Membrane-bound and soluble IL-2 receptors (p55 and p75 chains) on peripheral blood mononuclear cells from patients with solid malignancies

The aim of the investigation was to study directly the IL-2 receptor (IL-2 R) and its subunits, p55 and p75 chains, either membrane-bound or soluble, on PBMC of patients with solid malignancies and, indirectly, the same patients’ PBMC ability to produce IL-2. Fifty-eight cancer patients, 29 men and 29 women, were studied: their mean age was 57.3 yr, range 35–79. Twenty-two healthy age-sex-matched subjects served as controls. The tumors were the most common and the most representative among human cancers, i.e., breast, lung, head and neck, digestive tract and liver, prostate and gynecologic cancers: they were generally in advanced stages and in 23 cases metastatic. The PBMC proliferative response to PHA, PHA plus IL-2, and IL-2 was evaluated along with the response to PHA in the presence of anti-p55, anti-p75 monoclonal antibodies, or both. Moreover, membrane-bound IL-2 R (p55 and p75 chains) on PHA-stimulated PBMC was detected, along with soluble IL-2 R in the serum and in the culture supernatants. The conclusions suggest that in solid malignancies: the membrane-bound IL-2 Rs, both p55 and p75 chains, are expressed normally, there is an high serum level of soluble IL-2 R, there is a normal release of soluble IL-2 R in culture, and there is an indirect evidence of a lack of IL-2 production. Therefore, no primary impairment of IL-2 R was found in solid tumors. Moreover, in our study we have found no difference in any parameter studied between patients with and patients without metastases.     

Cyclic AMP directly inhibits IL-2 receptor expression in human T cells: expression of both p55 and p75 subunits is affected.

Using purified human T lymphocytes stimulated in serum-free media with adhered anti-CD3 + exogenous IL-2, we have shown that elevated [cAMP]i (mimicked by CPT-cAMP or induced by the physiological agonist PGE2) directly inhibits mitogen-induced 1) [3H]thymidine incorporation by PBMC, purified T cells, and isolated CD4+ and CD8+ T cell subpopulations; 2) expression of both high- and low-affinity IL-2 receptors; 3) plasma membrane expression of both p55 and p75 subunits of the IL-2 receptor; and 4) expression of p55 mRNA, but not p75 mRNA. The decrease in p55 mRNA is not due to enhanced mRNA metabolism. We conclude that elevated [cAMP]i, acting directly on T cells, inhibits mitogenesis by decreasing IL-2 receptor expression. We discuss the possible physiological relevance for the multiple stages of T cell activation that are sensitive to elevated [cAMP]i.     

Interleukin-4 downregulates the p70 chain of the IL-2 receptor on peripheral blood mononuclear cells

Data that support a differential regulation of the interleukin-2 receptor (IL-2R) alpha-(p55 or Tac) and beta-chain (p70) expression by IL-4 are presented. Cytofluorometric analysis performed on peripheral blood mononuclear cells, some of which had been nylon wool passed (enriched for T-cells), in the presence or absence of phytohemagglutinin (PHA) or OKT3, demonstrated that IL-4 has a dose-dependent capacity to inhibit beta-chain IL-2R expression, whereas the alpha-chain is nearly unaffected. We could also, as a consequence of the decreased p70 expression, detect a slight increase in the amounts of IL-2 obtained from PHA-stimulated cultures, when IL-4 was present. Further, the proliferative response, especially to IL-2, but also to PHA alone, was depressed in the presence of IL-4. These data thus give further support to the idea that not only the IL-2R complex as such, but also the two individual IL-2R chains, can be independently regulated.     

