IL-2R Alpha Chain Research Articles

Enhanced lymphokine production and lymphokine receptor expression in multiple antibody-stimulated human CD4+ peripheral blood lymphocytes.

Treatment of T lymphocytes with antibodies directed against the T cell receptor CD3 complex results in cellular activation that can be augmented by costimulation through other cell surface receptors. The activities of anti-CD3-stimulated human CD4+ PBL were compared to anti-CD3 plus anti-CD2-, anti-CD4-, or anti-CD11a (LFA-1)-stimulated cells. [3H]thymidine incorporation, lymphokine receptor expression, expansion of cell numbers, and lymphokine mRNA and protein were measured. Forty-eight hours after activation, costimulated CD4+ cells demonstrated increased numbers of cells positive for surface IL-2R alpha-chain, IL-2R beta-chain, IFN-gamma receptors, and TNF-alpha receptors. By day 6, costimulated cells exhibited a sevenfold greater expansion in cell numbers compared to cells stimulated with anti-CD3 alone. Anti-CD3 plus anti-CD11a stimulation consistently induced the highest secretion of IL-2 and IFN-gamma, whereas variation in secretion of TNF-alpha and IL-4 between different donors was noted. Analysis of lymphokine receptor mRNA demonstrated increased levels of mRNA for IL-2R alpha-chain and IFN-gamma receptor that preceded the phenotypic changes on the cell surface. In contrast, levels of IL-2R beta-chain and the TNF-alpha receptor mRNA decreased after stimulation. Amounts of IL-2, IL-4, and TNF-alpha, but not IFN-gamma, secreted also correlated with the levels of mRNA measured in the cells. Although costimulation through CD2, CD4, or LFA-1 appeared equally effective for the induction of the lymphokine receptors, these additional stimuli had a different impact on lymphokine secretion. These results indicate that specific control of lymphokine secretion and receptor induction can be another function of the CD2, CD4, and CD11a cell surface receptors. This control is evident at both transcriptional and post-transcriptional levels.

Prolonged IL-2 receptor alpha/CD25 expression after T cell activation via the adhesion molecules CD2 and CD28. Demonstration of combined transcriptional and post-transcriptional regulation.

The T cell adhesion molecule CD28 provides a costimulatory signal in combination with CD2 and CD3 mAb. CD28 regulates the expression of cytokines by T cells, not only IL-2, but also IL-1 alpha and CSF-1, usually synthesized by accessory cells. We have investigated the mechanisms through which CD28 modulates the expression of the IL-2R alpha chain. Whereas activation through CD2 or CD28 alone induced no or only low IL-2R alpha chain expression, activation through CD2 plus CD28 led to both a high and prolonged (greater than 14 days) cell surface and mRNA expression. In contrast, immobilized CD3 mAb-dependent activation induced a transient expression of the IL-2R alpha chain, which was neither further increased nor prolonged by CD28 costimulation. Upon CD2 plus CD28 stimulation, the half-lives of the two IL-2R alpha transcripts increased progressively between days 1 and 4, in contrast to each pathway alone. Whereas each activation pathway alone induced either no (CD2) or low (CD28) levels of IL-2R alpha gene transcription, the CD2 plus CD28 stimulation was associated with its increased transcription, which persisted at similar rates between 5 and 96 h post stimulation. The in vitro costimulation via the CD2 and CD28 molecules thus regulates the expression of the IL-2R alpha gene both at the transcriptional and post transcriptional levels. Our results therefore demonstrate a new immunoregulatory function of the CD28 molecule on IL-2R alpha expression, which, through its increased transcription rate and stabilization, could, together with high levels of cytokines secretion, be responsible for the prolonged T cell proliferation.

IL-2-induced signal transduction involves the activation of nuclear NF-kappa B expression.

