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The effects of IL12 on B-cell subset function
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COMMENTS
Germann et al. are absolutely right in supposing that IL12 might also play a role in the effector phase of CHS. Recent work from our laboratory indicates that injection of an antibody directed against IL12 into already sensitized mice before performance of ear challenge significantly suppresses the specific ear swelling response. This suggests that IL12 may also be critically involved in the effector phase of CHS. Thus, as suggested by Germann et al., our observations are in accordance with reports demonstrating IL12 as a potent costimulatory factor for already differentiated Thl cells. Whether IL12 is expressed in the skin during application of the challenging dose and whether it has an effect on the migration of specific T cells has not yet been studied. We agree that the term antagonist implies that IL12 inhibits activities of IL10 rather than just suppressing the release. Thus, probably the term cuunterplayer would have been more appropriate. The findings by Morris et al., that in vivo application of IL1 2 leads to dramatic increases in IL10 production indeed was surprising for us. Momentarily, we have no convincing data that IL12 interferes with the effects of UV-irradiation by suppressing IL10 production. This was just a speculation. We
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BY SCHWARZ absolutely agree that bearing in mind the data by Morris et al., the possibility of a strictly local effect has to be considered. Moreover, it is not yet absolutely clear which cell population functions as the major source for IL10 during UV-induced immunosuppression. Rivas and Ulhich demonstrated keratinocytes as the major source for IL10 (Rivas and Ullrich 1992), while Kang et al. identified macrophages to be primarily responsible for the release of ILlO. The latter study, however, has been performed in the human system (Kang et al., 1994).
References Rivas, J.M. & S.E., Uhich (1992). Systemic suppression of delayed type hypersensitivity by supematants from UV-irradiated keratinocytes. An essential role for keratinocyte-derived IL-lo. J. Immunol. 149, 386.5 3871. Kang, K., Hammerberg, C., Meunier, L. & Cooper, K.D. (1994) CD1 lb+ macrophages that infiltrate human epidermis after in vivo ultraviolet exposure potently produce IL10 and represent the major secretory source of epidermal IL-10 protein. J. Immunol. 153, 5256-5264.
The effects of IL12 on B-cell subset function D. W. Metzger cl), L.A.
Vogel (l), V.H. Van Cleavec2), T.L. Lester(‘), and J.M. Buchanan(‘)
(I’ Departmen t of Microbiology, Medical College of Ohio, Toledo, OH 43699 (USA), and ‘2) Preclinical Research, Genetics Institute, Andover, MA 01810 (USA)
It has been established by many groups of investhat interleukin(IL)12 plays a pivotal role in controlling cell-mediated immunity. Accumulating evidence now indicates that IL12 also strongly regulates B-lymphocyte function. It has been generally believed that IL12 does not interact directly with B cells, but mediates it effects on antibody production by stimulating Thl cells to secrete IFNy. IFNy is tigators
Correspondence: Toledo,
OH 43699-0008.
Dennis W. Metzger,
Ph.D.,
Department
known to enhance production of the Thl-associated isotype, IgG2a, and to inhibit expression of Th2associated isotypes such as IgGl and IgE (Snapper and Paul, 1987; Finkelman et al., 1988). Thus, it is predicted that IL12 would have similar effects on humoral immunity. In this paper, we review our recent work which shows that IL12 leads to long-term enhancement of conventional antibody
of Microbiology,
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responses, particularly IgG2a responses. Conversely, IL12 suppresses the function of Bl cells, a subset of B cells which are reported to be critically involved in the development of autoimmunity and chronic lymphocytic leukaemia (CLL). Finally, we discuss recent results which suggest that an IL12 receptor is present on B 1 cells and activated conventional B cells, indicating that IL12 indeed has direct effects on the function of both of these B-cell subsets. Role of IL12 in conventional
antibody
responses
Several laboratories have investigated the role of IL12 in humoral immune responses and have shown that IL12 suppresses secretion of Th2-associated isotypes (IgGl and IgE) in both mice and humans, but has variable effects on production of Thlassociated isotypes (IgG2a). M&night et al. (1994) examined antibody responses to the TNP hapten in C57BL/6 mice and reported that IL12 injected during initial antigen priming strongly suppressed IgGl levels that were measured 8- 10 days later. However, the treatment only slightly enhanced IgG2a antibody levels. The authors attributed the lack of significant IgG2a enhancement to the fact that the mice produced large amounts of IgG2a anti-TNP antibody in the absence of IL12 and thus the cytokine could not further increase IgG2a levels. Morris et al. (1994) examined polyclonal responses that were induced by anti-IgD antibody and showed that IL12 inhibited both IgGl and IgG2a secretion. A recent paper by Germann et al. (1995) reported that IL12 suppressed murine IgE anti-collagen and anti-phospholipase A, antibody levels 30 days after immunization, but increased production of all other isotypes, including IgGl, IgG2a and IgG2b. In humans, Kiniwa et al. (1992) found that IL12 inhibited in vitro IgE secretion by IL4-stimulated B cells, but had no effect on levels of IgM, IgG or IgA. Jelinek and Brataan (1995), on the other hand, reported that human B cells cultured with IL12 and the activating agents Staphlococcus aureus and IL2, exhibited enhanced production of IgM, IgG and IgA. We have examined in detail the effects of in vivo IL12 treatment on murine anti-hen eggwhite lysozyme (HEL) antibody responses in BALB/c mice. HEL as a model antigen offers two major advantages for studying the influence of cytokines on humoral immunity. First, it is a small protein (MW 14,400 Da) which is extremely well-characterized at both the structural and immunological levels. Its sequence and three-dimensional structure are known, and naturally occurring variants with defined amino acid changes are available for epitope mapping studies. A second advantage of using HEL is the fact that the murine response to this antigen is highly restricted to only IgGl antibody production,
IN IMMUNOLOGY even during primary responses (Metzger et al., 1984; Buchanan et al. 1995). Thus, any effects of IL12 on induction of isotypes other than IgGl can be readily observed. Although the normal response of mice to HEL involves little if any expression of specific IgM or IgG2a, significant amounts of IgG2a anti-HEL antibody can be induced by certain experimental manipulations. For example, sorted Bl cells can respond to HEL when transferred with primed T cells into SCID mice, and this response involves both IgGl and IgG2a antibodies (Vogel et al., 1995). In addition, in vivo IL12 treatment induces significant levels of IgG2a anti-HEL antibody (Buchanan et al., 1995). These results indicate that there is no inherent defect in the ability of mice to mount Thl-like responses to HEL and thus, this antigenic system offers a unique opportunity to readily monitor the influence of IL12 on humoral immunity. The observed IgGl restriction of typical antiHEL responses suggests that the T cells involved in the normal response of mice belong to the Th2 subset, while there is little contribution by Thl cells, the Th population thought to be responsible for inducing IgG2a production. Thus, it might be expected that administration of IL12 during induction of an antiHEL response would activate antigen-specific Thl cells and have significant effects on the isotype distribution of the secreted antibodies. To test this prediction, BALB/c mice were injected intraperitoneally with 1.Op.g rIL12 on days -1, 0 and 1, and immunized with 100 pg HEL on day 0. Seven days after immunization, ILlZtreated mice demonstrated greatly elevated HEL-specific IgG2a antibody levels and suppressed IgGl levels, while PBS-treated control mice showed a typical IgGl-restricted response (Buchanan et al., 1995). These results were consistent with the Thl-Th2 model of IL12 action. However, by day 28, ILlZtreated mice showed heightened serum antibody levels of both isotypes, similar to the results of Germann et al. (1995) who also used protein antigens in their study. Delaying cytokine treatment until after the typical IgGl anti-HEL response had already been established also led to significant elevation of serum IgG2a antibody levels. Results essentially identical to these have been obtained by us in two other B-cell stimulation systems - BALB/c in vivo responses to phosphorylcholine (PC) hapten and in vitro responses following LPS activation. In the case of the anti-PC response, treatment with IL12 at the time of immunization leads not only to increases in IgGl and IgG2a antibodies two weeks postimmunization but to a 2-5fold enhancement in levels of specific IgG3 and IgA antibodies as well. Because of the dramatically different effects of IL12 observed at days 7 and 28 after immunization, we performed a kinetic study in which serum anti-
IMMJNOREGULATION bodies from immunized and IL12 treated mice were measured on days 4-9 after priming and then weekly thereafter. This experiment demonstrated that IgGl suppression induced by IL12 occurs only very early in the response and does not correlate with enhancement of IgG2a production. Enhanced production of IgG2a actually correlates with loss of IgGl suppression and after day 10, expression of both isotypes is increased in mice treated with IL12 and antigen. Thus, the dominant effect of IL12 on antibody responsiveness depends upon the time of observation after treatment, a finding that explains the conflicting results of others who reported primarily either isotype suppression or enhancement after IL12 administration. Early in the response, the action of IL12 on humoral immunity is selective and reflects Thl cell regulation. However, this pattern is only temporary, and then long-lasting increases in expression of all antibody isotypes are observed. Other experimental variables including mouse strain and nature of the activating agent also likely play important roles in influencing the results of IL12 administration. The importance of IFNy in mediating the effects of IL12 on anti-HEL antibody responses was examined by in vivo administration of neutralizing antiIFNy mAb to IL12-treated mice. Surprisingly, it was found that treatment with anti-IFNy antibody reversed IL12-induced suppression of IgGl antibody production that is observed early in the response, but had no effect on IgG2a enhancement. Others have similarly reported that modification of Ig secretion caused by IL12 is either not affected (McKnight et al., 1994; Vezzio ef al., 1993) or incompletely inhibited (Morris et al., 1994) by antiIFNy antibodies. It therefore appears that the effects of IL12 on humoral immunity are at least partially independent of IFNy. Taken together, our findings in the HEL system indicate that (1) IL12 reproducibly induces large amounts of specific IgG2a antibodies in vivo, (2) it can alter isotype profiles of both primary and secondary antibody responses, and (3) its effects on humoral immunity are not completely explained by induction of Thl cell-derived IFNy.