IL-2-dependent proliferation of murine T cells requires expression of both the p55 and p70 subunits of the IL-2 receptor

An IL-4-dependent T cell clone (LD8) was isolated from the murine IL-2-dependent cytotoxic T cell line C30.1. This clone has lost the capacity to proliferate in response to IL-2 after long-term culture in IL-4. LD8 cells express the p70, but not the p55, subunit of the IL-2R on their cell surface. The number of p70 IL-2R molecules on LD8 cells is comparable with the number of high-affinity IL-2R on the parental C30.1 cell line. LD8 cells can efficiently internalize IL-2 through the p70 IL-2R subunit. Following stimulation by IL-2, LD8 cells up-regulate p70 IL-2R mRNA, but do not express p55 IL-2R mRNA. IL-2-dependent proliferation of LD8 cells was reconstituted after introduction and expression of a human p55 IL-2R cDNA. To further investigate the role of p70 IL-2R, we have measured IL-2-induced proliferation of C30.1 cells in the presence of three anti-p55 IL-2R mAb (5A2, PC61, and 7D4) that recognize different epitopes. Under the experimental conditions used, the combination of anti-p55 IL-2R mAb prevents the formation of high-affinity IL-2R, but does not affect the binding of IL-2 to p70 IL-2R or IL-2 internalization. However, these three mAb inhibit proliferation of C30.1 cells even in the presence of IL-2 concentrations sufficient to saturate p70 IL-2R. Together these results demonstrate that p70 IL-2R alone is not sufficient to transmit IL-2-induced growth signals and that formation of p55-p70 IL-2R complex is required for IL-2-dependent proliferation of murine T cells.     

Different induction of p55 and p70/75 interleukin-2 receptor subunits in human cells

There are two interleukin-2 receptor (IL-2R) subunits (p55 and p70/75) on human lymphocytes. Induction of the expressions of these IL-2R subunits was examined by the protein kinase-C (PK-C) activator (phorbol myristate acetate, PMA) and the calcium ionophore, ionomycine (IM). IM induced predominantly p70/75 expression on human T and B cells as indicated by the results of chemical crosslinking studies and binding assays. In contrast, PMA induced p55 expression significantly. These results suggest that the calcium-calmodulin and PK-C pathways regulate p70/75 and p55 expressions differently, and indicate that these intracellular signal messengers could control the responsiveness to IL-2, changing the affinity and number of receptors in vivo.     

Study of peripheral blood lymphocyte subset distribution and IL-2 receptor (IL-2 R) p55–p75 subunit expression in patients with cancer of different sites

The aim of the study was to evaluate the subset distribution and the IL-2 R p55–p75 subunit expression on unstimulated and phytohemagglutinin (PHA)-stimulated (at 3-d) peripheral blood mononuclear cells (PBMC), of patients with solid cancers of different sites. Indeed the expression of the two subunits of IL-2R is an essential prerequisite for The action of the IL-2 on CD8⁺, CD16⁺ lymphocytes as effectors in antitumor activity (LAK-cells). The subset distribution (CD3, CD4, CD8, CD16, DR) was assessed by cytofluorometry with specific monoclonal antibodies (MAbs); the p55 (CD25) and p75 subunit expression was evaluated by specific MAb (OKT26a and anti-p75). Ninety patients with advanced cancer (mainly non-small cell lung cancer [NSCLC], head and neck cancer, and gynecological cancer; mean age 55 yr; range 27–80) were studied. Thirty-five age- and sex-matched healthy subjects were studied as controls. Our data show that there is no significant difference in the subset distribution between cancer patients and controls. Furthermore, no difference has been found in the expression of p55 subunits on unstimulated PBMC between cancer patients and controls. No difference has been found in the expression of both p55 and p75 subunits on PHA-stimulated PBMC between cancer patients and controls. Our results can support the rationale for further clinical trials with IL-2 in solid malignancies.     

Differential expression of interferon-gamma,IL-4 and IL-10 in peripheral blood mononuclear cells during early pregnancy of the bovine

Maternal systemic immune response is regulated by conceptus-derived signals through peripheral blood mononuclear cells (PBMCs) via blood circulation during early pregnancy in cattle. In this study, the PBMCs from day 18 in non-pregnant cows and days 14, 18 and 30 in pregnant cows were used to explore the expression of interferon-gamma (IFN-γ), interleukin 4 (IL-4) and IL-10, and the plasma progesterone (P4) concentration was also determined. The results showed that the expression levels of mRNA and the protein of IFN-γ were lower and that IL-4 and IL-10 were higher in the PBMCs from the pregnant cows than in those of non-pregnant cows. From this study, early pregnancy induced a lower Th1 immunity (IFN-γ) and a higher Th2 immunity (IL-4 and IL-10) in the PBMCs, which may be related to interferon-tau and P4, thereby contributing to successful pregnancy in cattle.     

