274
Eur. J. Immunol. 1978.8: 274-278
W.J. Pichler, S. Broder, L. Muul et aL
23 Novogrodsky, A., Lotan, R., Ravid, A. and Sharon, N., J. Zmmunol. 1975.115: 1243. 24 Sela, B., Wang, J.L. and Edelman, G.M., . I Exp. . Med. 1976.143: 665. 25 Bennett, V., O'Keefe, E. and Cuatrecasas, P., R o c . Nat. Acad. Sci. US 1975. 72: 33. 26 Kornberg, R.D. and McConnell,H.M.,Proc. Nat. Acad. Sci. US 1971.68: 2564.
W.J. Pichler", S. Broder, Linda Muul, I. Magrath and T.A. Waldmann National Institutes of Health, National Cancer Institute, Metabolism and Pediatric Oncology Blanches, Bethesda
27 Rodbell, M., in Anfinsen, C.B., Goldberger, R.F. and Schechter, A.N. (Eds.), Current Topics in Biochemistry, Academic Press, New York 1972, p. 187. 28 de Petris, S. and Raff, M.C., Eur. J. Zmmunol. 1972.2: 523. 29 Karnovsky, M.J., Unanue, E.R. and Leventhal, M., J. Exp. Med. 1972.136: 907. 30 Stackpole, C.W., Jacobson, J.B. and Lardis, M.P., Nature 1974. 248: 232.
Fc IgM receptors on human lymphoblastoid B cell lines Five human lymphoblastoid cell lines have been investigated for their ability to secrete immunoglobulins (IgG, IgA, IgM) and for the presence of different cell surface markers, with special emphasis o n the F c IgM receptor, using a rosette technique with IgM-coated bovine red blood cells (EA-IgM). Four cell lines (Hu, 8432, SB, PA3) were characterized as having a B cell origin due t o the presence of surface immunoglobulins, complement receptors, mouse red blood cell rosette formation, low avidity F c IgG receptors and absence of sheep red blood cell rosette formation. Two of these B cell lines (Hu and PA3) secreted IgM, two cell lines (8432 and SB) secreted IgG, while the human T cell line Molt 4 did not secrete Ig. All four B cell lines exhibited F c IgM receptors (17-35 %) while the T cell line Molt 4 had no detectable F c IgM receptor. The receptor was specific for IgM. Neither aggregated IgG, soluble IgG immune complexes nor EDTA could abrogate the rosette formation. IgM immune complexes, pure human IgM, as well as normal human serum had an inhibitory effect o n EA-IgM rosette formation. The receptor was trypsin-sensitive and required protein synthesis for expression. There was n o correlation between the expression of F c IgM receptors and secretion of any given Ig class, indicating that B cells may express Fc IgM receptors independently of their commitment t o produce IgM o r IgG.
1 Introduction Recently, it has become apparent that not only human T cells [ l ] , but a subpopulation of normal human B cells and chronic lymphocytic leukemia (CLL) cells of clearly defined B cell origin also express receptors for IgM [2-41. This receptor was found o n a fraction of B cells which expressed Ia-like antigens [3, 41 and which had surface immunoglobulins (sIg) of the p
[I 19351
* On leave from the University of Vienna, Austria. Correspondence: Werner J. Pichler, National Institutes of Health, National Cancer Institute, Metabolism Branch, Building 10, Room 4N115, Bethesda, MD 20014, USA Abbreviations: ALL: Acute lymphoblastic leukemia BRBC: Bovine red blood cells BSA: Bovine serum albumin CLL: Chronic lymphocytic leukemia EBV: Epstein Ban Virus E k C : Sheep red cells coated with IgG and mouse complement EAMC: Sheep red cells coated with IgM and mouse complement EA-I&: Bovine red blood cells coated with IgG EA-IgM: Bovine red blood cells coated with IgM FCS: Fetal calf serum FITC Fluorescein isothiocyanate IC: Immune complex (es) MRBC: Mouse red blood cells OVA: Ovalbumin PWM: Pokeweed mitogen RF: Rosette formation RFT: Rosette-forming cells sIg: Surface immunoglobulin(s) SRBC: Sheep red blood cells T N P Trinitrophenyl PBS: Phosphate-buffered saline