Using electrophoretic mobility shift assays and DNA-protein cross-linking techniques, we demonstrate that IL-2 binding to functional forms of the human IL-2R activates nuclear expression of the eukaryotic transcription factor NF-kappa B. These inductive effects of IL-2 were observed in three different cellular systems including human Jurkat T cells stably transfected with IL-2R beta cDNA, mouse pro-B BA/F3 cells stably expressing human IL-2R beta chains either alone or in combination with human or murine IL-2R alpha chains, and purified primary resting human T cells constitutively displaying small numbers of IL-2R beta molecules. IL-2 activation of nuclear NF-kappa B expression is regulated in part at a post-translational level, involving the rapid translocation of both the 50- and 65-kDa subunits of NF-kappa B from the cytoplasm to the nucleus. However, IL-2 induction also produced an increase in mRNA for NF-kappa B p105, indicating an additional pretranslational component of regulation. In contrast, IL-2 exerts only minor effects on NF-kappa B p65 mRNA expression. IL-2 induction of NF-kappa B through the IL-2R beta subunit was both correlated with activation of the endogenous IL-2R alpha gene and critically dependent upon the presence of a serine-rich cytoplasmic domain within IL-2R beta (amino acid residues 267-312). This domain has previously been shown to be essential for IL-2-induced cell growth and may correspond to a binding site for an IL-2R beta-associated tyrosine kinase. Together, these findings suggest that NF-kappa B may play an important role as one intracellular second messenger relaying signals from the plasma membrane to cell nucleus that leads to IL-2-induced activation and growth.

IgM and IgG but not cytokine secretion is restricted to the CD27+ B lymphocyte subset.

In a recent study we reported that CD27 is expressed on a subpopulation of human B lymphocytes and presented circumstantial phenotypic evidence that CD27 expression may be acquired late during B cell differentiation. Here we present functional data showing that, after in vitro stimulation, CD27+ but not CD27- B cells secrete large amounts of both IgM and IgG. Using double immunofluorescence staining of CD27 and IgD, three functionally different B cell subsets representing distinct stages of B cell differentiation can be isolated: 1) the CD27- IgD+ B cells, which do not secrete appreciable Ig; 2) the CD27+IgD+ B cells, which exclusively secrete IgM; and 3) the CD27+IgD- B cells, which comprise the IgG-producing cells. Furthermore, costimulation of CD27- B cells with low m.w. B cell growth factor, in the presence or in the absence of a CD40 mAb, does not induce these cells to become Ig-secreting cells. Although CD27- B cells hardly secrete Ig of any isotype in response to Staphylococcus aureus+IL-2, these cells proliferate vigorously and express the IL-2R alpha chain (CD25) under these stimulatory conditions. Furthermore, both CD27- and CD27+ B cells are capable of producing similar amounts of IL-6 and TNF-alpha. Taken together, these findings indicate that CD27 is a unique non-Ig surface marker discriminating naive from primed B lymphocytes. Furthermore, the capacity to proliferate and to secrete the B cell differentiation factors IL-6 and TNF-alpha already exists at an early B cell differentiation stage at which the cells lack CD27 expression and are not induced to produce Ig.

Prostaglandin E2 and other cyclic AMP-elevating agents modulate IL-2 and IL-2R alpha gene expression at multiple levels.

cAMP is an intracellular second messenger that conveys inhibitory signals for T cell activation and clonal proliferation. cAMP also inhibits the production of IL-2 and IL-2R alpha-chain expression. To determine the mechanisms of this inhibition, human peripheral blood T lymphocytes were stimulated with anti-CD3 mAb, PHA, PMA, or ionomycin, alone or in combination. cAMP elevation by PGE2, cholera toxin, or the cell-permeable analogue 8-bromo-cAMP inhibited the tyrosine phosphorylation of a protein of 100 kDa. This inhibition was associated with decreased IL-2 production and IL-2R alpha expression at both the protein product and the mRNA levels. Nuclear run-off assays showed that the inhibitory effect of cAMP on IL-2 and IL-2R alpha gene expression is mediated at the transcriptional level. H-8, an inhibitor of protein kinase A, reversed the inhibitory effect of cAMP on nuclear transcription of the IL-2 gene, suggesting that this is mediated through activation of protein kinase A. Post-transcriptionally, cAMP elevation decreased the t1/2 of IL-2 mRNA by more than 50%. These data indicate that cAMP inhibits cell membrane, cytoplasmic, and nuclear events associated with T cell activation and highlight the complexities of its action of lymphocyte function.