不同诱导因子对人外周血单个核细胞P2X7受体表达的作用

ATP激活P2X7受体可产生一系列的白细胞功能反应,因此P2X7受体的表达调控引起我们的兴趣。然而P2X7受体在正常人外周血单个核细胞(peripheral blood mononuclear cells,PBMC)、单核细胞中的表达调控机制尚未阐明。本文用半定量RT-PCR方法检测多种细胞因子、细菌抗原、丝裂原对P2X7受体表达的诱导作用,探索P2X7受体的诱导表达模式。结果表明,单个核细胞和单核细胞可检出P2X7受体的表达;白细胞介素2、4、6(interleukin-2、-4、-6,IL-2、IL-4、IL-6)、肿瘤坏死因子仪(tumour necrosis factor-α,TNF-α)等细胞因子和金黄色葡萄球菌CowanⅠ株(Staphylococcus aureus Cowan strainⅠ,SAC)、脂多糖(lipopolysaccharide,LPS)能上调PBMC的P2X7受体表达,而γ干扰素(interferon-γ,IFN-γ)、粒-巨噬细胞集落刺激因子(granulocyte-macrophage colony-stimulating factor,GM-CSF)、巨噬细胞集落刺激因子(macmphage colony-stimulating factor,M-CSF)和植物血凝素(phytohemagglutinin-M,PHA-M)等则没有作用;LPS和M-CSF可以提高单核细胞的P2X7受体表达,IFN-γ、TNF-α、GM-CSF作用较弱,但是这些因子的预处理并不能增强LPS对P2X7受体表达的诱导。炎症因子促进P2X7受体的表达,提示P2X7受体可能在对抗细菌感染的免疫反应中起一定作用,这有待于进一步研究。     

Direct identification of the murine IL-2 receptor p55-p75 heterodimer in the absence of IL-2

The proteins cross-linked to the IL-2R p55 subunit were biochemically compared in distinct cell populations that varied in their capacity to express high affinity IL-2R. We directly cross-linked p75 to p55 in the absence of IL-2 for the cell populations that bear only high affinity IL-2R. Furthermore, although no endogenous IL-2 production was detected, p75 was readily cross-linked to p55 for EL4J-3.4, a p55 transfectant of EL4 that bears high affinity IL-2R. These results strongly suggest that high affinity IL-2R exist as a preformed heterodimer of p55 and p75 which do not require IL-2 for their association. Furthermore, cross-linking of three other proteins of apparent Mr of 100,000, 135,000, and 180,000 to p55 was also seen, raising the possibility of a more complex subunit composition for the IL-2R.     

IL-2 stimulates the production of IL-1 alpha and IL-1 beta by human peripheral blood mononuclear cells

Human PBMC were cultured in medium containing human rIL-2, and the supernatants and cell lysates were analyzed for IL-1 alpha and IL-1 beta using specific RIA. IL-2, but not the excipient detergents included in the rIL-2 preparation, induced the synthesis of both cytokines. The concentrations of IL-1 alpha and IL-1 beta in the cell lysates and supernatants depended on both the concentration of rIL-2 in the culture medium and the duration of the incubation. After 24 h of stimulation, IL-2-induced IL-1 alpha remained almost entirely cell-associated. In contrast, IL-1 beta was present in both cell lysates and supernatants and was more abundant in the latter. SDS-PAGE analysis after radioimmunoprecipitation with anti-IL-1 antibodies indicates that cell-associated IL-1 resulting from IL-2 stimulation was in the form of the 35 kDa IL-1 precursor whereas secreted IL-1 was almost entirely in the form of the mature 18 kDa product. Depletion of monocytes from the PBMC culture substantially reduced IL-2-induced IL-1 production. In addition, Leu M3+ monocytes obtained through FACS, but not CD16+ NK cells, produced both IL-1 alpha and IL-1 beta in response to IL-2. The low level of endotoxin present in the IL-2 preparation used in our studies and the selective inhibition by polymyxin B of LPS-induced, but not IL-2-induced, IL-1 production by PBMC indicate that IL-2-induced IL-1 production was not due to endotoxin contamination. Furthermore, an anti-IL-2 antiserum selectively inhibited IL-1 production in response to IL-2 stimulation. We conclude that IL-2 is a potent inducer of IL-1 synthesis and secretion in vitro and propose that IL-1 may be generated in vivo in patients undergoing IL-2 immunotherapy.     

