Interleukin 10 (IL-10) upregulates IL-1 receptor antagonist production from lipopolysaccharide-stimulated human polymorphonuclear leukocytes by delaying mRNA degradation

Polymorphonuclear leukocytes (PMN) have been identified as cells capable of producing a number of pro- and antiinflammatory cytokines in response to specific agonists. Previously, we showed that tumor necrosis factor (TNF), interleukin-1 beta (IL-1 beta), and IL-8, are produced by PMN after stimulation with agonists, such as lipopolysaccharide (LPS). In this study, we demonstrate that LPS is also a potent stimulus for the mRNA expression and release of the IL-1 receptor antagonist (IL-1ra). In addition, we show that the release of IL-1ra from LPS-stimulated PMN is markedly potentiated in the presence of IL-10 (from two to threefold after 18 h of stimulation). Moreover, we observed that this upregulation of IL-1ra production by IL-10 in LPS- stimulated PMN took place through IL-1ra mRNA stabilization. Indeed, the half-life of IL-1ra mRNA was prolonged in PMN stimulated in the presence of IL-10 and LPS, as compared with cells stimulated with LPS alone. That IL-10 selectively upregulates IL-1ra production in LPS- activated PMN, while it inhibits the production of IL-1 beta, TNF, and IL-8 under the same conditions, suggests that IL-10 may be an important physiologic regulator of cytokine production from PMN, and emphasizes the potential role of IL-10 in inflammatory responses.     

Interleukin 4 protects chronic lymphocytic leukemic B cells from death by apoptosis and upregulates Bcl-2 expression

B chronic lymphocytic leukemia (B-CLL) is characterized by the accumulation of slow-dividing and long-lived monoclonal B cells arrested at the intermediate stage of their differentiation. We previously showed that interleukin 4 (IL-4) not only inhibits but also prevents the proliferation of B-CLL cells. We report here that IL-4 protects the B-CLL cells from death by apoptosis (programmed cell death [PCD]). IL-4 inhibits spontaneous and hydrocortisone (HC)-induced PCD of highly purified B cells from 12 unselected CLL patients, as shown by sustained cell viability and lack of DNA fragmentation. IL-1, -2, -3, -5, -6, -7, tumor necrosis factor alpha, and transforming growth factor beta have no protective effect. The in vitro rescue from apoptosis by IL-4 is reflected by an increased expression of Bcl-2 protein, a proto-oncogene directly involved in the prolongation of cell survival in vivo and in vitro. Hence, IL-4-treated B-CLL cells express significantly more Bcl-2 than unstimulated, HC-treated, or fresh B-CLL cells. Furthermore, subcutaneous injection of IL-4 into one CLL patient enhances Bcl-2 protein expression in the leukemic B cells. These data may suggest that IL-4 prevents apoptosis of B-CLL cells using a Bcl-2-dependent pathway. Given our recent observations that fresh T cells from B-CLL patients express IL-4 mRNA, we propose that IL-4 has an essential role in the pathogenesis of CLL disease, by preventing both the death and the proliferation of the malignant B cells.     

Interleukin 4 inhibits in vitro proliferation of leukemic and normal human B cell precursors.

In the present study, we have investigated the effects of IL-4 on the proliferation and differentiation of leukemic and normal human B cell precursors (BCP). We have demonstrated that IL-4 significantly inhibited spontaneous [3H]thymidine ([3H]-TdR) incorporation by leukemic blasts from some B lineage acute lymphoblastic leukemia (BCP-ALL) patients (8 of 14). Furthermore, IL-4 was found to suppress the spontaneous and factor-dependent (IL-7 and IL-3) proliferation of normal BCP (CD10+ surface [s] IgM- cells) isolated from fetal bone marrow. Maximum growth inhibition of either leukemic or normal BCP was reached at low IL-4 concentrations (10 U/ml), and the effect was specifically neutralized by anti-IL-4 antibody. IL-4 was further found to induce the expression of CD20 antigen on BCP-ALL cells from a number of the cases examined (5 of 8), but in contrast to leukemic cells, IL-4 failed to induce CD20 antigen on normal BCP. Finally, IL-4 was found to induce neither the expression of cytoplasmic mu chain, nor the appearance of sIgM+ cells in cultures of normal or leukemic BCP. Our data indicate that IL-4 has the potential to inhibit cell proliferation in leukemic and normal human B lymphopoiesis but is unable to drive the transition from BCP to mature B cells.     

