230
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4 Winchester, R.J., Fu, S.M.,Hoffman, T.and Kunkel, H.C., J. Immunol. 1975.114: 1210.
13 Nisonoff, A.,Wissler, F.C., Lipman, L.N. and Woernely, D.L., Arch. Biochem. Biophys. 1960.89: 230.
5 Haustein, D., J. Immunol.Meth. 1975. 7 25.
14 Scheidegger,J.J.,Int. Arch. Allergy Appl. Immunol. 1965. 7: 103. 15 Levy, H.B. and Sober, H.A.,Proc. SOC.Exp. Biol. Med. 1960.103: 250.
6 Bergman, Y ., Blatt, C. and Haimovich, J., Isr. J. Med. Sci. 1976. 12: 1360. 7 Roelants, G.E., Mayor, K.S., Hag, L.B. and Loor, F., Eur. J. Immunol. 1916.6: 75. 8 Little, J.R. and Eisen, H.N., in Williams, C.A. and Chase, M.W.
(Eds.), Methods in Immunology and Immunochemistry, Vol. I, Academic Press, New York 1967, p. 128. 9 Axe'n, R., Porath, J. and Ernback, S . , Nuture 1967.214: 1302. 10 Porter, R.R., Biochem. J. 1959. 73: 119.
16 Davidson, W.F. and Parish, C.R., J. Immunol. Meth. 1975. 7: 291. 17 Cebra, J.J. and Goldstein, G.,J. Immunol. 1965. 95: 230. 18 Haughton, G. and McCehee, M.P., Immunology 1969. 16: 447. 19 HaranGhera, N., Kotler, M. and Meshorer, A., J. Nut. Cuncer Inst. 1967.39: 653.
11 Laemmli, U.K., Nuture 1970.227: 680.
20 Rubin, B., J@gensen, P.N. and HQier-Madsen,M.,Scund. J. Im. munol. 1976.5: 450.
12 Cohen,A. and Schlesinger,M., Trunspluntution 1970.10: 130.
21 Perlman, P. and Perlman, H., Cell. Immunol. 1970. I : 300.
J.L. Molenaar, M. van Galen, A.J. Hannema, W. Zeijlemaker and K.W. Pondman
Spontaneous release of Fc receptor-like material from human lymphoblastoid cell lines
Central Laboratory of the Netherlands Red Cross Blood Transfusion Service and University Laboratory of Experimental and Clinical 'Immunology, Amsterdam
In the culture medium of some human lymphoblastoid cell lines material is released with the following properties: (a) hemagglutination reaction of IgG-sensitized erythrocytes; ( b ) enhancement of precipitation of DNA-antiDNA complexes; (c) inhibition of binding of C l q to immune complexes; (d) inhibition of immune complex binding to lymphocytes; (e) inhibition of antibody-dependent lymphocytotoxicity The material is not identical with C l q o r rheumatoid factor, it is heat resistant (30 min at 56 "C); the molecular weight is about 100 000 daltons and it is capable of inhibiting antibody production in vitro. I t is suggested that this material consists of Fc receptors spontaneously shed from lymphocyte membranes.
1. Introduction Recently, a number of reports have appeared describing spontaneous release of lymphocyte receptors. For example, Ramseier [ 11 described spontaneous shedding of T lymphocyte receptors for alloantigens. Owen [2] reported the release of the T lymphocyte receptor for sheep erythrocytes (SRBC) in the supernatant o f phytohemagglutinin (PHA)stimulated lymphocytes. Fridman and coworkers suggested that Fc receptors ( F c R ) may be released from T lymphocytes [3-61. They pub[I 15821 Correspondence: J.L. Molenaar, Department of Immunochemistry, Central Laboratory of the Netherlands Red Cross Blood Transfusion Service, P.O. Box 9190, Amsterdam, The Netherlands Abbreviations: FcR: Fc receptor FcR-lm: Fc receptor-like material KLH: Keyhole limpet hemocyanin T lymphocyte: Thymusderived lymphocyte(s) SRBC: Sheep erythrocyte PHA: Phytohemagglutinin EA: Antibody-coated erythrocyte SLE: Systemic lupus erythematosus B lymphocyte: Bone marrowderived lymphocyte(s) FCS: Fetal calf serum RFC: Rosette-forming cell BSA: Bovine serum albumin PBS: Phosphate buffered saline E: Erythrocyte FITC: Fluorescein isothiocyanate ADL: Antibodydependent lymphocytotoxicity PFC: Plaque-forming cell RF: Rheumatoid factor
lished data about the spontaneous release of an immunoglobulin-binding factor from antigen-activated thymus-derived lymphocytes ( T lymphocytes). This material agglutinated antibody-coated erythrocytes (EA), it inhibited complement-dependent hemolysis of EA at the level of C 1 (therefore it has specificity for a region close t o o r identical with the complement binding site of IgG), it had Fc specificity for IgG, could be purified on Sepharose-IgG, and appeared capable of suppressing antibody synthesis in vitro [7]. The possibility o f release of FcR from lymphocytes in vivo has been suggested by Diaz-Jouanen [8] who demonstrated in sera of systemic lupus erythematosus (SLE) patients a factor that could specifically block antibody-dependent lymphocytotoxicity (ADL). Until recently it was thought that the membrane-bound FcR on lymphocytes had exclusive affinity t o immune complexes o r aggregates containing IgC. Such an IgG FcR was found to be present o n B as well as on T lymphocytes [9- 161. However, Moretta [ 171 and McConnell [ 181 recently demonstrated that a subpopulation of peripheral T lymphocytes had a receptor f o r antigendgM antibody complexes. Furthermore, Lamon [ 19, 201 and coworkers demonstrated that both IgM and IgG antibodies were able t o induce target cell destruction by normal mouse lymphocytes. The IgM FcR appeared t o be present on both B and T cells.
Eur. J. Immunol. 1977. 7: 230-236 It has also been reported that human lymphoblastoid cell lines carried FcR [2 1 ] and that such cell lines produced prcl teins which can bind to antigen-antibody complexes [ 221. The purpose of the present investigation was t o determine whether human T and B cell lines release material with properties of an FcR and t o see whether it has specificity for IgC, for IgM or for both. The data suggest the release of FcR-like material (FcR-lm) with Fc specificity for both I& and IgM.
2. Materials and methods 2.1. Cell lines
Established B cell lines (Raji, Daudi, P3HR1, EB3, TSI 1 and CCRF-SB) and T cell lines (CCRF-CEM, CCRF-HSB, Molt 4) were mainly obtained from several laboratories (Dr. G.Klein, Karolinska Institute, Stockholm, Sweden; Dr. 0. Schonherr, Department of Microbiology, Organon, Oss, The Netherlands; Dr. T.A.W. Splinter, Central Lab. of the Netherlands Red Cross Blood Transfusion Service, Amsterdam, The Netherlands). The cells were grown at 37 "C in a suspension culture in RPMI 1640 (Gibco, Grand Island, N.Y.) supplemented with 10 % heat-inactivated fetal calf serum (FCS), penicillin (100 pglml), and with streptomycin (50 pg/ml). 2.2. Preparation of cell line supernatants
(a) Cell lines were cultured for two days up to a density of about 2 x lo6 cells/ml in RPMI 1640 f 10 % heat-inactivated FCS. Afterwards the cells were centrifuged at 600 x g for 10 min and the supernatants were collected; (b) cell line cells washed five times with serum-free RPMI 1640 were cultured for a period of 3 h at 37 "C in serum-free RPMI 1640. Supernatants were collected after centrifugation for 10 min at 600 x g. In both cases the cell line suspensions contained more than 95 % viable cells as judged by trypan blue exclusion.
Fc receptor-like material from human lymphoblastoid cell lines
231
scribed above, followed by affinity chromatography as described by Dickler [ 1 I]. Human IgG was isolated from normal serum by precipitation in 50 % (NH&S04 (twice) and by chromatography on DEAE-cellulose in 0.02 M phosphate buffer pH 7.5. F(ab')2 fragments of human IgG were prepared by hydrolysis with 2 % pepsin (Worthington Biochemical Corp., Freehold, N.J.) in 0. I M acetate buffer, pH 4.5, for 20 h at 37 "C. The hydrolysis was stopped by addition of a saturated solution of Tris t o a pH of 7.5. The material was then applied to a calibrated Sephadex G l 5 0 column, equilibrated and eluted with 0.15 M phosphate buffered saline (PBS), pH 7.5. The F(ab')z-containing fractions were pooled and concentra ted. Highly purified C Iq was isolated from fresh human serum as described by Yonemasu et al. [26]. C l q appeared to be pure in immunodiffusion. The isolated protein was stored in a 0.1 M phosphate buffer, pH 7, at a concentration of 1 mg/ml at -70 "C until use. For use in the inhibition assay C l q was labeled with lZsI according t o the method of Hunter et al. [ 271. The specific radioactivity was about 0.5 pCi/pg protein. Bovine serum albumin (BSA) at a concentration of 1 mg/ml was added before storage at - 70 "C. 2.4. Detection of FcR on cell line lymphocytes 2.4.1. Rosette formation with EA cells
EA complexes were prepared by incubating 0 Rh erythrocytes ( 5 x lo8 cells/ml) with human anti-D serum (final dilution 1 :4) for 30 min at 37 "C. Only one batch of anti-D serum was used selected for optimal rosette formation. 0.4 x lo6 lymphocytes were mixed with lo7 EA (total volume 0.25 ml of BSS + 10 % heat-inactivated FCS). After centrifugation for 5 min at 200 x g the mixture was incubated for 30 rnin at 25 "C. After resuspending, the percentage of EA-RFC was scored by counting 200 cells in a hemocytometer. A cell with more than four adherent erythrocytes was regarded as a rosette. 2.4.2. Immunofluorescence with KLH-anti-KLH soluble complexes
2.3. Antibodies, immunoglobulins and C l q
Anti-Forssman sera were induced in rabbits [23]. IgG fractions were prepared by precipitation in 50 % (NH4)2S04 and chromatography on DEAE-cellulose with 0.02 M phosphate buffer, pH 7.5. IgM fractions were prepared by DEAE chromatography using a linear salt gradient from 0.02 M to 0.3 M phosphate buffer, pH 7.5 and gel filtration on Sephadex G-200 [ 241. Neither the IgG was contaminated by IgM, nor was the collected IgM contaminated by IgG, as was demonstra ted by immunoelectrophoresis. Anti-DNA IgG and IgM fractions were prepared from a serum of a patient with SLE with high anti-ds-DNA binding capacity by isokinetic sucrose density gradient centrifugation as described by Aarden [25]. Fractions containing IgM and IgG were separately pooled. No contaminating IgG could be detected in the IgM fractions as determined by immunodiffusion, and vice versa. Anti-mouse mastocytoma cell IgG antibody fractions were prepared from rabbit antisera by isokinetic sucrose density gradient centrifugation. Anti-keyhole limpet hemocyanin (KLH) IgG antibodies were purified by isokinetic Sucrose density centrifugation as de-
Lymphocytes ( 1 00 pl; 107/ml in Earle's- 1 % BSA, pH 7.5) were incubated with rabbit IgG antibodies to KLH (0.2 mg/ml) for 10 min at room temperature. Thereafter, fluorescein isothiocyanate (FITCtlabeled KLH (0.1 mg/ml) was added, followed by another incubation at room temperature for 30 min. The cells were then washed with Earle's buffer supplemented with 1 % BSA, fixed with paraformaldehyde ( 1 % in PBS, pH 7.4), and washed with PBS. The cells were mounted in 50 % glycerol and examined with fluorescence microscopy using a Leitz Orthoplan microscope [28]. 2.5. Sepharose-coupled protein
CNBr-activated Sepharose 4B was prepared as described previously [29]. Human IgG or F(ab')2'y was covalently bound t o the beads in a concentration of 10 mg/ml packed Sepharose. After coupling had taken place the remaining active groups were blocked by incubation with 0.2 M ethanolamine (pH 9.0). Noncovalently bound proteins were eluted by a short washing with 1 % glycine, 2 % citric acid, pH 2.5 until A28onrn was zero. The beads were finally washed and equilibrated in 0.02 M phosphate buffer, pH 7.0.