only and p + 6 classes [4]. We have also observed receptors for bovine red blood cells coated with IgM (EA-IgM) o n two patients with CLL having sIg of the 6 class only (unpublished). The receptor for IgM can b e blocked by the Fc piece of IgM but not b y its F(ab')* fragment [3]. Therefore, t o distinguish the receptor for IgM from the sIg of t h e IgM class which acts as antigen receptor, we propose the nomenclature FcIgM receptor for this surface receptor which is specific for the F c piece of IgM, and will use this terminology throughout this paper. The purpose of the current study was t o define whether human B cell lines express F c IgM receptors. Human hematopoietic cell lines represent a steadily proliferating resource of homogeneous cells expressing different surface receptors according t o their origin [5] and the procedure used for establishing the lines [6,7]. Most long-term human lymphoid cell lines show B cell characteristics, express Epstein Barr (EB) viral genome, and certain B cell lines secrete Ig of a specific class. I n this communication, we have analyzed several longterm human B cell lines for their capacity t o secrete Ig and t o express receptors for F c IgM. I n addition, we have used the restriction of Ig class production of certain B cell lines t o see whether there is an association between expression of receptors for Fc IgM and secretion of Ig of a specific class.
Eur. J. Immunol. 1978.8: 274-278
2 Materials and methods 2.1 Cell line cells
The cell line Hu was established according to usual techniques from a patient with cutaneous lymphoma [6]. The origin of the other investigated cell lines is given in Sect. 3.2, Table 2. All of the cell lines were cultured in RPMI 1640 medium supplemented with 20 % heat-inactivated fetal calf serum (FCS), penicillin, streptomycin and glutamine in 75 cm2 tissue culture flasks.
2.2 Measurement of Ig synthesis by lymphocytes in vitro To study spontaneous as well as lectin-induced Ig synthesis by the cell lines in vitro, 2 x l o 6 cells were cultured without pokeweed mitogen (PWM) or in the presence of PWM. In some experiments, cells were x-irradiated with 2000 R to inhibit mitosis. Secretion of IgG, IgA, and IgM into the medium was determined by double antibody radioimmunoassay techniques essentially identical t o those previously described for IgE [8].
B cell lines with Fc IgM receptors
275
these conditions no EA-IgM binding was observed. Latex phagocytosis and spontaneous sheep red blood cell (SRBC) RF was tested as described elsewhere [2].
2.4 Demonstration of Fc IgG receptors by soluble immune complexes Trinitrophenylated (TNP)-BSA and anti-TNP-BSA IgG antibodies were a generous gift from Dr. Warren Strober. The immune complexes (IC) were prepared essentially as described by Arbeit et al. [lo]. Briefly, 20 pl of TNP-BSA (0.6 mg/ml) were mixed with 50 pl rabbit anti-TNP IgG fraction and 30 pl phosphate-buffered saline (PBS), incubated for 1 h at room temperature and 10 pl of preformed IC added to 50 pl of the lymphocyte suspension (20 x 106/ml). The mixture was incubated for 30 rnin at room temperature, washed twice and either stained for IC binding with a goat anti-rabbit IgG (Cappel), or EA-IgM added to determine whether the EA-IgM binding was blocked by soluble IgG IC.