A role for protein tyrosine kinase activity in natural cytotoxicity as well as antibody-dependent cellular cytotoxicity. Effects of herbimycin A.

NK cells, CD3- large granular lymphocytes, have diverse means by which they lyse targets, including antibody-dependent cellular cytotoxicity. The low affinity receptor for the Fc portion of Ig (Fc gamma RIIIA), like the TCR, is a multimeric receptor complex coupled to a protein tyrosine kinase. In the present study, we observed that inhibition of tyrosine kinase activity by herbimycin A interferes with receptor-mediated phosphorylation of a variety of substrates and mobilization of intracellular calcium. Fc gamma RIIIA induced IL-2R alpha-chain expression was also extremely sensitive to herbimycin A as was antibody-dependent cellular cytotoxicity, in fact more so than receptor-mediated phosphorylation and calcium mobilization. In contrast to Fc gamma RIIIA, the surface molecules and biochemical mechanisms involved in NK cytotoxicity and lymphokine-activated killing are not well characterized. Interestingly, however, herbimycin A also blocks these modes of cytolysis, implicating a role for tyrosine kinase function in these processes. Whether FcR-mediated signaling and receptor-mediated signaling involved in NK activity share specific biochemical intermediates is not known, but the involvement of tyrosine kinase function in the latter means of cytotoxicity may provide novel avenues for understanding the biochemical basis of this perplexing cellular function.

Interleukin (IL)-2 and IL-3 induce distinct but overlapping responses in murine IL-3-dependent 32D cells transduced with human IL-2 receptor beta chain: involvement of tyrosine kinase(s) other than p56lck.

We have established IL-3-dependent 32D myeloid progenitor cells stably expressing the human IL-2 receptor beta chain (IL-2R beta). Whereas parental 32D cells proliferated only in response to IL-3, the transduced cells also proliferated in response to IL-2. Transduced cells expressed high- and intermediate-affinity IL-2Rs, resulting from expression of human IL-2R beta and murine IL-2R alpha chain (IL-2R alpha). IL-2 induced phenotypic changes not induced by IL-3, including the upregulated expression of endogenous murine IL-2R alpha and IL-2R beta and an increase in cell size. Therefore, the transduced IL-2R beta was not merely coupling with the IL-3 signaling pathway. IL-3 augmented several IL-2-induced responses including the up-regulation of IL-2R alpha. Both IL-2- and IL-3-induced proliferation and IL-2 induced IL-2R alpha expression were inhibited by the tyrosine kinase inhibitor herbimycin A. Thus, both IL-2- and IL-3-mediated effects required tyrosine kinase activity. The identity of the tyrosine kinase(s) mediating the IL-2 signals in these cells is not known but cannot be p56lck, a tyrosine kinase found in T cells, since 32D-IL-2R beta cells do not express p56lck.

Intracellular events involved in CD5-induced human T cell activation and proliferation.

In this report we describe a novel pathway of human T cell activation and proliferation involving the CD5 surface Ag. The CD5-specific Cris1 mAb induces by itself monocyte-dependent proliferation of PBMC. Among a panel of CD5-specific mAb (Leu1, OKT1, LO-CD5a, F101-1C5, and F145GF3), only the F145GF3 mAb shared this property with Cris1. The analysis of the biochemical pathway involved in this activation showed the lack of detectable hydrolysis of inositol phosphates or early increments in the intracellular cytosolic calcium concentration, after triggering cells with the mitogenic CD5 mAb. However, stimulation with CD5 induces activation of protein kinase C, as measured by phosphorylation of a specific peptide substrate (peptide GS), which can be inhibited by a pseudosubstrate peptide inhibitor. Stimulation with CD5 mAb induces also tyrosine kinase activity, with a substrate pattern that differs from that induced after triggering lymphocytes through the TCR-CD3 complex. On the other hand the IL-2/IL-2R pathway seems involved in the CD5-mediated proliferation of PBMC because anti-IL-2R-specific mAb inhibits CD5-induced proliferation, and stimulation with mitogenic CD5 mAb induces production of IL-2 and expression of IL-2R alpha and beta chains. Therefore, the triggering of the CD5 Ag can induce IL-2- and monocyte-dependent human T cell proliferation by a biochemical pathway that differs, at least in the first stages, from the one that mediates TCR-CD3 complex-induced T cell activation.

