Presence of a p70 IL-2-binding peptide on leukemic cells from various hemopoietic lineages

Recent studies have shown that IL-2R are composed of at least two polypeptide chains of 55 kDa (Tac or alpha-chain) and 70 to 75 kDa (p70 or beta-chain). The association of both chains forms high affinity IL-2R, whereas each chain alone binds IL-2 with a low (alpha-chain) or intermediate (beta-chain) affinity. So far, the p70 peptide has been found, in the absence of the Tac peptide, on the surface of lymphoid cells of T, B, or NK lineage. In this study, we investigated whether leukemic cells of various hemopoietic lineages expressed the p70 IL-2-binding protein. We found that both fresh leukemic cells obtained from patients, and cells from established leukemic lines of T cells, B cell, and myeloid origin constitutively expressed a p70 IL-2-binding protein on their surface, as detected by affinity cross-linking of radioiodinated IL-2. IL-2 binding and cross-linking to these cells was completely inhibited in the presence of an excess unlabeled rIL-2, but not with an anti-Tac mAb. Binding experiments on pre-B and myeloid cell lines revealed intermediate affinity IL-2R, whereas both high and intermediate affinity IL-2R were detected in T leukemic cells. The intermediate affinity binding of 125I-rIL-2 to the leukemic cell lines MOLT4 and Reh6 was inhibited by the TU27 mAb, which recognized the p75 chain of IL-2R. Moreover, the TU27 mAb could stain the K562, KM3, and MOLT4 (weakly) cell lines by indirect immunofluorescence. A high dose of rIL-2 (400 U/ml) enhanced the proliferation of cells from one out of three patients with acute myeloblastic leukemia, but it did not induce differentiation of the cells in any of three cases. Thus the finding of p70 IL-2-binding molecules on immature lymphoid and nonlymphoid hemopoietic cells should disclose new biologic functions for IL-2.     

Impaired expression of high affinity interleukin 2 receptor on activated lymphocytes from patients with systemic lupus erythematosus

Peripheral lymphocytes stimulated with phytohemagglutinin (PHA-blasts) were examined for their responsiveness to exogenous interleukin 2 (IL-2). The proliferative response of PHA-blasts to IL-2 was significantly lower in patients with systemic lupus erythematosus (SLE) than in normal subjects. To clarify the reason for this defect, the expression of IL-2 receptor (IL-2R) on PHA-blasts was investigated using anti-Tac antibody and purified IL-2. Cytofluorometric analysis showed no statistical differences in the Tac positivity of PHA-blasts among normal subjects and patients with active and inactive SLE. Scatchard analysis using 125I-labeled anti-Tac monoclonal antibody revealed that the number of Tac epitopes on PHA-blasts was also not different among them. Next, the affinity of IL-2R expressed on PHA-blasts was determined by Scatchard analysis using radiolabeled IL-2 as a ligand. The number of high affinity IL-2R on the PHA-blasts was significantly decreased in patients with active and inactive SLE, as compared with normal subjects. The responsiveness of PHA-blasts to exogenous IL-2 was well correlated to the number of high affinity IL-2R, but not to the number of Tac epitopes or total IL-2R. Inasmuch as high affinity components of IL-2R are functionally active, the defective expression of high affinity IL-2R may be responsible for the T cell dysfunctions in SLE.     

Immunomodulatory effects of transforming growth factor-beta on T lymphocytes. Induction of CD8 expression in the CTLL-2 cell line and in normal thymocytes.