Activation by IL-2, but not IL-4, up-regulates the expression of the p55 subunit of the IL-2 receptor on IL-2- and IL-4-dependent T cell lines

We have investigated the effects of IL-2 and IL-4 on different parameters of T cell activation using three T cell lines. The Th cell line L14 and the cytotoxic T cell line C30.1, both grown in IL-2-containing medium, and a line derived from C30.1 cells (line 1) cultured in IL-4 for a prolonged period were studied. All three cell lines could be activated with IL-2 or IL-4. T cell stimulation by either IL-2- or IL-4-induced identical patterns of cell size enlargement and transferrin receptor expression. However, only IL-2 up-regulated cell-surface expression of the p55 subunit of the IL-2R (p55 IL-2R) as measured by flow cytometry and RIA. This difference was also reflected by the accumulation of soluble p55 IL-2R in the culture medium. No significant increase in expression of membrane or soluble forms of p55 IL-2R was detected after IL-4 stimulation. mAb specific for p55 IL-2R which block IL-2-induced T cell growth did not affect IL-4-mediated T cell proliferation indicating that p55 IL-2R is not involved in IL-4-mediated T cell growth. Analysis of IL-4R expression performed on line 1 using biotinylated IL-4 revealed that IL-4, but not IL-2, is capable of increasing IL-4R expression. Together these results suggest that during IL-2- or IL-4-induced T cell proliferation, each lymphokine specifically up-regulates its own receptor.     

Increased cytotoxicity and CD16 (Leu 11) expression in long-term, IL-2-activated human peripheral blood mononuclear cells

CD16 (Leu 11) positive cells are believed to be the effector cells for the so-called LAK phenomenon. Current evidence suggests that this cell population is comprised predominantly of IL-2-activated CD3 negative Leu 11+ NK cells and a minor proportion of Leu 11+ CD3+ MHC unrestricted type II cytotoxic T cells. The current study demonstrates a continuous increase in the frequency of Leu 11+ (and CD8+) cells and a decline of CD3 and CD4 positive cells during prolonged culture of human PMBL with high levels of rIL-2. Cytotoxicity also increases in this time period parallel with Leu 11 to a maximum of activity on the twelfth day of culture. This correlation suggests that the long-term activated killer cells generated in this period are Leu 11+, CD8+, CD3-, CD4- activated NK cells. With regard to tumor therapy, the long-term culture of PMBL in rIL-2 may be of advantage over short-term activation protocols. If the Leu 11+ cells are in fact the mediators of the therapeutic response, the long-term culture generates up to six times more effector cells. In addition, this method allows significant savings in the expense for leukophoresis, cell culture, and laboratory personnel. The efficacy of long-term, cultured rIL-2-activated Leu 11+ cells for tumor therapy is currently being investigated in clinical trails.     

Increased cyclooxygenase-2 expression in peripheral blood mononuclear cells of smokers and hyperlipidemic subjects

Cyclooxygenase (COX)-2 is expressed in macrophages of arteriosclerotic lesions and promotes inflammation. We investigated whether COX-2 is already expressed in peripheral blood mononuclear cells (PBMCs) of subjects possessing risk-related factors, such as in smokers and hyperlipidemics. PBMCs were isolated from the venous blood of normolipidemic nonsmokers (NL-NSM; n = 15), normolipidemic smokers (NL-SM; n = 12), hyperlipidemic nonsmokers (HL-NSM; n = 10), and hyperlipidemic smokers (HL-SM; n = 10). RNA from PBMCs was used for RT-PCR. Plasma concentrations of oxidized low-density lipoproteins (oxLDL) were measured by ELISA, those of glutamate and cystine by HPLC. The results show that COX-2 expression in PBMCs was significantly increased in the groups with cardiovascular risk factors (NL-SM, HL-SM, HL-NSM) compared with NL-NSM. COX-2 expression in PBMCs was positively correlated with concentrations of total serum cholesterol, oxLDL, glutamate, or cystine. We suggest that the elevated COX-2 expression indicates a priming of PBMCs as a response to a systemic pro-oxidative and proinflammatory shift in subjects with cardiovascular risk factors, which might also contribute to growth and instability of arteriosclerotic lesions.     