Activated T lymphocytes from mice infected by lymphocytic choriomeningitis virus display high affinity IL-2 receptors but do not proliferate in response to IL-2.

The i.v. injection of mice with lymphocytic choriomeningitis virus (LCMV) initiates a rapid and long lasting immunodepression which can be monitored in vivo or in vitro. Splenic T lymphocytes taken from mice infected for 7 days with LCMV are characterized by a low proliferative capacity in response to Con A stimulation in vitro. In an initial attempt to understand the molecular mechanisms regulating the general anergy induced by the viral infection, we have analyzed the transcription of IL-2 and of p55 IL-2R alpha gene, two genes involved in T cell proliferation. IL-2 gene transcripts were hardly detected after Con A activation of spleen cells from LCMV-infected mice. In contrast, the expression of the gene encoding IL-2R alpha chain was induced as in control noninfected cells. In addition, the expression of the p75 IL-2R beta chain was not modified. The transcripts of the IL-2R alpha and of the IL-2R beta genes were normally translated as high affinity. IL-2R were expressed on the membrane of T lymphocytes from LCMV-infected mice. Despite the finding that these receptors could also internalize IL-2, the exogenous addition of this growth factor did not induce cell proliferation, indicating that the virus-induced blockade is multifocal.

Postnatal Development of Murine Spleen Cell Proliferative Responses Induced by Interleukin 2 or Phorbol Ester and Calcium Ionophore A23187

To determine the kinetics of the ontogeny of polygenically controlled proliferative responses, spleen cells from 5, 10 and 20 day-old mice, previously identified as high (B10.PL-Thy- 1 a , AKR.M) and low (C3H.A) responders were tested. Cells (10 5) were stimulated in vitro with human recombinant IL-2 (35 U), a mixture of either IL-2 (0.7 U) and 13-myristate 12-acetate phorbol (5 ng) or phorbol and calcium ionophore A23187 (5 ng). Two parameters were assessed; proliferation measured by 3H-thymidine incorporation and the synergistic effect expressed as a synergism coefficient (SC): the ratio of proliferation induced by a mixture to the sum of proliferations induced by each component alone. At the age of 5 days, all three strains, regardless of stimulation, produced similar proliferation and showed no synergistic effect (SC = 1.0). At the age of 10 days, proliferation of high responder cells was 3-5 times higher than that of the low responder, but only high responders displayed marginal synergistic effect (2>SC>1.5). At the age of 20 days, proliferative responses of the high responders increased somewhat and a definitive synergistic effect became clearly demonstrable (SC>3.0). This effect increased further after the age of 20 days to reach the level of SC>5.0 in adult high responders. The delay in expression of synergistic effect was due to a substantial proliferation induced by phorbol alone in 10 day-old animals. The available data allow the conclusion that phenotypic strain variation in proliferative responses is first detectable at the age of 10 days and becomes fully developed only after attaining the age of 20 days or more. Since the magnitude of proliferative responses was not associated with frequencies of IL-2R alpha + cells it seems that differences in responsiveness can not be attributed to the induction of IL-2R alpha chain, and they may reflect intrinsic abilities of cells to proliferate.

Interleukin-3-induced downregulation of the expression of interleukin-2 receptor beta chain in human T cells

We examined the effect of interleukin-3 (IL-3) on human CD4+ cloned T cells, P607 and 1C2. By flow cytometric analysis, we found that IL-3 downregulated the surface expression of IL-2 receptor (R) beta chain in a dose-dependent manner but had little effect on that of IL-2R alpha chain. A simultaneous 125I-labeled IL-2 binding assay showed a decrease in the number of high-affinity, but not of low-affinity, IL-2Rs by IL- 3. The downregulation of the IL-2R beta chain began 3 hours after culture initiation, increased further thereafter, and was completely inhibited by anti-IL-3 antibodies. Expression of mRNA for either alpha or beta chain was not reduced by IL-3, and this suggests that the reduction of surface beta chain expression was not caused by the reduction of beta chain mRNA. IL-3-accelerating internalization of IL- 2R beta chain appeared to be one of the mechanisms for IL-3-induced downregulation of surface IL-2R beta chain expression. IL-3 alone increased the proliferation of T-cell clones but decreased the existing increment of their proliferation by IL-2. Accordingly, IL-3 may be one of the factors acting as a liaison between the hematopoietic and immune systems.