We investigated the role of transforming growth factor-beta 1 (TGF-beta) in regulation of T cell growth and differentiation. Treatment of CTLL-2 cells with TGF-beta inhibited IL-2-dependent proliferation and caused morphologic changes as well as increased adherence. A major change of phenotype in TGF-beta-treated cells was the de novo expression of CD8 alpha chain in 35% of cells, which required the continuous presence of TGF-beta. Of the CD8 alpha+ cells, 20 to 30% co-expressed CD8 beta chain. Increased CD8 expression occurred even in the total absence of cell growth, was not a consequence of growth inhibition, and was not a result of selective growth or survival of CD8+ cells. New RNA synthesis was required for TGF beta-induced CD8 alpha surface expression, inasmuch as this was prevented by treatment with actinomycin D. Northern blot analysis demonstrated that cells treated with IL-2 + TGF-beta rapidly accumulated mRNA encoding both chains of the CD8 dimer, to a level fourfold greater than control by 6 to 12 h. In contrast, the IL-2-dependent increases in IL-2R alpha, IL-2R beta, and Granzyme B mRNA levels in these cultures were profoundly inhibited by TGF-beta. When unfractionated murine thymocytes were stimulated with phorbol dibutyrate plus ionomycin and cultured with IL-2 + TGF-beta, an increase in CD8 alpha mRNA was seen and greater numbers of CD8+ cells with higher levels of CD8 alpha and CD8 beta surface expression resulted, as compared to controls treated with IL-2 alone. Furthermore, similar treatment of CD4-CD8-(double negative) thymocytes with TGF-beta induced de novo CD8 alpha expression by a substantial number of cells, and the majority of these CD8+ cells lacked TCR/CD3. These data suggest that TGF-beta has both positive and negative regulatory effects on the expression of gene products important for T lymphocyte differentiation and function.     

The third molecule associated with interleukin 2 receptor alpha and beta chain.

It is known that the affinity cross-linking study of the human high-affinity Interleukin 2 (IL-2) receptor reveals triplet bands consisting of 70 kDa alpha chain(Tac)-IL-2 and the 90/80 kDa doublet. We found the cell lines lacking the lower band of the doublet in spite of the expression of both alpha and beta chains. No IL-2 binding was detectable in the presence of anti-Tac antibody in these cells. Immunoprecipitation from the cell extract of [125 I] IL-2-cross-linked T cells with anti-beta chain polyclonal IgG detected the upper band, but not lower band of the doublet. These data suggest that the lower band of the doublet represents an unknown IL-2-binding protein (p65) distinct from the beta chain and this molecule may be involved in the intermediate-affinity IL-2 binding together with the beta chain.     

Interleukin 5 up-regulates high-affinity interleukin 2 receptor expression by human resting peripheral T cells: a comparison with the effect of interleukin 4 on B cells.

The separate regulation mechanisms of cytokines on two classes of interleukin 2 receptors (IL-2R) on human peripheral T and B cells were analyzed by a flow cytometer using a double stain with IL-2R alpha (55 kilodalton Tac) and IL-2R beta (75 kilodalton mik beta 1, mik beta 3). Although the expression of IL-2R alpha by T cells was slightly enhanced by IL-2 and IL-4, expression of the beta chain was diminished by both cytokines. IL-5 by itself did not alter the expression of either IL-2R alpha or beta, but preculturing with IL-2 for 24 h followed by IL-5 for another 24 h induced an increase in IL-2R alpha expression and in simultaneous alpha/beta chain expression. Increased numbers of high-affinity IL-2R were confirmed by 125I binding assays. On B cells, IL-4 increased alpha, beta, and simultaneous alpha/beta chain expression, but IL-4-treated B cells did not show an increased number of high-affinity IL-2R.     

IL-4 inhibits IL-2 synthesis and IL-2-induced up-regulation of IL-2R alpha but not IL-2R beta chain in CD4+ human T cells.