IL12 inhibits
Bl-cell
function
We have also examined the influence of IL12 on the subset of B cells known as Bl cells. Bl cells are thought to play an important role in the development of autoimmunity and CLL, the most common adult leukaemia in Western society (reviewed in Kipps, 1989). These cells can be distinguished from conventional (B2) cells by the presence of small amounts of the pan-T-cell marker CD5, in conjunction with the typical B-cell markers IgM,
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CD45R(B220), class II MHC and FcyR. However, they lack T-cell markers such as CD3, CD4 and CD8. Bl cells are also the B-cell population that constitutively expresses the receptor for IL5. In normal mice, Bl cells are found in large percentages early in ontogeny and then as the animal ages, their representation decreases as B2 cells infiltrate the various lymphoid organs. By adulthood, Bl cells are rare in spleen (about 2% of total B cells in BALB/c spleens), lymph nodes and peripheral blood. However, they remain present in large numbers in the peritoneal and pleural cavities where they exist as CD5 +‘- CD23 - MAC-l + cells. It is generally believed that B2 cells are the primary lymphocytes involved in responses against T-dependent exogenous antigens, but the normal function of Bl cells remains unresolved. Bl cells may be ligand-driven to secrete large amounts of IgM which reacts with low affinity to T-independent antigens, including self antigens. Bl cell numbers are significantly increased in human autoimmune conditions (Kipps, 1989; Lydyard et al., 1987) and in autoimmune strains of mice (Hayakawa et al., 1983). Although they are thought to contribute little to typical Tdependent antibody responses, Bl cells can clearly be influenced by T-cell-derived cytokines (Wetzel, 1989; Waldschmidt et al., 1991). Furthermore, we have found that under appropriate conditions, Bl cells can be induced to mount T-dependent IgG antibody responses (Vogel et al., 1995). Bl cells are also the primary source of B-cellderived ILlO, a cytokine that inhibits IL12-induced IFNy secretion (D’Andrea et al., 1993; Tripp et al., 1993). Treatment of mice with anti-IL10 mAb causes specific depletion of Bl cells, an effect apparently mediated by the increased levels of IFNy in anti-1110~treated mice (Ishida et aZ., 1992). Interestingly, it has been found that numbers of ILlOsecreting cells in mouse spleens are significantly increased by IL12 treatment (Morris et al., 1994). To examine the potential influence of IL12 on B 1 cells, we first investigated the idiotypic profile of anti-PC antibodies after immunization of BALB/c mice with PC coupled to KLH and treatment with IL12. Anti-PC responses in this mouse strain are dominated by antibodies that bear the T15 idiotype which are reported to be secreted solely by Bl cells (Masmoudi et al., 1990). Thus, measuring T15 idiotype expression during an anti-PC response provides a convenient method for monitoring B l-cell function. T15 idiotype levels in the sera of the treated animals were assessed by an inhibition ELISA in which dilutions of serum were tested for the ability to compete with TEPC 15 myeloma protein for binding to an anti-T15 idiotype mAb. Total anti-PC antibody levels on day 7 were comparable between the IL12- and PBS-treated groups, and were increased on day 17 in cytokine-treated mice as
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compared to control animals. Nonetheless, it was found that sera from mice immunized with PC and treated with IL12 contained undetectable levels of T15 idiotype, while immunized and vehicle-treated animals contained significant T15 idiotype activity. This result suggests that IL12 mediates specific suppression of Bl cell activity. Some studies have disputed the idea that B 1 cells are the sole source of T15 idiotype-bearing antibodies. Our further experiments therefore directly tested whether IL12 reduces in viva Bl-cell expression. In fact, we found by flow cytometric analyses that a single course of IL12 treatment caused a remarkable and nearly complete loss of B cells in the peritoneum, an organ rich in Bl cells. Small numbers of B cells only began to reappear in the peritoneal cavity 30 days post-IL12 treatment and were restored to normal numbers by day 45. Interestingly, both Bl (MAC-l+, CD45R(B220)+, IgMh@, CD23-) and B2 (MAC-l-, CD45R(B220)+, IgM’OW, CD23+) peritoneal cells were decreased by IL12 treatment. Upon reap earance of B cells on day 45, B2 cells (CD5-, IgM P“,) were present at frequencies similar to those observed in PBS-treated mice, but neither Bla cells (CD5+, IgM hish) nor Blb cells (CD5-, IgMhish) could be detected. This loss was reflected not only in percentages of B cells but also in absolute numbers of B cells recovered from the peritoneum. In contrast, exposure to IL12 had no detectable effect on levels of splenic B cells. Thus, intraperitoneal injection of IL12 leads to a localized depletion of B cells, with a more permanent effect on the Bl-cell subset. A preliminary report by Ham and colleagues (Palanivei et al., 1994) has similarly described reversal of Schisrosoma mansoni-induced B l-cell recruitment into the peritoneal cavities of mice after IL12 treatment. Interestingly, loss of peritoneal B cells was observed by us only upon simultaneous treatment with antigen, but not when IL12 was injected alone. This suggests that antigen challenge induces a signal which acts in concert with IL12 to deplete B cells at this site. Our findings may help explain the results of Honjo and colleagues (Murakami et al, 1992), who reported that antigen administered intraperitoneally depletes Bl cells bearing transgenic Ig receptors. These results were originally paradoxical because surface Ig ligation of B cells in vitro does not lead to cell death, but in fact causes increased production of Bl cells (Ying-zi et al., 1991). It is possible that antigen challenge in vivo provokes IL12 secretion by peritoneal macrophages which then changes the population dynamics of peritoneal B cells. IL12 was also found to suppress ILS-induced in vitro proliferation of Bl cells, but had no effect on cell survival compared to control cultures, demonstrating that the cytokine does not directly cause Bl cell death. Therefore, administration of IL12 may
IN IMMUNOLOGY cause in vivo changes in Bl cell function and disappearance from the peritoneum not by cell death but through increased trafficking to other organs. Chung et al. (1995) recently reported that injection of IL12 into mice induces a 50% increase in numbers of lymph node CD45R(B220)+ B cells two days after cytokine administration. However, the nature of these lymph node B cells (Bl versus B2) and their source has not yet been determined. An alternative explanation for the in viva disappearance of Bl cells is the possibility that IL12 stimulates accessory cells in the peritoneum to mediate B-cell-specific cytotoxicity. Such cytotoxic accessory cells would apparently not be stimulated in our in vitro conditions. Clearly, further work will be required to investigate the mechanism for the effects of IL12 on Bl cell function. Nevertheless, our results suggest that IL12 would be a useful treatment for inhibiting inappropriate Bl-cell activity such as is seen during autoimmune disease and CLL. Presence
of an IL12 receptor
on B cells
The receptor for IL12 is thus far poorly characterized, with only a low affinity human IL12 receptor subunit having been described. Desai et al. (1992) have found a human IL12 receptor on activated CD56+ NK cells and on activated T cells of both the CD4+ and CD8+ subsets using flow cytometry and direct IL12 binding analyses. Resting PBL and tonsillar cells, as well as activated tonsillar B cells, lacked detectable IL12 binding activity. Furthermore, several B-cell lines failed to demonstrate IL12 binding, as did most T-cell lines. Scatchard analysis identified a single binding site on PHA-activated T lymphoblasts with an equilibrium dissociation constant of 100 to 600 pmol/l (Chizzonite et al., 1992) although more recent analyses have demonstrated three binding sites with apparent affinities of 5 to 20 pmol/l, 50 to 200 pmol/l, and 2 to 6 nmol/l (Chua et al., 1994). Screening of a cDNA expression library with an antibody capable of precipitating complexes of IL12 and IL12 receptor from cell lysates identified a lOO-kDa component of the human IL12 receptor with an affinity for IL12 of 2 to 5 nM (Chua et al., 1994). This protein, termed the 81 subunit, is a member of the haemopoietin receptor family and shows homology to gp130. It appears that the 100~kDa protein represents the low affinity IL12 binding site on human lymphoblasts and that another, as of yet unidentified subunit(s), is required to generate high affinity IL12 receptors. An 8%kDa receptor-associated component was reported by Chizzonite et al. (1992) in an early paper, but this subunit has not been further characterized. The ~35 chain of bioactive IL12 has been found to have sequence similarity to IL6 and the p40 chain has similarity to the IL6 receptor (Merberg et al.,