232
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2.6. Purification of FcR-lm
A column was prepared of 150 ml Sephadex G 2 5 containing 2 0 ml IgG-bound Sepharose. 200 ml of a cell line supernatant dialyzed against 0.02 M phosphate buffer, pH 7.0, was applied t o the column and washed with the same buffer. The I&bound FcR-lm was eluted with 1 % glycine, 2 % citric acid, pH 2.5. The Sepharose G-25 caused a quick separation of the FcR-lm from the acid buffer. Effluents and eluates were collected, concentrated and tested. As control preparations, supernatants of cell lines, in which n o FcR-lm could be detected, were processed on Sepharose-IgG. Analogous fractions were collected, concentrated and used. Sepharose-IgC used for purification of the FcR-lm could not be used more than once. After acid elution the bound IgC quickly lost the property t o bind FcR-lrn. Since the Sepharose-IgG had t o be prewashed with an acid buffer of pH 2.5, it had only a relatively low capacity to bind FcR-lm. 2.7. Detection of FcR-lm 2.7.1. Hemagglutination of EA(IgC)
20 "C for 30 min; (c) 1 pg of 12SI-labeledC l q in 10 pl was added, followed by a second incubation for 30 min at 2 0 "C; (d) 200 pl of the reaction mixture was layered on 150 pl of a 40 7% sucrose solution in 0.05 M sodium phosphate buffer, pH 7.0 in a 400 p l polyethylene microfuge tube (Beckman Instruments Inc., Fullerton, CA.). Centrifugation was performed at about 1 3 000 x g in a Beckman microfuge 152 for 5 min. The tip of the microfuge tube containing the pelleted cells was cut off with a razor blade and collected in a tube. The upper portion of the microfuge tube, its contents included, was added t o a second tube. The radioactivity in both tubes was measured. The 12sI-labeledC Iq uptake by EA is calculated as described by Sobel [30]. To determine inhibition of lZSI-labeledC l q binding t o E A by FcR-lm, uptake of C l q by E A in the absence of FcR-lm was quantitated simultaneously. The inhibition percentage of uptake was calculated from: % uptake in control - % uptake in test x 100 % uptake in control 2.7.4. Inhibition of immune complex binding t o lymphocytes
by FcR-lm
SRBC (E) were suspended at a concentration of lo9 cells/ml. These cells were coated with purified rabbit anti-Forssman IgG by incubating the cells with a sub-agglutinating concentration of antibodies for 30 min at 3 7 "C in barbital buffer, pH 7.5. Microtiter plates (Cooke) were filled with 50 pl 1 % BSA in PBS, 50 /A EA (5 x 1 07/ml) and 50 pl of an FcR-lm preparation in two-fold dilutions. The hemagglutination titer was expressed as the reciprocal of the highest dilution of FcR-lm causing agglutination.
Lymphocytes ( l o 6 in Earle's medium - 1 % BSA, pH 7.5) were incubated with 100 pl FcR-lm and 100 pl rabbit IgG antibody preparation (0.2 mg/ml) t o KLH for 10 min at room temperature, followed by addition of FITC-labeled KLH (0.1 mg/ml) and another incubation at room temperature for 2 0 min. The cells were washed, fixed and examined as described in Sect. 2.4.2.
2.7.2. Enhancement of DNA-anti-DNA precipitation
A micro assay sytem for detection of ADL was used as described by Zeijlemaker e t al. [31]. 2 x l o 6 mouse P815-X2 mastocytoma cells radiolabeled with SICr were optimally sensitized with rabbit anti-mastocytoma serum or the purified IgG fractions thereof. The cytotoxicity tests and their inhibition were performed in round-bottom microtiter plates (Cooke). Each well contained 2 x 1 O3 target cells and various amounts of effector cells, starting at 2 x l o 5 (ratio effector/target = 100). For inhibition experiments mainly a ratio of 50 was used. The target cells were first incubated with 5 0 p l of a FcR-lm preparation during 15 min at room temperature. Next, 50 pl of a lymphocyte suspension was added. T h e plates were then incubated for 3 h at 37 "C. Controls included target cells with FcR-lm alone, and unsensitized target cells in the presence of FcR-lm and effector cells. The latter two controls usually gave a 51Cr release between 5 and 1 0 %. From the raw data, the net 5'Cr release could be calculated after correction for spontaneous and maximal "Cr release [29]. The percentage of inhibition of ADL was calculated from:
Purified anti-ds-DNA of both Ig classes was mixed with jHlabeled PM2-DNA [25] (20 ng; 60 000 cpm/pg) and incubated for 60 min in a total volume of 150 pl at 3 7 "C. Next, 50 p l purified FcR-lm was added t o the DNA-anti-DNA complexes, followed by another incubation of 120 min at 4 "C. Precipitating DNA-anti-DNA complexes were then spun down at 2000 x g. To 100 /A of the supernatant 0.5 ml of Soluene 350 was added, followed by 10 ml o f scintillation liquid (toluene - PPO - POPOP). The radioactivity was counted in a Packard liquid scintillation counter, model 3320, at a counting efficiency of 40 %. All determinations were carried out in duplicate. The amounts of IgG and IgM-anti-DNA were chosen in such a manner that without addition of FcR-lm about 15-20 % of the labeled DNA was spun down. The percentage of precipitate was calculated from: total counts - measured counts x 100 (%) total counts 2.7.3. Inhibition of C l q binding
This test is a modification of the immune complex detection method described by Sobel [30] and consisted of the following steps: (a) 50 pl of a n FcR-lm-containing sample, 100 p l saline and 100 p l of a sucrose barbital buffer (3 mmho/cm) were mixed in a plastic test tube (final pH 7.2, conductivity 9 mmho/cm). (b) 4 x lo7 EA were added and the mixture was incubated at
2.7.5. Inhibition of ADL
% net "Cr release - % net "Cr release (+FcR-lm) x 100 (%) % net " ~ release r
2.7.6. Suppression of in vitro antibody synthesis by FcR-lm
Spleen cells o f B6D2P1 mice (8 x 1 O6 - 10 x 1 06/ml) were cultured i n t h e presence of 1 % of SRBC for 5 days. To some cultures graded doses of purified FcR-Im o r control preparations were added a t time zero of incubation. Direct plaque forming cells (PFC) were assayed as described by Gisler [7].
Eur. J. Immunol. 1977.7: 230-236
Fc receptor-like material from human lymphoblastoid cell lines
233
2.8. Assays for detection of C l q or Ig in FcR-lm preparations
3.3. Purification of FcR-lm
2.8.1. Quantitative immunofluorescence
In order to purify FcR-lm, 200 ml crude CCRFCEM supernatant (medium RPMI 1640 + 1 0 % heat-inactivated FCS or serum-free RPMI 1640) was introduced onto a column consisting of 25 ml Sepharose-IgC and 150 ml Sephadex (3-25. The elution profiles obtained with different test systems after uncoupling the FcR-lm from Sepharose-IgG with the acid buffer are demonstrated in Fig. 1. The peak fraction showed an inhibition of C 1q binding of 6 %, an inhibition of 20 % of the ADL, and a 15 % enhancement in precipitation of DNA-anti-DNA complexes. Agglutination of EA(1gG) (with a titer of 4) and inhibition of KLH-anti-KLH binding to the respective cells (18 %) could only be demonstrated in the peak fraction. The slight increase in precipitation of DNAanti-DNA or inhibition of C l q binding in the late fractions were artefacts due to the decrease of the pH during elution with the acid buffer necessary to uncouple the FcR-lm from Sepharose-IgC. Analogous elution profiles could be obtained with supernatants of the T S l 1 cell line. The positive fractions from the eluates were pooled, concentrated and used for further experiments. Fractions obtained after elution of heatinactivated FCS or after elution of the supernatants from the P3HR1, Molt4 or the CCRFSB were negative as to biological activity. In this case unspecific binding of proteins from the control supernatants occurred. Acid eluates of such controls demonstrated an A~SO,,,,,in the peak fraction of about 0.4. Acid eluates obtained from supernatants of CCRFCEM or TSl 1 processed on F(ab')lcoated Sepharose columns were also negative.