There is growing evidence to suggest a regulatory role of IL-4 in the immune system affecting both proliferation and lymphokine production. In the present work we have analyzed the effect of IL-4 on IL-2 and IFN-gamma synthesis by stimulating CD4+ human T cells (+10% accessory cells) with Con A in the presence of several doses (1 to 100 U/ml) of human rIL-4. The results showed an impaired IL-2 and IFN-gamma synthesis in the presence of IL-4. This inhibition was dose dependent and was evident only when IL-4 was added in the first 2 h of culture. Moreover, the external addition of IL-2 did not revert the inhibitory effect of IL-4 on IL-2 and IFN-gamma synthesis induced by Con A. We have also analyzed the effect of IL-4 on the expression of both alpha- and beta-chains of the IL-2R. Although the expression of IL-2R alpha mRNA was not modified after 6 h in culture in the presence of IL-4, a decrease was observed at 24 and 48 h. The addition of rIL-2 showed that the inhibition in IL-2R alpha expression could be explained by an impairment in the up-regulatory signal transmitted through the IL-2R. In addition to this, IL-4 did not modify the IL-2R beta mRNA expression at 6 and 24 h although a decreased expression was observed at 48 h which could be explained by the defective IL-2 production. The differential effect of IL-4 on the up-regulatory effect of IL-2 in the expression of IL-2R alpha and IL-2R beta suggest the existence of different regulatory mechanisms acting on the expression of both chains.     

Regulation of beta2-adrenergic receptors on CD4 and CD8 positive lymphocytes by cytokines in vitro

Increasing evidence points to a close relationship between the autonomic nervous system and the immune system. To further investigate mechanisms regulating beta2-adrenergic receptor (beta2R) expression in lymphocytes, the influence of cytokines on the density of beta2R on purified CD4+ and CD8+ lymphocytes was determined in vitro. beta2R were determined by means of a radioligand binding assay with (125I)iodocyanopindolol. CD4+ and CD8+ lymphocytes were incubated with catecholamines, interleukin 1beta (IL-1beta) and interleukin 2 (IL-2) for 6-72 h. The results demonstrate declining beta2R numbers on CD4+ and CD8+ lymphocytes in vitro augmented by epinephrine. IL-1beta has no effect on beta2R expression compared to medium. However, incubation with IL-2 resulted in an up-regulation of beta2R on CD8+ lymphocytes. Thus, the study demonstrates a differential regulation of beta2R on T-lymphocyte subpopulations with CD8+ lymphocytes being more susceptible to mechanisms of beta2R modulation than CD4+ lymphocytes. The findings further strengthen the concept of a close interplay between the autonomic nervous system and the immune system.     

IL-2-R beta expression and function within resting CD8+ T cells preferentially segregate with the CD45R0+ subset.

Human peripheral blood CD8+ T cells constitutively express a low level of IL-2-R beta chains which were shown in this study to be preferentially carried by the CD45R0+ subset. Such receptors can transduce signals for in vitro IL-2-induced cytolytic function and for the initiation of soluble anti-CD3 and IL-2-induced cell proliferation. Using these stimulation models, a comparison was made between the responsiveness of resting, small CD45R0+ and CD45RA+ subpopulations of CD8+ T cells, both of them being isolated by negative selection and rigorously depleted of monocytes and of IL-2-inducible non-MHC-restricted CTL. Strong proliferation was induced in CD8+/CD45R0+ cells in response to IL-2 and soluble anti-CD3 (each of these stimuli being by itself ineffective), while in contrast, CD8+/CD45RA+ cells manifested, in this system, little reactivity. Accordingly, no conversion to the CD45R0 phenotype occurred in single stained CD45RA+ T cells following their incubation with the stimuli. A similar restriction of reactivity to CD8+/CD45R0+ T cells was observed with respect to IL-2-induced targetable T cell cytotoxicity. The CTL activity induced by IL-2 alone occurred without cell division. In contrast, the additional increase in CTL activity occurring upon the synergistic actions of anti-CD3 mAb and IL-2 coincided with intense cell proliferation, with no generation of LAK activity. The inhibition exerted by anti-IL-2-R beta mAb in the cytolytic and the proliferative activities induced by these stimuli in resting CD8+/CD45R0+ T cells emphasizes the importance of constitutive IL-2-R beta chains in the biology of these cells.     