IMMUNOREGULATION
1992). Thus, it has been hypothesized that IL12 is evolutionarily related to a primordial cytokine and representsa complex between this molecule and one chain of a probable multichain receptor. According to this model, there remains a strong enough attraction between the complex and remaining chains of the receptor to allow binding of IL12. Given the broad range of biological activity of IL12, it is likely that these putative remaining chains differ between different target cells. This could explain the observed heterogeneity in binding affinity as discussed above. Furthermore, the known receptor components cannot be detected on some cells that are clearly responsive to IL12, e.g., resting T cells and NK cells (Desai et al., 1992). Recently it was shown that both Thl and Th2 cells bind IL12 with similar affinities and express similar levels of mRNA for the cloned pl subunit of the IL12 receptor (Szabo et al., 1995). This result was surprising, since Thl cells but not Th2 cells are responsive to IL12; however, it was found that Th2 cells have a specific receptor-associated defect in intracellular signaling (Szabo et al., 1995). In addition, a preliminary report suggeststhat at least some human B-cell lines express the pl IL12 receptor subunit, but these cell lines are incapable of binding radiolabelled IL 12 (Wu et al., 1995). These various findings indicate the existence of multiple forms of the IL12 receptor. Published studies which have examined the potential binding of IL12 to human B cells were hampered by the fact that the B-cell stimulating agents failed to induce significant B-cell activation as assessedby cell proliferation. Furthermore, not all B-cell subsetswere examined. Jelinek and Brataan (1995) have now reported that IL12 in fact directly promotes the growth of activated human B cells. Because of this finding and the questionable role of IFNy in mediating the effects of IL12 on humoral immunity, we considered the possibility that IL12 interacts directly with a B-cell IL12 receptor. To detect an IL12 receptor on murine B cells, a threestep staining method was employed : incubation with IL12 followed by biotinylated rabbit anti-mouse IL12, and streptavidin conjugated to allophycocyanin. Flow cytometric analysis revealed saturable staining of fresh peritoneal B cells, but no staining of resting T cells or macrophages.Positively stained peritoneal cells included both Bl cells [CD45R(B220)+ CD23- MAC-l+] and B2 cells [CD45R(B220)+ CD23+ MAC- 1-l. Fresh splenic B and T cells failed to demonstrate positive staining, but spleencell blasts obtained after LPS stimulation did show expression of the IL12 receptor. All of the IL12R+ cells in this case were activated B cells as judged by staining for IgM, CD45R(B220) and the early activation marker CD69. Positive staining was ablated by omitting IL12 from the procedure, showing that it was not due to detection of endogenousB cell IL12. Staining was also lost by the use of biotin-
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ylated normal rabbit Ig instead of anti-IL12 or by the use of human IL12 instead of murine IL12, results that demonstrate the specificity of the reaction. Staining was not inhibited by murine IL6, which has been shown to have homology to IL12 (Merberg et al., 1992), nor by IL5. Recently, we found that human CD19+ PBL B cells stimulated with Staphylococcus aweus and IL2 also bind human IL12 as determined by a similar flow cytometric technique. Taken together, our studies and those of others have shown that IL12 significantly influences humoml immunity and that many of its effects on B-cell function are likely mediated directly rather than through the activity of Thl-associated cytokines. The provocative finding of enhanced B2-cellmediated humoral responsesto protein and hapten antigens in the face of suppressedBl cell activity suggests the involvement of membrane signalling mechanisms that may be distinct from those in T cells and which may vary between different B-cell subsets.
References Buchanan, J.M., Vogel, L.A., Van Cleave, V.H. & Metzger, D.W. (1995) Interleukin-12 alters the isotyperestricted antibody response of mice to hen eggwhite lysozyme. ht. Immunol., 7, 1519-1528. Chizzonite, R., Truitt, T., Desai, B.B., Nunes, P., Podlaski, F.J., Stem, A.S. & Gately, M.K. (1992), IL-12 receptor. -1. Characterization of the receptor on phytohemagglutinin-activated human lymphoblasts. J. ImmunoZ., 148, 3117-3124. Chua, A.O., Chizzonite, R., Desai, B.B., Truitt, T.P., Nunes, P., Minetti, L.J., Warrier, R.R., Presky, D.H., Levine, J.F., Gately, M.K. & Gubler, U. (1994), Expression cloning of a human IL-12 receptor component. A new member of the cytokine receptor superfamily with strong homology to gp130. J. Immunol., 153, 128-136. Chung, CL., Schopf, L.R., Goldman, S.J., Leonard, J.P. & Sypek, J.P. (1995), Interleukin-12 expands B220+ cell populations in mice infected with Leishmania major, in “9th Int. Congress Immunol.“, p. 894. D’Andrea, A., Aste-Amezaga, M., Valiante, N.M., Ma, X., Kubin, M. & Trinchieri, G. (1993), Interleukin-10 inhibits human lymphocyte IFN-y production by suppressing natural killer cell stimulatory factor/interleukin-12 synthesis in accessory cells. J. Exp. Med., 178, 1041-1048. Desai, B.B., Quinn, P.M., Wolitzky, A.G., Mongini, P.K.A., Chizzonite, R. & Gately, M.K. (1992) IL-12 receptor. II. Distribution and regulation of receptor expression. J. Immunol., 148, 3125-3132. Finkelman, F.D., Katona, I.M., Mosmann, T.R. & Coffman, R.L. (1988), II++ regulates the isotypes of Ig secreted during in vivo humoral immune responses. J. Immunol., 140, 1022-1027. Germann, T., Bongartz, M., Dlugonska, H., Hess, H., Schmitt, E., Kolbe, L., Kolsch, E., Podlaski, F.J., Gately, M.K. & Rude, E. (1995), Interleukin-12 pro-
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foundly upregulates the synthesis of antigen-specific complement-fiing IgG2a, IgG2b, and IgG3 antibody subclasses in vivo. Eur. .I. Immunol., 25, 823-829. Hayakawa, K., Hardy, R.R., Parks, D.R. & Herzenberg, L.A. (1983), The “Ly-1 B” cell subpopulation in normal, immunodefective, and autoimmune mice. J. Exp. Med., 157, 202-218. Ishida, H., Hastings, R., Keamey, I. & Howard, M. (1992) Continuous anti-interleukin 10 antibody administration depletes mice of Ly-1 B cells but not conventional B cells. J. fip. Med., 175, 1213-1220. Jelinek, D.F. & Braaten, J.K. (1995), Role of IL-12 in human B lymphocyte proliferation and differentiation. J. Immunol., 154, 1606-1613. Kiniwa, M., Gately, M., Gubler, U., Fargeas, C. & Delespesse, G. (1992), Recombinant interleukin-12 suppresses the synthesis of immunoglobulin E by interleukin-4 stimulated human lymphocytes. J. Clin. Invest., 90, 262-266. Kipps, T.J. (1989), The CD5 B cell. Adv. ImmunoZ., 47, 117-186. Lydyard, P.M., Youinou, P.Y. & Cooke, A. (1987), CD5positive B cells in rheumatoid arthritis and chronic lymphocytic leukemia. Immunol. Today, 8, 37-38. Masmoudi, H., Mota-Santos, T., Huetz, F., Coutinho, A. & Cazenave, P.A. (1990), All T15 Id-positive antibodies (but not the majority of VhTlS+ antibodies) are produced by peritoneal CD5+ B lymphocytes. Znt. Immunol., 2, 515-520. M&night, A.J., Zimmer, G.J., Fogelman, I., Wolf, SF. & Abbas, A.K. (1994). Effects of IL-12 on helper T cell-dependent immune responses in vivo. .I. Immunol., 152, 2172-2179. Merberg, D.M., Wolf, S.F. & Clark, S.C. (1992), Sequence similarity between NKSF and the IL-6/GCSF family. Immurwl. Today, 13,77-78. Metzger, D.W., Ch’ng, L.-K., Miller, A. & Sercarz, E.E. (1984). The expressed lysozyme-specific B-cell repertoire. I. Heterogeneity in the monoclonal anti-HEL specificity repertoire, and its difference from the in situ repertoire. Eur. J. Immunol., 14, 87-93. Morris, S.C., Madden, K.B., Adamovicz, J.J., Gause, W.C., Hubbard, B.R., Gately, M.K. & Finkelman, F.D. (1994), Effects of IL-12 on in vivo cytokine gene expression and Ig isotype selection. J. Immunol., 152, 1047-1056.