Human recombinant interleukin 4 induces Fc epsilon receptors (CD23) on normal human B lymphocytes

Human rIL-4 is able to induce the expression of low-affinity receptors for IgE (Fc epsilon RL/CD23) on resting B lymphocytes, as determined by the binding of either the anti Fc epsilon RL/CD23-specific mAb 25 or IgE. Stimulation of B cells with insolubilized anti-IgM antibody increases the number of cells expressing Fc epsilon RL/CD23 upon culturing with IL-4 and enhances the level of Fc epsilon RL/CD23 expression on these cells. Fc epsilon RL/CD23 induction is specific for IL-4 since IL-1 alpha, IL-2, IFN-gamma, B cell-derived B cell growth factor (BCGF), and a low-molecular-weight BCGF were ineffective. IFN-gamma strongly inhibited the induction of Fc epsilon RL/CD23 by IL-4.     

Expression of functional high affinity immunoglobulin E receptors (Fc epsilon RI) on monocytes of atopic individuals

Suggestive evidence indicates that immunoglobulin E (IgE)-dependent activation of mononuclear phagocytes plays an important pathogenic role in allergic tissue inflammation. Prevailing opinion holds that low affinity IgE receptors are the relevant IgE-binding structures on monocytes/macrophages and that functional events occurring after cross- linking of membrane-bound IgE on these cells are mediated by these receptors. Here we demonstrate that peripheral blood monocytes can bind monomeric IgE via the high affinity IgE receptor (Fc epsilon RI) and that Fc epsilon RI expression on these cells is upregulated in atopic persons. Further, we demonstrate that, upon monocyte adherence to substrate, bridging of monocyte Fc epsilon RI is followed by cell activation. We propose that direct interaction of multivalent allergen with Fc epsilon RI(+)-bound IgE on mononuclear phagocytes results in cell signaling via Fc epsilon RI and that the biological consequences of this event may critically influence the outcome of allergic reactions.     

Pseudo-high affinity interleukin 2 (IL-2) receptor lacks the third component that is essential for functional IL-2 binding and signaling

Functional studies of the interleukin 2 receptor (IL-2R) of two (ED515-D and Kit225) IL-2-dependent and three (ED515-I, 3T3-alpha beta 11, and Hut102) IL-2-independent cell lines were done. All of these cell lines appeared to express high as well as low affinity IL-2R. However, ED515-I and 3T3-alpha beta 11, which expressed the IL-2R beta chain, did not bind IL-2 at all when IL-2 binding to their IL-2R alpha chain was blocked with anti-Tac monoclonal antibody, whereas the intermediate affinity binding in ED515-D, Kit225, and Hut102 cells remained. We tentatively called the high affinity IL-2R of the former cells pseudo-high affinity IL-2R. The dissociation constant of pseudo-high affinity IL-2R was higher than that of ordinary high affinity IL-2R. Internalization of cell-bound 125I-IL-2 into ED515-I and 3T3-alpha beta 11 cells was less efficient than that into ED515-D cells. The addition of IL-2 neither promoted cell growth nor upregulated IL-2R alpha chain expression in ED515-I and 3T3-alpha beta 11 cells. Furthermore, tyrosine phosphorylation of the cellular proteins (p120, p98, p96, p54, and p38) was induced or enhanced in response to the addition of IL-2 in ED515-D and Kit225 cells, but not in the cell lines expressing pseudo-high affinity IL-2R. Finally, 125I-IL-2 crosslinking followed by SDS-PAGE analysis showed an 80-kD band corresponding to p65 + IL-2, in addition to bands corresponding to IL-2R alpha and beta chain + IL-2 in cells bearing ordinary high affinity IL-2R but not in cells with pseudo-high affinity IL-2R. Taken together, we consider that another protein whose molecular mass is approximately 65 kD is functionally important in IL-2 binding and subsequent signal transduction and may be the third component of IL-2R.     

