Regulation of the interleukin 4 signal in human B-lymphocytes

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264,14503-14507 93. Scechter, Y., Yaish, P., Chorev, M., Gilon, C., Braun, S. & Levitzki, A. (1989) EMBO J. 8, 1676-1680 94. Roifman, C., Mills, G. B., Pan, Z., Gazit, A. & Levitzki, A. (1990)J. Immunol. in the press 95. Padeh, S., Levitzki, A., Mills, G. B. & Roifman. C. H. (1990)J. Clin. Invest. in the press 96. Shiraishi, T., Owada, M. K., Tatsuka, M., Yamashita, T., Watanabe, K. & Kakunaga, T. (1989) Cancer Res. 49,2374-2379 97. Kumar, A., Moreau, J.-L., Gilbert, M. & Theze, J. (1987)J. Immunol. 139,3680-3684 98. Fung, M., Ju, G. & Greene, W. C. (1988) J. Exp. Med. 168,1923-1928 99. Mills, G. B., Schmandt, R., McGill, M., Amendola, T., Hill, M., Jacobs, K., May, C., Rodricks, A. & Hogg, D. (1990) Unpublished work 100. Hanks, S. K.. Quinn, A. M. & Hunter, T. (1988) Science 241,42-49 101. Morrison, D. K., Kaplan, D. R., Escobedo, J. A. & Rapp, U. R. (1989) Cell (Cambridge, Mass.) 58, 649-657 102. Murray, A. W. & Kirschner, M. W. (1989) Science 246,614-621 103. Anderson, N. G., Maller, J. L., Tonks, N. K. & Sturgill, T. W. (1990) Nature (London) 343,651-653 104. Cosman, D., Lyman, S. D., Idzerda, R. L., Beckmann, M. P., Park, L. s.,Goodwin, R. G. & March,

C. J. (1990) Trends Biochem. Sci. 15,265-270 105. Florkeiwicz, R. Z. & Sommer, A. (1989) Proc. Natl. Acad. Sci. U.S.A. 86,3978-3981 106. Wilson, T. & Treisman, R. (1988) Nature (London) 336,396-399 107. Jenkins, M. K., Pardoll, D. M., Mizuguchi, J., Chused, T. M. & Schwartz, R. H. (1987) Proc. Natl. Acad. Sci. USA. 84,5409-541 3 108. Otten, G., Wilde, D. B., Prystowsky, M. B., Olshan, J. S., Rabin, H., Henderson, L. E. & Fitch, F. W. (1986) Eur. J. Immunol. 16,217-224 109. Otten, G., Herold, K. C. & Fitch, F. W. (1987) J. Immunol. 139,1348-1352 110. Schell, S. & Fitch, F. (1989) J. Immunol. 143, 1499-1 505 1 1 1 . Williams, M. E., Lichtman, A. H. & Abbas, A. (1990)J. Immunol. 144,1208-1214 112. Gajewski, T., Schell, S. R. & Fitch, F. W. (1990) J. Immunol. 144,4110-4120 113. Mills, G. B., May, C., Hill, M., Ebanks, R., Mellors, A. & Gelfand, E. W. (1989) J. Immunol. 142, 1995-2003 114. Mary, D., Aussel, C., Ferrua, B. & Fehlmann, M. (1987)J. Immunol. 139,1179-1 183 115. Johnson, K. W., Davis, B. H. & Smith, K. A. (1988) Proc. Natl. Acad. Sci. USA. 85,6072-6076 ~~~

~

Received 3 1 December 1990

Regulation of the interleukin 4 signal in human B-lymphocytes Michael Finney,* Robert H. Michell,t Steven Gillis$ and John Gordon*$ *Department of Immunology,The Medical School, Vincent Drive, Birmingham, tDepartment of Biochemistry, University of Birmingham, Birmingham BI 5 2TT, U.K. and Slmmunex Corporation, 51 University Street, Seattle, WA 98 I0 I, U.S.A. ~

~~~

Introduction Interleukin 4 (IL-4), a 20 kDa glycoprotein product of T-helper cells, influences the activation, clonal expansion, and isotype-selective differentiation of mammalian B-lymphocytes. In both man and mouse, IL-4 appears to be a necessary component for the switching of uncommitted B-cells to the production of IgE [l]. When combined with signals delivered through the CD40 cell-surface glycoprotein, IL-4 is able to sustain the growth cycle of already-cycling human B-cells [2], and these two factors, in conjunction with appropriate feeder systems, have been used successfully to generate longterm B-cell lines [2a].