IN IMMUNOLOGY Murakami, M., Tsubata, T., Okamoto, M., Shimizu, A., Kumagai, S., Imura, H. & Honjo, T. (1992), Antigeninduced apoptotic death of Ly-1 B cells responsible for autoimmune disease in transgenic mice. Nature (Lond.), 357, 77-80. Palanivei, V., Ham, D.A. & Sypek, J. (1994), Recombinant murine IL- 12 inhibits ligand-specific recruitment of peritoneal B220+, CD5+, (B-l) cells. J. Immunol., 152, 3220. (Abstract) Snapper, C.M. & Paul, W.E. (1987), Interferon-y and B cell stimulatory factor-l reciprocally regulate Ig isotype production. Science, 236,944,947. Szabo, S.J., Jacobson, N.G., Dighe, A.S., Gubler, U. & Murphy, K.M. (1995), Developmental commitment to the Th2 lineage by extinction of IL-12 signaling. Immunity, 2,665-675. Tripp, C.S., Wolf, S.F. & Unanue, E.R. (1993), Interleukin 12 and tumor necrosis factor c1 are costimulators of interferon 7 production by natural killer cells in severe combined imrnunodeficiency mice with listeriosis, and interleukin 10 is a physiologic antagonist. Proc. Natl. Acad. Sci. USA, 90, 3725-3729. Vezzio, N., Gately, M., Kiniwa, M., Wu, C.Y. & Delespesse, G. (1993). IFN-y-independent suppression of IgE synthesis by IL-12. J. Immunol., 150,61A. Vogel, L.A., Sercarz, E.E. & Metzger, D.W. (1995), Antibody response of murine Bl cells to hen eggwhite lysozyme. Cell. Immunol., 161, 88-97. Waldschmidt, T.J., Kroese, F.G.M., Tygrett, L.T., Conrad, D.H. & Lynch, R.G. (1991), The expression of B cell surface receptors. III. The murine low-affinity IgE Fc receptor is not expressed on Ly 1 or “Ly l-like” B cells. Int. Immunol., 3, 305-315. Wetzel, G.D. (1989), Interleukin 5 regulation of peritoneal Ly-1 B lymphocyte proliferation, differentiation and autoantibody secretion. Eur. J. Immunol., 19, 17011707. Wu, C.Y., Wartier, R., Carvajal, D., Chua, A., Minetti, L., Mongini, P., Chizzonite, R., Presky, D., Gubler, U. & Gately, M. (1995). Biological function and distribution of human IL-12R B chain, in “9th Int. Congr. Immunol.“, p. 299. Ying-zi, C., Rabin, E. & Wortis, H.H. (1991). Treatment of murine CD5- B cells with anti-Ig, but not LPS, induces surface CD5: two B-cell activation pathways. Int, Immunol., 3,467-476.
COMMENTARY ONMETZGERETAL. By Presky et al.: We have also observed the presence of IL12R on murine B cells which, in our experiments, were activated by culture with SAC plus IL2. IL12 binding was detected by a flow cytometry method similar to that described by Metzger et al. It appears that this method detects primarily low affinity IL12 binding sites and thus does not necessarily imply the presence on activated B cells of high affinity receptors
comparable to those present on activated T and NK cells. We have observed upregulation of IL12RPl expression on human tonsillar and peripheral blood B cells activated by a variety of stimuli (Wu et a/., submitted). However, in our experiments, these activated human B cells did not bind or make a detectable biologic response to IL12. It is possible that because of technical differences between our experiments and those of Metzger et al. and of Jelinek and Braaten (19951, the B cells in our studies failed
IMMUNOREGULATION to achieve a comparable state of activation resulting in the observed absence of IL12 binding and responsiveness. Reference Jelinek, D.F. & Braaten, J.K. (19951, Role of IL-12 in human B lymphocyte proliferation and differentiation. .I. Immunol., 154, 1606-1613.
By Germann, Riide and Schmitt: It was suggested that IL12 might have some direct (IFNyindependent) effect on the upregulation of IgG2a synthesis because administration of anti-IFNy mAb’s in combination with IL12 failed to inhibit the increase in IgG2a in some in wivo systems. In order to investigate the involvement of IFN in the IL12-induced upregulation of antigen-specific IgG2a production, we have performed experiments using IFNy receptor knockout mice immunized with protein antigens. In the sera of such animals, there was almost no increase in antigen-specific lgG2a synthesis upon injections of IL12 (unpublished observation). This shows that the (strong) upregulation of antigen-specific IgG2a synthesis is largely due to lL12-induced IFNy and that the failure of some groups to inhibit the effect of IL12 on IgG2a synthesis by anti-IFNy mAbs (as discussed in this chapter) is probably due to incomplete neutralization of IFNy.
By Trinchieri and Showe: The role of IL12 in regulating, directly or indirectly, B-cell functions is of central importance for the in vivo immunoregulatory effects of IL12. The recent studies by the authors and others reviewed by Metzger et a/. are providing important information which will advance our understanding of such a role. In the studies of immunization with HEL or PC that Metzger et al. review, at 28 days after immunization, unlike at 7 days, an enhancement of all isotypes including IgA was observed. However,
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in a model of lung gene therapy using replication-defective adenovirus vectors, we (Yang et a/., 1995) observed that if the mice were treated with IL12 at the time of the infection, an almost complete suppression of neutralizing IgA antibodies in the bronchoalveolar lavage fluid was observed at day 35, with no significant effect on IgG antibodies. This effect of IL12 had important biological and therapeutic implications, because it completely suppressed the mucosal immunity against the viral vector, allowing a second infection with the same vector, impossible in animals not treated with IL12 in which the neutralizing IgA antibodies prevented the reinfection. These data suggest that not only the time after immunization, but possibly also the site of immunization and the nature of the antigen may modify the effect of IL12. Similarly to the data discussed by Metzger et a/., we detected expression of mRNA for the IL12RP, chain in all the lines of a very large panel of human B lymphoblastoid cell lines, whether EBV-positive or not (Benjamin et a/., submitted). Although the levels of accumulation of IL12Rj3, chain mRNA were for many cell lines comparable to those observed in activated T cells, the large majority of cell lines did not display detectable IL12 binding; one of the cell lines, however, bound 1251-lL12with both low and high affinity, similarly to what was observed in activated T cells. We also showed that the mRNA for the mouse IL12RP,.chain was expressed in the spleen of mice Injected with LPS LPS within 2 or 3 h and that B cells contributed at least in part to this expression. Because the structure and function of the IL12R have not been completely elucidated and these studies analysed only expression of one of the chains of the receptor, further investigation is required to fully understand the possible physiological role of IL12 receptor on B cells. Reference Yang, Y., Trinchieri, G. &Wilson, J.M. (1995), Recombinant IL-12 prevents formation of blocking IgA antibodies to recombinant adenovirus and allows repeated gene therapy to mouse lung. Nature Medicine, 1, 890-893.