The IL-2 receptor alpha-chain alters the binding of IL-2 to the beta-chain.

The binding of IL-2 to its high affinity receptor results in the formation of the ternary complex consisting of IL-2, alpha-chain (p55, Tac) and beta-chain (p75). We studied the role of alpha-chain in IL-2 binding to the high affinity receptor using IL-2 analog Lys20 which was made by the substitution of Lys for Asp20 of wild-type rIL-2. Lys20 bound to MT-1 cells solely expressing alpha-chain at low affinity, but did not bind to YT-2C2 cells which solely expressed beta-chain. However, direct binding of radiolabeled Lys20 to ED515-D cells, an HTLV-I-infected and IL-2-dependent T cell line, revealed both high affinity and low affinity binding although the Kd value of high affinity binding was 50 to 100 times higher than that of the high affinity binding of wild-type rIL-2. High affinity binding of Lys20 was completely blocked by 2R-B mAb recognizing IL-2R beta-chain. Anti-Tac mAb recognizing IL-2R alpha-chain abolished all of the specific Lys20 bindings. In contrast to the replacement of cell bound 2R-B mAb with wild-type rIL-2 at 37 degrees C, the addition of an excess of Lys20 did not cause the detachment of cell-bound radiolabeled or FITC-labeled 2R-B mAb. Consistent with the results of binding studies, Lys20 induced the proliferation of ED515-D cells, but not large granular lymphocyte leukemic cells. The growth of ED-515D cells was completely suppressed by either anti-Tac mAb or 2R-B mAb. These results strongly suggest that coexpression of the IL-2R alpha- and beta-chains alters the binding affinity of Lys20 and that the interaction between IL-2 and the alpha-chain is a key event in the formation of the IL-2/IL-2R ternary complex.

The integrity of the conserved ‘WS motif’ common to IL-2 and other cytokine receptors is essential for ligand binding and signal transduction.

Recent studies have identified a new family of cytokine receptors, which is primarily characterized by the conservation of periodically interspersed four cysteine residues and the W-S-X-W-S sequence ('WS motif') within the extracellular domain. However, the role of such conserved structures still remains elusive, in particular that of the WS motif. Interleukin-2 (IL-2) is known to play a critical role in the clonal expansion of antigen-stimulated T lymphocytes, and the IL-2 signal is delivered by one of the receptor components, the IL-2 receptor beta (IL-2R beta) chain. The IL-2R beta chain, unlike the IL-2R alpha chain, belongs to this receptor family. In the present study, we analyzed the function of the WS motif of IL-2R beta (Trp194-Ser195-Pro196-Trp197-Ser198) with the use of site-directed mutagenesis. Our results indicate the critical role of the two Trp residues in the proper folding of the IL-2R beta extracellular domain and point to the general functional importance of the WS motif in the new cytokine receptor family.

Mucosal T-Cell Function

Lymphocytes in the intestinal lamina propria probably differ from lymphocyte populations in the circulation or in other tissue sites in a number of ways. First, lamina propria lymphocytes are phenotypically distinct in that few of these cells normally express the Leu-8 or CD45RA antigens. The presence of these molecules on CD4 T cells correlates with suppressor and suppressor-inducer function, and therefore the majority of CD4 T cells in the intestinal lamina propria have the phenotype associated with high helper activity. A substantial proportion of lamina propria lymphocytes also have evidence of activation, based on expression of the IL-2R alpha chain and HLA-DR molecules. Lymphocytes in the intestinal lamina propria are different in their potential for expression of lymphokine gene products, because activated cells from the lamina propria have high expression of mRNA for IL-2, IL-4, IL-5, and IFN-gamma in comparison to circulating lymphocytes. Mesenteric lymph node T cells also differ from circulating lymphocytes in their high expression of IL-4 and IL-5 mRNA. A further difference between mesenteric lymph node and lamina propria T cells is that the former can proliferate in response to IL-4, whereas the latter cannot. These phenotypic and mRNA differences of lamina propria lymphocytes also correlate well with their high helper activity in vitro for immunoglobulin synthesis in the pokeweed mitogen system. T cells with the potential for cytolytic activity are present in the intestinal lamina propria, although there is no definitive evidence that they are cytolytically active under physiologic or pathologic conditions. Finally, in a model system of intestinal inflammation, lymphogranuloma venereum in nonhuman primates, lamina propria T cells at the site of inflammation were unable to respond to specific antigens with proliferation but did respond with high helper activity. These observations are all consistent with the conclusion that T cells in the lamina propria are pleomorphic but are highly enriched for subpopulations of activated memory cells that are geared for effector functions such as helper and cytolytic functions. These functions are likely to be critical in maintaining normal host defense in the mucosal environment.