2.5 Addition of different substances to inhibit EA-IgM binding
2.3 Surface marker characterization The techniques used to detect surface markers have been previously described [2]. Cells were washed 3 times in medium containing 10 % FCS and were finally suspended in Medium 199 (Gibco, Grand Island Biological Co, NY) to give a cell concentration of 10' cells/ml. SIg were determined with a fluorescein isothiocyanate (F1TC)-conjugated F(ab'), fragment of an anti-F(ab')2 antiserum (Cappel Labs., Cochranville, PA). Immunofluorescence was evaluated in a Leitz microscope using Ploem illumination. Mouse red blood cell rosette formation (MRBC RF) was done according to Gupta [9], using BALB/c mice as a red blood cell source and MRBC-absorbed FCS. EA-IgG and EA-IgM preparations were prepared as previously described [2]. Briefly, bovine red blood cells (BRBC) were washed three times in Veronalbuffered saline containing 0.01 M EDTA, sensitized with antiBRBC IgG fraction (EA-IgG), diluted 1 :SO (hemolysis titer 1 :16 000, protein concentration 4 mg/ml) or with an antiBRBC IgM fraction (EA-IgM), diluted 1 :25 (hemolysis titer 1: 128 000, protein concentration 5 mg/ml) by incubation at 37 OC for 30 min. The IgG and IgM fractions were prepared from an inactivated rabbit hyperimmune serum, and the purity tested as described previously [2]. The cells were washed 3 times and resuspended in Medium 199/1 % bovine serum albumin (BSA) to give a l % suspension. Fifty p1 of cell line cell suspension ( 107/ml) and 50 pl of coated BRBC were mixed, spun down at 200 x g and kept at 0 OC for 1-3 h. Resuspension was done very gently. One drop of resuspended cells was placed on a slide, and at least 200 cells were counted. Adherence of 3 or more erythrocytes was considered positive. Uncoated BRBC, or BRBC coated with F(ab')? fragments of the anti-BRBC IgG, sensitized with the same quantity of antibody as for the EA-IgG preparations, served as controls. Complement receptors were detected by EAC rosettes using SRBC coated with IgM or IgG antibody (Cordis Labs., Miami, FL) and mouse serum as a complement source ( EAMC or EAGC). Equal volumes of cell line lymphocytes, and of the EAC suspension were rotated at 37 OC for 1/2 h and the rosette formation (RF) evaluated immediately thereafter. Under
Fifty pl of the cell suspension (1 0 x 106/ml) was mixed with 50 pl of various substances, incubated at 37 OC for 10 min and at 4 OC for 50 min. Following this, 50 pl of EA-IgM suspension was added and the rosette assay performed as described above. The following substrates were tested: 0.33 M EDTA human serum, heat-inactivated and absorbed with BRBC, containing 1 m d m l IgM; aggregated IgG, (10 mglml), prepared by incubating Cohn fraction I1 (Miles Labs., Elkhart, IN) for 20 min at 63 OC; purified human IgM, prepared by starch block electrophoresis and Sephadex G 2 0 0 separation of two Waldenstrom sera. The concentrations added, varied between 2.4 mg/ml and 0.025 mg/ml. Anti-Clq and anti-C3, FITC-conjugated, were diluted 1 :3 (Behringwerke, Marburg, FRG). Soluble ovalbumin (OVA) IgM IC were prepared in antigen excess by mixing and incubating (30 min, room temperature) equal volumes of an OVA solution (1 mg protein/ml in PBS) with the IgM fraction of a rabbit anti-OVA semm (1 mg protein/ml in PBS). The isolation and characterization of the IgM fraction has been performed as described for the IgM antiBRBC fraction [2]. Twenty pl of the IC, of the OVA solution or of 1 :2 diluted IgM antibody solution were tested for their inhibitory effect. The receptor was further tested for its enzymatic sensitivity and protein synthesis requirement. One ml of 0.5 % trypsin solution (Gibco) was mixed with 1 ml of the cell line suspension, incubated at 37 OC for 30 min, washed twice with medium containing 20 % FCS, and either checked for EAIgM RF immediately or after 20 h cultivation at 37 "C in a 5.% C 0 2 atmosphere. Fifty pg/ml of the protein synthesis inhibitor cycloheximide (Sigma Chemical Co., St. Louis, MO) was added to untreated or trypsin-treated cells (2 x lo6/ ml) and the cells cultured for 20 h in Medium 199/20 % FCS. 2.6 Absorption of IgM anti-BRBC antibody fraction with cell line cells To rule out a rheumatoid factor-like activity of the sIg [ 111, anti-BRBC IgM antibody diluted 1 :25 was absorbed with washed, packed Hu and PA3 cells for 1 h at 4 "C and for 1 h at room temperature and then used to prepare EA-IgM.
276
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W.J. Pichler, S. Broder, L. Muul et al.