Human purified C 1q was covalently linked to Sepharose4B at a concentration of 1 mg/ml packed Sepharose as described in Sect. 2.5. Rabbit anti-human C l q (0.5 ml) were incubated in a dilution of 1 :40 at room temperature with 10' SepharoseC l q beads for 30 min in the presence o r absence of purified FcR-lm. After washing three times with PBS the beads were finally incubated with 0.5 ml FITC-labeled horse anti-rabbit Ig at a dilution of 1:40 at room temperature for another 30 min. After additional washing the liquid was removed and the beads were mounted in 50 % glycerol in PBS. Quantitative measurement of the fluorescence was performed as described by Cape1 [32]. 2.8.2. Hemagglutination inhibition Human C l q or Ig were coupled to SRBC at a concentration of 10 c(g/5 x l o 7 E using the CrCl3 technique (0.07 % CtC13). The agglutination of the coated cells by serially diluted antiC l q or anti-Ig was tested in the presence o r absence of purified FcR-lm.
3. Results 3.1. Production of FcR-lm by B and T lymphoblastoid cell lines The various human T and B cell lines were tested for release of FcR-lm activity into their supernatant. For rough screening, the capacity of the different supernatants to agglutinate EA(IgG) cells was investigated. From the B cell lines the TS 1 1 supernatant had the strongest activity with an agglutination titer of 16. Supernatants of the Daudi, Raji, EB were slightly positive with a titer of 4. Supernatants from P3HR1, CCRFSB were negative. Among the T cell lines, only the supernatant of CCRFCEM had activity (titer of 16). It made no difference whether FCS was present in the medium or not. The positive supernatants also had the capacity to inhibit C l q binding to EA (6- 10 %), to inhibit KLH-anti-KLH binding to lymphocytes (20 %) and to inhibit antibody-dependent lymphocytotoxicity (5-8 %). On the basis of the results mentioned above, the CCRFCEM and TSl 1 cell lines and their supernatants were selected for further investigation. the negative supernatant of the P3HR line served as a control. 3.2. Presence of FcR on the lymphoblastoid cell surface The hypothesis of these experiments is that FcR from the cell surface are spontaneously shed into the supernatant. Therefore, the lymphoblastoid cell line cells were investigated for the presence of FcR on the cell surface, using EA rosette formation and immunofluorescence with KLHanti-KLH soluble complexes. Detection of FcR o n the cell surface using KLH-anti-KLH complexes appeared to be more sensitive than rosette formation, as will be described elsewhere (in preparation). Using the immunofluorescence test with KLH-anti-KLH IgG complexes 17 f 2 % of the CCRFCEM and 20 f 2 % of TSl 1 cells were positive for FcR. Rosetting percentages were 5 and 12 %, respectively.
O.'
r I
0.5 k
I
I
,2044
Figure I. An example of acid elution profiles of FcR-lm when crude CCRFCEM supernatant (medium RPMI 1640 + 10 % heat-inactivated FCS) WBS processed on Sepharose-IgG. The volume of the eluted frao tions was 2 mL (.----0 )Absorbance at 280 nm; ( O w )enhancement DNAanti-DNA precipitation (%); (.--.) inhibition of ADL (%); (A-*-*-A)inhibition of Clq binding to EA (96).
--
3.4. Demonstration of FcR-lm activities 3.4.1. Enhancement of immune complex precipitation
When a dose response of purified pooled and concentrated FcR-lm was performed by testing its capacity to enhance the precipitation of DNA-anti-DNA IgG complexes, curves were obtained as in Fig. 2. Surprisingly, enhancement of precipitation could also be observed when purified IgM anti-DNA-
234
Eur. J. immunol. 1977. 7: 230-236
J.L.Molenaar, M.van Galen, A.J. Hannema et. al.
DNA complexes were used (Fig. 2). Both effects could be obtained with purified FcR-lm from either CCRFCEM or TSl 1 supernatants. The P3HRl control appeared t o be negative with both IgM as well as the IgG anti-DNA-DNA complexes. Maximal precipitation was obtained for FcR-lm (CCRFCEM) at a dilution of 1 :4, and for FcR-lm (TSl 1) at a dilution of 1:2. Since we have so far no exact information about the mechanism of enhanced precipitation, it should be noted that the curves give a qualitative rather than a quantitative impression of the phenomenon. The course of the curves suggests a bivalent character which also explains the agglutination of EA. In the absence of antibodies to DNA both FcR-lm preparations did not precipitate DNA d o n e when tested at a 1 :4 dilution.
3.4.3. Inhibition of the binding of KLH-anti-KLH-IgC to lymphocytes
Addition of different amounts of purified FcR-lm obtained form CCRFCEM or TSl 1 supernatants (two-fold dilutions) to their respective cell line cells in the presence of FITCKLH-anti-KLH complexes reduced the number of fluorescing cells. Only with the undiluted FcR-lm preparations a reduction from 17 % to 10 % for the CCRFCEM cell line, and from 20 % to 1 1 % for the TSl 1 cell line cells was observed. 3.4.4. Inhibition of ADL
A dose response of both purified FcR-lm preparations, with respect t o inhibitisn of antibody-dependent lymphocytotoxicity, is given in Fig. 4. In these ADL experiments the target cell antibodies were of the I g C class. The FcR-lm of CEM supernatant appeared to be a more efficient inhibitor than the TSl 1-FcR-lm. Experiments with purified IgM mastocytoma cell antibodies will be carried out to demonstrate whether, under conditions at which the effector cells were cultured overnight in 10 % FCS, an IgM-dependent lymphocytotoxicity could occur, since only under these conditions the IgM-FcR could be detected on the lymphocyte surface. Inhibition of such an IgM-dependent cytotoxicity will be tried to show with purified FcR-lm.
Figure 2. Enhancement of DNA-anti-DNA precipitation by purified FcR-lm (c---o) IgM anti-DNA: CCRF-CEM; (0-0) IgC anti-DNA: CCRF-CEM; ( 0 - 4 ) IgM anti-DNA TS11; ( ~ m I&) anti-DNA: IgG anti-DNA: TS11; (A---A) IgM anti-DNA: P3HR1; @-A) P3HR1.
3.4.2. Inhibition of C l q binding
Since FcR on lymphocytes bind t o approximately the same site on Ig as does C l q [33], purified pooled and concentrated FcR-lm was further tested for its capacity to inhibit C 1q binding t o EA coated with both IgG or IgM antibodies. It was found that C l q binding to both antibody types was inhibited. The results of a dose response of the same preparations as shown in Fig. 2 with both IgG and IgM antibodies are demonstrated in Fig. 3. Both FcR-lm preparations caused somewhat higher inhibition of C 1q binding t o EA-IgM than to EA-IgG.
Figure 4. Inhibition of ADL by purified FcR-lm Target cells sensitized CCRF-CEM; @-A) TS11; (0-a) with IgG antibodies. (0-0) P3HR1.
3.4.5. Inhibition of in vitro antibody synthesis by FcR-lm
I . .
8
h'ra b
+4
a
'4 urnpaam
Figure 3. Inhibition of Clq binding to EA by purified FcR-lm (x-X) (0-0)
EA-IgM: CCRFCEM; (0- 0 ) EA-IgG: CCRF-CEM; EA-IgM: TS11; (v-V) EA-IgG: TS11; (+O):P3HR1.
The possible interference of purified FcR-lm with antibody synthesis in vitro was also investigated. Addition of different amounts of FcR-lm together with SRBC to a mouse spleen cell culture at the initiation of the response, significantly suppressed the PFC response as demonstrated in Table 1 Suppression of the PFC response was not due to a decrease in the number of viable cells. FcR-lm from CCRFCEM and TSl 1 were both inhibitory, whereas material obtained after adsorption and acid elution of the control P3HRl supernatants on Sepharose-IgC did not inhibit the antibody production in vitro. Also, CCRFCEM o r TS11 supernatants similarly processed on Sepharose-F(ab')yy columns did not result in preparations that contained suppressor material.
Eur. J. Immunol. 1977. 7: 230-236
Fc receptor-like material from human lymphoblastoid cell lines
Table 1. Effect of purified FcR-lm on the response of spleen cell cultures to SRBC added at day zero Additions
Microliters
No. of PFO)
Inhibition
(96) None
-
30 CCRF-CEM 90 TSl 1 30 TSll 90 120 P3HR1 CCRF-CEM~) 120 120 TSl lb) System without SRBC
CCRF-CEM
2 080 f 224 1 1 4 0 f 85 700 f 283 1 565 f 247 1 195 f 276 2 m * m 2040f186 1900f230 305 f 106
53 78 29 49
a) Arithmetic means (+standard deviation) of PFC counts (day 5)
from four independent experiments. b) Processed on Sepharose-F(ab')zy.