FINAL
COMMENTS
Germann et al. are absolutely right in supposing that IL12 might also play a role in the effector phase of CHS. Recent work from our laboratory indicates that injection of an antibody directed against IL12 into already sensitized mice before performance of ear challenge significantly suppresses the specific ear swelling response. This suggests that IL12 may also be critically involved in the effector phase of CHS. Thus, as suggested by Germann et al., our observations are in accordance with reports demonstrating IL12 as a potent costimulatory factor for already differentiated Thl cells. Whether IL12 is expressed in the skin during application of the challenging dose and whether it has an effect on the migration of specific T cells has not yet been studied. We agree that the term antagonist implies that IL12 inhibits activities of IL10 rather than just suppressing the release. Thus, probably the term cuunterplayer would have been more appropriate. The findings by Morris et al., that in vivo application of IL1 2 leads to dramatic increases in IL10 production indeed was surprising for us. Momentarily, we have no convincing data that IL12 interferes with the effects of UV-irradiation by suppressing IL10 production. This was just a speculation. We
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BY SCHWARZ absolutely agree that bearing in mind the data by Morris et al., the possibility of a strictly local effect has to be considered. Moreover, it is not yet absolutely clear which cell population functions as the major source for IL10 during UV-induced immunosuppression. Rivas and Ulhich demonstrated keratinocytes as the major source for IL10 (Rivas and Ullrich 1992), while Kang et al. identified macrophages to be primarily responsible for the release of ILlO. The latter study, however, has been performed in the human system (Kang et al., 1994).
References Rivas, J.M. & S.E., Uhich (1992). Systemic suppression of delayed type hypersensitivity by supematants from UV-irradiated keratinocytes. An essential role for keratinocyte-derived IL-lo. J. Immunol. 149, 386.5 3871. Kang, K., Hammerberg, C., Meunier, L. & Cooper, K.D. (1994) CD1 lb+ macrophages that infiltrate human epidermis after in vivo ultraviolet exposure potently produce IL10 and represent the major secretory source of epidermal IL-10 protein. J. Immunol. 153, 5256-5264.
The effects of IL12 on B-cell subset function D. W. Metzger cl), L.A.
Vogel (l), V.H. Van Cleavec2), T.L. Lester(‘), and J.M. Buchanan(‘)
(I’ Departmen t of Microbiology, Medical College of Ohio, Toledo, OH 43699 (USA), and ‘2) Preclinical Research, Genetics Institute, Andover, MA 01810 (USA)
It has been established by many groups of investhat interleukin(IL)12 plays a pivotal role in controlling cell-mediated immunity. Accumulating evidence now indicates that IL12 also strongly regulates B-lymphocyte function. It has been generally believed that IL12 does not interact directly with B cells, but mediates it effects on antibody production by stimulating Thl cells to secrete IFNy. IFNy is tigators
Correspondence: Toledo,
OH 43699-0008.
Dennis W. Metzger,
Ph.D.,
Department
known to enhance production of the Thl-associated isotype, IgG2a, and to inhibit expression of Th2associated isotypes such as IgGl and IgE (Snapper and Paul, 1987; Finkelman et al., 1988). Thus, it is predicted that IL12 would have similar effects on humoral immunity. In this paper, we review our recent work which shows that IL12 leads to long-term enhancement of conventional antibody
of Microbiology,
Medical
College
of Ohio,
3000
Arlington
Avenue,
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responses, particularly IgG2a responses. Conversely, IL12 suppresses the function of Bl cells, a subset of B cells which are reported to be critically involved in the development of autoimmunity and chronic lymphocytic leukaemia (CLL). Finally, we discuss recent results which suggest that an IL12 receptor is present on B 1 cells and activated conventional B cells, indicating that IL12 indeed has direct effects on the function of both of these B-cell subsets. Role of IL12 in conventional
antibody
responses
Several laboratories have investigated the role of IL12 in humoral immune responses and have shown that IL12 suppresses secretion of Th2-associated isotypes (IgGl and IgE) in both mice and humans, but has variable effects on production of Thlassociated isotypes (IgG2a). M&night et al. (1994) examined antibody responses to the TNP hapten in C57BL/6 mice and reported that IL12 injected during initial antigen priming strongly suppressed IgGl levels that were measured 8- 10 days later. However, the treatment only slightly enhanced IgG2a antibody levels. The authors attributed the lack of significant IgG2a enhancement to the fact that the mice produced large amounts of IgG2a anti-TNP antibody in the absence of IL12 and thus the cytokine could not further increase IgG2a levels. Morris et al. (1994) examined polyclonal responses that were induced by anti-IgD antibody and showed that IL12 inhibited both IgGl and IgG2a secretion. A recent paper by Germann et al. (1995) reported that IL12 suppressed murine IgE anti-collagen and anti-phospholipase A, antibody levels 30 days after immunization, but increased production of all other isotypes, including IgGl, IgG2a and IgG2b. In humans, Kiniwa et al. (1992) found that IL12 inhibited in vitro IgE secretion by IL4-stimulated B cells, but had no effect on levels of IgM, IgG or IgA. Jelinek and Brataan (1995), on the other hand, reported that human B cells cultured with IL12 and the activating agents Staphlococcus aureus and IL2, exhibited enhanced production of IgM, IgG and IgA. We have examined in detail the effects of in vivo IL12 treatment on murine anti-hen eggwhite lysozyme (HEL) antibody responses in BALB/c mice. HEL as a model antigen offers two major advantages for studying the influence of cytokines on humoral immunity. First, it is a small protein (MW 14,400 Da) which is extremely well-characterized at both the structural and immunological levels. Its sequence and three-dimensional structure are known, and naturally occurring variants with defined amino acid changes are available for epitope mapping studies. A second advantage of using HEL is the fact that the murine response to this antigen is highly restricted to only IgGl antibody production,
IN IMMUNOLOGY even during primary responses (Metzger et al., 1984; Buchanan et al. 1995). Thus, any effects of IL12 on induction of isotypes other than IgGl can be readily observed. Although the normal response of mice to HEL involves little if any expression of specific IgM or IgG2a, significant amounts of IgG2a anti-HEL antibody can be induced by certain experimental manipulations. For example, sorted Bl cells can respond to HEL when transferred with primed T cells into SCID mice, and this response involves both IgGl and IgG2a antibodies (Vogel et al., 1995). In addition, in vivo IL12 treatment induces significant levels of IgG2a anti-HEL antibody (Buchanan et al., 1995). These results indicate that there is no inherent defect in the ability of mice to mount Thl-like responses to HEL and thus, this antigenic system offers a unique opportunity to readily monitor the influence of IL12 on humoral immunity. The observed IgGl restriction of typical antiHEL responses suggests that the T cells involved in the normal response of mice belong to the Th2 subset, while there is little contribution by Thl cells, the Th population thought to be responsible for inducing IgG2a production. Thus, it might be expected that administration of IL12 during induction of an antiHEL response would activate antigen-specific Thl cells and have significant effects on the isotype distribution of the secreted antibodies. To test this prediction, BALB/c mice were injected intraperitoneally with 1.Op.g rIL12 on days -1, 0 and 1, and immunized with 100 pg HEL on day 0. Seven days after immunization, ILlZtreated mice demonstrated greatly elevated HEL-specific IgG2a antibody levels and suppressed IgGl levels, while PBS-treated control mice showed a typical IgGl-restricted response (Buchanan et al., 1995). These results were consistent with the Thl-Th2 model of IL12 action. However, by day 28, ILlZtreated mice showed heightened serum antibody levels of both isotypes, similar to the results of Germann et al. (1995) who also used protein antigens in their study. Delaying cytokine treatment until after the typical IgGl anti-HEL response had already been established also led to significant elevation of serum IgG2a antibody levels. Results essentially identical to these have been obtained by us in two other B-cell stimulation systems - BALB/c in vivo responses to phosphorylcholine (PC) hapten and in vitro responses following LPS activation. In the case of the anti-PC response, treatment with IL12 at the time of immunization leads not only to increases in IgGl and IgG2a antibodies two weeks postimmunization but to a 2-5fold enhancement in levels of specific IgG3 and IgA antibodies as well. Because of the dramatically different effects of IL12 observed at days 7 and 28 after immunization, we performed a kinetic study in which serum anti-
IMMJNOREGULATION bodies from immunized and IL12 treated mice were measured on days 4-9 after priming and then weekly thereafter. This experiment demonstrated that IgGl suppression induced by IL12 occurs only very early in the response and does not correlate with enhancement of IgG2a production. Enhanced production of IgG2a actually correlates with loss of IgGl suppression and after day 10, expression of both isotypes is increased in mice treated with IL12 and antigen. Thus, the dominant effect of IL12 on antibody responsiveness depends upon the time of observation after treatment, a finding that explains the conflicting results of others who reported primarily either isotype suppression or enhancement after IL12 administration. Early in the response, the action of IL12 on humoral immunity is selective and reflects Thl cell regulation. However, this pattern is only temporary, and then long-lasting increases in expression of all antibody isotypes are observed. Other experimental variables including mouse strain and nature of the activating agent also likely play important roles in influencing the results of IL12 administration. The importance of IFNy in mediating the effects of IL12 on anti-HEL antibody responses was examined by in vivo administration of neutralizing antiIFNy mAb to IL12-treated mice. Surprisingly, it was found that treatment with anti-IFNy antibody reversed IL12-induced suppression of IgGl antibody production that is observed early in the response, but had no effect on IgG2a enhancement. Others have similarly reported that modification of Ig secretion caused by IL12 is either not affected (McKnight et al., 1994; Vezzio ef al., 1993) or incompletely inhibited (Morris et al., 1994) by antiIFNy antibodies. It therefore appears that the effects of IL12 on humoral immunity are at least partially independent of IFNy. Taken together, our findings in the HEL system indicate that (1) IL12 reproducibly induces large amounts of specific IgG2a antibodies in vivo, (2) it can alter isotype profiles of both primary and secondary antibody responses, and (3) its effects on humoral immunity are not completely explained by induction of Thl cell-derived IFNy.