3 Results
3.2 Surface characteristics of cell line cells (Table 2)
3.1 Ig production by the cell line cells (Table 1)
Spontaneous RF with untreated SRBC was not detected in the cell line cells Hu, PA3 and 8432, but 66 % of the Molt 4 line cells formed rosettes. Of the Hu and PA3 cells 98 %, 81 % of the SB cell line, 81 % and 60 % of the 8432 cell line cells exhibited sIg. N o sIg were detected on Molt 4 cell line cells. Another presumptive B cell marker, MRBC RF, was found to be positive in 5 % of Hu, 12 % of the PA3, 14 % of the SB, and in 27 % of the 8432 cell line cells; Molt 4 cell line cells did not bind MRBC.
The Hu cell line and the cell line PA3 could secrete IgM but not IgG or IgA in vitro, while the cell line 8432 and SB secreted IgG but no IgM or IgA. No Ig was found in the supernatant of cultured Molt 4 cells. The Ig production by the long-term cell lines tested was not radiosensitive and appeared independent of the addition of PWM. This implies that under the conditions tested, Ig secretion did not require cell division. These results contrast with the marked radiosensitivity of Ig production by normal lymphocytes. Table 1. Ig secretory capacity of cell lines Cell liner
Days of culture
IgG
IgA
IgM
Complement receptors were found on all 5 cell lines studied. Molt 4 cells, considered to be of T cell origin due to their ability to form spontaneous rosettes with SRBC, also had 82 % complement receptors. This finding has already been described and discussed [ 151. To exclude the possible participation of Fc IgM receptors in RF with EA-IgM complement complexes, we tested the presence of complement receptors with IgG and complement-coated SRBC as well. The results obtained with EAGC were similar to the results obtained with EAMC,the slightly higher value of EAMCrosetting may be attributable to better complement fixation of the EA-IgM complex.
(ng/2 x 106 cells/culture) Hu + PWM”) + 2000 r + PWM
13 13 13
< 14 < 14 < 14
< 32
PA-3 + Pwhl + 2000r + PWM
7 1 7
Eur. J. Immunol. 1978.8: 274-278
W.J. Pichler, S. Broder, L. Muul et aL
23 Novogrodsky, A., Lotan, R., Ravid, A. and Sharon, N., J. Zmmunol. 1975.115: 1243. 24 Sela, B., Wang, J.L. and Edelman, G.M., . I Exp. . Med. 1976.143: 665. 25 Bennett, V., O'Keefe, E. and Cuatrecasas, P., R o c . Nat. Acad. Sci. US 1975. 72: 33. 26 Kornberg, R.D. and McConnell,H.M.,Proc. Nat. Acad. Sci. US 1971.68: 2564.
W.J. Pichler", S. Broder, Linda Muul, I. Magrath and T.A. Waldmann National Institutes of Health, National Cancer Institute, Metabolism and Pediatric Oncology Blanches, Bethesda
27 Rodbell, M., in Anfinsen, C.B., Goldberger, R.F. and Schechter, A.N. (Eds.), Current Topics in Biochemistry, Academic Press, New York 1972, p. 187. 28 de Petris, S. and Raff, M.C., Eur. J. Zmmunol. 1972.2: 523. 29 Karnovsky, M.J., Unanue, E.R. and Leventhal, M., J. Exp. Med. 1972.136: 907. 30 Stackpole, C.W., Jacobson, J.B. and Lardis, M.P., Nature 1974. 248: 232.