3.4.6. Properties of FcR-lm
Gel filtration on Sephadex (3-200 of the SepharosaIgG purified FcR-lm and testing the fractions on their biological activity using human C3b and albumin as markers, demonstrated a molecular weight of about 100 000- 1 10 000 daltons for the FcR-lm. Furthermore, the purified FcR-lm seemed to be heat-resistant (30 min, 56 "C). The properties of both the T and B FcR-lm are summarized in Table 2. The purified T and B FcR-lm preparations used in the above test systems were extensively screened for the presence of C l q or Ig. Addition of FcR-lm to a n t i c l q did not decrease its binding to Sepharose-bound C 1q, as was tested in a quantitative immunofluorescence assay, or by agglutination of C lq-CrC13-erythrocytes. With these test systems it was possible to detect 1 - 10 ng/ml C 1q in control experiments. Absence of C l q was also demonstrated in hemolytic assays using EA, C 1r, C l s and functionally purified C2-C9. In control experiments it appeared that about 10 ng/ml C l q was detectable in such a biological assay. Absence of Ig in the purified FcR-lm preparations was demonstrated by the failure to inhibit the agglutination of Ig-coated erythrocytes with anti-Ig. In controls it appeared that about 1 ng Ig was detectable in such an hemagglutination inhibition assay. Table 2. Properties of purified FcR-lm Bivalent
235
and inhibits both immune complex binding to FcR-positive lymphocytes and ADL. The presence of FCS in the incubation medium did not alter the results. The release of this material in detectable amounts by only some cell lines may be due t o differences in growth rate and turnover of membrane proteins. Such material can, however, not be detected in supernatants of nonstimuiated human peripheral blood lymphocytes. This suggests that only stimulated [3, 41 or transformed lymphocytes can release such membrane structures. The released FcR-lm is not identical with C l q o r rheumatoid factor for the following reasons: (a) unlike C 1q, FcR-lm appeared t o be heat-resistant (30 min, 56 "C); (b) it does not precipitate at low ionic strength and at pH 7.5 whereas C l q does [34]; (c) FcR-lm inhibits ADL whereas C l q does not (unpublished observations); (d) Sepharose-IgG columns do not bind FcR-lm after repeated acid elutions whereas C 1 q does bind normally; (e) FcR-lm appeared not t o be related to the rheumatoid factor (RF) since it does not fix complement and upon binding t o Ig it blocks the C l q binding whereas R F is able t o fix complement [35]; (f) the molecular weight determined by Sephadex G-200 gel filtration was about 100 000 daltons whereas C 1q o r R F have a molecular weight of 385 000 and at least 160 000, respectively. Furthermore C l q or Ig can neither be detected in the FcR-lm preparations nor influence the test systems at concentrations near the lowest detection level. As to its properties, FcR-lm seems to be bivalent since it agglutinates EA and precipitates DNA-anti-DNA complexes. The FcR-lm has much in common with the Ig-binding factor (IBF) described by Fridman [3-71, except for its reactivity with IgM complexes, but perhaps such differences are due to methodology. The molecular weight of 100 000 - 1 10 000 daltons is in agreement with that observed for IBF (Fridman, personal communication). However, this Sepharose-IgG binding material has t o be fractionated by more refined methods and its purity has to be defined by radioactive labeling followed by gel electrophoresis or density sedimentation and by producing antibodies against the shed receptors before definitively characterizing the molecular properties. IgG and IgM FcR have been demonstrated now on both B and T cells [9, 10, 12-20]. No cross-reactivity between these receptors on the membrane level could be demonstrated so far. It is known, however, that they have properties in commons such as trypsin resistance, sensitivity for pronase treatment and the fact that they do not react with monomer IgM or IgG [36,37].
Mol.wt. 100 000 daltons Heat resistant (30min 5 6 "c) Not related to Clq Binds to IgG and IgM Inhibits primary immune response in vltm.
4. Discussion
The present report shows that some lymphoblastoid cell lines release in their culture medium material which agglutinates EA which has Fc specificity, enhances the precipitation of immune complexes, inhibits C 1q binding t o EA
The FcR-lm preparations we obtained enhance the precipitation of both IgG and IgM-DNA complexes and inhibit the C l q binding t o both EA(1gG) as well as EA(1gM). Contamination of the different IgM preparations by IgG used for testing the FcR-lm activity seems unlikely. First, the separation by isokinetic sucrose gradients was found t o be very effective, in that IgM was only recovered i n fractions 6-1 1, whereas IgG was found in fractions 17-25. In the tests, only material from fractions 6-9 was used for IgM and from fractions 21-25 for IgG. Using immunodiffusion with anti-IgG and anti-IgM, no crosscontamination of the two preparations could be demonstrated. Second, the IgM when complexed with antigen showed no reaction with RF.
236
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If the shedded, purified FcR-lm contained two types of receptors, one for IgG and one for IgM, they must have about the same molecular weight, since their biological properties are present in the same fractions of the Sephadex G-200 gel filtration. The possibility that cell line lymphocyte FcR after shedding have different characteristics from membrane-bound FcR or FcR from normal cells, cannot be excluded and needs further investigation. Experiments are in progress to directly demonstrate IgM FcR on the membrane of lymphoblastoid cell line cells and try t o show inhibition of IgM complex binding or inhibition of IgM-dependent cytotoxicity by FcR-lm.
Eur. J. Immunol. 1977. 7: 230-236 9 Basten, A., Miller, J.F.A.P., Sprent, J. and Pye, J., J. Exp. Med. 1972.135: 610. 10 Dickler, H.B. and Kunkel, H.G., J. Exp. Med. 1972.136: 191. 11 Dickler, H.B., J. Exp. Med. 1974.140: 508. 12 Paraskevas, F., Lee, S.T., Om, K.B. and Israels, L.G., 1. Immunol. 1972.108: 1319. 13 Yoshida, T.O. and Anderson, B.,Sccmd. J. ImmunoL 1972. I: 401. 14 Boxel, J.A. van, Rosenstreich. D.C., J. Exp. Med. 1974.139: 1002. 15 Andersson,C.L. andGrey, H.M.,J. Exp. Med. 1974.139: 1175. 16 Stout, R.D. and Henenberg, L.A., J. Exp. Med. 1975.142: 611. 17 Moretta, L., Ferrarini, M., Durante, M.L. and Mingari, M.C., Eur. J. Immunol. 1975.5: 565. 18 McConnell, I. and Hurd, C.M.,Immunology 1976.30: 835.
As to the biological activity, the assumption that both the
FcR-lm from the T and B cell line supernatant are suppressors of antibody production in vitro is strengthened by the data that Sepharose coated with F(ab’)z fragments is unable to bind the inhibitor. Since small amounts of material were sufficient and washing procedures were followed during the assays, it appeared that the FcR-lm itself does not influence the plaque formation. The fact that human FcR-lm could influence the in vitro antibody production in the mouse system indicates a relatively low species specificity of the material. If the released material is indeed identical with an FcR, these data would indicate that the FcR probably plays an important role in the regulation of humoral antibody production. The authors wish t o thank Dr. W.H. Fridman for performing the experiments on the response of antibody production in vitro by C.P. Engelfriet, P.Th.A. Schellekens and LA. Loos FcR-lm and h. for their stimulating discussions. Received November 4,1976; in revised form February 14,1977.
19 Lamon, E.W., Skurzak,H.M., Anderson, B.,Whitten,H.D. and Klein,E.,J. Immunol.1975.114: 1171. 20 Lamon, E.W., Andersson, B., Whitten, H.D.,Hunt, M.M. and Ghanta, V.,J. Immunol.1976.116: 1199. 21 Theophilopoulos, A.N., Dixon, F.J. and Bokisch, V.A., J. Exp. Med. 1974.140: 877. 22 Premkumar. E., Singer, P.A. and Williamson, A.R., Cell 1975.5: 87. 23 Kabat, E.A. and Mayer, M.M., Experimental Immunochemisny, Sec. Ed., C. Thomas Publisher, Springfield Ill. 1961, p. 150. 24 Weir, D.M., Handbook of Experimental Immunology, Blackwell Scientific PubL, Sec. Ed. 1973, chapter 7.7. 25 Aarden, L.A., Lakmaker, F. and Groot, E.R. de, J. Immunol.Meth. 1976.11: 153. 26 Yonemasu, K., Stroud, R.M., J. Immunol. 1971.106: 304. 27 Hunter, W.M. and Greenwoods, F.C., Nature 1962.194: 495. 28 Veen, J.H. ten and Feltkamp-Vroom, Th.M., clin. Exp. Immunol. 1973.15: 591. 29 Molenaar, J.L.. Helder, A.W., Muller, MAC., GorbMulder, M..
Jonker, L.S., Brouwer. M. and Pondman, K.W., Immunochemistry
5. References 1 Ramseier, H., Eur. J. Immunol.1975.5: 23. 2 Owen, F.L. and Fanger, M.W., J. Immunol. 1975.115: 765. 3 Fridman, W.H. and Golstein, P., Cell. Immunol. 1974. 11: 442. 4 Fridman, W.H., Nelson, R.A. and Liabeuf, A., J. Immunol.1974. 113: 1008. 5 Neauport-Sautes, C., Dupuis, D. and Fridman, W.H., Eur. J. Immunol. 1975.5: 849.
6 Guimezanes, A.. Fridman, W.H., Gisler, R.H. and Kourilsky, F.M., Eur. J. Immunol. 1976.6: 69.
1975.12: 359. 30 Sobel, AT., Bokisch, V.A. and Miiller-Eberhard, H.J., J. Exp. Med. 1975.142: 139. 31 Zeijlemaker. W.P., Roos, M.Th.L.,Schellekens,P.Th.A. and Eijsvoogel, V.P., Eur. J. Immunol.1975.5: 579. 32 Capel, P.J.A., J. Immunol.Methods 1974.5: 165. 33 Michaelsen, T.E., Wisldff, F. and Natvirg, J.B., Scand. J. Immunol. 1975.4: 71. 34 Tamura, N. and Nelson, R.A., J. Immunol. 1968.101: 1333. 35 Tesar, J.T. and Schmid, F.S., J. Immunol.1973.110: 993.
7 Gisler, R.H. and Fridman, W.H., J. Exp. Med. 1975.142: 507.
36 Basten, A., Miller, J.F.A.P., Abraham, R., Gamble, J. and Chia, E., Int. Arch. Allergy Appl. Immunol. 1976.50: 1309.
8 Diaz-Jouanen, E., Bankhurst. A.D., Messner, R.P. and Williams, R.C., Arthritis Rheum. 1976.19: 142.
37 Ferrarini, M., Moretta, L., Mingari, M.C., Tonda, P. and Pernis, B., Eur. J. Immunol. 1976.6: 520.
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4 Winchester, R.J., Fu, S.M.,Hoffman, T.and Kunkel, H.C., J. Immunol. 1975.114: 1210.