Abbreviations used: IL-4, interleukin 4; TGF/?, transforming growth factor /?;MHC, major histocompatibility complex. $To whom correspondence should be addressed.

IL-4 can exert direct effects on resting B-lymphocytes which, in vivo, would probably occur after cognate interactions between B- and T-cells. For murine B-lymphocytes, cell activation is exemplified by an up-regulation of Ia antigen, but a corresponding increase in major histocompatibility complex (MHC) class I1 expression has not been so readily observed in human B-cells [l]. This may largely be due to the already high expression of MHC class I1 on human B-cells. In contrast, expression of the CD23 cell-surface glycoprotein, which is normally barely detectable on resting human B-cells, is dramatically enhanced after their exposure to IL-4 [3]. CD23 binds IgE with low affinity and has been implicated in the regulation of IgE production. Moreover, its extracellular domain can be released from cells by proteolytic cleavage, and the resulting soluble fragments are endowed with cytokine-like activities [3]. Both the induction of CD23 and the promotion of IgE synthesis by IL-4

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can be antagonized by type I and type I1 interferons. In addition to CD23, IL-4 also enhances the expression of other functional molecules on the human B-cell surface: these include CD40, CD72 and IgM. Clearly, it would be of interest to define the biochemical signals which are responsible for delivering the IL-4 message to human B-cells. As part of a three-centre study, we recently described the ability of IL-4 to turn on an apparently novel second messenger cascade in quiescent human B-cells [4]. This initially involves a transient activation of PtdIns(4,5)Pz hydrolysis which is followed, several minutes later, by a sustained elevation in intracellular cyclic AMP: this complete sequence of events is necessary for the induction of CD23 expression several hours later. Surprisingly, neither of these second messenger systems appears to be involved in the activation of murine B cells by IL-4. In the present communication, we report new data which give further insight into the involvement of the phosphoinositide pathway in the delivery of the IL-4 signal to human B-cells. We also describe investigations of the actions of several agents that interfere with IL-4-promoted CD23 induction and conclude that each appears to be acting via a different pathway. Finally, we report that the influence of each of these physiological antagonists of IL-4 action can be effectively counteracted by delivering additional signals to the B-cell through other defined surface glycoproteins.

IL-4 and other CaZ+-mobilizingagents promote the phosphorylation of a 38 kDa (pl 4.5) protein Given that the sequential activation of the two second messenger systems that are turned on in human B-cells by 1L-4 would lead to the activation of several protein kinases, it was somewhat surprising that no enhanced phosphorylation of cell-associated proteins had previously been observed. It seemed possible that previous analyses of phosphoproteins, based only upon size separation, provided insufficient resolution to allow detection of these events. We therefore turned to two-dimensional analysis incorporating separations based on charge in addition to size. Now it could be observed that, after 20 min exposure to IL-4, there was an enhanced phosphorylation of a 38 kDa (PI 4.5) polypeptide: this was accompanied by a reduction in the phosphorylation of a 76 kDa protein (PI 5.3). The former, but not the latter, change was also observed in resting B-cells which had been stimulated with ionomycin or anti-p (antibody to cellsurface IgM), either of which provoke an increase in

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[Ca2+Ii.As would be anticipated, cells that had been treated with (low doses of) a phorbol ester 12-0tetradecanoylphorbol 13-acetate that causes persistent activation of protein kinase C showed enhanced phosphorylation of several proteins: the phosphorylation status of the 38 kDa molecule remained unchanged.