Interleukin (IL)-4 inhibits IL-10 to promote IL-12 production by dendritic cells

Interleukin (IL)-4 is known to be the most potent cytokine that can initiate Th2 cell differentiation. Paradoxically, IL-4 instructs dendritic cells (DCs) to promote Th1 cell differentiation. We investigated the mechanisms by which IL-4 directs CD4 T cells toward the Th1 cell lineage. Our study demonstrates that the IL-4-mediated induction of Th1 cell differentiation requires IL-10 production by DCs. IL-4 treatment of DCs in the presence of lipopolysaccharide or CpG resulted in decreased production of IL-10, which was accompanied by enhanced IL-12 production. In IL-10-deficient DCs, the level of IL-12 was greatly elevated and, more importantly, the ability of IL-4 to up-regulate IL-12 was abrogated. Interestingly, IL-4 inhibited IL-10 production by DCs but not by B cells. The down-regulation of IL-10 gene expression by IL-4 depended on Stat6 and was at least partly caused by decreased histone acetylation of the IL-10 promoter. These data indicate that IL-4 plays a key role in inducing Th1 cell differentiation by instructing DCs to produce less IL-10.     

P2z受体在介导慢性淋巴细胞白血病细胞凋亡中的作用

目的：研究嘌呤能Ｐ２ｚ受体在介导慢性淋巴细胞白血病（ＣＬＬ）细胞凋亡中的作用。方法：将表达Ｐ２ｚ受体［Ｐ２ｚ（＋）］与不含Ｐ２ｚ受体［Ｐ２ｚ（－）］的两组ＣＬＬ细胞分别同１．０ｍｍｏｌ／Ｌ三磷酸腺苷（ＡＴＰ）体外培养８小时，以电镜、ＤＮＡ凝胶电泳和定量ＤＮＡ３′端ＴｄＴ法检测细胞凋亡；并对ＡＴＰ、苯甲酰苯甲酸ＡＴＰ（ＢｚＡＴＰ）、２甲基硫ＡＴＰ（２ＭｅＳＡＴＰ）、γ硫代ＡＴＰ（ＡＴＰγＳ）及其它核苷的诱导效应和氧化型ＡＴＰ（ＯｘＡＴＰ）、１［Ｎ，Ｏ二（５异喹啉碘酰基）Ｎ甲基Ｌ酪氨酰］４苯哌嗪（ＫＮ６２）的抑制效应做定量研究。结果：①Ｐ２ｚ＋细胞能在ＡＴＰ诱导下呈现典型的细胞凋亡形态和ＤＮＡ梯形条带；②不同的Ｐ２ｚ受体激活剂可通过Ｐ２ｚ受体诱导细胞凋亡并产生不同量的ＤＮＡ降解片段，诱导能力的强弱顺序是ＢｚＡＴＰ＞ＡＴＰ＞２ＭｅＳＡＴＰ，而ＡＴＰγＳ或其它核苷几乎无诱导能力；③ＯｘＡＴＰ和ＫＮ６２可完全阻止诱导剂诱导的细胞凋亡。结论：Ｐ２ｚ受体在介导由ＡＴＰ等诱导ＣＬＬ细胞凋亡的过程中起着十分重要的作用     

Interleukin 8 (IL-8) selectively inhibits immunoglobulin E production induced by IL-4 in human B cells

