Effects of hyperlipaemic serum on interleukin-2 (IL-2) production, IL-2 receptor expression, and T cell proliferation induced by IL-2 in cynomolgus monkeys

The effect of hyperlipaemic serum on mitogen-induced T lymphocyte proliferation was investigated with cynomolgus monkeys. The mitogen-induced blastogenesis was remarkably inhibited when either hyperlipaemic or normal monkey lymphocytes were incubated with hyperlipaemic sera. Hyperlipaemic serum also inhibited ConA-induced interleukin 2 (IL-2) production as well as IL-2 receptor (IL-2R) expression of normal monkey lymphocytes. On the other hand, it showed slight inhibition of T-cell proliferation induced by adding recombinant human IL-2 to IL-2R-positive normal monkey lymphocytes. These results indicate that hyperlipaemic serum inhibited an early stage of T-cell autocrine activation pathway including IL-2 production and IL-2R expression.     

Defective production of and response to IL-2 in acute human falciparum malaria

Patients with acute Plasmodium falciparum malaria have defective cell-mediated immune responses to malaria-specific Ag (MA). This immunologic defect may partially explain the difficulty with which natural immunity to falciparum malaria develops and may have important implications for the efficacy of potential malaria vaccines in endemic areas. To investigate the basis of this immune defect, we have examined the capacity of PBMC from patients with acute falciparum malaria to produce IL-2 and to express I1-2R in response to Ag stimulation. The effect of exogenous IL-1 and IL-2 on lymphocyte proliferation was studied. Soluble IL-2R levels were measured in acute and convalescent sera. Our results showed that no detectable IL-2 was produced and no IL-2R were expressed by PBMC in response to MA during the acute infection. IL-2 production and IL-2R expression were also depressed when PBMC were exposed to streptococcal Ag. The specific immune defect was not reconstituted by the addition of graded doses of purified human IL-1 or IL-2 and could not be attributed to suppressor adherent cells. In contrast to the absence of IL-2 and cell-bound IL-2R, circulating soluble IL-2R was elevated in acute sera. These findings suggest that the lack of IL-2, through either a defect in its production or inhibition of its activity, may be the basis of the Ag-specific immune unresponsiveness in acute P. falciparum malaria.     

The IL-2A receptor pathway and its role in lymphocyte differentiation and function

Murine myeloid cells are developed from hemopoietic stem/progenitor cells. Different types of progenitor cells have variable differentiation potentials. Among the ten main types of cells differentiated from lymphoid progenitor cells, regulatory T cells (Tregs), an important cell subpopulation regulating immune and inflammatory responses, arise from the hematopoietic stem cells in the bone marrow. Tregs then differentiate into T lymphocytes and migrate to the thymus and finally generate Treg subsets, which are subsequently activated and regulated by inflammatory cytokines in the peripheral blood. Tregs also have different phenotypes and immunomodulatory functions. The cytokine interleukin-2/interleukin-2 receptor (IL-2/IL-2R) pathway is an important regulatory signaling pathway of Tregs. Besides, different types of CD4⁺ and CD8⁺ cells have different immune effects in the absence of IL-2. IL-2R consists of three subunits, α chain (CD25), β chain (CD122), and γ chain (CD132). Different subunit combinations have different effects on the activation of immune cells. Multiple studies have shown that IL2RA deficiency has various effects on the immune function in mice. This article reviews the subunit composition and signaling pathway of IL-2R, the classification of Tregs in a murine myeloid cell line and the regulatory effect of IL-2/IL-2R on them, the regulatory impact and signaling mechanism of IL-2/IL-2R on CD4⁺/CD8⁺ lymphocyte differentiation, the primary manifestations and molecular mechanism of immune dysfunction in IL-2- and IL-2R-deficient mice, soluble IL-2Rα as a biomarker for diagnosis, prognosis and therapeutic efficacy of treatment in immune system disorders, and the development and clinical application of IL-2 mutants.     

IL-2 and polyoma BK virus infection; A systematic review article

It has been demonstrated that IL-2 plays a dual role in induction/suppression of immune responses via activation of conventional and regulatory T lymphocytes, respectively. IL-2 contacts complete IL-2 receptor (IL-2R) which contains CD25 (α chain) on the antigen specific activated T helper and cytotoxic lymphocytes and also T regulatory cells. Additionally, previous investigations revealed that polyoma BK virus (PBK) reactivation and induction of PBK associated nephropathy (PBKAN) is a main complication following renal transplantation. Based on the important dual roles played by IL-2 in the immune responses, it may be hypothesized that IL-2/IL-2R interaction could be considered a potential mechanism against/toward PBK reactivation and also PBKAN. Accordingly, the aim of the current review article is to determine the roles of IL-2 IL-2/IL-2R interaction in PBK reactivation and PBKAN complications.     