Anti-p, but not IL-4, stimulates production of Ins( I,3,4,5)P, and a larger and more sustained [Caz+], elevation Removal of Ca2+from the extracellular medium by the addition of 2 mM-EGTA did not affect the rapid and transient Ca2+flux generated in human B-cells upon the addition of IL-4. By contrast, and as reported previously, the more sustained rise in [Ca2+Ii provoked by anti-p was curtailed upon removal of extracellular ions, even though the initial peak response was unaffected. Even in the absence of extracellular Caz+, the Ca2+ signal induced by anti-,u was usually greater and invariably more prolonged than that provoked by IL-4. Indeed, although the generation of Ins( 1,4,5)P3 upon IL-4 stimulation was consistently detected in our experiments, we were not always able to detect the accompanying elevation in [Ca2+Ii. Detailed comparison of the inositol phosphates generated upon the addition of the two signals revealed some interesting differences which might help to explain the sometimes elusive Ca2+ response. Two methods were used to analyse inositol phosphates: h.p.1.c. analysis of water-soluble components acutely labelled with ['H]inositol and a competitive binding assay for Ins( 1,4,5)P3. The former has the advantage of providing information about changes in the labelling of a variety of inositol phosphates, whereas the latter allows a more rigorous examination of the time course of changes in the cellular content of Ins( 1,4,5)P,. The major changes observed following stimulation of quiescent B-cells with IL-4 or anti-,u are summarized in Table 1. First, although the maximum change in Ins( 1,4,5)P3levels were of similar magnitude for the two stimuli, the kinetics of the IL-4-provoked change were invariably more rapid, both peaking and returning to basal levels at earlier times than that driven by anti-p. Perhaps most interestingly, while anti-p provoked an approximate 7-fold increase in Ins( 1,3,4,5)P4production, IL-4 was able to muster, at best, an increase of 75% in the level of this inositol phosphate. IL-4 consistently turns on the production of the Ca2+-mobilizing messenger Ins( 1,4,5)P3 and

lntercellular Cross-Talk

Table I . ~

Comparison between anti-p and IL-4-promoted stimulation of quiescent human B-cells IL-4

Anti-p

+

+ Phosphorylation of 38 kDa protein Ins( I ,4,5)P, production 5-fold 5-fold I .5-f0ld 7-fold Ins( I ,3,4,5)P4production [Ca2+],elevation f/+++ transient sustained [Cyclic AMP], accumulation

+++

+++ +++

the induction of CD23 by IL-4 has been shown to be a Ca2+-dependentphenomenon. However, it has been difficult to measure reproducibly the change in [Ca2+Ii which accompanies the transient rise in Ins( 1,4,S)P3.The observations just described may help to explain this inconsistency. Ins( 1,3,4,5)P4 appears to have an important function, at least in some cells, in increasing the size of the Ca2+ pool that is available to Ins(1,4,5)P3[ S ] . Cullen and colleagues [6] have recently used 1ns(2,4,S)P3to study this question more rigorously: this isomer of InsP, mobilizes Ca2+ in the same way as Ins( 1,4,S)P,, but it cannot be metabolized to Ins(1,3,4,S)P4.In the mouse lymphoma line L1210, Ins( 1,3,4,S)P4alone did not mobilize Ca2+,but it synergized effectively with Ins(2,4,5)P3 in releasing Ca2+, particularly when sub-optimal doses of the Ins(2,4,5)P3 isomer were applied and/or the basal [Ca2+Iiwas relatively low. Under these conditions, Ins(2,4,S)P3 alone could mobilize approximately 10% of the level of Ca2+ that could be released by a combination of Ins(2,4,S)P3 and Ins(1,3,4,S)P,. The observation that IL-4 stimulation of human B-cells causes a significant rise in Ins( 1,4,5)P, production, but little Ins( 1,3,4,S)P4 generation, would therefore imply that it can only release a fraction of the available intracellular Ca2+,with little possibility of drawing on extracellular sources of Ca2+ to boost the response.

Inhibitors of IL-4-promoted CD23 production in human B-cells The interferons are now well-established physiological antagonists of IL-Ctriggered CD23 production and of the subsequent drive toward IgE synthesis. We have recently examined in detail this antagonism on three parameters of CD23 induction: surface expression as measured by fluorescence-activated cell sorting (f.a.c.s) analysis; total

cell content as assessed by enzyme-linked immunosorbent assay (e.1.i.s.a.); and the release of soluble CD23, again determined by e.1.i.s.a. In our studies, which used highly purified Go B-cells as the targets for IL-4 action, either a type I (a)or type I1 ( y ) interferon could inhibit all three measures of CD23 induction equally, but only by a maximum of 40-50%. When both were added together, inhibition of the response to IL-4 approached loo%, thus indicating that each class of interferon used a different mechanism to suppress the induction of CD23 expression by IL-4. Two other suppressors of B-cell activation, transforming growth factor B (TGFB) and an antibody to the CD19 membrane protein, were similarly assessed for their ability to interfere with the pathway of CD23 induction initiated by IL-4. The increase in cell-associated CD23, whether determined by f.a.c.s. analysis of surface expression or by e.1.i.s.a. measurement of cell lysates, was inhibited by SO-70% with both agents, with TGFB usually being the more potent. IL-4-promoted release of soluble CD23 was similarly diminished in the presence of TGFB. However, the antibody to CD19, which had no influence when presented alone, increased the amount of soluble CD23 accumulating in the culture medium in response to IL-4. Although we do not yet know how the engagement of CD19 causes enhanced CD23 release, the overall influence of CD19 activation can still be considered antagonistic towards IL-Cpromoted CD23 production because the total amount of CD23 induced (i.e. cellassociated plus released) was reduced in the presence of CD19 antibody. All the inhibitors of IL4-promoted CD23 expression appeared to be working differently, inasmuch as their combined effects were always greater than when each was used alone.