Fc IgM receptors on human lymphoblastoid B cell lines Five human lymphoblastoid cell lines have been investigated for their ability to secrete immunoglobulins (IgG, IgA, IgM) and for the presence of different cell surface markers, with special emphasis o n the F c IgM receptor, using a rosette technique with IgM-coated bovine red blood cells (EA-IgM). Four cell lines (Hu, 8432, SB, PA3) were characterized as having a B cell origin due t o the presence of surface immunoglobulins, complement receptors, mouse red blood cell rosette formation, low avidity F c IgG receptors and absence of sheep red blood cell rosette formation. Two of these B cell lines (Hu and PA3) secreted IgM, two cell lines (8432 and SB) secreted IgG, while the human T cell line Molt 4 did not secrete Ig. All four B cell lines exhibited F c IgM receptors (17-35 %) while the T cell line Molt 4 had no detectable F c IgM receptor. The receptor was specific for IgM. Neither aggregated IgG, soluble IgG immune complexes nor EDTA could abrogate the rosette formation. IgM immune complexes, pure human IgM, as well as normal human serum had an inhibitory effect o n EA-IgM rosette formation. The receptor was trypsin-sensitive and required protein synthesis for expression. There was n o correlation between the expression of F c IgM receptors and secretion of any given Ig class, indicating that B cells may express Fc IgM receptors independently of their commitment t o produce IgM o r IgG.
1 Introduction Recently, it has become apparent that not only human T cells [ l ] , but a subpopulation of normal human B cells and chronic lymphocytic leukemia (CLL) cells of clearly defined B cell origin also express receptors for IgM [2-41. This receptor was found o n a fraction of B cells which expressed Ia-like antigens [3, 41 and which had surface immunoglobulins (sIg) of the p
[I 19351
* On leave from the University of Vienna, Austria. Correspondence: Werner J. Pichler, National Institutes of Health, National Cancer Institute, Metabolism Branch, Building 10, Room 4N115, Bethesda, MD 20014, USA Abbreviations: ALL: Acute lymphoblastic leukemia BRBC: Bovine red blood cells BSA: Bovine serum albumin CLL: Chronic lymphocytic leukemia EBV: Epstein Ban Virus E k C : Sheep red cells coated with IgG and mouse complement EAMC: Sheep red cells coated with IgM and mouse complement EA-I&: Bovine red blood cells coated with IgG EA-IgM: Bovine red blood cells coated with IgM FCS: Fetal calf serum FITC Fluorescein isothiocyanate IC: Immune complex (es) MRBC: Mouse red blood cells OVA: Ovalbumin PWM: Pokeweed mitogen RF: Rosette formation RFT: Rosette-forming cells sIg: Surface immunoglobulin(s) SRBC: Sheep red blood cells T N P Trinitrophenyl PBS: Phosphate-buffered saline
only and p + 6 classes [4]. We have also observed receptors for bovine red blood cells coated with IgM (EA-IgM) o n two patients with CLL having sIg of the 6 class only (unpublished). The receptor for IgM can b e blocked by the Fc piece of IgM but not b y its F(ab')* fragment [3]. Therefore, t o distinguish the receptor for IgM from the sIg of t h e IgM class which acts as antigen receptor, we propose the nomenclature FcIgM receptor for this surface receptor which is specific for the F c piece of IgM, and will use this terminology throughout this paper. The purpose of the current study was t o define whether human B cell lines express F c IgM receptors. Human hematopoietic cell lines represent a steadily proliferating resource of homogeneous cells expressing different surface receptors according t o their origin [5] and the procedure used for establishing the lines [6,7]. Most long-term human lymphoid cell lines show B cell characteristics, express Epstein Barr (EB) viral genome, and certain B cell lines secrete Ig of a specific class. I n this communication, we have analyzed several longterm human B cell lines for their capacity t o secrete Ig and t o express receptors for F c IgM. I n addition, we have used the restriction of Ig class production of certain B cell lines t o see whether there is an association between expression of receptors for Fc IgM and secretion of Ig of a specific class.
Eur. J. Immunol. 1978.8: 274-278
2 Materials and methods 2.1 Cell line cells
The cell line Hu was established according to usual techniques from a patient with cutaneous lymphoma [6]. The origin of the other investigated cell lines is given in Sect. 3.2, Table 2. All of the cell lines were cultured in RPMI 1640 medium supplemented with 20 % heat-inactivated fetal calf serum (FCS), penicillin, streptomycin and glutamine in 75 cm2 tissue culture flasks.
2.2 Measurement of Ig synthesis by lymphocytes in vitro To study spontaneous as well as lectin-induced Ig synthesis by the cell lines in vitro, 2 x l o 6 cells were cultured without pokeweed mitogen (PWM) or in the presence of PWM. In some experiments, cells were x-irradiated with 2000 R to inhibit mitosis. Secretion of IgG, IgA, and IgM into the medium was determined by double antibody radioimmunoassay techniques essentially identical t o those previously described for IgE [8].