Overexpression of interleukin-2 receptor alpha in a human squamous cell carcinoma of the head and neck cell line is associated with increased proliferation,drug resistance,and transforming ability

It has been previously demonstrated that human carcinomas express interleukin-2 receptor (IL-2R) alpha, beta, and gamma chains. The beta and gamma chains of IL-2R have intermediate binding affinity for IL-2 and are responsible for the intracellular signaling cascades after IL-2 stimulation. IL-2Ralpha lacks the cytoplasmic domain, but is essential for increasing the IL-2-binding affinity of other receptors. Overexpression of IL-2Ralpha in tumor cells is associated with tumor progression and a poor patient prognosis. To define molecular mechanisms responsible for the effects associated with IL-2Ralpha expression, ex vivo experiments were performed with the squamous cell carcinoma head-and-neck cancer line, PCI-13, which was genetically engineered to overexpress the IL-2Ralpha chain. While IL-2Ralpha-overexpressing PCI-13 cells were capable of forming colonies in soft agar, PCI-13 cells transfected with the control vector or those expressing IL-2Rgamma did not. Consistently, IL-2Ralpha-expressing tumor cells proliferated more rapidly than the control or IL-2Rgamma+ cells, associated with increased levels of cyclins A and D1 and cyclin-dependent kinase (cdk(s)) 2 and 4 proteins. In addition, IL-2Ralpha-expressing cells were significantly more resistant to apoptosis induction by a tripeptidyl proteasome inhibitor (ALLN) and two chemotherapeutic drugs (VP-16 and taxol) than the control or IL-2Rgamma+ cells. Accompanying the drug resistance, high levels of anti-apoptotic Bcl-X(L) and Bcl-2 proteins were found in the mitochondria-containing fraction of IL-2Ralpha-expressing tumor cells. Treatment of IL-2Ralpha-expressing cells with a specific Janus kinase 3 (Jak3) inhibitor decreased expression of cyclin A, cyclin D1, Bcl-X(L), and Bcl-2 proteins. Finally, high levels of ubiquitinated proteins were detected in the proliferating IL-2Ralpha-expressing cells. Our data suggest that increased proliferation rates and decreased drug sensitivity of IL-2Ralpha-expressing tumor cells are responsible for the enhanced tumor aggressiveness and poor clinical prognosis of patients whose tumors express IL-2Ralpha.     

Interleukin-1 receptors on human thyroid cells and on the rat thyroid cell line FRTL-5.

Cellular binding of interleukin-1 (IL-1) was tested on monolayers of human thyrocytes in secondary culture, on long-term cultures of human thyrocytes, and on the rat thyroid cell line FRTL-5. The human thyrocytes in secondary culture showed specific binding of human 125I-rIL-1 alpha. Scatchard plots of data obtained at 4 degrees C indicated the presence of a single population of receptors with a Kd of 30 to 170 pM and 2,000 to 6,000 receptors per cell. Incubation at room temperature resulted in internalization of the receptor-ligand complex. Parallel experiments were performed with the IL-1 receptor-positive murine T-cell lines EL-4 and NOB-1. The IL-1 receptors on these cells had Kd values one fifth to one tenth those on human thyroid cells in secondary culture. Both rIL-1 alpha and rIL-1 beta inhibited 125I-rIL-1 alpha binding to human thyrocytes and the murine T cells. In contrast to the cells in secondary culture, there was no specific binding of 125I-rIL-1 alpha to long-term cultivated human thyroid cells or to the FRTL-5 cells. We concluded that recently described differences in the response to IL-1 of different thyroid cell culture systems are most likely caused by differences in expression of IL-1 receptors.     