1,25-Dihydroxyvitamin D3 modulates the effects of interleukin 2 independent of IL-2 receptor binding

Previous studies have shown that 1,25-dihydroxyvitamin D3 (calcitriol) is a macrophage-derived cytokine and a potent inhibitor of IL-2 and interferon-gamma (IFN-gamma) production and T lymphocyte proliferation. The growth inhibitory effect of calcitriol is only partially reversed by IL-2 addition, suggesting IL-2 independent effects. In this report we characterize the IL-2-independent effects of calcitriol on lymphocyte activation. Calcitriol inhibited cellular transition from early to late G1 (G1A-G1B transition) in both the absence and presence of IL-2. Exogenous IL-2 did not increase either IFN-gamma production or transferrin receptor (TfR) expression in the presence of calcitriol despite increases in cell entry into late G1 and proliferation. Calcitriol treatment reduced TfR expression by activated T lymphocytes independent of their location in the cell cycle, further suggesting its independence from IL-2-mediated events. Combinations of rIL-2 and rIL-4 did not reverse calcitriol-dependent inhibition of proliferation and TfR expression to any greater degree than rIL-2 alone. Northern blot analysis demonstrated the decrease in IFN-gamma and TfR mRNA accumulation with calcitriol treatment was unaffected by exogenous IL-2. In contrast, IL-2R mRNA and protein were increased by IL-2, with superinduction in the presence of calcitriol, demonstrating that the lack of effect on IFN-gamma and TfR was not due to IL-2 insensitivity. Moreover, equivalent numbers of high-affinity IL-2R were expressed by both control and calcitriol-treated T lymphoblasts. Thus, lectin-activated T lymphocyte responsiveness to IL-2, as measured by IL-2R expression and proliferation, can be partly to completely dissociated from IFN-gamma production and TfR expression in the presence of calcitriol. Finally, IL-2-induced proliferation of unstimulated mononuclear cells and purified T lymphocytes was inhibited by calcitriol. These data indicate that local production of calcitriol by activated macrophages is capable of regulating T lymphocyte activation not only through suppression of IL-2 production, but also through additional mechanism(s), that are mediated at a post-IL-2R level.     

Novel mechanism for Trypanosoma cruzi-induced suppression of human lymphocytes. Inhibition of IL-2 receptor expression

Co-culture of blood forms of Trypanosoma cruzi, the causative agent of Chagas' disease, with human PBMC impaired the capacity of T lymphocytes to express surface receptors for IL-2. This effect was evidenced by marked reductions in both the proportion of Tac+ cells and the density of Tac Ag on the surface of the positive cells, determined by flow cytometry. The extent of the inhibition increased with parasite concentration. Under optimal or suboptimal conditions of stimulation with either PHA or monoclonal anti-CD3, specific for an epitope of the T3-Ti human T cell Ag receptor complex, the presence of T. cruzi curtailed the capacity of T lymphocytes to proliferate and express Il-2R but did not affect IL-2 production. Furthermore, the addition of exogenous IL-2 did not restore the responsiveness of suppressed human lymphocytes but did when mouse lymphocytes were used instead. Therefore, unlike mouse lymphocytes, human lymphocyte suppression by T. cruzi did not involve deficient IL-2 production and was accompanied by impaired IL-2 utilization. Co-culture of human monocytes/macrophages with suppressive concentrations of T. cruzi increased IL-1 production, and the parasite did not decrease IL-1 secretion stimulated by a bacterial LPS. Therefore, the suppression of IL-2R expression and lymphoproliferation is not likely to have been an indirect consequence of insufficient IL-1 production due to infection of monocytes or macrophages. We have shown that suppression of human lymphocyte proliferation by T. cruzi is not caused by nutrient consumption, absorption of IL-2, lymphocyte killing, or mitogen removal by the parasite. Therefore, these results uncover a novel suppressive mechanism induced by T. cruzi, involving inhibited expression of IL-2R after lymphocyte activation and rendering T cells unable to receive the IL-2 signal required for continuation of their cell cycle and mounting effective immune responses.     