The effect of interleukin 8 (IL-8) on IL-4-induced immunoglobulin E (IgE) production was studied. IL-4 induced IgE and IgG4 production by tonsillar mononuclear cells (MNC) without affecting IgM, IgG1, IgA, IgG2, or IgG3 production. IL-8 inhibited IL-4-induced IgE and IgG4 production, whereas it had no effect on IgM, IgG1, IgA, IgG2, and IgG3 production. The inhibitory effect by IL-8 was specific, since it was blocked by anti-IL-8 mAb, but not by control IgG1. Although interferon gamma (IFN-gamma) also inhibited IgE and IgG4 production by MNC stimulated with IL-4, the inhibitory effect of IL-8 was not mediated by IFN-gamma, since the IL-8-induced inhibition could not be blocked by anti-IFN-gamma. Furthermore, anti-IL-8 mAb had no effect on IFN-gamma-induced inhibition. Moreover, addition of IL-5 or IL-6 did not reverse IL-8-induced inhibition of IgE production. In contrast to these observations with MNC, IL-4 failed to induce IgE and IgG4 production by purified B cells. However, combined treatment of purified B cells cells with IL-4 and anti-CD40 antibody resulted in IgE but not IgG4 production. IL-8 inhibited this IgE production without affecting IgM, IgG1, IgG2, IgG3, IgG4, or IgA production, whereas IFN-gamma, IFN-alpha, or prostaglandin E2 (PGE2) failed to do so. These results indicate that IL-8 antagonizes IL-4-induced IgE production by directly affecting B cells through a specific mechanism that is different from IFN-gamma, IFN-alpha, or PGE2.     

An Interleukin (IL)-10/IL-12 Immunoregulatory Circuit Controls Susceptibility to Autoimmune Disease

Cells of the innate immune system secrete cytokines early in immune responses that guide maturing T helper (Th) cells along appropriate lineages. This study investigates the role of cytokine networks, bridging the innate and acquired immune systems, in the pathogenesis of an organ specific autoimmune disease. Experimental allergic encephalomyelitis (EAE), a demyelinating disease of the central nervous system, is widely used as an animal model for multiple sclerosis. We demonstrate that interleukin (IL)-12 is essential for the generation of the autoreactive Th1 cells that induce EAE, both in the presence and absence of interferon γ. The disease-promoting effects of IL-12 are antagonized by IL-10 produced by an antigen nonspecific CD4⁺ T cell which, in turn, is regulated by the endogenous production of IL-12. This unique immunoregulatory circuit appears to play a critical role in controlling Th cell differentiation and provides a mechanism by which microbial triggers of the innate immune system can modulate autoimmune disease.     

Interleukin 10 (IL-10) inhibits human lymphocyte interferon gamma- production by suppressing natural killer cell stimulatory factor/IL-12 synthesis in accessory cells

Natural killer cell stimulatory factor or interleukin 12 (NKSF/IL-12) is a heterodimeric cytokine produced by monocytes/macrophages, B cells, and possibly other accessory cell types primarily in response to bacteria or bacterial products. NKSF/IL-12 mediates pleiomorphic biological activity on T and NK cells and, alone or in synergy with other inducers, is a powerful stimulator of interferon gamma (IFN- gamma) production. IL-10 is a potent inhibitor of monocyte-macrophage activation, that inhibits production of tumor necrosis factor alpha (TNF-alpha), IL-1 and also IFN-gamma from lymphocytes acting at the level of accessory cells. Because TNF-alpha and IL-1 are not efficient inducers of IFN-gamma, the mechanism by which IL-10 inhibits IFN-gamma production is not clear. In this paper, we show that IL-10 is a potent inhibitor of NKSF/IL-12 production from human peripheral blood mononuclear cells activated with Staphylococcus aureus or lipopolysaccharide (LPS). Both the production of the free NKSF/IL-12 p40 chain and the biologically active p70 heterodimer are blocked by IL- 10. NKSF/IL-12 p40 chain mRNA accumulation is strongly induced by S. aureus or LPS and downregulated by IL-10, whereas the p35 mRNA is constitutively expressed and only minimally regulated by S. aureus, LPS, or IL-10. Although IL-10 is able to block the production of NKSF/IL-12, a powerful inducer of IFN-gamma both in vitro and in vivo, the mechanism of inhibition of IFN-gamma by IL-10 cannot be explained only on the basis of inhibition of NKSF/IL-12 because IL-10 can partially inhibit IFN-gamma production induced by NKSF/IL-12, and also, the IFN-gamma production in response to various stimuli in the presence of neutralizing antibodies to NKSF/IL-12. Our findings that antibodies against NKSF/IL-12, TNF-alpha, or IL-1 beta can significantly inhibit IFN-gamma production in response to various stimuli and that NKSF/IL-12 and IL-1 beta can overcome the IL-10-mediated inhibition of IFN-gamma, suggest that IL-10 inhibition of IFN-gamma production is primarily due to its blocking production from accessory cells of the IFN-gamma- inducer NKSF/IL-12, as well as the costimulating molecule IL-1 beta.     

