml or less) (Fig. 1 and [7]). These data suggest that only IL-2 and C3 complexes of certain composition can bind effectively to both CR1 and IL-2R on the helper T cells, thus triggering the cells for proliferation. C3 and its fragments bind non-covalently to various molecules, such as IgG [23, 24] and LPS [25]. Investigation of the interaction of C3 with IL-2 revealed that the presence of C3a was not important for the binding. We found no significant difference between the IL-2 binding capacity of C3 (M r approx. 190000) and its high-molecular-weight fragments C3b (M r approx. 180000) and C3c (Mr approx. 145 000). However, C3d, the final degradation product of C3 (M r approx. 25 000) reacted with IL-2 to a lesser extent. C3d-K, (kallikrein-cleavage fragments of iC3b) and a C3-fragment preparation probably containing the active component of C3d-K [12] suppress the IL2-dependent proliferation o f T cells [11, 12]. To elucidate the differential effect of the larger (aggrC3, C3b-like C3) and smaller (C3d-K, the C3-fragment preparation) derivatives of C3 on the IL-2-

d e p e n d e n t T cell g r o w t h a q u a n t i t a t i v e a s s e s m e n t o f the interaction between the growth factor and the c o m p l e m e n t p r o t e i n is r e q u i r e d . We s h o w e d t h a t IL-2 c o m p e t i t i v e l y i n h i b i t s t h e b i n d i n g o f C3 to R B C (Fig. 3). IL-2 i n t e r a c t e d w i t h C3 (Fig. 4) b u t n o t w i t h CR1 ( d a t a n o t s h o w n ) . O n the other hand, experiments with the IL-2R-bearing C o n A - b l a s t s a n d w i t h t h e d e t e r g e n t extracts o f t h e s e cells s h o w e d t h a t C3 d o e s n o t b i n d to I L - 2 R (Table 2 a n d Fig. 2). T h e s e d a t a p r o v e t h a t , in spite o f t h e fact t h a t the IL-2R contains two of the homologous repeating units c h a r a c t e r i s t i c to C 3 - b i n d i n g p r o t e i n s [7], it d o e s n o t react w i t h t h e c o m p l e m e n t p r o t e i n . O n the o t h e r h a n d , IL-2 i n h i b i t s l i g a n d - b i n d i n g to CR1 as a consequence of the interaction between the ligands in t h e f l u i d phase, b u t n o t o n t h e c e l l - m e m b r a n e .

References [1] Schreiber, R. D. (1984) Springer Semin. Immunopathol. 7, 221 - 249. [2] Ross, G. D. and Medof, M. E (1985) Adv. Immunol. 37, 217 - 267. [3] BOttger, E. C. and Bitter-Suermann, D. (1987) Immunol. Today 8, 261-264. [4] Melchers, E, Erdei, A., Corbel, C., Leptin, M., Schulz, T. E and Dierich, M. P. (1986) Mol. lmmunol. 23, 1173-1176. [5] Dierich, M. P., Erdei, A., Huemer, H., Petzer, A., Stauder, R., Schulz, T. E and Gergely, J. (1987) Immunol. Lett. 14, 235 - 242. [6] Pepys, M. B. (1974) J. Exp. Med. 140, 126-145. [7] Erdei, A., Spaeth, E., Alsenz, J., Ri.ide,E., Schulz, T., Gergely, J. and Dierich, M.P. (1984) Mol. Immunol. 21, 1215-1221. [8] Reid, K. B. M., Bently, D. R., Cambell, R. D., Chung, L. P., Sire, R. B., Kristensen, T. and Tack, B. E (1986) Immunol. Today 7, 230-234. [9] Wilson, J. G., Andriopoulos, N. A. and Fearon, D. T. (1987) Complement 6, 192-209.

[10] Dierich, M. P., Schulz, T. F., Eigentler, A., Huemer, H. and Schwfible, W. (1988) Mol. Immunol. (Special Issue: Signals and Signal Processing V) 25, 1043-1051. [11] Meuth, J. L., Morgan, E. L., Di Scipio, R. G. and Hugli, T. E. (1983) J. Immunol. 130, 2605-2611. [12] Walker, C., Kristensen, E, Bitter-Suerman, D., Stadler, B. M. and Weck, A. L. (1986) J. lmmunol. 136, 3396-3401. [13] Hammer, C. H., Wirtz, G. H., Renfer, L., Gresham, H. D. and Tack, B. E (1981) J. Biol. Chem. 256, 3995-4003. [14] Papamichail, M. and Pepys, M. B. (1970) Immunology 36, 461 - 468. [15] Bokisch, V.A., Mfiller-Eberhard, H.J. and Cochrane, C. G. (1969) J. Exp. Med. 129, 1109-1113. [16] Schulz, T. E, Scharfenberger, H., Lambris, J. D., Rieber, P., Riethmfiller, G. and Dierich, M. P. (1985) Immunology 54, 791-800. [17] Vik, D.P. and Fearon, D.T. (1985) J. Immunol. 134, 2571-2579. [18] Diamantstein, T. and Osawa, H. (1984) Mol. Immunol. 21, 1229-1236. [19] Weir, D. M. (1986) Handbook of Experimental Immunology, Vol. 2, Blackwell Scientific Publications. [20] Osawa, H., Josimovic-Alasevic, O. and Diamantstein, T. (1986) J. Immunol. Meth. 92, 109-115. [21] Caulfield, M. J. and Shaffer, D. (1984) J. Immunol. Methods 74, 205-215. [22] Wilson, J. G., Tedder, T. E and Fearon, D. T. (1983) J. Immunol. 131, 684-689. [23] Hautanen, A. (1981) Scand. J. Immunol. 13, 245-254. [24] Kulics, J., Rajnav61gyi, t~, Ffist, G. and Gergely, J. (1983) Mol. Immunol. 20, 805-810. [25] Beuscher, H. U. and Brade, V. (1986) Immunobiology 173, 41-55. [26] Janatova, J., Lorenz, P. E., Schechter, A. N., Prahl, J. W. and Tack, B. E (1980) Biochemistry 19, 4471-4478. [27] Markwell, M. A. K. and Fox, E (1978) Biochemistry 17, 4807-4817. [28] Schlessinger, J. (1979) in: Physical-Chemical Aspects of Cell-surface Events in Cellular Regulation (C. De Lisi and R. Blumenthal, Eds.), Elsevier Science Publishing Co., New York. [29] Osawa, H. and Diamantstein, T. (1984) J. Immunol. 132, 2445 - 2450. [30] Watson, J. (1979) J. Exp. Med. 150, 1510.

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