Eotaxin activates T cells to chemotaxis and adhesion only if induced to express CCR3 by IL-2 together with IL-4

The transmigration and adherence of T lymphocytes through microvascular endothelium are essential events for their recruitment into inflammatory sites. In the present study, we investigated the expression of CC chemokine receptor CCR3 on T lymphocytes and the capacities of the CC chemokine eotaxin to induce chemotaxis and adhesion in T lymphocytes. We have observed a novel phenomenon that IL-2 and IL-4 induce the expression of CCR3 on T lymphocytes. We also report that CC chemokine eotaxin is a potent chemoattractant for IL-2- and IL-4-stimulated T lymphocytes, but not for freshly isolated T lymphocytes. Eotaxin attracts T lymphocytes via CCR3, documented by the fact that anti-CCR3 mAb blocks eotaxin-mediated T lymphocyte chemotaxis. In combination with IL-2 and IL-4, eotaxin enhances the expression of adhesion molecules such as ICAM-1 and several integrins (CD29, CD49a, and CD49b) on T lymphocytes and thus promotes adhesion and aggregation of T lymphocytes. The eotaxin-induced T lymphocyte adhesion could be selectively blocked by a specific cAMP-dependent protein kinase inhibitor, H-89, indicating that eotaxin activates T lymphocytes via a special cAMP-signaling pathway. Our new findings all point toward the fact that eotaxin, in association with the Th1-derived cytokine IL-2 and the Th2-derived cytokine IL-4, is an important T lymphocyte activator, stimulating the directional migration, adhesion, accumulation, and recruitment of T lymphocytes, and paralleled the accumulation of eosinophils and basophils during the process of certain types of inflammation such as allergy.     

Constitutive expression of IL-2Rbeta chain and its effects on IL-2-induced vascular leak syndrome

IL-2-induced vascular leak syndrome (VLS) is an important mechanism explaining the toxic effects of this cytokine and limiting its therapeutic use. We previously characterized a mouse model of IL-2-induced pulmonary VLS used to demonstrate that NK lymphocytes are involved in early/acute phase VLS (after one IL-2 injection). We also showed that NK cells and polymorphonuclear neutrophils (PMN) are involved in the late/chronic phase of the syndrome (after four daily IL-2 injections). In this study we use our mouse model to evaluate the role played by the IL-2 receptor (IL-2R) in VLS induction. Mouse and human IL-2R are different since the mouse IL-2Rbeta chain does not recognize IL-2. Here, we compare the acute and late VLS responses in human IL-2Rbeta transgenic and C57BL/6 wild type mice. Parameters linked to early phase VLS (bronchoconstriction and PMN mobilization) are enhanced in human IL-2Rbeta transgenic mice. By contrast, parameters used to measure late events (protein leakage and edema) are similar in human IL-2Rbeta transgenic mice and C57BL/6 wild type animals. However, after four IL-2 injections, the cellular content of the bronchoalveolar lavage fluids was different between the two types of animals. This study also characterizes a humanized animal model that could be further used to study human IL-2 activity and side effects in vivo.     

IL-4 induces loss of B lymphocyte Fc gamma R II ligand binding capacity

Murine B lymphocytes cultured for 24 h with rIL-4 lost (mean reduction of 88%, range 81 to 96%) the capacity to bind Ag-IgG antibody complexes to B lymphocytes as assessed by flow microfluorometry. This effect was specific in that it was not seen with IL-1, IL-2, or IFN-gamma; IL-4 did not have a similar effect on other B lymphocyte membrane molecules; and the effect was completely prevented by anti-IL-4 (mAb 11B11). More than 60% inhibition of the binding of complexes was seen with as little as 1 U/ml of IL-4 although maximal inhibition was seen with greater than or equal to 30 U/ml. IL-4-induced inhibition of the binding of complexes was time dependent (the effect was first seen after 8 h and was not maximal until 24 h), temperature dependent (it did not occur at 4 degrees C), and reversible (B lymphocytes that had lost the ability to bind complexes due to IL-4 regained this capacity when re-cultured for 24 h in the absence of IL-4). The effect could be partially prevented by IFN-gamma. The inability to bind complexes appeared to be mainly due to an alteration of Fc gamma R (Fc Receptors) II rather than down-regulation of receptor expression because IL-4 induced only a moderate reduction in the binding of two Fc gamma R II specific mAb (20% for 2.4G2 and 32% for K9.361). The IL-4-induced loss of binding of complexes to B lymphocyte Fc gamma R II appears to be a novel form of receptor regulation (function rather than expression), and likely plays a role in the up-regulation of B lymphocytes by IL-4 by preventing Fc gamma R II-mediated inhibition of B lymphocyte responses.     