Interleukin 2 receptors on human B cells. Implications for the role of interleukin 2 in human B cell function

In the present study, we examined the expression of interleukin 2 (IL-2) receptors on normal human B cells as well as established B cell lines. Anti-Tac monoclonal antibody did not bind to freshly separated normal human B cells. Unexpectedly, with the appropriate activation of the normal B cells by anti-mu antibody, phorbol myristate acetate, or Staphylococcus aureus Cowan I (SAC), Tac antigen was induced on the activated B cells. Anti-Tac antibody showed consistent reactivity with two B cell lines that were infected by human T cell leukemia virus (HTLV) and some reactivity with two out of eight Epstein-Barr virus-transformed B cell lines established from normal adult donors. Immunoprecipitation analysis revealed that antigens of similar size with a molecular weight of 50,000-60,000 can be precipitated with anti-Tac antibody from phytohemagglutinin-stimulated normal T cell blasts and normal activated B cells, as well as a cloned B cell line. Binding assays of IL-2 on normal activated B cells and on the cloned B cell (HS1) revealed that B cells have significantly fewer sites and lower-affinity IL-2 receptors compared with phytohemagglutinin-stimulated normal T cell blasts. Finally, biological properties of the IL-2 receptor on B cells were examined by incubating B cells with recombinant IL-2. It was found that moderate concentrations of IL-2 induce significant enhancement of proliferation and differentiation in SAC-activated normal B cells. These results suggest that normal B cells may express functional IL-2 receptors or closely related proteins and thus IL-2 may play a significant role in the modulation of B cell function.     

Inability of mitogen-activated lymphocytes obtained from patients with malignant primary intracranial tumors to express high affinity interleukin 2 receptors.

Patients with primary malignant brain tumors manifest a variety of abnormalities in cell-mediated and humoral immunity. Diminished T cell reactivity has been shown in these patients to be linked to deficiencies in interleukin 2 (IL-2) production that cannot be overcome by exogenous IL-2. In this study, specific binding of radiolabeled IL-2 to PHA-stimulated lymphocytes from brain tumor patients demonstrates that the number of high affinity interleukin 2 receptors (IL-2R) is greatly reduced. FACS analysis indicates that the relative density of the p55 protein (Tac protein) is lower on the mitogen-activated lymphocytes obtained from patients than on comparably treated lymphocytes from normal individuals. These data indicate that mitogen-stimulated lymphocytes obtained from patients have fewer functional high affinity IL-2R principally because of the failure to express sufficient levels of the p55 protein for association with the p75 protein. Northern analysis of total RNA isolated from mitogen-stimulated T cells from patients demonstrates normal levels of steady state mRNA, which codes for the p55 protein. Moreover, there is no defect in the postranslational processing of the primary translation product of this mRNA suggesting that normal levels of the p55 protein are produced in activated T cells from patients.     