13 Nisonoff, A.,Wissler, F.C., Lipman, L.N. and Woernely, D.L., Arch. Biochem. Biophys. 1960.89: 230.
5 Haustein, D., J. Immunol.Meth. 1975. 7 25.
14 Scheidegger,J.J.,Int. Arch. Allergy Appl. Immunol. 1965. 7: 103. 15 Levy, H.B. and Sober, H.A.,Proc. SOC.Exp. Biol. Med. 1960.103: 250.
6 Bergman, Y ., Blatt, C. and Haimovich, J., Isr. J. Med. Sci. 1976. 12: 1360. 7 Roelants, G.E., Mayor, K.S., Hag, L.B. and Loor, F., Eur. J. Immunol. 1916.6: 75. 8 Little, J.R. and Eisen, H.N., in Williams, C.A. and Chase, M.W.
(Eds.), Methods in Immunology and Immunochemistry, Vol. I, Academic Press, New York 1967, p. 128. 9 Axe'n, R., Porath, J. and Ernback, S . , Nuture 1967.214: 1302. 10 Porter, R.R., Biochem. J. 1959. 73: 119.
16 Davidson, W.F. and Parish, C.R., J. Immunol. Meth. 1975. 7: 291. 17 Cebra, J.J. and Goldstein, G.,J. Immunol. 1965. 95: 230. 18 Haughton, G. and McCehee, M.P., Immunology 1969. 16: 447. 19 HaranGhera, N., Kotler, M. and Meshorer, A., J. Nut. Cuncer Inst. 1967.39: 653.
11 Laemmli, U.K., Nuture 1970.227: 680.
20 Rubin, B., J@gensen, P.N. and HQier-Madsen,M.,Scund. J. Im. munol. 1976.5: 450.
12 Cohen,A. and Schlesinger,M., Trunspluntution 1970.10: 130.
21 Perlman, P. and Perlman, H., Cell. Immunol. 1970. I : 300.
J.L. Molenaar, M. van Galen, A.J. Hannema, W. Zeijlemaker and K.W. Pondman
Spontaneous release of Fc receptor-like material from human lymphoblastoid cell lines
Central Laboratory of the Netherlands Red Cross Blood Transfusion Service and University Laboratory of Experimental and Clinical 'Immunology, Amsterdam
In the culture medium of some human lymphoblastoid cell lines material is released with the following properties: (a) hemagglutination reaction of IgG-sensitized erythrocytes; ( b ) enhancement of precipitation of DNA-antiDNA complexes; (c) inhibition of binding of C l q to immune complexes; (d) inhibition of immune complex binding to lymphocytes; (e) inhibition of antibody-dependent lymphocytotoxicity The material is not identical with C l q o r rheumatoid factor, it is heat resistant (30 min at 56 "C); the molecular weight is about 100 000 daltons and it is capable of inhibiting antibody production in vitro. I t is suggested that this material consists of Fc receptors spontaneously shed from lymphocyte membranes.
1. Introduction Recently, a number of reports have appeared describing spontaneous release of lymphocyte receptors. For example, Ramseier [ 11 described spontaneous shedding of T lymphocyte receptors for alloantigens. Owen [2] reported the release of the T lymphocyte receptor for sheep erythrocytes (SRBC) in the supernatant o f phytohemagglutinin (PHA)stimulated lymphocytes. Fridman and coworkers suggested that Fc receptors ( F c R ) may be released from T lymphocytes [3-61. They pub[I 15821 Correspondence: J.L. Molenaar, Department of Immunochemistry, Central Laboratory of the Netherlands Red Cross Blood Transfusion Service, P.O. Box 9190, Amsterdam, The Netherlands Abbreviations: FcR: Fc receptor FcR-lm: Fc receptor-like material KLH: Keyhole limpet hemocyanin T lymphocyte: Thymusderived lymphocyte(s) SRBC: Sheep erythrocyte PHA: Phytohemagglutinin EA: Antibody-coated erythrocyte SLE: Systemic lupus erythematosus B lymphocyte: Bone marrowderived lymphocyte(s) FCS: Fetal calf serum RFC: Rosette-forming cell BSA: Bovine serum albumin PBS: Phosphate buffered saline E: Erythrocyte FITC: Fluorescein isothiocyanate ADL: Antibodydependent lymphocytotoxicity PFC: Plaque-forming cell RF: Rheumatoid factor
lished data about the spontaneous release of an immunoglobulin-binding factor from antigen-activated thymus-derived lymphocytes ( T lymphocytes). This material agglutinated antibody-coated erythrocytes (EA), it inhibited complement-dependent hemolysis of EA at the level of C 1 (therefore it has specificity for a region close t o o r identical with the complement binding site of IgG), it had Fc specificity for IgG, could be purified on Sepharose-IgG, and appeared capable of suppressing antibody synthesis in vitro [7]. The possibility o f release of FcR from lymphocytes in vivo has been suggested by Diaz-Jouanen [8] who demonstrated in sera of systemic lupus erythematosus (SLE) patients a factor that could specifically block antibody-dependent lymphocytotoxicity (ADL). Until recently it was thought that the membrane-bound FcR on lymphocytes had exclusive affinity t o immune complexes o r aggregates containing IgC. Such an IgG FcR was found to be present o n B as well as on T lymphocytes [9- 161. However, Moretta [ 171 and McConnell [ 181 recently demonstrated that a subpopulation of peripheral T lymphocytes had a receptor f o r antigendgM antibody complexes. Furthermore, Lamon [ 19, 201 and coworkers demonstrated that both IgM and IgG antibodies were able t o induce target cell destruction by normal mouse lymphocytes. The IgM FcR appeared t o be present on both B and T cells.
Eur. J. Immunol. 1977. 7: 230-236 It has also been reported that human lymphoblastoid cell lines carried FcR [2 1 ] and that such cell lines produced prcl teins which can bind to antigen-antibody complexes [ 221. The purpose of the present investigation was t o determine whether human T and B cell lines release material with properties of an FcR and t o see whether it has specificity for IgC, for IgM or for both. The data suggest the release of FcR-like material (FcR-lm) with Fc specificity for both I& and IgM.
2. Materials and methods 2.1. Cell lines
Established B cell lines (Raji, Daudi, P3HR1, EB3, TSI 1 and CCRF-SB) and T cell lines (CCRF-CEM, CCRF-HSB, Molt 4) were mainly obtained from several laboratories (Dr. G.Klein, Karolinska Institute, Stockholm, Sweden; Dr. 0. Schonherr, Department of Microbiology, Organon, Oss, The Netherlands; Dr. T.A.W. Splinter, Central Lab. of the Netherlands Red Cross Blood Transfusion Service, Amsterdam, The Netherlands). The cells were grown at 37 "C in a suspension culture in RPMI 1640 (Gibco, Grand Island, N.Y.) supplemented with 10 % heat-inactivated fetal calf serum (FCS), penicillin (100 pglml), and with streptomycin (50 pg/ml). 2.2. Preparation of cell line supernatants
(a) Cell lines were cultured for two days up to a density of about 2 x lo6 cells/ml in RPMI 1640 f 10 % heat-inactivated FCS. Afterwards the cells were centrifuged at 600 x g for 10 min and the supernatants were collected; (b) cell line cells washed five times with serum-free RPMI 1640 were cultured for a period of 3 h at 37 "C in serum-free RPMI 1640. Supernatants were collected after centrifugation for 10 min at 600 x g. In both cases the cell line suspensions contained more than 95 % viable cells as judged by trypan blue exclusion.
Fc receptor-like material from human lymphoblastoid cell lines
231
scribed above, followed by affinity chromatography as described by Dickler [ 1 I]. Human IgG was isolated from normal serum by precipitation in 50 % (NH&S04 (twice) and by chromatography on DEAE-cellulose in 0.02 M phosphate buffer pH 7.5. F(ab')2 fragments of human IgG were prepared by hydrolysis with 2 % pepsin (Worthington Biochemical Corp., Freehold, N.J.) in 0. I M acetate buffer, pH 4.5, for 20 h at 37 "C. The hydrolysis was stopped by addition of a saturated solution of Tris t o a pH of 7.5. The material was then applied to a calibrated Sephadex G l 5 0 column, equilibrated and eluted with 0.15 M phosphate buffered saline (PBS), pH 7.5. The F(ab')z-containing fractions were pooled and concentra ted. Highly purified C Iq was isolated from fresh human serum as described by Yonemasu et al. [26]. C l q appeared to be pure in immunodiffusion. The isolated protein was stored in a 0.1 M phosphate buffer, pH 7, at a concentration of 1 mg/ml at -70 "C until use. For use in the inhibition assay C l q was labeled with lZsI according t o the method of Hunter et al. [ 271. The specific radioactivity was about 0.5 pCi/pg protein. Bovine serum albumin (BSA) at a concentration of 1 mg/ml was added before storage at - 70 "C. 2.4. Detection of FcR on cell line lymphocytes 2.4.1. Rosette formation with EA cells
EA complexes were prepared by incubating 0 Rh erythrocytes ( 5 x lo8 cells/ml) with human anti-D serum (final dilution 1 :4) for 30 min at 37 "C. Only one batch of anti-D serum was used selected for optimal rosette formation. 0.4 x lo6 lymphocytes were mixed with lo7 EA (total volume 0.25 ml of BSS + 10 % heat-inactivated FCS). After centrifugation for 5 min at 200 x g the mixture was incubated for 30 rnin at 25 "C. After resuspending, the percentage of EA-RFC was scored by counting 200 cells in a hemocytometer. A cell with more than four adherent erythrocytes was regarded as a rosette. 2.4.2. Immunofluorescence with KLH-anti-KLH soluble complexes
2.3. Antibodies, immunoglobulins and C l q
Anti-Forssman sera were induced in rabbits [23]. IgG fractions were prepared by precipitation in 50 % (NH4)2S04 and chromatography on DEAE-cellulose with 0.02 M phosphate buffer, pH 7.5. IgM fractions were prepared by DEAE chromatography using a linear salt gradient from 0.02 M to 0.3 M phosphate buffer, pH 7.5 and gel filtration on Sephadex G-200 [ 241. Neither the IgG was contaminated by IgM, nor was the collected IgM contaminated by IgG, as was demonstra ted by immunoelectrophoresis. Anti-DNA IgG and IgM fractions were prepared from a serum of a patient with SLE with high anti-ds-DNA binding capacity by isokinetic sucrose density gradient centrifugation as described by Aarden [25]. Fractions containing IgM and IgG were separately pooled. No contaminating IgG could be detected in the IgM fractions as determined by immunodiffusion, and vice versa. Anti-mouse mastocytoma cell IgG antibody fractions were prepared from rabbit antisera by isokinetic sucrose density gradient centrifugation. Anti-keyhole limpet hemocyanin (KLH) IgG antibodies were purified by isokinetic Sucrose density centrifugation as de-
Lymphocytes ( 1 00 pl; 107/ml in Earle's- 1 % BSA, pH 7.5) were incubated with rabbit IgG antibodies to KLH (0.2 mg/ml) for 10 min at room temperature. Thereafter, fluorescein isothiocyanate (FITCtlabeled KLH (0.1 mg/ml) was added, followed by another incubation at room temperature for 30 min. The cells were then washed with Earle's buffer supplemented with 1 % BSA, fixed with paraformaldehyde ( 1 % in PBS, pH 7.4), and washed with PBS. The cells were mounted in 50 % glycerol and examined with fluorescence microscopy using a Leitz Orthoplan microscope [28]. 2.5. Sepharose-coupled protein
CNBr-activated Sepharose 4B was prepared as described previously [29]. Human IgG or F(ab')2'y was covalently bound t o the beads in a concentration of 10 mg/ml packed Sepharose. After coupling had taken place the remaining active groups were blocked by incubation with 0.2 M ethanolamine (pH 9.0). Noncovalently bound proteins were eluted by a short washing with 1 % glycine, 2 % citric acid, pH 2.5 until A28onrn was zero. The beads were finally washed and equilibrated in 0.02 M phosphate buffer, pH 7.0.