Antagonism of IL-4-promoted CD23induction can be selectively interrupted by signals delivered through three discrete surface receptors Interferons appear not to affect either the binding of IL-4 to its receptor or the expression of IL-4 receptors [7]. This suggests that they must be influencing post-receptor events, probably by interrupting a key step in the sequence of intracellular changes that ultimately leads to enhanced transcription from the CD23 gene. This raises the possibility that positive signals delivered via alternative routes could be capable of by-passing those blocked by interferongenerated signals, thereby allowing the final stages

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in the sequence of events that is normally initiated by IL-4 to be completed. We have explored this possibility not only for the interferon-mediated inhibition but also for the turn-off of IL-4-promoted CD23 expression elicited with TGFB and antibody to CD19. The results are summarized in Table 2. Three surface molecules were identified which, upon their engagement by monoclonal antibodies, transmitted information to the B-cell such that the influence of the inhibitors of CD23 induction were now overcome. Importantly, the ability of each of these alternative positive signals to override inhibition was unique: engagement of CD40 successfully counteracted the ability of all the antagonists to inhibit IL-4-promoted CD23 production; ligation of surface IgM rescued the cells from inhibition by TGFB or by the interferons, but left that delivered through CD19 untouched; and a monoclonal antibody to CD72 prevented TGF B-driven antagonism, but not that of the interferons or CD19antibody. These observations support the notion that each of the antagonists prevented IL-4 from promoting optimal CD23 production through a different mechanism.

Concluding remarks The above observations point not only to the complexity of the signalling pathways activated in B-cells by IL-4 but also to the variety of mechanisms employed by agents that counteract the effects of IL-4. The ability to override selectively the influence of the inhibitors of IL-4 action through the delivery of discrete positive signals to the B-cell underpins the notion that detailed receptor crosstalk within precise microenvironments will largely dictate the final outcome of any initial response. T o date. we have no detailed information as to the level Table 2

Complexity of receptor cross-talk in the regulation of IL-4-promoted CD23 production The inhibitors of IL-Cpromoted CD23 production and also the influence on their action of stimulating 6-cells through the surface receptors indicated are summarized.

Inhibition overriden after ligation of (?): Inhibitor

IgM

CD40

CD72

Interferons CD 19-antibody TGFP

Yes No Yes

Yes Yes

No No Yes

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Yes

at which the antagonists interrupt the IL-4-signal leading to CD23 expression. All the antagonists appear to be selective for this phenotypic response to IL-4; the IL-Cinduced up-regulation of IgM, for example, is only marginally affected by interferons and TGFB and not at all by CD19 antibodies. Even before the complexities of antagonism and counter-antagonism begin to be unravelled, we are still left with several important questions on 'simple' IL-4 action unanswered. Most perplexing at present is why the second messenger systems activated upon IL-4 binding should appear to be so different in mouse and man. The recently cloned receptors from the two species are highly homologous and the major biological consequences of IL-4 action are very similar; the apparent minor differences reported between the two species may well be trivial. We previously suggested that the ways in which B-cells are isolated from human and murine tissue may account for some of the apparent discrepancies in the signalling pathways. The source of B-cells may also be contributory. It is also possible that the techniques by which second messenger changes are measured might account for some of the apparent differences. This may be particularly pertinent in assessing inositol lipid metabolism, given the transitory nature of InsP3 generation in human B-cells stimulated with IL-4 and the failure to proceed to InsP, production. Similarly, the failure to register Ca2+ elevations in murine B-cells responding to IL-4 may also reflect the inefficiency of mobilizing Ca2+ stores by Ins(1,4,5)P3 in the absence of significant Ins(1,3,4,5)P4generation. It is of interest to note the study by Ashida and colleagues [a], who showed that agents capable of inhibiting release of intracellular Ca2+ stores, but not agonists of extracellular Ca" channels, blocked the capacity of IL-4 to promote enhanced expression of MHC class I1 (Ia) antigen in murine B-lymphocytes. We are unclear at present whether the second messenger cascade shown to be necessary for CD23 production is also responsible for other phenotypic changes induced by IL-4. The up-regulation of IgM expression, for example, requires lower doses of IL-4 and is not blocked by antibody to CD19. While these observations raise the possibility of two IL-4 receptors or of two signal transduction pathways coupled to a single type of receptor, they would equally be compatible with a bifurcation in a single pathway relatively far downstream so leading to independent up-regulation of IgM and of CD23. It should be noted that, in the human B-cell line Jijoye, an IL-4-responsive ele-