Interleukin 12 (IL-12) induces tyrosine phosphorylation of JAK2 and TYK2: differential use of Janus family tyrosine kinases by IL-2 and IL- 12

Interleukin (IL-12) has many effects on the function of natural killer and T cells, and is important in the control of cell-mediated immunity. IL-2 and IL-12 display many similar activities, yet each also induces a distinct set of responses. A human IL-12 receptor subunit has recently been cloned and, like the IL-2R beta and IL-2R gamma, is a member of the hematopoietic receptor superfamily; however, the molecular mechanisms of IL-12 action are unknown. In this report we show that IL- 12 and IL-2 induce tyrosine phosphorylation of distinct members of the Janus (JAK) family of protein tyrosine kinases in human T lymphocytes. IL-12, but not IL-2, stimulates the tyrosine phosphorylation of TYK2 and JAK2, whereas JAK1 and JAK3, which are phosphorylated in response to IL-2, are not phosphorylated after IL-12 treatment. The use of distinct but related JAK family tyrosine kinases by IL-12 and IL-2 may provide a biochemical basis for their different biological activities.     

Prolactin releasing peptide has high affinity and efficacy at neuropeptide FF2 receptors

Neuropeptide FF (NPFF) and prolactin-releasing peptide (PrRP) are two members of the RFamide peptide family. In this study we investigated whether these RFamide peptides, which have common structural features in their C-terminal RFamide motif and share several physiologically important functions, could exert their effects through the same set of receptors. The affinity and functional activity of several related RFamide peptides were determined at the human neuropeptide FF receptor subtype 2 (hNPFF2) and the human prolactin-releasing peptide (hPrRP) receptors. The full-length human prolactin releasing peptide 31 (hPrRP31) had significantly higher efficacy compared with NPFF and its stable analog, (1DMe)Y8Fa, at the hNPFF2 receptor. In contrast, NPFF and (1DMe)Y8Fa were not efficacious at the hPrRP receptor. Our study indicated a generally relatively low level of discrimination for RFamide peptides at the NPFF receptor, whereas the hPrRP receptor clearly preferred PrRP or very closely related peptides. The seemingly promiscuous binding of the RFamide peptides to the NPFF receptor was further confirmed by receptor autoradiography. PrRP may thus signal through the NPFF receptors in vivo.     

Detection and characterization of human high molecular weight B cell growth factor receptors on leukemic B cells in chronic lymphocytic leukemia.

Human high molecular weight-B cell growth factor (HMW-BCGF) (60 kD) stimulates activated normal B cells, B cell precursor acute lymphoblastic leukemia (BCP-ALL) cells, hairy cell leukemia (HCL) cells, prolymphocytic leukemia (PLL) cells, and chronic lymphocytic leukemia (CLL) cells. The expression of human high molecular weight B cell growth factor (HMW-BCGF) receptors (R) on clonal populations of leukemic B cells in CLL was studied by ligand binding assays using 125I-labeled HMW-BCGF as well as by immunofluorescence/flow cytometry and Scatchard analyses using an anti-HMW-BCGF R monoclonal antibody (MAb), designated BA-5. There was a high correlation between HMW-BCGF R expression and responsiveness to HMW-BCGF. 60% of CLL cases constitutively expressed HMW-BCGF R and showed a marked proliferative response to HMW-BCGF in [3H]TdR incorporation assays as well as colony assays. Similarly, HCL cells, PLL cells, and activated normal B cells expressed functional HMW-BCGF R, as determined by ligand binding assays using 125I-HMW-BCGF, [3H]TdR incorporation assays, and reactivity with BA-5 MAb. Scatchard analyses indicated the existence of approximately 3,000 HMW-BCGF R/cell on HMW-BCGF responsive CLL cells with an apparent Ka value of 4.6 X 10(7) M-1. The concentrations of HMW-BCGF required for maximum stimulation of CLL cells were two to three orders of magnitude lower than those needed for half maximal receptor occupancy, indicating that only a small fraction of HMW-BCGF R need to be occupied to stimulate leukemic CLL B cells. Crosslinking of surface bound 125I-HMW-BCGF (60 kD) with the bivalent crosslinker DTSSP to its binding site on fresh CLL cells identified a 150-kD HMW-BCGF/HMW-BCGF R complex, suggesting an apparent molecular weight of 90 kD for the receptor protein. The growth stimulatory effects of HMW-BCGF on clonogenic CLL cells did not depend on accessory cells or costimulant factors. The anti-HMW-BCGF R monoclonal antibody BA-5 disrupted HMW-BCGF/HMW-BCGF R interactions at the level of clonogenic CLL cells and inhibited HMW-BCGF-stimulated CLL colony formation in vitro. To our knowledge, this study represents the first detailed analysis of expression, function, and structure of HMW-BCGF R on B lineage CLL cells.     