Regulation of CD3- lymphocyte function with an antibody against the IL-2 beta chain receptor: modulation of NK and LAK activity and production of IFN gamma

To elucidate the role of interleukin 2 (IL-2) activation in CD3- lymphocytes, we examined the ability of monoclonal antibody (MAb) TU27, developed against the IL-2 receptor (IL-2R) p75 protein (IL-2R beta), to block lymphocyte activation with exogenous IL-2, as well as its innate ability to activate lymphocytes as a result of its surface ligand interaction. The binding of the TU27 MAb and the results of 125I-IL-2 cross-linking experiments suggest that the IL-2R beta chain is expressed primarily on CD3-, CD56+ lymphocytes; although the protein was also detected in a small portion of CD3+ cells, its expression appeared to be donor dependent. In the present study, we found that TU27 totally blocked natural killer (NK) cell activation in a 4-h assay but had no effect on basal levels of NK activity. When treatment was extended to 24 to 72 h, the MAb was able to block the induction of both NK and lymphokine-activated killer (LAK) activity. Of interest was the observation that MAb treatment alone augmented NK activity and subsequent interferon gamma (IFN gamma) production in CD3- lymphocytes but did not activate LAK activity or induce cell growth. Collectively, these results indicate that TU27 not only reacts with p70-75 IL-2R beta but can abrogate IL-2 binding and subsequent activation events. In addition, some CD3- lymphocyte functions (e.g., NK activity and IFN gamma secretion) are directly induced by the binding of MAb to p70-75 through signals that only partially mimic IL-2.     

Interleukin-4 receptors on human blood mononuclear cells

We have studied regulation of the expression of the interleukin-4 receptor (IL-4R) on human blood mononuclear cells (PBMC) using both 125I-IL-4 binding assay and flow cytometric analysis of biotinylated IL-4 (B-IL-4) binding. PBMC express approximately 300 high-affinity IL-4R per cell (Kd = 25-100 pM). Activation of PBMC for 60-80 hr by phytohemagglutinin (PHA) or concanavalin A (Con A) results in a 2- to 4.5-fold increase of IL-4R number without alteration of IL-4R affinity for IL-4. Binding of B-IL-4 showed that IL-4R expression is upregulated on virtually all PHA-stimulated PBMC, whereas it mostly concerns larger cells among Con A-activated PBMC. Reculture of PHA-blasts with 1 nM IL-4 further upregulates IL-4R expression to a level approximately 10-fold higher than observed on freshly isolated PBMC. Interestingly, IL-4 is able to reinduce high IL-4R levels on cells that have been deprived of IL-4 for 20 hr and IL-2 is almost as efficient. Finally, SDS-PAGE analysis of IL-4-binding molecules on unstimulated, PHA- and PHA/IL-4-activated PBMC revealed the same three peptides of MW 140-130, 80-75, and 70-65 kDa, as shown on human cell lines.     

Depressed IL-12-mediated signal transduction in T cells from patients with Sézary syndrome is associated with the absence of IL-12 receptor beta 2 mRNA and highly reduced levels of STAT4.

Sézary syndrome (SS) is the leukemic phase of cutaneous T cell lymphoma characterized by the proliferation of clonally derived CD4+ T cells that release cytokines of the Th2 T cell phenotype (IL-4, IL-5, IL-10), whereas Th1 T cell cytokines (IL-2, IFN-gamma) are markedly depressed as is expression of IL-12, a pivotal cytokine for Th1 cell differentiation. Normal Th1 cells express both the beta 1 and beta 2 chains of the IL-12 receptor (IL-12R) and tyrosine phosphorylate STAT4 in response to IL-12. Th2 T cells express only the IL-12R beta 1 and thus do not tyrosine phosphorylate STAT4 in response to IL-12. To determine whether SS cells are Th2-like at the level of IL-12 signal transduction, we analyzed RNA from seven patients for the presence of message for the IL-12R beta 1 and beta 2 genes using RNase protection assays and assessed whether IL-12 induced tyrosine-phosphorylation of STAT4 by immunoblotting. In PBL from six of seven SS patients tested, beta 2 message was expressed at low to undetectable levels and its expression could not be stimulated by either IFN-alpha or IFN- gamma, which stimulated beta 2 expression in control PBL. The absence of beta 2 expression is further supportive evidence for the Th2 lineage of SS cells. However, unlike normal Th2 cells, SS cells also showed severely reduced levels of STAT4, suggesting that they have a depressed response to any inducer of the STAT4 signal transduction pathway, including IFN-alpha. This is the first observation linking STAT4 gene expression with a human disease and suggests that dysregulation of STAT4 expression may be significant to the development and/or progression of SS.     