232
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2.6. Purification of FcR-lm
A column was prepared of 150 ml Sephadex G 2 5 containing 2 0 ml IgG-bound Sepharose. 200 ml of a cell line supernatant dialyzed against 0.02 M phosphate buffer, pH 7.0, was applied t o the column and washed with the same buffer. The I&bound FcR-lm was eluted with 1 % glycine, 2 % citric acid, pH 2.5. The Sepharose G-25 caused a quick separation of the FcR-lm from the acid buffer. Effluents and eluates were collected, concentrated and tested. As control preparations, supernatants of cell lines, in which n o FcR-lm could be detected, were processed on Sepharose-IgG. Analogous fractions were collected, concentrated and used. Sepharose-IgC used for purification of the FcR-lm could not be used more than once. After acid elution the bound IgC quickly lost the property t o bind FcR-lrn. Since the Sepharose-IgG had t o be prewashed with an acid buffer of pH 2.5, it had only a relatively low capacity to bind FcR-lm. 2.7. Detection of FcR-lm 2.7.1. Hemagglutination of EA(IgC)
20 "C for 30 min; (c) 1 pg of 12SI-labeledC l q in 10 pl was added, followed by a second incubation for 30 min at 2 0 "C; (d) 200 pl of the reaction mixture was layered on 150 pl of a 40 7% sucrose solution in 0.05 M sodium phosphate buffer, pH 7.0 in a 400 p l polyethylene microfuge tube (Beckman Instruments Inc., Fullerton, CA.). Centrifugation was performed at about 1 3 000 x g in a Beckman microfuge 152 for 5 min. The tip of the microfuge tube containing the pelleted cells was cut off with a razor blade and collected in a tube. The upper portion of the microfuge tube, its contents included, was added t o a second tube. The radioactivity in both tubes was measured. The 12sI-labeledC Iq uptake by EA is calculated as described by Sobel [30]. To determine inhibition of lZSI-labeledC l q binding t o E A by FcR-lm, uptake of C l q by E A in the absence of FcR-lm was quantitated simultaneously. The inhibition percentage of uptake was calculated from: % uptake in control - % uptake in test x 100 % uptake in control 2.7.4. Inhibition of immune complex binding t o lymphocytes
by FcR-lm
SRBC (E) were suspended at a concentration of lo9 cells/ml. These cells were coated with purified rabbit anti-Forssman IgG by incubating the cells with a sub-agglutinating concentration of antibodies for 30 min at 3 7 "C in barbital buffer, pH 7.5. Microtiter plates (Cooke) were filled with 50 pl 1 % BSA in PBS, 50 /A EA (5 x 1 07/ml) and 50 pl of an FcR-lm preparation in two-fold dilutions. The hemagglutination titer was expressed as the reciprocal of the highest dilution of FcR-lm causing agglutination.
Lymphocytes ( l o 6 in Earle's medium - 1 % BSA, pH 7.5) were incubated with 100 pl FcR-lm and 100 pl rabbit IgG antibody preparation (0.2 mg/ml) t o KLH for 10 min at room temperature, followed by addition of FITC-labeled KLH (0.1 mg/ml) and another incubation at room temperature for 2 0 min. The cells were washed, fixed and examined as described in Sect. 2.4.2.
2.7.2. Enhancement of DNA-anti-DNA precipitation
A micro assay sytem for detection of ADL was used as described by Zeijlemaker e t al. [31]. 2 x l o 6 mouse P815-X2 mastocytoma cells radiolabeled with SICr were optimally sensitized with rabbit anti-mastocytoma serum or the purified IgG fractions thereof. The cytotoxicity tests and their inhibition were performed in round-bottom microtiter plates (Cooke). Each well contained 2 x 1 O3 target cells and various amounts of effector cells, starting at 2 x l o 5 (ratio effector/target = 100). For inhibition experiments mainly a ratio of 50 was used. The target cells were first incubated with 5 0 p l of a FcR-lm preparation during 15 min at room temperature. Next, 50 pl of a lymphocyte suspension was added. T h e plates were then incubated for 3 h at 37 "C. Controls included target cells with FcR-lm alone, and unsensitized target cells in the presence of FcR-lm and effector cells. The latter two controls usually gave a 51Cr release between 5 and 1 0 %. From the raw data, the net 5'Cr release could be calculated after correction for spontaneous and maximal "Cr release [29]. The percentage of inhibition of ADL was calculated from:
Purified anti-ds-DNA of both Ig classes was mixed with jHlabeled PM2-DNA [25] (20 ng; 60 000 cpm/pg) and incubated for 60 min in a total volume of 150 pl at 3 7 "C. Next, 50 p l purified FcR-lm was added t o the DNA-anti-DNA complexes, followed by another incubation of 120 min at 4 "C. Precipitating DNA-anti-DNA complexes were then spun down at 2000 x g. To 100 /A of the supernatant 0.5 ml of Soluene 350 was added, followed by 10 ml o f scintillation liquid (toluene - PPO - POPOP). The radioactivity was counted in a Packard liquid scintillation counter, model 3320, at a counting efficiency of 40 %. All determinations were carried out in duplicate. The amounts of IgG and IgM-anti-DNA were chosen in such a manner that without addition of FcR-lm about 15-20 % of the labeled DNA was spun down. The percentage of precipitate was calculated from: total counts - measured counts x 100 (%) total counts 2.7.3. Inhibition of C l q binding
This test is a modification of the immune complex detection method described by Sobel [30] and consisted of the following steps: (a) 50 pl of a n FcR-lm-containing sample, 100 p l saline and 100 p l of a sucrose barbital buffer (3 mmho/cm) were mixed in a plastic test tube (final pH 7.2, conductivity 9 mmho/cm). (b) 4 x lo7 EA were added and the mixture was incubated at
2.7.5. Inhibition of ADL
% net "Cr release - % net "Cr release (+FcR-lm) x 100 (%) % net " ~ release r
2.7.6. Suppression of in vitro antibody synthesis by FcR-lm
Spleen cells o f B6D2P1 mice (8 x 1 O6 - 10 x 1 06/ml) were cultured i n t h e presence of 1 % of SRBC for 5 days. To some cultures graded doses of purified FcR-Im o r control preparations were added a t time zero of incubation. Direct plaque forming cells (PFC) were assayed as described by Gisler [7].
Eur. J. Immunol. 1977.7: 230-236
Fc receptor-like material from human lymphoblastoid cell lines
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2.8. Assays for detection of C l q or Ig in FcR-lm preparations
3.3. Purification of FcR-lm
2.8.1. Quantitative immunofluorescence
In order to purify FcR-lm, 200 ml crude CCRFCEM supernatant (medium RPMI 1640 + 1 0 % heat-inactivated FCS or serum-free RPMI 1640) was introduced onto a column consisting of 25 ml Sepharose-IgC and 150 ml Sephadex (3-25. The elution profiles obtained with different test systems after uncoupling the FcR-lm from Sepharose-IgG with the acid buffer are demonstrated in Fig. 1. The peak fraction showed an inhibition of C 1q binding of 6 %, an inhibition of 20 % of the ADL, and a 15 % enhancement in precipitation of DNA-anti-DNA complexes. Agglutination of EA(1gG) (with a titer of 4) and inhibition of KLH-anti-KLH binding to the respective cells (18 %) could only be demonstrated in the peak fraction. The slight increase in precipitation of DNAanti-DNA or inhibition of C l q binding in the late fractions were artefacts due to the decrease of the pH during elution with the acid buffer necessary to uncouple the FcR-lm from Sepharose-IgC. Analogous elution profiles could be obtained with supernatants of the T S l 1 cell line. The positive fractions from the eluates were pooled, concentrated and used for further experiments. Fractions obtained after elution of heatinactivated FCS or after elution of the supernatants from the P3HR1, Molt4 or the CCRFSB were negative as to biological activity. In this case unspecific binding of proteins from the control supernatants occurred. Acid eluates of such controls demonstrated an A~SO,,,,,in the peak fraction of about 0.4. Acid eluates obtained from supernatants of CCRFCEM or TSl 1 processed on F(ab')lcoated Sepharose columns were also negative.