TGF-beta does not inhibit IL-12- and IL-2-induced activation of Janus kinases and STATs

The immune system is an important target for the cytokine TGF-beta1, whose actions on lymphocytes are largely inhibitory. TGF-beta has been reported to inhibit IL-12- and IL-2-induced cell proliferation and IFN-gamma production by T cells and NK cells; however, the mechanisms of inhibition have not been clearly defined. It has been suggested by some studies that TGF-beta blocks cytokine-induced Janus kinase (JAK) and STAT activation, as in the case of IL-2. In contrast, other studies with cytokines like IFN-gamma have not found such an inhibition. The effect of TGF-beta on the IL-12-signaling pathway has not been addressed. We examined this and found that TGF-beta1 did not have any effect on IL-12-induced phosphorylation of JAK2, TYK2, and STAT4 although TGF-beta1 inhibited IL-2- and IL-12-induced IFN-gamma production. Similarly, but in contrast to previous reports, we found that TGF-beta1 did not inhibit IL-2-induced phosphorylation of JAK1, JAK3, and STAT5A. Furthermore, gel shift analysis showed that TGF-beta1 did not prevent activated STAT4 and STAT5A from binding to DNA. Our results demonstrate that the inhibitory effects of TGF-beta on IL-2- and IL-12-induced biological activities are not attributable to inhibition of activation of JAKs and STATs. Rather, our data suggest the existence of alternative mechanisms of inhibition by TGF-beta.     

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.     

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.     

Enforced expression of Bcl-2 restores the number of NK cells,but does not rescue the impaired development of NKT cells or intraepithelial lymphocytes,in IL-2/IL-15 receptor beta-chain-deficient mice

IL-2/IL-15Rbeta-deficient mice display impaired development of NK cells, NKT cells, and intraepithelial lymphocytes of the intestine and skin. To determine the role of survival signals mediated by IL-2/IL-15R in the development of these innate lymphocytes, we introduced a bcl-2 transgene into IL-2/IL-15Rbeta-deficient mice. Enforced expression of Bcl-2 restored the number of NK cells in IL-2/IL-15Rbeta-deficient mice, but the rescued NK cells showed no cytotoxic activity. The numbers of NKT cells and intestinal intraepithelial lymphocytes did not increase significantly, and skin intraepithelial lymphocytes remained undetectable in the bcl-2 transgenic IL-2/IL-15Rbeta-deficient mice. These results indicate an essential role of IL-2/IL-15R-mediated survival signals in the development of NK cells, but they also show that additional nonsurvival signals from IL-2/IL-15R are necessary for innate lymphocyte development.     

IL-4 inhibits IL-2 receptor expression and IL-2-dependent proliferation of human T cells

Recent studies have shown that IL-4 can affect lymphocyte responses to IL-2. To evaluate the effects of IL-4 on T cell responses to physiologically relevant stimuli, we studied normal human T cells cultured with a low concentration of anti-CD3 mAb and IL-2 in the presence and absence of added IL-4. The addition of IL-4 to cultures of T cells stimulated with anti-CD3 mAb and IL-2 reduced the proliferative response by 49 to 59%. The inhibitory effect was observed in 3-, 5-, and 7-day cultures. Inhibition was dose-dependent with maximal inhibition at concentrations greater than or equal to 5 to 10 U/ml IL-4. IL-4-mediated inhibition occurred early during the T cell response, inasmuch as addition of IL-4 after stimulation for 24 h did not result in significant inhibition. Phenotypic analyses of cells cultured in the presence of anti-CD3 mAb, IL-2, and IL-4 suggested that the mechanism of regulation by IL-4 involves the inhibition of IL-2R expression. The proportion of both CD4+ and CD8+ cells that expressed IL-2R in response to IL-2 was diminished in the presence of IL-4, although HLA-DR levels were unaffected. Soluble IL-2R was also reduced in supernatants of cultures stimulated with anti-CD3 mAb, IL-2, and IL-4 as compared to cultures stimulated with anti-CD3 mAb and IL-2. These findings indicate that when normal human T cells are stimulated in vitro in a manner that approximates a physiologic interaction with Ag in vivo, rIL-4 provides a potent inhibitory signal to IL-2 responsive cells that is likely mediated by IL-4-induced inhibition of IL-2R expression.     