Expression of murine IL-2 receptor beta-chain on thymic and splenic lymphocyte subpopulations as revealed by the IL-2-induced proliferative response in human IL-2 receptor alpha-chain transgenic mice

Lymphocytes from the human (h) IL-2R alpha chain transgenic mice (TGM) constitutively express high affinity binding sites for hIL-2, consisting of transgenic h-IL-2R alpha and endogenous murine IL-2R beta, and therefore easily proliferate in vitro in response to hIL-2. Our study was undertaken to clarify the hIL-2-responsive lymphocyte subsets in the TGM, which should most likely reflect the normal distribution of m IL-2R beta expression. In both thymus and spleen, the majority of expanded cells by hIL-2 was CD3+CD4-CD8+ TCR alpha beta+ cells. The proliferation of CD4+ cells was not observed at all from either organ despite the expression of transgenic hIL-2R alpha. Potent cellular proliferation was also observed from the thymocytes that had been depleted of CD8+ cells, the expanded cells consisting of CD3- (15-40%) and CD3+ populations (60-85%). Among CD3+ cells, approximately the half portion expressed TCR alpha beta, whereas the other half was suggested to express TCR gamma delta. A variable portion (5-20%) of the CD3+ cells expressed CD8 (Lyt-2) in the absence of Lyt-3, and the CD3+CD8+ cells were confined preferentially to the TCR alpha beta- (TCR gamma delta+) population. In the culture of splenocytes depleted of CD8+ cells, however, the proliferated cells were mostly CD3-CD4-CD8-TCR-Mac1-, whereas a minor portion (10-30%) was CD3+CD4-CD8-TCR alpha beta- (TCR gamma delta+. Analysis of TCR genes at both DNA and mRNA levels confirmed the phenotypical observations. These results strongly suggested that IL-2R beta was constitutively and selectively expressed on the primary murine thymocytes and splenic T and NK cells, except for CD4+ cells in both organs.     

IL-12 receptor. II. Distribution and regulation of receptor expression.

IL-12 is a heterodimeric lymphokine that induces IFN-gamma production by resting PBMC, enhances the lytic activity of NK/lymphokine activated killer cells, and causes the proliferation of activated T cells and NK cells. In this report, we have investigated the expression of IL-12R on mitogen- and IL-2-activated PBMC or tonsillar lymphocytes as well as on a variety of cell lines. The results of radiolabeled IL-12-binding assays indicated that high affinity IL-12R are present on PBMC activated by various T cell mitogens or by IL-2. High affinity IL-12R were also found to be expressed constitutively on a transformed marmoset NK-like cell line HVS.SILVA 40. At the time of peak IL-12R expression, mitogen- or IL-2-activated cells displayed approximately 1000 to 9000 IL-12 binding sites/cell with an apparent Kd of 100 to 900 pM. Kinetic studies revealed that maximum expression of IL-12R occurred earlier on PHA-activated PBMC as compared with PBMC activated by IL-2, and that expression of IL-12R on these cells correlated with their ability to proliferate in response to IL-12. Although IL-2 could up-regulate IL-12R expression on resting PBMC, the ability of mitogen-activated PBMC to up-regulate IL-12R was found to be independent of IL-2. Analysis of IL-12R expression by flow cytometry revealed that receptors for IL-12 are present on activated T cells of both the CD4+ and CD8+ subsets and on activated CD56+ NK cells. In contrast, neither resting PBMC or tonsillar B cells nor tonsillar B cells activated by anti-IgM/Dx, anti-IgM/Dx + IL-2, or SAC + IL-2 displayed IL-12R detectable by flow cytometry or by the radiolabeled IL-12-binding assay. In summary, these results indicate that activation of T cells or NK cells results in up-regulation of IL-12R expression; on the other hand, B cell activation, at least under some circumstances, appears not to be associated with enhanced expression of IL-12R.     