Human purified C 1q was covalently linked to Sepharose4B at a concentration of 1 mg/ml packed Sepharose as described in Sect. 2.5. Rabbit anti-human C l q (0.5 ml) were incubated in a dilution of 1 :40 at room temperature with 10' SepharoseC l q beads for 30 min in the presence o r absence of purified FcR-lm. After washing three times with PBS the beads were finally incubated with 0.5 ml FITC-labeled horse anti-rabbit Ig at a dilution of 1:40 at room temperature for another 30 min. After additional washing the liquid was removed and the beads were mounted in 50 % glycerol in PBS. Quantitative measurement of the fluorescence was performed as described by Cape1 [32]. 2.8.2. Hemagglutination inhibition Human C l q or Ig were coupled to SRBC at a concentration of 10 c(g/5 x l o 7 E using the CrCl3 technique (0.07 % CtC13). The agglutination of the coated cells by serially diluted antiC l q or anti-Ig was tested in the presence o r absence of purified FcR-lm.
3. Results 3.1. Production of FcR-lm by B and T lymphoblastoid cell lines The various human T and B cell lines were tested for release of FcR-lm activity into their supernatant. For rough screening, the capacity of the different supernatants to agglutinate EA(IgG) cells was investigated. From the B cell lines the TS 1 1 supernatant had the strongest activity with an agglutination titer of 16. Supernatants of the Daudi, Raji, EB were slightly positive with a titer of 4. Supernatants from P3HR1, CCRFSB were negative. Among the T cell lines, only the supernatant of CCRFCEM had activity (titer of 16). It made no difference whether FCS was present in the medium or not. The positive supernatants also had the capacity to inhibit C l q binding to EA (6- 10 %), to inhibit KLH-anti-KLH binding to lymphocytes (20 %) and to inhibit antibody-dependent lymphocytotoxicity (5-8 %). On the basis of the results mentioned above, the CCRFCEM and TSl 1 cell lines and their supernatants were selected for further investigation. the negative supernatant of the P3HR line served as a control. 3.2. Presence of FcR on the lymphoblastoid cell surface The hypothesis of these experiments is that FcR from the cell surface are spontaneously shed into the supernatant. Therefore, the lymphoblastoid cell line cells were investigated for the presence of FcR on the cell surface, using EA rosette formation and immunofluorescence with KLHanti-KLH soluble complexes. Detection of FcR o n the cell surface using KLH-anti-KLH complexes appeared to be more sensitive than rosette formation, as will be described elsewhere (in preparation). Using the immunofluorescence test with KLH-anti-KLH IgG complexes 17 f 2 % of the CCRFCEM and 20 f 2 % of TSl 1 cells were positive for FcR. Rosetting percentages were 5 and 12 %, respectively.
O.'
r I
0.5 k
I
I
,2044
Figure I. An example of acid elution profiles of FcR-lm when crude CCRFCEM supernatant (medium RPMI 1640 + 10 % heat-inactivated FCS) WBS processed on Sepharose-IgG. The volume of the eluted frao tions was 2 mL (.----0 )Absorbance at 280 nm; ( O w )enhancement DNAanti-DNA precipitation (%); (.--.) inhibition of ADL (%); (A-*-*-A)inhibition of Clq binding to EA (96).
--
3.4. Demonstration of FcR-lm activities 3.4.1. Enhancement of immune complex precipitation
When a dose response of purified pooled and concentrated FcR-lm was performed by testing its capacity to enhance the precipitation of DNA-anti-DNA IgG complexes, curves were obtained as in Fig. 2. Surprisingly, enhancement of precipitation could also be observed when purified IgM anti-DNA-
234
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J.L.Molenaar, M.van Galen, A.J. Hannema et. al.
DNA complexes were used (Fig. 2). Both effects could be obtained with purified FcR-lm from either CCRFCEM or TSl 1 supernatants. The P3HRl control appeared t o be negative with both IgM as well as the IgG anti-DNA-DNA complexes. Maximal precipitation was obtained for FcR-lm (CCRFCEM) at a dilution of 1 :4, and for FcR-lm (TSl 1) at a dilution of 1:2. Since we have so far no exact information about the mechanism of enhanced precipitation, it should be noted that the curves give a qualitative rather than a quantitative impression of the phenomenon. The course of the curves suggests a bivalent character which also explains the agglutination of EA. In the absence of antibodies to DNA both FcR-lm preparations did not precipitate DNA d o n e when tested at a 1 :4 dilution.
3.4.3. Inhibition of the binding of KLH-anti-KLH-IgC to lymphocytes
Addition of different amounts of purified FcR-lm obtained form CCRFCEM or TSl 1 supernatants (two-fold dilutions) to their respective cell line cells in the presence of FITCKLH-anti-KLH complexes reduced the number of fluorescing cells. Only with the undiluted FcR-lm preparations a reduction from 17 % to 10 % for the CCRFCEM cell line, and from 20 % to 1 1 % for the TSl 1 cell line cells was observed. 3.4.4. Inhibition of ADL
A dose response of both purified FcR-lm preparations, with respect t o inhibitisn of antibody-dependent lymphocytotoxicity, is given in Fig. 4. In these ADL experiments the target cell antibodies were of the I g C class. The FcR-lm of CEM supernatant appeared to be a more efficient inhibitor than the TSl 1-FcR-lm. Experiments with purified IgM mastocytoma cell antibodies will be carried out to demonstrate whether, under conditions at which the effector cells were cultured overnight in 10 % FCS, an IgM-dependent lymphocytotoxicity could occur, since only under these conditions the IgM-FcR could be detected on the lymphocyte surface. Inhibition of such an IgM-dependent cytotoxicity will be tried to show with purified FcR-lm.
Figure 2. Enhancement of DNA-anti-DNA precipitation by purified FcR-lm (c---o) IgM anti-DNA: CCRF-CEM; (0-0) IgC anti-DNA: CCRF-CEM; ( 0 - 4 ) IgM anti-DNA TS11; ( ~ m I&) anti-DNA: IgG anti-DNA: TS11; (A---A) IgM anti-DNA: P3HR1; @-A) P3HR1.
3.4.2. Inhibition of C l q binding
Since FcR on lymphocytes bind t o approximately the same site on Ig as does C l q [33], purified pooled and concentrated FcR-lm was further tested for its capacity to inhibit C 1q binding t o EA coated with both IgG or IgM antibodies. It was found that C l q binding to both antibody types was inhibited. The results of a dose response of the same preparations as shown in Fig. 2 with both IgG and IgM antibodies are demonstrated in Fig. 3. Both FcR-lm preparations caused somewhat higher inhibition of C 1q binding t o EA-IgM than to EA-IgG.
Figure 4. Inhibition of ADL by purified FcR-lm Target cells sensitized CCRF-CEM; @-A) TS11; (0-a) with IgG antibodies. (0-0) P3HR1.
3.4.5. Inhibition of in vitro antibody synthesis by FcR-lm
I . .
8
h'ra b
+4
a
'4 urnpaam
Figure 3. Inhibition of Clq binding to EA by purified FcR-lm (x-X) (0-0)
EA-IgM: CCRFCEM; (0- 0 ) EA-IgG: CCRF-CEM; EA-IgM: TS11; (v-V) EA-IgG: TS11; (+O):P3HR1.
The possible interference of purified FcR-lm with antibody synthesis in vitro was also investigated. Addition of different amounts of FcR-lm together with SRBC to a mouse spleen cell culture at the initiation of the response, significantly suppressed the PFC response as demonstrated in Table 1 Suppression of the PFC response was not due to a decrease in the number of viable cells. FcR-lm from CCRFCEM and TSl 1 were both inhibitory, whereas material obtained after adsorption and acid elution of the control P3HRl supernatants on Sepharose-IgC did not inhibit the antibody production in vitro. Also, CCRFCEM o r TS11 supernatants similarly processed on Sepharose-F(ab')yy columns did not result in preparations that contained suppressor material.
Eur. J. Immunol. 1977. 7: 230-236
Fc receptor-like material from human lymphoblastoid cell lines
Table 1. Effect of purified FcR-lm on the response of spleen cell cultures to SRBC added at day zero Additions
Microliters
No. of PFO)
Inhibition
(96) None
-
30 CCRF-CEM 90 TSl 1 30 TSll 90 120 P3HR1 CCRF-CEM~) 120 120 TSl lb) System without SRBC
CCRF-CEM
2 080 f 224 1 1 4 0 f 85 700 f 283 1 565 f 247 1 195 f 276 2 m * m 2040f186 1900f230 305 f 106
53 78 29 49
a) Arithmetic means (+standard deviation) of PFC counts (day 5)
from four independent experiments. b) Processed on Sepharose-F(ab')zy.