Physiologic activation of T cells via the T cell receptor induces NF-kappa B.

The transcription factor NF-kappa B has been implicated in the mitogen-induced expression of several genes that are critical for the immunologic function of T cells such as those encoding IL-2 and the IL-2R alpha chain (IL-2R alpha). We show here that NF-kappa B is induced in T cells activated by Ag, anti-CD3 antibody, or allogeneic stimulation. The induction of NF-kappa B via the TCR was dependent on protein kinase C. IL-2, which also activates IL-2R alpha expression and proliferation in T cells, was not able to induce NF-kappa B. TCR-mediated induction of NF-kappa B suggests a central role for this factor in activated T cells and also provides a mechanism for activation of latent HIV provirus during the normal immune response.

IL-7 induces human lymphokine-activated killer cell activity and is regulated by IL-4.

Induction of lymphokine-activated killer (LAK) activity by IL-2 has been described and characterized as broadly cytolytic activity against both fresh and cultured tumors. rIL-7 in the absence of IL-2 also induces LAK activity in human cells. This activity is unique for IL-7, because it is not shared by other cytokines including IL-1, IL-4, IL-6, and TNF-alpha. IL-7 also induces either de novo or increased expression of the surface markers CD25 (Tac, IL-2R alpha-chain), CD54 (ICAM-1), Mic beta 1 (IL-2R beta-chain) and CD69 (early T cell activation Ag). IL-7-induced LAK activity is independent of IL-2 secretion, because it is not abrogated by IL-2 antisera. The LAK precursor responding to IL-7 stimulation is enriched in the null cell fraction as has been demonstrated for IL-2-induced LAK cells. TGF-beta and IL-4 interfere with generation of LAK activity by IL-7. Anti-IL-4 antiserum enhances IL-7-induced LAK activity and augments induction of surface marker expression by IL-7. This may be indirect evidence that IL-7 stimulation leads to induction of IL-4 activity. Our results describe the activation of mature lymphoid cells by IL-7. This and the previously described role of IL-7 in lymphohemopoiesis makes it a cytokine of potential therapeutic value for treatment of immunodeficiency states and possibly the immunotherapy of cancer.

The expression of IL-2R alpha-chain is enhanced by activation of adenylate cyclase in large granular lymphocytes and natural killer cells.

An increase in intracellular cAMP level induced the expression of IL-2R alpha-chain, the 55-kDa component of IL-2R complex, in a human NK-like cell line, YT. We show here that forskolin also induces the expression of IL-2R alpha-chain on mouse large granular lymphocytes (LGL) but not on T cells. In contrast, treatment with a combination of phorbol ester and calcium ionophore, which is a strong inducer of IL-2R alpha-chain on T cells, does not induce the expression of the alpha-chain on LGL cells. Forskolin was shown to activate the transcription of IL-2R alpha-chain gene in YT cells as revealed by the chloramphenicol acetyltransferase assay. Chemical cross-linking experiments using radio-iodinated IL-2 also supported the enhanced expression of IL-2R alpha-chain by treatment with forskolin. In contrast to the alpha-chain, IL-2R beta-chain was not induced by forskolin as revealed by flow cytofluorometry with a mAb against the beta-chain molecule. These results indicate that the activation of adenylate cyclase induces or/and enhance the expression of IL-2R alpha-chain at the transcriptional level in LGL/NK cells including mouse LGL and human YT cell, which leads to the enhanced expression of high affinity IL-2 receptors.