IL12 inhibits
Bl-cell
function
We have also examined the influence of IL12 on the subset of B cells known as Bl cells. Bl cells are thought to play an important role in the development of autoimmunity and CLL, the most common adult leukaemia in Western society (reviewed in Kipps, 1989). These cells can be distinguished from conventional (B2) cells by the presence of small amounts of the pan-T-cell marker CD5, in conjunction with the typical B-cell markers IgM,
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CD45R(B220), class II MHC and FcyR. However, they lack T-cell markers such as CD3, CD4 and CD8. Bl cells are also the B-cell population that constitutively expresses the receptor for IL5. In normal mice, Bl cells are found in large percentages early in ontogeny and then as the animal ages, their representation decreases as B2 cells infiltrate the various lymphoid organs. By adulthood, Bl cells are rare in spleen (about 2% of total B cells in BALB/c spleens), lymph nodes and peripheral blood. However, they remain present in large numbers in the peritoneal and pleural cavities where they exist as CD5 +‘- CD23 - MAC-l + cells. It is generally believed that B2 cells are the primary lymphocytes involved in responses against T-dependent exogenous antigens, but the normal function of Bl cells remains unresolved. Bl cells may be ligand-driven to secrete large amounts of IgM which reacts with low affinity to T-independent antigens, including self antigens. Bl cell numbers are significantly increased in human autoimmune conditions (Kipps, 1989; Lydyard et al., 1987) and in autoimmune strains of mice (Hayakawa et al., 1983). Although they are thought to contribute little to typical Tdependent antibody responses, Bl cells can clearly be influenced by T-cell-derived cytokines (Wetzel, 1989; Waldschmidt et al., 1991). Furthermore, we have found that under appropriate conditions, Bl cells can be induced to mount T-dependent IgG antibody responses (Vogel et al., 1995). Bl cells are also the primary source of B-cellderived ILlO, a cytokine that inhibits IL12-induced IFNy secretion (D’Andrea et al., 1993; Tripp et al., 1993). Treatment of mice with anti-IL10 mAb causes specific depletion of Bl cells, an effect apparently mediated by the increased levels of IFNy in anti-1110~treated mice (Ishida et aZ., 1992). Interestingly, it has been found that numbers of ILlOsecreting cells in mouse spleens are significantly increased by IL12 treatment (Morris et al., 1994). To examine the potential influence of IL12 on B 1 cells, we first investigated the idiotypic profile of anti-PC antibodies after immunization of BALB/c mice with PC coupled to KLH and treatment with IL12. Anti-PC responses in this mouse strain are dominated by antibodies that bear the T15 idiotype which are reported to be secreted solely by Bl cells (Masmoudi et al., 1990). Thus, measuring T15 idiotype expression during an anti-PC response provides a convenient method for monitoring B l-cell function. T15 idiotype levels in the sera of the treated animals were assessed by an inhibition ELISA in which dilutions of serum were tested for the ability to compete with TEPC 15 myeloma protein for binding to an anti-T15 idiotype mAb. Total anti-PC antibody levels on day 7 were comparable between the IL12- and PBS-treated groups, and were increased on day 17 in cytokine-treated mice as
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compared to control animals. Nonetheless, it was found that sera from mice immunized with PC and treated with IL12 contained undetectable levels of T15 idiotype, while immunized and vehicle-treated animals contained significant T15 idiotype activity. This result suggests that IL12 mediates specific suppression of Bl cell activity. Some studies have disputed the idea that B 1 cells are the sole source of T15 idiotype-bearing antibodies. Our further experiments therefore directly tested whether IL12 reduces in viva Bl-cell expression. In fact, we found by flow cytometric analyses that a single course of IL12 treatment caused a remarkable and nearly complete loss of B cells in the peritoneum, an organ rich in Bl cells. Small numbers of B cells only began to reappear in the peritoneal cavity 30 days post-IL12 treatment and were restored to normal numbers by day 45. Interestingly, both Bl (MAC-l+, CD45R(B220)+, IgMh@, CD23-) and B2 (MAC-l-, CD45R(B220)+, IgM’OW, CD23+) peritoneal cells were decreased by IL12 treatment. Upon reap earance of B cells on day 45, B2 cells (CD5-, IgM P“,) were present at frequencies similar to those observed in PBS-treated mice, but neither Bla cells (CD5+, IgM hish) nor Blb cells (CD5-, IgMhish) could be detected. This loss was reflected not only in percentages of B cells but also in absolute numbers of B cells recovered from the peritoneum. In contrast, exposure to IL12 had no detectable effect on levels of splenic B cells. Thus, intraperitoneal injection of IL12 leads to a localized depletion of B cells, with a more permanent effect on the Bl-cell subset. A preliminary report by Ham and colleagues (Palanivei et al., 1994) has similarly described reversal of Schisrosoma mansoni-induced B l-cell recruitment into the peritoneal cavities of mice after IL12 treatment. Interestingly, loss of peritoneal B cells was observed by us only upon simultaneous treatment with antigen, but not when IL12 was injected alone. This suggests that antigen challenge induces a signal which acts in concert with IL12 to deplete B cells at this site. Our findings may help explain the results of Honjo and colleagues (Murakami et al, 1992), who reported that antigen administered intraperitoneally depletes Bl cells bearing transgenic Ig receptors. These results were originally paradoxical because surface Ig ligation of B cells in vitro does not lead to cell death, but in fact causes increased production of Bl cells (Ying-zi et al., 1991). It is possible that antigen challenge in vivo provokes IL12 secretion by peritoneal macrophages which then changes the population dynamics of peritoneal B cells. IL12 was also found to suppress ILS-induced in vitro proliferation of Bl cells, but had no effect on cell survival compared to control cultures, demonstrating that the cytokine does not directly cause Bl cell death. Therefore, administration of IL12 may
IN IMMUNOLOGY cause in vivo changes in Bl cell function and disappearance from the peritoneum not by cell death but through increased trafficking to other organs. Chung et al. (1995) recently reported that injection of IL12 into mice induces a 50% increase in numbers of lymph node CD45R(B220)+ B cells two days after cytokine administration. However, the nature of these lymph node B cells (Bl versus B2) and their source has not yet been determined. An alternative explanation for the in viva disappearance of Bl cells is the possibility that IL12 stimulates accessory cells in the peritoneum to mediate B-cell-specific cytotoxicity. Such cytotoxic accessory cells would apparently not be stimulated in our in vitro conditions. Clearly, further work will be required to investigate the mechanism for the effects of IL12 on Bl cell function. Nevertheless, our results suggest that IL12 would be a useful treatment for inhibiting inappropriate Bl-cell activity such as is seen during autoimmune disease and CLL. Presence
of an IL12 receptor
on B cells
The receptor for IL12 is thus far poorly characterized, with only a low affinity human IL12 receptor subunit having been described. Desai et al. (1992) have found a human IL12 receptor on activated CD56+ NK cells and on activated T cells of both the CD4+ and CD8+ subsets using flow cytometry and direct IL12 binding analyses. Resting PBL and tonsillar cells, as well as activated tonsillar B cells, lacked detectable IL12 binding activity. Furthermore, several B-cell lines failed to demonstrate IL12 binding, as did most T-cell lines. Scatchard analysis identified a single binding site on PHA-activated T lymphoblasts with an equilibrium dissociation constant of 100 to 600 pmol/l (Chizzonite et al., 1992) although more recent analyses have demonstrated three binding sites with apparent affinities of 5 to 20 pmol/l, 50 to 200 pmol/l, and 2 to 6 nmol/l (Chua et al., 1994). Screening of a cDNA expression library with an antibody capable of precipitating complexes of IL12 and IL12 receptor from cell lysates identified a lOO-kDa component of the human IL12 receptor with an affinity for IL12 of 2 to 5 nM (Chua et al., 1994). This protein, termed the 81 subunit, is a member of the haemopoietin receptor family and shows homology to gp130. It appears that the 100~kDa protein represents the low affinity IL12 binding site on human lymphoblasts and that another, as of yet unidentified subunit(s), is required to generate high affinity IL12 receptors. An 8%kDa receptor-associated component was reported by Chizzonite et al. (1992) in an early paper, but this subunit has not been further characterized. The ~35 chain of bioactive IL12 has been found to have sequence similarity to IL6 and the p40 chain has similarity to the IL6 receptor (Merberg et al.,