3.4.6. Properties of FcR-lm
Gel filtration on Sephadex (3-200 of the SepharosaIgG purified FcR-lm and testing the fractions on their biological activity using human C3b and albumin as markers, demonstrated a molecular weight of about 100 000- 1 10 000 daltons for the FcR-lm. Furthermore, the purified FcR-lm seemed to be heat-resistant (30 min, 56 "C). The properties of both the T and B FcR-lm are summarized in Table 2. The purified T and B FcR-lm preparations used in the above test systems were extensively screened for the presence of C l q or Ig. Addition of FcR-lm to a n t i c l q did not decrease its binding to Sepharose-bound C 1q, as was tested in a quantitative immunofluorescence assay, or by agglutination of C lq-CrC13-erythrocytes. With these test systems it was possible to detect 1 - 10 ng/ml C 1q in control experiments. Absence of C l q was also demonstrated in hemolytic assays using EA, C 1r, C l s and functionally purified C2-C9. In control experiments it appeared that about 10 ng/ml C l q was detectable in such a biological assay. Absence of Ig in the purified FcR-lm preparations was demonstrated by the failure to inhibit the agglutination of Ig-coated erythrocytes with anti-Ig. In controls it appeared that about 1 ng Ig was detectable in such an hemagglutination inhibition assay. Table 2. Properties of purified FcR-lm Bivalent
235
and inhibits both immune complex binding to FcR-positive lymphocytes and ADL. The presence of FCS in the incubation medium did not alter the results. The release of this material in detectable amounts by only some cell lines may be due t o differences in growth rate and turnover of membrane proteins. Such material can, however, not be detected in supernatants of nonstimuiated human peripheral blood lymphocytes. This suggests that only stimulated [3, 41 or transformed lymphocytes can release such membrane structures. The released FcR-lm is not identical with C l q o r rheumatoid factor for the following reasons: (a) unlike C 1q, FcR-lm appeared t o be heat-resistant (30 min, 56 "C); (b) it does not precipitate at low ionic strength and at pH 7.5 whereas C l q does [34]; (c) FcR-lm inhibits ADL whereas C l q does not (unpublished observations); (d) Sepharose-IgG columns do not bind FcR-lm after repeated acid elutions whereas C 1 q does bind normally; (e) FcR-lm appeared not t o be related to the rheumatoid factor (RF) since it does not fix complement and upon binding t o Ig it blocks the C l q binding whereas R F is able t o fix complement [35]; (f) the molecular weight determined by Sephadex G-200 gel filtration was about 100 000 daltons whereas C 1q o r R F have a molecular weight of 385 000 and at least 160 000, respectively. Furthermore C l q or Ig can neither be detected in the FcR-lm preparations nor influence the test systems at concentrations near the lowest detection level. As to its properties, FcR-lm seems to be bivalent since it agglutinates EA and precipitates DNA-anti-DNA complexes. The FcR-lm has much in common with the Ig-binding factor (IBF) described by Fridman [3-71, except for its reactivity with IgM complexes, but perhaps such differences are due to methodology. The molecular weight of 100 000 - 1 10 000 daltons is in agreement with that observed for IBF (Fridman, personal communication). However, this Sepharose-IgG binding material has t o be fractionated by more refined methods and its purity has to be defined by radioactive labeling followed by gel electrophoresis or density sedimentation and by producing antibodies against the shed receptors before definitively characterizing the molecular properties. IgG and IgM FcR have been demonstrated now on both B and T cells [9, 10, 12-20]. No cross-reactivity between these receptors on the membrane level could be demonstrated so far. It is known, however, that they have properties in commons such as trypsin resistance, sensitivity for pronase treatment and the fact that they do not react with monomer IgM or IgG [36,37].
Mol.wt. 100 000 daltons Heat resistant (30min 5 6 "c) Not related to Clq Binds to IgG and IgM Inhibits primary immune response in vltm.
4. Discussion
The present report shows that some lymphoblastoid cell lines release in their culture medium material which agglutinates EA which has Fc specificity, enhances the precipitation of immune complexes, inhibits C 1q binding t o EA
The FcR-lm preparations we obtained enhance the precipitation of both IgG and IgM-DNA complexes and inhibit the C l q binding t o both EA(1gG) as well as EA(1gM). Contamination of the different IgM preparations by IgG used for testing the FcR-lm activity seems unlikely. First, the separation by isokinetic sucrose gradients was found t o be very effective, in that IgM was only recovered i n fractions 6-1 1, whereas IgG was found in fractions 17-25. In the tests, only material from fractions 6-9 was used for IgM and from fractions 21-25 for IgG. Using immunodiffusion with anti-IgG and anti-IgM, no crosscontamination of the two preparations could be demonstrated. Second, the IgM when complexed with antigen showed no reaction with RF.
236
J.L. Molenaar, M. van Galen, A.J. Hannema et. al.
If the shedded, purified FcR-lm contained two types of receptors, one for IgG and one for IgM, they must have about the same molecular weight, since their biological properties are present in the same fractions of the Sephadex G-200 gel filtration. The possibility that cell line lymphocyte FcR after shedding have different characteristics from membrane-bound FcR or FcR from normal cells, cannot be excluded and needs further investigation. Experiments are in progress to directly demonstrate IgM FcR on the membrane of lymphoblastoid cell line cells and try t o show inhibition of IgM complex binding or inhibition of IgM-dependent cytotoxicity by FcR-lm.
Eur. J. Immunol. 1977. 7: 230-236 9 Basten, A., Miller, J.F.A.P., Sprent, J. and Pye, J., J. Exp. Med. 1972.135: 610. 10 Dickler, H.B. and Kunkel, H.G., J. Exp. Med. 1972.136: 191. 11 Dickler, H.B., J. Exp. Med. 1974.140: 508. 12 Paraskevas, F., Lee, S.T., Om, K.B. and Israels, L.G., 1. Immunol. 1972.108: 1319. 13 Yoshida, T.O. and Anderson, B.,Sccmd. J. ImmunoL 1972. I: 401. 14 Boxel, J.A. van, Rosenstreich. D.C., J. Exp. Med. 1974.139: 1002. 15 Andersson,C.L. andGrey, H.M.,J. Exp. Med. 1974.139: 1175. 16 Stout, R.D. and Henenberg, L.A., J. Exp. Med. 1975.142: 611. 17 Moretta, L., Ferrarini, M., Durante, M.L. and Mingari, M.C., Eur. J. Immunol. 1975.5: 565. 18 McConnell, I. and Hurd, C.M.,Immunology 1976.30: 835.
As to the biological activity, the assumption that both the
FcR-lm from the T and B cell line supernatant are suppressors of antibody production in vitro is strengthened by the data that Sepharose coated with F(ab’)z fragments is unable to bind the inhibitor. Since small amounts of material were sufficient and washing procedures were followed during the assays, it appeared that the FcR-lm itself does not influence the plaque formation. The fact that human FcR-lm could influence the in vitro antibody production in the mouse system indicates a relatively low species specificity of the material. If the released material is indeed identical with an FcR, these data would indicate that the FcR probably plays an important role in the regulation of humoral antibody production. The authors wish t o thank Dr. W.H. Fridman for performing the experiments on the response of antibody production in vitro by C.P. Engelfriet, P.Th.A. Schellekens and LA. Loos FcR-lm and h. for their stimulating discussions. Received November 4,1976; in revised form February 14,1977.
19 Lamon, E.W., Skurzak,H.M., Anderson, B.,Whitten,H.D. and Klein,E.,J. Immunol.1975.114: 1171. 20 Lamon, E.W., Andersson, B., Whitten, H.D.,Hunt, M.M. and Ghanta, V.,J. Immunol.1976.116: 1199. 21 Theophilopoulos, A.N., Dixon, F.J. and Bokisch, V.A., J. Exp. Med. 1974.140: 877. 22 Premkumar. E., Singer, P.A. and Williamson, A.R., Cell 1975.5: 87. 23 Kabat, E.A. and Mayer, M.M., Experimental Immunochemisny, Sec. Ed., C. Thomas Publisher, Springfield Ill. 1961, p. 150. 24 Weir, D.M., Handbook of Experimental Immunology, Blackwell Scientific PubL, Sec. Ed. 1973, chapter 7.7. 25 Aarden, L.A., Lakmaker, F. and Groot, E.R. de, J. Immunol.Meth. 1976.11: 153. 26 Yonemasu, K., Stroud, R.M., J. Immunol. 1971.106: 304. 27 Hunter, W.M. and Greenwoods, F.C., Nature 1962.194: 495. 28 Veen, J.H. ten and Feltkamp-Vroom, Th.M., clin. Exp. Immunol. 1973.15: 591. 29 Molenaar, J.L.. Helder, A.W., Muller, MAC., GorbMulder, M..
Jonker, L.S., Brouwer. M. and Pondman, K.W., Immunochemistry
5. References 1 Ramseier, H., Eur. J. Immunol.1975.5: 23. 2 Owen, F.L. and Fanger, M.W., J. Immunol. 1975.115: 765. 3 Fridman, W.H. and Golstein, P., Cell. Immunol. 1974. 11: 442. 4 Fridman, W.H., Nelson, R.A. and Liabeuf, A., J. Immunol.1974. 113: 1008. 5 Neauport-Sautes, C., Dupuis, D. and Fridman, W.H., Eur. J. Immunol. 1975.5: 849.
6 Guimezanes, A.. Fridman, W.H., Gisler, R.H. and Kourilsky, F.M., Eur. J. Immunol. 1976.6: 69.
1975.12: 359. 30 Sobel, AT., Bokisch, V.A. and Miiller-Eberhard, H.J., J. Exp. Med. 1975.142: 139. 31 Zeijlemaker. W.P., Roos, M.Th.L.,Schellekens,P.Th.A. and Eijsvoogel, V.P., Eur. J. Immunol.1975.5: 579. 32 Capel, P.J.A., J. Immunol.Methods 1974.5: 165. 33 Michaelsen, T.E., Wisldff, F. and Natvirg, J.B., Scand. J. Immunol. 1975.4: 71. 34 Tamura, N. and Nelson, R.A., J. Immunol. 1968.101: 1333. 35 Tesar, J.T. and Schmid, F.S., J. Immunol.1973.110: 993.
7 Gisler, R.H. and Fridman, W.H., J. Exp. Med. 1975.142: 507.
36 Basten, A., Miller, J.F.A.P., Abraham, R., Gamble, J. and Chia, E., Int. Arch. Allergy Appl. Immunol. 1976.50: 1309.
8 Diaz-Jouanen, E., Bankhurst. A.D., Messner, R.P. and Williams, R.C., Arthritis Rheum. 1976.19: 142.
37 Ferrarini, M., Moretta, L., Mingari, M.C., Tonda, P. and Pernis, B., Eur. J. Immunol. 1976.6: 520.