IL-2 and IL-6 synergize to augment the pore-forming protein gene expression and cytotoxic potential of human peripheral blood T cells

Our previous studies have demonstrated that high dose IL-2 (1000 U/ml) alone can induce human peripheral blood T cell pore-forming protein (PFP) mRNA expression and cytotoxic potential. We now report that the levels of IL-2 needed to induce these effects in T cells can be significantly reduced in the presence of IL-6. IL-6 and suboptimal doses of IL-2 (10 U/ml) were found to costimulate PFP mRNA expression and cytotoxic potential in resting human peripheral blood T cells, whereas IL-6 or low dose IL-2 alone had no effect. The induction of T cell PFP mRNA by IL-2/IL-6 was extremely rapid and increases in both PFP mRNA expression and cytotoxic potential were IL-6 dose dependent. The costimulatory effect of IL-6 did not appear to involve the IL-2/IL-2R pathway in as much as IL-6 did not induce IL-2 production or detectably increase IL-2R surface expression in T cells. These findings, in addition to the rapid induction of PFP mRNA by IL-2/IL-6, suggested that IL-6 can directly and independently provide an additional signal to augment the differentiation of CTL. In contrast to the results observed in T cells, IL-6 and IL-2 could enhance CD3- large granular lymphocyte (LGL) NK activity, but IL-6 either alone or in combination with IL-2 had no effect on constitutive PFP mRNA expression in resting LGL. These data further confirm that different mechanisms may be responsible for lymphokine activation of CTL and LGL in human peripheral blood. In particular it appears that IL-6 acts as a costimulatory signal with IL-2 in generating CTL and that IL-6 functions in part by acting in synergy with IL-2 to induce PFP, a major lytic protein involved in lymphocyte cytotoxicity.     

IL-33/ST2 contributes to severe symptoms in Plasmodium chabaudi-infected BALB/c mice

It has been reported that IL-33 contributes to potentiation of Th2 inflammatory diseases and protection against helminth infection. Increased plasma IL-33 levels have been observed in patients with severe falciparum malaria, however, the role of IL-33 in malaria remains unclear. Here we report that IL-33 enhances inflammatory responses in malaria infection. ST2-deficiency altered severity of inflammation in the liver and serum levels of pro-inflammatory cytokines such as TNF-α and IL-6, and IL-13 that is a Th2 cytokine during Plasmodium chabaudi infection. IL-13-deficient mice have similar phenotype with ST2-deficient mice during P. chabaudi infection. Furthermore, ST2- and IL-13-deficiency reduced mortality from P. chabaudi infection. These results indicate that IL-33/ST2 can induce production of proinflammatory cytokines, such as TNF-α and IL-6, through production of IL-13 in P. chabaudi-infected BALB/c mice, suggesting that IL-33/ST2 play a critical role in inflammatory responses to malaria infection. Thus, these findings may define a novel therapeutic target for patients with severe malaria.     

The synergistic effects of interleukin 2 and interleukin 7 on the proliferation and autologous tumor cell lysis of tumor-associated lymphocytes

Interleukin-7 (IL-7) has an ability to stimulate the proliferation of pre-B cells. It has been shown that IL-7 can also activate T lymphocytes. We here demonstrate that IL-7 in combination with interleukin-2 (IL-2) can drive cell proliferation and enhance the autologous tumor cell lysis by peripheral blood mononuclear cells (PBMC) and autologous mixed lymphocyte tumor cell culture (MLTC)-derived effector cells (MLTC cells). These synergistic effects of IL-2 and IL-7 on the proliferation and the augmentation of autologous tumor cell lysis were found for both effector cells. These effects were inhibited by neutralizing antibodies to IL-2 or IL-7, and by a combination of both antibodies, significantly. In terms of phenotypical expression, CD3 positive cells comprised the vast majority of MLTC cells after culture in medium containing IL-2 and IL-7 with an increase of IL-2 receptor positive cells.Abbreviations CD cluster differentiation - IFN interferon - IL interleukin - JRU Japanese Reference Unit - LAK lymphokine activated killer - mAb monoclonal antibody - MLTC mixed lymphocyte tumor cell culture - PBMC peripheral blood mononuclear cells - TILs tumor infiltrating lymphocytes