B cell lines with Fc IgM receptors
275
these conditions no EA-IgM binding was observed. Latex phagocytosis and spontaneous sheep red blood cell (SRBC) RF was tested as described elsewhere [2].
2.4 Demonstration of Fc IgG receptors by soluble immune complexes Trinitrophenylated (TNP)-BSA and anti-TNP-BSA IgG antibodies were a generous gift from Dr. Warren Strober. The immune complexes (IC) were prepared essentially as described by Arbeit et al. [lo]. Briefly, 20 pl of TNP-BSA (0.6 mg/ml) were mixed with 50 pl rabbit anti-TNP IgG fraction and 30 pl phosphate-buffered saline (PBS), incubated for 1 h at room temperature and 10 pl of preformed IC added to 50 pl of the lymphocyte suspension (20 x 106/ml). The mixture was incubated for 30 rnin at room temperature, washed twice and either stained for IC binding with a goat anti-rabbit IgG (Cappel), or EA-IgM added to determine whether the EA-IgM binding was blocked by soluble IgG IC.
2.5 Addition of different substances to inhibit EA-IgM binding
2.3 Surface marker characterization The techniques used to detect surface markers have been previously described [2]. Cells were washed 3 times in medium containing 10 % FCS and were finally suspended in Medium 199 (Gibco, Grand Island Biological Co, NY) to give a cell concentration of 10' cells/ml. SIg were determined with a fluorescein isothiocyanate (F1TC)-conjugated F(ab'), fragment of an anti-F(ab')2 antiserum (Cappel Labs., Cochranville, PA). Immunofluorescence was evaluated in a Leitz microscope using Ploem illumination. Mouse red blood cell rosette formation (MRBC RF) was done according to Gupta [9], using BALB/c mice as a red blood cell source and MRBC-absorbed FCS. EA-IgG and EA-IgM preparations were prepared as previously described [2]. Briefly, bovine red blood cells (BRBC) were washed three times in Veronalbuffered saline containing 0.01 M EDTA, sensitized with antiBRBC IgG fraction (EA-IgG), diluted 1 :SO (hemolysis titer 1 :16 000, protein concentration 4 mg/ml) or with an antiBRBC IgM fraction (EA-IgM), diluted 1 :25 (hemolysis titer 1: 128 000, protein concentration 5 mg/ml) by incubation at 37 OC for 30 min. The IgG and IgM fractions were prepared from an inactivated rabbit hyperimmune serum, and the purity tested as described previously [2]. The cells were washed 3 times and resuspended in Medium 199/1 % bovine serum albumin (BSA) to give a l % suspension. Fifty p1 of cell line cell suspension ( 107/ml) and 50 pl of coated BRBC were mixed, spun down at 200 x g and kept at 0 OC for 1-3 h. Resuspension was done very gently. One drop of resuspended cells was placed on a slide, and at least 200 cells were counted. Adherence of 3 or more erythrocytes was considered positive. Uncoated BRBC, or BRBC coated with F(ab')? fragments of the anti-BRBC IgG, sensitized with the same quantity of antibody as for the EA-IgG preparations, served as controls. Complement receptors were detected by EAC rosettes using SRBC coated with IgM or IgG antibody (Cordis Labs., Miami, FL) and mouse serum as a complement source ( EAMC or EAGC). Equal volumes of cell line lymphocytes, and of the EAC suspension were rotated at 37 OC for 1/2 h and the rosette formation (RF) evaluated immediately thereafter. Under