Allergen rush immunotherapy increases interleukin (IL)-12 production and IL-12 receptor beta2 chain expression in patients with allergic asthma

Interleukin (IL)-12 production and IL-12 receptor (IL-12R) beta2 chain expression were investigated in patients with allergic asthma successfully treated with rush immunotherapy (RIT) and control patients with mild allergic asthma. Peripheral blood mononuclear cells (PBMCs) were stimulated with Dermatophagoides farinae (Der f), and production of cytokines was measured. Furthermore, the effects of cytokines on IL-12R beta2 chain expression on CD4(+) T cells were investigated. Production by PBMCs of IL-12 and IFN-gamma was significantly higher and production of IL-4 was significantly lower after stimulation with Der f allergen in RIT-treated patients than in control patients. Significant increases in the expression of IL-12R beta2 chain before and after stimulation of CD4(+) T cells with IL-12 or IFN-gamma were observed in RIT-treated patients compared with that in control patients. Allergen RIT increases IL-12 production and IL-12R beta2 chain expression and thus may convert cytokine production from Th2 to Th1 or Th0 type in allergic asthma.     

Plasticity of IL-2 and IL-2 receptor chains in rat lymphoid tissues in situ after stimulation with staphylococcal enterotoxin A

Although the effects of mitogens on the synthesis of interleukin-2 (IL-2) and IL-2 receptor (IL-2r) have been described, a detailed in situ analysis of the spatio-temporal changes of the expression of the IL-2 gene and the three IL-2r components in lymphoid tissues is still missing. Therefore, we analyzed the IL-2 and IL-2r expression after a staphylococcal enterotoxin A (SEA)-induced T cell activation on a cellular and anatomical basis in the Wistar rat. SEA caused a rapid induction of IL-2 mRNA in T cells of spleen, lymph node, and thymus, followed by the appearance of high systemic IL-2 serum levels (5 ng/ml), and a significant increase of CD25 on CD4+ and CD8+ lymphocytes. The histotopographic analysis of the IL-2r chains revealed a strong upregulation of IL-2r alpha (alpha) and IL-2r beta (beta) mRNAs in similar T cell specific compartments of spleen, lymph node, and thymus as seen for IL-2 mRNA. The abundant constitutive expression of IL-2r gamma (gamma) mRNA was unaffected by SEA. The parallel upregulation of IL-2, IL-2ralpha, and beta chains in conjunction with the continuous presence of the IL-2rgamma chain predominantly in T cell regions of immune organs suggests that the biological effects of IL-2 are essentially limited to T cells, at least after superantigen stimulation.     

Expression of CD5 regulates responsiveness to IL-1

The role of the CD5 surface molecule in T cell responsiveness to IL-1 was examined. A CD5-mutant Jurkat cell line was generated from a CD5+ parent cell line. This CD5- mutant subclone was infected with a defective retrovirus containing the CD5 cDNA and/or the neo gene encoding G418 resistance. The CD5+ wild type Jurkat produced IL-2 in response to anti-CD3 mAb, OKT3, cross-linked to a solid surface. IL-2 production was enhanced by co-culture with IL-1 or anti-CD5 Mab. Neither the CD5- mutant nor the CD5- G418-resistant infectant responded to anti-CD5 mAb or to IL-1. Responsiveness to IL-1 was restored by cell surface expression of CD5 in the CD5+ infectant. Both the CD5+ wild type Jurkat and the CD5+ infectant responded equivalently to purified IL-1, IL-1 alpha and rIL-1 beta. Optimal concentrations of IL-1 and anti-CD5 mAb had an additive effect on the enhancement of IL-2 production stimulated with cross-linked anti-CD3 mAb suggesting that IL-1 and CD5 act through distinct, complementary pathways to augment T cell activation. The correlation of CD5 expression and specific binding of rIL-1 beta was examined in these cell lines. Both the specific binding (at 4 degrees C) and subsequent internalization (at 37 degrees C) of 125I labeled rIL-1 beta was equivalent in the CD5+ infectant and the CD5+ wild type Jurkat cell, whereas specific binding of 125I-labeled rIL-1 beta was markedly decreased in the CD5-G418-resistant infectant. These observations strongly suggest that cell surface expression of CD5 regulates binding of and responsiveness to IL-1.     

