T-cell and mast cell lines respond to B-cell stimulatory factor 1

Transcription

1 Proc. Natl. Acad. Sci. USA Vol. 83, pp , August 1986 Immunology T-cell and mast cell lines respond to B-cell stimulatory factor 1 (lymphokine/interleukin 4/B-cell growth factor 1/mast celi growth factor 2/T-celH growth factor 2) TIMOTHY R. MOSMANN*, MARTHA W. BOND*, ROBERT L. COFFMAN*, JUNICHI OHARAt, AND WILLIAM E. PAULt *DNAX Research Institute of Molecular and Cellular Biology, 91 California Avenue, Palo Alto, CA 9434; and tlaboratory of Immunology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 2892 Contributed by William E. Paul, April 8, 1986 ABSTRACT The murine lymphokine B-cell stimulatory factor 1 (BSF-1) has been described previously in terms of its action on B lymphocytes. We now provide evidence that BSV-1 is also responsible for two additional biological activities. The first of these is the stimulation or maintenance of a state of activation in mouse T-cell lines. The second activity is the increase in the proliferative rate of certain mast cell lines costimulated with interleukin 3. The T-cell and mast cell activities are mediated by purified BSF-1 and copurify with BSF-1 from supernatants of certain T-cell lines. Each of these activities is inhibited by monoclonal anti-bsf.1 but not by monoclonal anti-interleukin 2 antibody. The'antibody inhibition results also indicate that BSF-1 is the major or only source of these two activities in the activated T-cell supernatants that we have tested. Activated T lymphocytes synthesize and secrete a number of lymphokines that activate B lymphocytes, resulting in proliferation and secretion of antibody. The best characterized of these is B-cell stimulatory factor 1 (BSF-1, formerly called B-cell growth factor, BCGF-1, ref. 1). BSF-1 was originally described by its ability to synergize with anti-igm antibody to induce proliferation of B cells (2). More recently, BSF-1 was also found to be responsible for inducing an increase in Ta antigen expression on resting B cells (3, 4), preparing B cells to enter S phase more promptly in response to several stimuli (5, 6), and enhancing the production of IgG1 (7) and IgE (8, 9) by lipopolysaccharide (LPS)-stimulated B cells. Two activities have been described recently in the supernatants of a subset of activated T helper cell clones (1, 11). One activity (initially designated mast cell growth factor 2, MCGF2) is the ability to synergize with interleukin 3 (IL-3) to cause optimal growth of certain mast cell-like lines, and the other activity (initially designated T-cell growth factor 2, TCGF2) is the induction or maintenance of a state of activation in T-cell lines. We observed (11) that MCGF2 and TCGF2 activities were produced by a subset of helper T-cell lines (TH2) that also produced BSF-1 but not interleukin 2 (IL-2) or interferon-y (IFN-y). Another subset of helper T-cell lines (TH1) produced IL-2 and IFN-y but not BSF-1, MCGF2, or TCGF2 activities (11). The concordance of expression of the last three activities raised the possibility that all three were mediated by the same lymphokine. In this communication, we present evidence that all three of these activities are mediated by one entity, currently known as BSF-1. These findings extend the range of cell types known to be affected by BSF-1 to include mast cell and T-lymphocyte lines in addition to B lymphocytes. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C solely to indicate this fact MATERIALS AND METHODS Cell Lines. The HT2 mouse T-cell line (12) was obtained from S. Strober, and the DA-1 cell line was obtained from J. Ihle through D. Rennick. The MB2-1 and LB2-1 T-cell lines and the MM3 mast cell line were established as described (11, 13). LB2-1 is an example of a type 1 T helper cell (TH1) clone and synthesizes IL-2, IL-3, granulocyte-macrophage colonystimulating factor (GM-CSF), and IFN-y in response to stimulation by antigen or Con A (13). MB2-1 is an example of a type 2 T helper cell (TH2) clone and produces IL-3, GM-CSF, BSF-1, TCGF2, and MCGF2 activities after stimulation (11). Recombinant Lymphokines. The cdna clone encoding mouse IL-2 (14) was modified and expressed in Escherichia coli (S. M. Zurawski, T.R.M., M. Benedik and G. Zurawski, unpublished data). Recombinant mouse IL-3 was obtained from the supernatants of COS cells transfected with the cdna clone of mouse IL-3 (15). Monoclonal Antibodies. The lib11 monoclonal anti-bsf-1 antibody has been described (16). The S4B6 monoclonal antimouse IL-2 antibody was obtained by fusing Sp2/ mouse hybridoma cells with spleen cells from rats immunized with concentrated supernatant proteins from LB2-1 T cells (unpublished data). Proliferation Assays. Bioassays for IL-2, IL-3, TCGF2, and MCGF2 have been described (11). IL-2 and TCGF2 were assayed on the HT2 T-cell line, IL-3 was assayed on the DA-1 cell line, and MCGF2 activity was measured by stimulation of the MM3 cell line in the presence of saturating amounts of IL-3. Supernatant from the LB2-1 T-cell line was used as a source of IL-3 (11). Under these conditions, MM3 cells respond additionally only to the MCGF2 activity present in TH2 supernatants. In all cases, the 3-(4,5-dimethylthiazol-2- yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay (17) was used. In this method, surviving and/or proliferating cells are detected at the end of the assay by their ability to cleave the tetrazolium salt MTU and produce a colored product. This cleavage is the result of the activity of dehydrogenase enzymes and hence is dependent on energy metabolism in intact cells. The presence of such activity correlates with the state of activation and number of living cells and is often used in place of [3H]thymidine incorporation in assays of lymphocyte proliferation (11, 17). Since this assay measures metabolic activity rather than DNA synthesis, the results of the MUT and [3H]thymidine assays can theoretically differ in some cases, and both assays can potentially differ from measurements of cell proliferation. In Abbreviations: BSF-1, B-cell stimulatory factor 1; GM-CSF, granulocyte-macrophage colony-stimulating factor; IFN-y, interferon-y; IL-2, interleukin 2; IL-3, interleukin 3; LPS, lipopolysaccharide; MCGF2, mast cell growth factor 2; MTT, 3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyltetrazolium bromide; TCGF2, T-cell growth factor 2; TH1, T helper cell type 1; TH2, T helper cell type 2.

2 Immunology: Mosmann et al. allmtt assays, 1 unit of activity is defined as the amount of factor that, in.1 ml, results in 5% of the maximal signal in the assay under the specified conditions of cell number and time. IgE-Enhancing Activity. T-cell supernatants or purified proteins were assayed for the ability to induce increased IgE secretion in cultures of splenic B cells stimulated with LPS as described (8). Total IgE in the supernatants was measured by an isotype-specific ELISA assay. Protein Separation. MB2-1 cells were stimulated with 1,tg of Con A per ml at 5 x 16 cells per ml in RPMI 164 medium lacking serum. After 24 hr, supernatant proteins were concentrated by Amicon ultrafiltration and applied to a C4 reverse-phase HPLC column (11). Proteins were eluted by a gradient from 3% to 8% aqueous acetonitrile with.1% trifluoroacetic acid as the mobile phase modifier. One-minute (.4 ml) fractions were assayed for biological activity. RESULTS BSF-1, TCGF2, and MCGF2 Activities Cochromatograph on HPLC. We first separated lymphokine activities by reverse-phase HPLC using total supernatant proteins from the T-cell clone MB2-1, which produces IL-3, GM-CSF, BSF-1, Proc. Natl. Acad. Sci. USA 83 (1986) 5655 MCGF2, and TCGF2 activities after Con A stimulation (11). BSF-1 was assayed by its IgE-enhancing activity (8); TCGF2 was measured