Expression of Recombination Activating Genes in Germinal Center B Cells: Involvement of Interleukin 7 (IL-7) and the IL-7 Receptor

Mouse germinal center (GC) B cells have been shown to undergo secondary V(D)J (V, variable; D, diversity; J, joining) recombination (receptor editing) mediated by the reexpressed products of recombination activating gene (RAG)-1 and RAG-2. We show here that interleukin (IL)-7 as well as IL-4 was effective in inducing functional RAG products in mouse IgD⁺ B cells activated via CD40 in vitro. Blocking of the IL-7 receptor (IL-7R) by injecting an anti– IL-7R monoclonal antibody resulted in a marked suppression of the reexpression of RAG-2 and subsequent V(D)J recombination in the draining lymph node of immunized mice, whereas RAG-2 expression was not impaired in immunized IL-4–deficient mice. Further, these peripheral B cells activated in vitro or in vivo were found to express IL-7R. These findings indicate a novel role for IL-7 and IL-7R in inducing receptor editing in GC B cells.     

Characterization of the interleukin 2 receptors (IL-2R) expressed on human natural killer cells activated in vivo by IL-2: association of the p64 IL-2R gamma chain with the IL-2R beta chain in functional intermediate-affinity IL-2R

Interleukin 2 (IL-2) receptors expressed on the surface of activated T cells and natural killer (NK) cells exhibit a variety of affinity states depending on their subunit composition. Low-affinity binding is associated with a 55-kDa alpha chain, intermediate-affinity binding with a 70-75-kD beta chain, and high-affinity binding with a bimolecular complex of the alpha and beta subunits. In a previous study of the IL-2 receptors expressed on NK cells obtained from cancer patients after in vivo IL-2 therapy, we documented a discrepancy between the level of beta chain and the level of intermediate-affinity IL-2 binding sites expressed on the cell surface. Based on this result, we postulated that formation of intermediate-affinity receptor sites required a component in addition to the beta chain, and that this component was present at limiting levels in the patient NK cells. In the present study we have examined the structure of the intermediate-affinity receptor complex using monoclonal antibodies that recognize the beta chain, but that do not interfere with its ability to bind IL-2. Evidence is presented establishing the physical association of a novel protein of 64 kD with the beta chain in intermediate-affinity IL-2 binding sites. This molecule, termed IL-2R gamma chain, coprecipitated with beta chains prepared from cells that had been incubated with IL-2, but was undetectable in immunoprecipitates prepared in the absence of IL-2. Examination of gamma chain expression in post-IL-2 therapy NK cells, where only low levels of intermediate-affinity IL-2 binding were detectable, revealed that the gamma chain was associated with, on average, only 10-12% of the beta chains expressed on such cells. This contrasted with approximately equal levels of beta and gamma chain expression on YT cells, a cell line that has both high levels of cell surface beta chain expression and high levels of IL-2 binding. Thus, the ratio of gamma chain to beta chain present in the immunoprecipitates roughly correlated with the proportion of beta chain involved in intermediate-affinity receptor sites. This result suggests that the 64-kD gamma chain is the component responsible for regulating the affinity of IL-2 association with the beta subunit. By further defining the structural components necessary for IL-2 receptor formation, these studies provide additional insight into mechanisms whereby lymphocytes might regulate their responsiveness to IL-2.