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ment has been found which is unique to the promoter region of CD23 [9]. Clearly, much remains to be done in unravelling the details of IL4-dependent change in B-cells, but the fulfilment of this goal should not only lend insight into the intricacies of immunological regulation, but might also provide a paradigm for elucidating some of the general mechanisms prevailing in systems which are finely tuned by detailed receptor cross-talk. 1. Paul, W. E. & Ohara, J. (1987) Annu. Rev. Immunol. 5, 429-457 2. Gordon, J., Millsum, M. J., Flores-Romo, L. & Gillis, S. (1989) Immunology 68,526-531 2a. Banchereav, J., de Paoli, P., Valli, A., Garcia, E. & Rousset, F. ( 199 1) Science 25 1,70-72 3. Gordon, J., Flores-Romo, L., Cairns, J. A., Millsum,

M. J., Lane, P. J., Johnson, G. D. & MacLennan, I. C. M. (1989) Immunol. Today 10,153-156 4. Finney, M., Guy, G. R., Michell, R. H., Gordon, J., Dugas, B., Rigley. K. P. & Callard, R. E. (1 990) Eur. J. Immunol. 20,15 1- 156 5. Irvine, R. F. (1990) FEBS Lett. 263,5-9 6. Cullen, P. J., Irvine, R F., Drobak, B. R. & Dawson, A. P. (1989) Biochem.J. 259,931-933 7. Galizzi, J. P., Cabrillat. H., Rousset, F., Menetrier, C., de Vries, J. E. & Banchereau, J. (1988) J. Immunol. 141, 1982- 1989 8. Ashida, T., Kubo, K.-I., Kawabata, I., Katagiri, M., Ogimot, M. & Yakura, H. (1990) Cell. Immunol. 126, 233-238 9. Suter, U., Texido, G. & Hofstetter, H. (1989) J. Immunol. 143,3087-3092 Received 20 December 1990

Role of oncogenes in the regulation of MHC antigen expression D. John Maudsley Cancer Research Campaign, Interferon and Cellular Immunity Research Group, Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, U.K.

Summary Class I and class I1 major histocompatibility complex (MHC) antigens are required for CD8+ cytotoxic T cells and CD4+ helper T-cells, respectively, to recognize foreign antigen. Regulating the levels of expression of these MHC antigens regulates the T-cell responses [l]. This regulation is mainly carried out by the interferons (IFN), which are produced in the disease state. Type I IFN (IFNa or IFNB; collectively ‘IFNa p ) up-regulates class I MHC and IFNy up-regulates class I and class I1 MHC. We and others [l-31 have shown that transfection of cells with a variety of oncogenes including ras and myc affects the level of MHC antigen expression. This and other data provide evidence for a scheme in which the signal transduction mechanisms whereby IFN up-regulates MHC antigens involve several (proto)oncogenes.

Introduction The regulation of MHC antigen expression is highly complex, with different tissues showing different basal levels of expression of MHC gene products, different levels of expression during different stages of development and different changes in the levels of expression in response to MHC regulatory signals. However, broadly speaking, most cells conAbbreviations used: MHC, major histocompatibility complex; IFN, interferon; PKC, protein kinase C.

stitutively express class I MHC antigens (H-2K, D and L in mouse, HLA-A, B and C in man), but not class I1 MHC antigens (I-A and I-E in mouse, and HLA-DP, DQ and DR in man). There are some exceptions to this in that, for example, hepatocytes, erythrocytes and cells of neural origin generally do not express class I antigens constitutively (but can be induced to do so). In addition, B-lymphocytes, dendritic cells and some tumour cells (e.g. melanomas) do express class I1 antigens constitutively. Most cells (if not all normal cells) on stimulation with IFN respond with increased class I MHC antigen expression: this is true for both I F N a S and IFNy. In many cell types, induction or enhancement of class I1 MHC antigen expression is stimulated by IFNy, but not by IFNaS. Indeed IFNaS, in certain circumstances, inhibits the induction by IFNy of class I1 MHC antigens. Additional stimuli, such as tumour necrosis factor, may enhance this induction of class I1 and, in cells that constitutively express class I1 antigens, such stimuli may be more effective. The disparity between the effects of IFN y and IFNaB on class I1 MHC antigen expression is at least partially explained by distinct receptors for the two classes of IFN [4-61. However, there is clearly much overlap between signals produced via the receptor for I F N a S and those produced via the receptor for IFNy in that both induce the antiviral response, decrease cell growth and induce class I

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