by its ability to stimulate HT2 cells (11); IL-3 and GM-CSF activities were measured by their ability to stimulate the DA-1 cell line; MCGF2 was measured by its ability to costimulate, with IL-3, the MM3 mast cell line (11). For all assays except IgE enhancement, the colorimetric assay (17) was used. Fig. 1 shows that the activities of BSF-1, MCGF2, and TCGF2 all centered on fraction 44, whereas IL-3 activity centered on fraction 42. Although IL-3 activity could not be well resolved from the other three activities by reverse-phase HPLC, it could readily be separated by other methods (ref. 1 and unpublished results). A second peak of activity (centered on fraction 53) detected on the DA-1 cell line was probably GM-CSF, since DA-1 responds to GM- CSF, and recombinant GM-CSF elutes in this position on this column (T.R.M. and M.W.B., unpublished results). In parallel column separations using supernatants from induced LB2-1 cells, IL-2 and IFN-y eluted at positions corresponding to fractions 7 and 73, respectively (T.R.M. and M.W.B., unpublished results). Purified BSF-1 also Has MCGF2 and TCGF2 Activities. Extensively purified BSF-1, obtained from EL4 cells (18), was then tested for activity in the MCGF2 and TCGF2 3- TCGF 2 4H MCGF2 V I D 2k 6 o x 1- 'E cx U- U- 2k 1 - -I-.- -a _ O x E E c uj -P > cd Fraction number Fraction number FIG. 1. BSF-1, TCGF2, and MCGF2 activities cochromatograph on HPLC. Supernatant proteins from Con A-induced MB2-1 cells were concentrated and chromatographed on a C4 reverse-phase HPLC column. Fractions were assayed for activity on the following cell lines: HT2 (detecting TCGF2 activity), DA-1 (detecting IL-3 and GM-CSF), and MM3 (detecting MCGF2 activity). The fractions were also tested for BSF-1 by their ability to enhance IgE secretion by LPS-activated B lymphocytes.

3 5656 Immunology: Mosmann et al. Proc. Natl. Acad. Sci. USA 83 (1986).6.5 ( 6 LOn LO.4.2 / 4'(# IFI._L_ _>1< =.- / I: I~~~~~~~~~~~~o fb. MCGF2 CoY/ ( log2 Dilution FIG. 2. Purified BSF-1 has MCGF2 and TCGF2 activities. Dilutions of purified BSF-1 (starting at 5 units/ml, refs. 16 and 18) were tested for TCGF2 and MCGF2 activities and compared with recombinant mouse IL-2 and supernatant from the MB2-1 cell line. (A) TCGF2 activity was tested on HT2 cells. (B) MCGF2 was tested on MM3 mast cells in the presence of saturating levels (-4 units/ml) of IL-3. In the MM3 assay, the background due to IL-3 has been subtracted. The means ± SEM of triplicates are plotted. -, BSF-1; , MB2-1 supernatant;, recombinant IL-2. assays. Fig. 2A shows that HT2 cells responded to purified BSF-1 and that the slope of the response curve was similar to the titration curve of MB2-1 supernatant but different from that obtained with IL-2. The maximum MTT signal in response to either BSF-1 or MB2-1 supernatant was lower than the stimulation due to IL-2. In the MCGF2 assay, MB2-1 supernatants and purified BSF-1 induced a response above the background level induced by IL-3 (Fig. 2B), whereas T-cell supernatants that do not contain BSF-1 do not induce a response in this assay (11). IL-3 and BSF-1 Are Required for Optimal Growth of MM3. MM3 cells were also tested for their response to purified BSF-1, recombinant IL-3, or a combination of the two factors. Fig. 3 shows that both are required for optimal stimulation of the MM3 cell line, as shown for MCGF2 activity (1, 11). Supernatant from induced MB2-1 cells, which contains IL-3 and MCGF2 activities (11), was able to induce a high level of proliferation without supplementation with IL-3. Monoclonal Anti-BSF-1 Antibody Inhibits MCGF2 and TCGF2 Activities. Inhibition by monoclonal antibodies specific for IL-2 or BSF-1 provided further evidence for the identity of the molecules responsible for BSF-1, MCGF2, and TCGF2 activities. In the HT2 assay, the anti-bsf-1 antibody inhibited the stimulation induced by MB2-1 supernatant (containing TCGF2 activity but not IL-2) or BSF-1 (Fig. 4 A log2 dilution FIG. 3. MM3 cells require IL-3 and BSF-1 for optimal response. BSF-1 (starting at 5 units/ml) was titrated on MM3 cells in the presence or absence of a constant amount of recombinant mouse IL-3 (2 units/ml, ref. 15). Titration curves of recombinant IL-3 and Con A-induced MB2-1 supernatant are also shown. -, BSF-1; MB2-1 supernatant;., recombinant IL-3; BSF-1 + constant IL-3. and B) but not by IL-2. In contrast, the anti-il-2 antibody inhibited the stimulation caused by recombinant IL-2 (Fig. 4C) but not by MB2-1 supernatant or purified BSF-1. The monoclonal antibodies were also tested for their ability to inhibit the growth of MM3 cells stimulated by T-cell supernatant containing MCGF2 and IL-3 or by a combination of purified BSF-1 and recombinant IL-3. Fig. 5 shows that the anti-bsf-1 antibody inhibited the increased response due to MCGF2 or BSF-1, whereas the anti-il-2 antibody had no effect. DISCUSSION The results described here provide strong evidence that BSF-1 is responsible for the activity in the supernatants of TH2 helper T cells that stimulates HT2 T cells in the calorimetric MTT assay. The evidence for the identity of TCGF2 and BSF-1 is particularly compelling since each of the two monoclonal antibodies inhibits only the stimulation due to the appropriate factor, using the same target cell line in both cases. This argues against possible direct effects of either monoclonal antibody on the target cell line. The TCGF2 activity neither inhibits nor enhances the stronger stimulation given by IL-2 (11). All other IL-2-dependent cell lines that we have examined also respond to saturating amounts of TCGF2 at a lower level than the response to IL-2 (T.R.M., unpublished data). The exact nature of the effect of BSF-1 on T cells is not yet understood. IL-2 and BSF-1 induce cell survival and thymidine incorporation (ref. 1 and unpublished results). However, the effects of IL-2 and BSF-1 are qualitatively different, since the slopes and saturation levels of the dose-response curves of these two lymphokines are noticeably different. The biologically relevant effect of BSF-1 on T cells may relate to differentiation or maintenance of viability, and the partial signal obtained in two "proliferation" assays might then be secondary to this effect. The MM3 as well as the MC/9 (19) mast cell lines require IL-3 and MCGF2 activities for high growth rate (1, 11). Purified BSF-1 can provide the MCGF2 activity, and monoclonal anti-bsf-1 inhibits MCGF2 activity in TH2 supernatants and also purified BSF-1. Unlike several other BSF-1

4 Immunology: Mosmann et al. Proc. Natl. Acad. Sci. USA 83 (1986) 5657 ( o c.) 1 \ Nl- LA 192 dilution dilution fl) ( C. C 192 dilution Constant IL-2.2- rl.) ( N- o.1 B. Constant BSF-1...,.5~ '\4 \\I\ dilution FIG. 4. TCGF2 activity is inhibited by monoclonal anti-bsf-1 antibody. S4B6 anti-il-2 and ilb11 anti-bsf-1 monoclonal antibodies were titrated on HT2 cells in the presence of constant amounts of T-cell supernatant (Sup.) containing TCGF2 activity (A), purified BSF-1 (1.2 units/ml) (B), or recombinant IL-2 (4 units/ml) (C). The starting dilution for both monoclonal antibody ascites fluids was 1:1. -, lib11 anti-bsf-1; , S4B6 anti-il-2. activities that are inhibited by IFN-y (8, 2), the enhanced growth of MM3 cells in response to BSF-1 is neither inhibited nor replaced by any other lymphokines that we have tested, including IFN-y, IL-2, IL-3, GM-CSF, and other lymphokines present in T helper clone supernatants (11). This provides a sensitive assay for BSF-1 that is not obscured even in the presence of supernatants from the subset of helper T cells that synthesize IL-2 and IFN-y (11). The experiments described above provide strong evidence that BSF-1 can mediate TCGF2 and MCGF2 activities. Since log2 dilution FIG. 5. MCGF2 activity is inhibited by