IMMUNOREGULATION
1992). Thus, it has been hypothesized that IL12 is evolutionarily related to a primordial cytokine and representsa complex between this molecule and one chain of a probable multichain receptor. According to this model, there remains a strong enough attraction between the complex and remaining chains of the receptor to allow binding of IL12. Given the broad range of biological activity of IL12, it is likely that these putative remaining chains differ between different target cells. This could explain the observed heterogeneity in binding affinity as discussed above. Furthermore, the known receptor components cannot be detected on some cells that are clearly responsive to IL12, e.g., resting T cells and NK cells (Desai et al., 1992). Recently it was shown that both Thl and Th2 cells bind IL12 with similar affinities and express similar levels of mRNA for the cloned pl subunit of the IL12 receptor (Szabo et al., 1995). This result was surprising, since Thl cells but not Th2 cells are responsive to IL12; however, it was found that Th2 cells have a specific receptor-associated defect in intracellular signaling (Szabo et al., 1995). In addition, a preliminary report suggeststhat at least some human B-cell lines express the pl IL12 receptor subunit, but these cell lines are incapable of binding radiolabelled IL 12 (Wu et al., 1995). These various findings indicate the existence of multiple forms of the IL12 receptor. Published studies which have examined the potential binding of IL12 to human B cells were hampered by the fact that the B-cell stimulating agents failed to induce significant B-cell activation as assessedby cell proliferation. Furthermore, not all B-cell subsetswere examined. Jelinek and Brataan (1995) have now reported that IL12 in fact directly promotes the growth of activated human B cells. Because of this finding and the questionable role of IFNy in mediating the effects of IL12 on humoral immunity, we considered the possibility that IL12 interacts directly with a B-cell IL12 receptor. To detect an IL12 receptor on murine B cells, a threestep staining method was employed : incubation with IL12 followed by biotinylated rabbit anti-mouse IL12, and streptavidin conjugated to allophycocyanin. Flow cytometric analysis revealed saturable staining of fresh peritoneal B cells, but no staining of resting T cells or macrophages.Positively stained peritoneal cells included both Bl cells [CD45R(B220)+ CD23- MAC-l+] and B2 cells [CD45R(B220)+ CD23+ MAC- 1-l. Fresh splenic B and T cells failed to demonstrate positive staining, but spleencell blasts obtained after LPS stimulation did show expression of the IL12 receptor. All of the IL12R+ cells in this case were activated B cells as judged by staining for IgM, CD45R(B220) and the early activation marker CD69. Positive staining was ablated by omitting IL12 from the procedure, showing that it was not due to detection of endogenousB cell IL12. Staining was also lost by the use of biotin-
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ylated normal rabbit Ig instead of anti-IL12 or by the use of human IL12 instead of murine IL12, results that demonstrate the specificity of the reaction. Staining was not inhibited by murine IL6, which has been shown to have homology to IL12 (Merberg et al., 1992), nor by IL5. Recently, we found that human CD19+ PBL B cells stimulated with Staphylococcus aweus and IL2 also bind human IL12 as determined by a similar flow cytometric technique. Taken together, our studies and those of others have shown that IL12 significantly influences humoml immunity and that many of its effects on B-cell function are likely mediated directly rather than through the activity of Thl-associated cytokines. The provocative finding of enhanced B2-cellmediated humoral responsesto protein and hapten antigens in the face of suppressedBl cell activity suggests the involvement of membrane signalling mechanisms that may be distinct from those in T cells and which may vary between different B-cell subsets.
References Buchanan, J.M., Vogel, L.A., Van Cleave, V.H. & Metzger, D.W. (1995) Interleukin-12 alters the isotyperestricted antibody response of mice to hen eggwhite lysozyme. ht. Immunol., 7, 1519-1528. Chizzonite, R., Truitt, T., Desai, B.B., Nunes, P., Podlaski, F.J., Stem, A.S. & Gately, M.K. (1992), IL-12 receptor. -1. Characterization of the receptor on phytohemagglutinin-activated human lymphoblasts. J. ImmunoZ., 148, 3117-3124. Chua, A.O., Chizzonite, R., Desai, B.B., Truitt, T.P., Nunes, P., Minetti, L.J., Warrier, R.R., Presky, D.H., Levine, J.F., Gately, M.K. & Gubler, U. (1994), Expression cloning of a human IL-12 receptor component. A new member of the cytokine receptor superfamily with strong homology to gp130. J. Immunol., 153, 128-136. Chung, CL., Schopf, L.R., Goldman, S.J., Leonard, J.P. & Sypek, J.P. (1995), Interleukin-12 expands B220+ cell populations in mice infected with Leishmania major, in “9th Int. Congress Immunol.“, p. 894. D’Andrea, A., Aste-Amezaga, M., Valiante, N.M., Ma, X., Kubin, M. & Trinchieri, G. (1993), Interleukin-10 inhibits human lymphocyte IFN-y production by suppressing natural killer cell stimulatory factor/interleukin-12 synthesis in accessory cells. J. Exp. Med., 178, 1041-1048. Desai, B.B., Quinn, P.M., Wolitzky, A.G., Mongini, P.K.A., Chizzonite, R. & Gately, M.K. (1992) IL-12 receptor. II. Distribution and regulation of receptor expression. J. Immunol., 148, 3125-3132. Finkelman, F.D., Katona, I.M., Mosmann, T.R. & Coffman, R.L. (1988), II++ regulates the isotypes of Ig secreted during in vivo humoral immune responses. J. Immunol., 140, 1022-1027. Germann, T., Bongartz, M., Dlugonska, H., Hess, H., Schmitt, E., Kolbe, L., Kolsch, E., Podlaski, F.J., Gately, M.K. & Rude, E. (1995), Interleukin-12 pro-
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foundly upregulates the synthesis of antigen-specific complement-fiing IgG2a, IgG2b, and IgG3 antibody subclasses in vivo. Eur. .I. Immunol., 25, 823-829. Hayakawa, K., Hardy, R.R., Parks, D.R. & Herzenberg, L.A. (1983), The “Ly-1 B” cell subpopulation in normal, immunodefective, and autoimmune mice. J. Exp. Med., 157, 202-218. Ishida, H., Hastings, R., Keamey, I. & Howard, M. (1992) Continuous anti-interleukin 10 antibody administration depletes mice of Ly-1 B cells but not conventional B cells. J. fip. Med., 175, 1213-1220. Jelinek, D.F. & Braaten, J.K. (1995), Role of IL-12 in human B lymphocyte proliferation and differentiation. J. Immunol., 154, 1606-1613. Kiniwa, M., Gately, M., Gubler, U., Fargeas, C. & Delespesse, G. (1992), Recombinant interleukin-12 suppresses the synthesis of immunoglobulin E by interleukin-4 stimulated human lymphocytes. J. Clin. Invest., 90, 262-266. Kipps, T.J. (1989), The CD5 B cell. Adv. ImmunoZ., 47, 117-186. Lydyard, P.M., Youinou, P.Y. & Cooke, A. (1987), CD5positive B cells in rheumatoid arthritis and chronic lymphocytic leukemia. Immunol. Today, 8, 37-38. Masmoudi, H., Mota-Santos, T., Huetz, F., Coutinho, A. & Cazenave, P.A. (1990), All T15 Id-positive antibodies (but not the majority of VhTlS+ antibodies) are produced by peritoneal CD5+ B lymphocytes. Znt. Immunol., 2, 515-520. M&night, A.J., Zimmer, G.J., Fogelman, I., Wolf, SF. & Abbas, A.K. (1994). Effects of IL-12 on helper T cell-dependent immune responses in vivo. .I. Immunol., 152, 2172-2179. Merberg, D.M., Wolf, S.F. & Clark, S.C. (1992), Sequence similarity between NKSF and the IL-6/GCSF family. Immurwl. Today, 13,77-78. Metzger, D.W., Ch’ng, L.-K., Miller, A. & Sercarz, E.E. (1984). The expressed lysozyme-specific B-cell repertoire. I. Heterogeneity in the monoclonal anti-HEL specificity repertoire, and its difference from the in situ repertoire. Eur. J. Immunol., 14, 87-93. Morris, S.C., Madden, K.B., Adamovicz, J.J., Gause, W.C., Hubbard, B.R., Gately, M.K. & Finkelman, F.D. (1994), Effects of IL-12 on in vivo cytokine gene expression and Ig isotype selection. J. Immunol., 152, 1047-1056.