Fifty pl of the cell suspension (1 0 x 106/ml) was mixed with 50 pl of various substances, incubated at 37 OC for 10 min and at 4 OC for 50 min. Following this, 50 pl of EA-IgM suspension was added and the rosette assay performed as described above. The following substrates were tested: 0.33 M EDTA human serum, heat-inactivated and absorbed with BRBC, containing 1 m d m l IgM; aggregated IgG, (10 mglml), prepared by incubating Cohn fraction I1 (Miles Labs., Elkhart, IN) for 20 min at 63 OC; purified human IgM, prepared by starch block electrophoresis and Sephadex G 2 0 0 separation of two Waldenstrom sera. The concentrations added, varied between 2.4 mg/ml and 0.025 mg/ml. Anti-Clq and anti-C3, FITC-conjugated, were diluted 1 :3 (Behringwerke, Marburg, FRG). Soluble ovalbumin (OVA) IgM IC were prepared in antigen excess by mixing and incubating (30 min, room temperature) equal volumes of an OVA solution (1 mg protein/ml in PBS) with the IgM fraction of a rabbit anti-OVA semm (1 mg protein/ml in PBS). The isolation and characterization of the IgM fraction has been performed as described for the IgM antiBRBC fraction [2]. Twenty pl of the IC, of the OVA solution or of 1 :2 diluted IgM antibody solution were tested for their inhibitory effect. The receptor was further tested for its enzymatic sensitivity and protein synthesis requirement. One ml of 0.5 % trypsin solution (Gibco) was mixed with 1 ml of the cell line suspension, incubated at 37 OC for 30 min, washed twice with medium containing 20 % FCS, and either checked for EAIgM RF immediately or after 20 h cultivation at 37 "C in a 5.% C 0 2 atmosphere. Fifty pg/ml of the protein synthesis inhibitor cycloheximide (Sigma Chemical Co., St. Louis, MO) was added to untreated or trypsin-treated cells (2 x lo6/ ml) and the cells cultured for 20 h in Medium 199/20 % FCS. 2.6 Absorption of IgM anti-BRBC antibody fraction with cell line cells To rule out a rheumatoid factor-like activity of the sIg [ 111, anti-BRBC IgM antibody diluted 1 :25 was absorbed with washed, packed Hu and PA3 cells for 1 h at 4 "C and for 1 h at room temperature and then used to prepare EA-IgM.
276
Eur. J. Immunol. 1978.8: 274-278
W.J. Pichler, S. Broder, L. Muul et al.
3 Results
3.2 Surface characteristics of cell line cells (Table 2)
3.1 Ig production by the cell line cells (Table 1)
Spontaneous RF with untreated SRBC was not detected in the cell line cells Hu, PA3 and 8432, but 66 % of the Molt 4 line cells formed rosettes. Of the Hu and PA3 cells 98 %, 81 % of the SB cell line, 81 % and 60 % of the 8432 cell line cells exhibited sIg. N o sIg were detected on Molt 4 cell line cells. Another presumptive B cell marker, MRBC RF, was found to be positive in 5 % of Hu, 12 % of the PA3, 14 % of the SB, and in 27 % of the 8432 cell line cells; Molt 4 cell line cells did not bind MRBC.
The Hu cell line and the cell line PA3 could secrete IgM but not IgG or IgA in vitro, while the cell line 8432 and SB secreted IgG but no IgM or IgA. No Ig was found in the supernatant of cultured Molt 4 cells. The Ig production by the long-term cell lines tested was not radiosensitive and appeared independent of the addition of PWM. This implies that under the conditions tested, Ig secretion did not require cell division. These results contrast with the marked radiosensitivity of Ig production by normal lymphocytes. Table 1. Ig secretory capacity of cell lines Cell liner
Days of culture
IgG
IgA
IgM
Complement receptors were found on all 5 cell lines studied. Molt 4 cells, considered to be of T cell origin due to their ability to form spontaneous rosettes with SRBC, also had 82 % complement receptors. This finding has already been described and discussed [ 151. To exclude the possible participation of Fc IgM receptors in RF with EA-IgM complement complexes, we tested the presence of complement receptors with IgG and complement-coated SRBC as well. The results obtained with EAGC were similar to the results obtained with EAMC,the slightly higher value of EAMCrosetting may be attributable to better complement fixation of the EA-IgM complex.
(ng/2 x 106 cells/culture) Hu + PWM”) + 2000 r + PWM
13 13 13
< 14 < 14 < 14
< 32
PA-3 + Pwhl + 2000r + PWM
7 1 7