monoclonal anti-bsf-1 antibody. S4B6 anti-il-2 and libli anti-bsf-1 monoclonal antibodies were titrated on MM3 cells in the presence of constant amounts of T-cell supernatant (Sup.) containing MCGF2 activity (A) or purified BSF-1 (1.2 units/ml) (B). All assay wells also contained 4 units of recombinant IL-3 per ml. The background due to IL-3 alone has been subtracted from all values. The starting dilution for both monoclonal antibody ascites fluids was 1:1. -, lib11 anti-bsf-1; , S4B6 anti-il-2. these studies were completed, we have isolated a recombinant cdna clone coding for mouse BSF-1 and confirmed that recombinant BSF-1 can mediate MCGF2 and TCGF2 activities (21). In addition, the antibody inhibition data described here suggest that BSF-1 is the major or only lymphokine responsible for these activities in activated TH2 T-cell supernatants, since the monoclonal anti-bsf-1 antibody is able to completely inhibit both activities in TH2 supernatants at the concentrations tested. Including the present information, BSF-1 appears to have several biological activities, including synergy in the induction of proliferation of B cells (2) and mast cell lines (this communication), early activation of resting B cells (3-6), activation of T-cell lines (this communication), and selective enhancement of IgG1 (7) and IgE (9) production by mitogenactivated B cells. These diverse activities could be mediated by action through different receptors. Alternatively, the same biochemical signal may produce different functional results in cells of distinct differentiated states. The availability of

5 5658 Immunology: Mosmann et al. purified recombinant BSF-1 and the identification of the receptor(s) will be required to resolve these and other questions. We thank S. Strober and D. Rennick for providing the HT2 and DA-1 cell lines, respectively, and we gratefully acknowledge the excellent technical assistance of H. Cherwinski, J. Tomasello, and J. Carty. 1. Paul, W. E. (1983) Immunol. Today 4, Howard, M., Farrar, J., Hilfiker, M., Johnson, B., Takatsu, K., Hamaoka, T. & Paul, W. E. (1982) J. Exp. Med. 155, Roehm, N. W., Liebson, H. J., Zlotnik, A., Kappler, J., Marrack, P. & Cambier, J. C. (1984) J. Exp. Med. 16, Noelle, R., Krammer, P. H., Ohara, J., Uhr, J. W. & Vitetta, E. S. (1984) Proc. Nati. Acad. Sci. USA 81, Oliver, K., Noelle, R. J., Uhr, J' W., Krammer, P. H. & Vitetta, E. S. (1985) Proc. Nati. Acad. Sci. USA 82, Rabin, E. M., Ohara, J. & Paul, W. E. (1985) Proc. Nati. Acad. Sci. USA 82, Vitetta, E. S., Ohara, J., Myers, C. D., Layton, J. E., Krammer, P. H. & Paul, W. E. (1985) J. Exp. Med. 162, Coffman, R. L. & Carty, J. (1986) J. Immunol. 136, Proc. Natl. Acad. Sci. USA 83 (1986) 9. Coffman, R. L., Ohara, J., Bond, M. W., Carty, J., Zlotnik, A. & Paul, W. E. (1986) J. Immunol. 136, Rennick, D. M. & Smith, C. A. (1986) Proc. Natl. Acad. Sci. USA 83, Mosmann, T. R., Cherwinski, H., Bond, M. W., Giedlin, M. A. & Coffman, R. L. (1986) J. Immunol., in press. 12. Watson, J. (1979) J. Exp. Med. 15, Giedlin, M. A., Longenecker, B. M. & Mosmann, T. R. (1986) Cell. Immunol. 97, Yokota, T., Arai, N., Lee, F., Rennick, D., Mosmann, T. & Arai, K. (1985) Proc. Natl. Acad. Sci. USA 82, Yokota, T., Lee, F., Rennick, D., Hall, C., Arai, N., Mosmann, T., Nabel, G., Cantor, H. & Arai, K. (1984) Proc. Natl. Acad. Sci. USA 81, Ohara, J. & Paul, W. E. (1985) Nature (London) 315, Mosmann, T. R. (1983) J. Immunol. Methods 65, Ohara, J., Lahet, S., Inman, J. & Paul, W. E. (1985) J. Immunol. 135, Nabel, G., Galli, S. J., Dvorak, A. M., Dvorak, H. F. & Cantor, H. (1981) Nature (London) 291, Mond, J. J., Finkelman, F. D., Sarma, C., Ohara, J. & Serrate, S. (1985) J. Immunol. 135, Lee, F., Yokota, T., Otsuka, T., Meyerson, P., Villaret, D., Coffman, R. L., Mosmann, T. R., Rennick, D. M., Roehm, N., Smith, C. A., Zlotnik, A. & Arai, K. (1986) Proc. Natl. Acad. Sci. USA 83,