IN IMMUNOLOGY Murakami, M., Tsubata, T., Okamoto, M., Shimizu, A., Kumagai, S., Imura, H. & Honjo, T. (1992), Antigeninduced apoptotic death of Ly-1 B cells responsible for autoimmune disease in transgenic mice. Nature (Lond.), 357, 77-80. Palanivei, V., Ham, D.A. & Sypek, J. (1994), Recombinant murine IL- 12 inhibits ligand-specific recruitment of peritoneal B220+, CD5+, (B-l) cells. J. Immunol., 152, 3220. (Abstract) Snapper, C.M. & Paul, W.E. (1987), Interferon-y and B cell stimulatory factor-l reciprocally regulate Ig isotype production. Science, 236,944,947. Szabo, S.J., Jacobson, N.G., Dighe, A.S., Gubler, U. & Murphy, K.M. (1995), Developmental commitment to the Th2 lineage by extinction of IL-12 signaling. Immunity, 2,665-675. Tripp, C.S., Wolf, S.F. & Unanue, E.R. (1993), Interleukin 12 and tumor necrosis factor c1 are costimulators of interferon 7 production by natural killer cells in severe combined imrnunodeficiency mice with listeriosis, and interleukin 10 is a physiologic antagonist. Proc. Natl. Acad. Sci. USA, 90, 3725-3729. Vezzio, N., Gately, M., Kiniwa, M., Wu, C.Y. & Delespesse, G. (1993). IFN-y-independent suppression of IgE synthesis by IL-12. J. Immunol., 150,61A. Vogel, L.A., Sercarz, E.E. & Metzger, D.W. (1995), Antibody response of murine Bl cells to hen eggwhite lysozyme. Cell. Immunol., 161, 88-97. Waldschmidt, T.J., Kroese, F.G.M., Tygrett, L.T., Conrad, D.H. & Lynch, R.G. (1991), The expression of B cell surface receptors. III. The murine low-affinity IgE Fc receptor is not expressed on Ly 1 or “Ly l-like” B cells. Int. Immunol., 3, 305-315. Wetzel, G.D. (1989), Interleukin 5 regulation of peritoneal Ly-1 B lymphocyte proliferation, differentiation and autoantibody secretion. Eur. J. Immunol., 19, 17011707. Wu, C.Y., Wartier, R., Carvajal, D., Chua, A., Minetti, L., Mongini, P., Chizzonite, R., Presky, D., Gubler, U. & Gately, M. (1995). Biological function and distribution of human IL-12R B chain, in “9th Int. Congr. Immunol.“, p. 299. Ying-zi, C., Rabin, E. & Wortis, H.H. (1991). Treatment of murine CD5- B cells with anti-Ig, but not LPS, induces surface CD5: two B-cell activation pathways. Int, Immunol., 3,467-476.
COMMENTARY ONMETZGERETAL. By Presky et al.: We have also observed the presence of IL12R on murine B cells which, in our experiments, were activated by culture with SAC plus IL2. IL12 binding was detected by a flow cytometry method similar to that described by Metzger et al. It appears that this method detects primarily low affinity IL12 binding sites and thus does not necessarily imply the presence on activated B cells of high affinity receptors
comparable to those present on activated T and NK cells. We have observed upregulation of IL12RPl expression on human tonsillar and peripheral blood B cells activated by a variety of stimuli (Wu et a/., submitted). However, in our experiments, these activated human B cells did not bind or make a detectable biologic response to IL12. It is possible that because of technical differences between our experiments and those of Metzger et al. and of Jelinek and Braaten (19951, the B cells in our studies failed
IMMUNOREGULATION to achieve a comparable state of activation resulting in the observed absence of IL12 binding and responsiveness. Reference Jelinek, D.F. & Braaten, J.K. (19951, Role of IL-12 in human B lymphocyte proliferation and differentiation. .I. Immunol., 154, 1606-1613.
By Germann, Riide and Schmitt: It was suggested that IL12 might have some direct (IFNyindependent) effect on the upregulation of IgG2a synthesis because administration of anti-IFNy mAb’s in combination with IL12 failed to inhibit the increase in IgG2a in some in wivo systems. In order to investigate the involvement of IFN in the IL12-induced upregulation of antigen-specific IgG2a production, we have performed experiments using IFNy receptor knockout mice immunized with protein antigens. In the sera of such animals, there was almost no increase in antigen-specific lgG2a synthesis upon injections of IL12 (unpublished observation). This shows that the (strong) upregulation of antigen-specific IgG2a synthesis is largely due to lL12-induced IFNy and that the failure of some groups to inhibit the effect of IL12 on IgG2a synthesis by anti-IFNy mAbs (as discussed in this chapter) is probably due to incomplete neutralization of IFNy.
By Trinchieri and Showe: The role of IL12 in regulating, directly or indirectly, B-cell functions is of central importance for the in vivo immunoregulatory effects of IL12. The recent studies by the authors and others reviewed by Metzger et a/. are providing important information which will advance our understanding of such a role. In the studies of immunization with HEL or PC that Metzger et al. review, at 28 days after immunization, unlike at 7 days, an enhancement of all isotypes including IgA was observed. However,
BY INTERLEUKIN-12
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in a model of lung gene therapy using replication-defective adenovirus vectors, we (Yang et a/., 1995) observed that if the mice were treated with IL12 at the time of the infection, an almost complete suppression of neutralizing IgA antibodies in the bronchoalveolar lavage fluid was observed at day 35, with no significant effect on IgG antibodies. This effect of IL12 had important biological and therapeutic implications, because it completely suppressed the mucosal immunity against the viral vector, allowing a second infection with the same vector, impossible in animals not treated with IL12 in which the neutralizing IgA antibodies prevented the reinfection. These data suggest that not only the time after immunization, but possibly also the site of immunization and the nature of the antigen may modify the effect of IL12. Similarly to the data discussed by Metzger et a/., we detected expression of mRNA for the IL12RP, chain in all the lines of a very large panel of human B lymphoblastoid cell lines, whether EBV-positive or not (Benjamin et a/., submitted). Although the levels of accumulation of IL12Rj3, chain mRNA were for many cell lines comparable to those observed in activated T cells, the large majority of cell lines did not display detectable IL12 binding; one of the cell lines, however, bound 1251-lL12with both low and high affinity, similarly to what was observed in activated T cells. We also showed that the mRNA for the mouse IL12RP,.chain was expressed in the spleen of mice Injected with LPS LPS within 2 or 3 h and that B cells contributed at least in part to this expression. Because the structure and function of the IL12R have not been completely elucidated and these studies analysed only expression of one of the chains of the receptor, further investigation is required to fully understand the possible physiological role of IL12 receptor on B cells. Reference Yang, Y., Trinchieri, G. &Wilson, J.M. (1995), Recombinant IL-12 prevents formation of blocking IgA antibodies to recombinant adenovirus and allows repeated gene therapy to mouse lung. Nature Medicine, 1, 890-893.