Monoclonal antibody against the human peripheral lymph node homing receptor homologue (Leu 8) inhibits B cell differentiation but not B cell proliferation.

Previous studies have shown that a subpopulation of circulating human B cells expresses the Leu 8 peripheral lymph node homing receptor homologue and that these B cells are capable of producing Ig in response to staphylococcus A Cowan I (SAC). In the present study the effect of a signal delivered via the Leu 8 molecule (using anti-Leu 8 mAb) on B cells was examined. Initially, it was shown that immobilized anti-Leu 8 suppressed IgM and IgG secretion of B cells activated by SAC + IL-2 but not that by PWM-prestimulated B cells or B cells stimulated with PWM in the presence of CD4+, Leu 8- T cells (a source of helper cells). It was also shown that anti-Leu 8 did not suppress SAC + IL-2-stimulated B cell proliferation or expression of IL-2R alpha-chain or c-myc mRNA in B cells. The addition of T cells, monocytes, purified IL-2, rIL-1, rIL-6, or human B cell growth factor did not overcome the inhibitory effect of anti-Leu 8 on SAC-stimulated B cell Ig production, and the inhibitory effect of anti-Leu 8 was not blocked by anti-TGF-beta. Finally, inhibition of B cell differentiation occurred even when anti-Leu 8 was added up to 72 hrs after initiation of cell culture. Thus, anti-Leu 8 is unique among inhibitors of B cell function in that it can down-regulate immunoglobulin synthesis without affecting B cell proliferation. These findings suggest that a natural ligand for Leu 8 could affect not only homing of B cells, but also B cell differentiation.

Cyclosporin A inhibits the interleukin 2 receptor α chain gene transcription but not its cell surface expression: the α chain stability can explain this discrepancy

The effect of the immunosuppressor cyclosporin A (CsA) on the expression of interleukin (IL) 2 receptors was investigated in a human T cell line IARC301 which constitutively expresses such receptors. This cell line also spontaneously secretes IL2 which supports its autocrine growth. We have previously shown that CsA prevents the constitutive transcription of the IL2 gene in these cells. Here we show that as soon as 4 h after CsA addition, the transcription of the gene encoding the alpha chain (p55) of IL2R was inhibited. IL2 can transiently increase the expression of this gene. CsA did not prevent this transient IL2-dependent induction of IL2R alpha, but could still partially inhibit it. Once IL2 induction was over, CsA exerted its full inhibition. Thus, CsA does not seem to inhibit IL 2R alpha gene transcription simply by inhibition of IL2 synthesis. However, no modification of IL2R alpha expression on the cell surface could be detected after 48 h in the presence of CsA. This discrepancy between the effect of CsA on IL2R alpha expression as probed at the mRNA or the protein level can be accounted for by the stability of the IL2R alpha protein after synthesis. Indeed, the half-life of IL2R alpha chain is longer than 40 h. This suggests that the alpha chain, after it is endocytosed together with the beta chain as a component of high-affinity IL2R, might recycle back to the cell surface.

Control of IL-2 receptor-alpha expression by IL-1, tumor necrosis factor, and IL-2. Complex regulation via elements in the 5' flanking region.

We have analyzed the mechanisms by which IL-1, IL-2, and TNF regulate expression of IL-2R alpha chain in a rodent T cell line. All three cytokines induce detectable IL-2R alpha mRNA by themselves, but there is strong synergy between IL-1 or TNF, on the one hand, and IL-2, on the other. The earliest phase of induction by IL-1 is independent of protein synthesis. IL-1, but not TNF, also stimulates transient secretion of IL-2. This leads to an autocrine stimulation of a further increase in IL-2R alpha mRNA levels. When IL-2 secretion has dropped off, continued IL-2R alpha expression requires both IL-2 and IL-1. Most or all of this regulation is due to changes in the rate of transcription of the IL-2R alpha gene. The response to IL-1 and IL-2 depends on a segment in the IL-2R alpha 5' flanking region, upstream of all cis-acting regulatory elements previously identified in the human gene.