Differential effect of IL-4 and IL-13 on the expression of recombination-activating genes in mature B cells from human peripheral blood

We examined the expression of recombination-activating genes (RAG-1 and RAG-2) and activation-induced cytidine deaminase (AID) by mature human blood B cells stimulated with anti-CD40 in the presence of IL-4 or IL-13. IL-4 was an effective cofactor for RAG-1 and RAG-2 expression, whereas IL-13 was not. In addition, IL-4-dependent RAG expression combined with AID and IgE expression allowed predominant expression of newly rearranged lambda light chains on IgE+ cells generated from kappa+ cells. Although the magnitudes of IL-4- and IL-13-dependent AID and IgE expression were related to expression levels of binding subunits of the IL-4 and IL-13 receptors, IL-13 was ineffective for light chain replacement in the induced IgE+ cells due to the failure in RAG expression. Our studies using mature blood B cells indicate that IL-4-responsive cells, unlike IL-13-responsive cells, undergo lambda gene rearrangement leading to replacement in parallel with RAG expression and suggest that this replacement may contribute to the regulation of affinity maturation of IgE antibodies.     

Synergistic action of monoclonal antibodies directed at p55 and p75 chains of the human IL-2-receptor

TU27, a mouse IgG1 mAb directed at the p75 chain of the human IL-2R, was analyzed for its ability to interact with IL-2 binding on isolated p75 chains (YT-2C2 cells) and high affinity p55/p75 receptors (human alloreactive T cell clone 4AS), to inhibit IL-2-induced proliferation (4AS cells) and to cooperate with an anti-p55 chain mAb (33B3.1) for inhibiting IL-2 binding and proliferation. TU27 and IL-2 bound to the isolated p75 chain expressed by YT-2C2 cells with respective dissociation constants (Kd) of 1.3 and 1 nM. They cross-inhibited each other for binding with inhibition constants (Ki) in agreement with their respective Kd values. The nature of the interaction was, however, not purely competitive and suggested nonidentical epitopes for the two ligands on the p75 chain. On 4AS cells, IL-2 bound with high affinity (Kd = 50 pM) and TU27 with an affinity similar to that found on YT-2C2 cells. The binding of TU27 and IL-2 were also mutually exclusive on 4AS cells. However, the mechanism of interaction of TU27 with IL-2 was complex since the inhibitory potency of the antibody depended on temperature, antibody preincubation and time of assay. Data obtained at 4 degrees C in the presence of suboptimal, tracer-like concentrations of IL-2 indicated that the intrinsic affinity of TU27 for the high affinity configuration was 15-fold lower than for the isolated p75 chain and argued in favor of the affinity-conversion model (as opposed to the preformed complex model) in which p55 and p75 are dissociated in the absence of IL-2. At 37 degrees C, TU27 inhibited IL-2 binding only on short time assays (6 min). Longer time (30 min) of IL-2 binding resulted in an almost complete disappearance of the effect of TU27, suggesting that internalization of the high affinity p55/p75/IL2 complex enables the cells to escape from the inhibitory effect of TU27. In the presence of the 33B3.1 mAb, the interaction of TU27 with IL-2 resembled the one observed on YT-2C2 cells, suggesting that 33B3.1 is able to inhibit the IL-2-induced association of p55 and p75. Both antibody were found to synergize on 4AS cells, as a result of a cooperative mechanism in which 33B3.1 blocks the formation of the high affinity complex hence allowing TU27 to bind with higher affinity, and TU27 blocks IL-2 binding to the p75 chain. Proliferation studies corroborated the binding experiments.(ABSTRACT TRUNCATED AT 400 WORDS)