Role of the interleukin 5 receptor system in hematopoiesis: molecular basis for overlapping function of cytokines.

Interleukin 5 (IL-5) is a kind of peptide hormone released from T lymphocytes of mammals infected with microorganisms or parasites. It is an acidic glycoprotein with a molecular mass of 40 to 50 kDa that consists of a homodimer of polypeptides. It controls hematopoiesis so that it increases natural immunity. In the mouse, IL-5 acts on committed B cells to induce differentiation into Ig-producing cells and on common progenitors for CD5+ pre-B cells and CD5+ macrophages to support their survival. The antibodies secreted by CD5+ B cells seem to be responsible for the primary protection against the infection with microorganisms or parasites. It also supports the growth and/or differentiation of eosinophil precursor and mature eosinophils, which can be effective for the removal of parasites in combination with the antibodies against them. Murine IL-5 receptor (IL-5R) consists of two different polypeptide chains; alpha chain and beta chain. The IL-5R alpha chain is 60 kDa protein that binds IL-5 with low affinity. The IL-5R beta chain is a 130 kDa protein which does not bind IL-5 by itself but is necessary to form the high affinity IL-5R. The beta chain was identified by using one of the anti-IL-5R mAb and anti-IL-3R mAb as the IL-3R homologue. This beta chain is also used as the beta chain of GM-CSF receptor. This fact suggests that there is a common signaling mechanism among these cytokines and efficient cooperation among them. At the same time, these findings may explain the overlapping role of these cytokines in the development of granulocytes.     

Functional reconstitution and regulation of IL-18 activity by the IL-18R beta chain

IL-18 and IL-12 are major IFN-gamma-inducing cytokines but the unique synergism of IL-18 and IL-12 remains unclear. In the human NK cell line NKO, IL-18R alpha, and IL-18R beta are expressed constitutively but IL-18 did not induce IFN-gamma unless IL-12 was present. COS-1 fibroblasts, which produce the chemokine IL-8 when stimulated by IL-1 beta or TNF-alpha, do not respond to IL-18, despite abundant expression of the IL-18R alpha chain. COS-1 cells lack expression of the IL-18R beta chain. The IL-18R beta cDNA was cloned from a human T-B lymphoblast cDNA library and COS-1 cells were transiently transfected with the IL-18R beta chain and a luciferase reporter. In transfected COS-1 cells, IL-18 induced IL-8 and luciferase in the absence of IL-12 and independently of IL-1 and TNF. Ab against the IL-18R alpha chain, however, prevented IL-18 responsiveness in COS-1 cells transfected with the IL-18R beta chain, suggesting that both chains be functional. In NKO cells and PBMC, IL-12 increased steady-state mRNA levels of IL-18R alpha and IL-18R beta; the production of IFN-gamma corresponded to IL-12-induced IL-18R alpha and IL-18R beta chains. We conclude that functional reconstitution of the IL-18R beta chain is essential for IL-12-independent proinflammatory activity of IL-18-induced IL-8 in fibroblasts. The synergism of IL-18 plus IL-12 for IFN-gamma production is, in part, due to IL-12 up-regulation of both IL-18R alpha and IL-18R beta chains, although postreceptor events likely contribute to IFN-gamma production.