Expression of high-affinity IL-4 receptors on human melanoma,ovarian and breast carcinoma cells

It has previously been shown that murine sarcoma cells express high-affinity IL-4 receptors (1L-4R) which are internalized after binding to the ligand (Puri et al., Cancer Res 1991; 51:3011-7). We have also reported that human renal cell carcinoma cells express high-affinity IL-4R. and IL-4 inhibits tumour growth in vitro (Obiri et al, J Clin Invest 1993; 91:88). In this study we investigated the expression and function of IL-4R on other human solid tumours. Human melanoma, ovarian carcinoma and breast carcinoma cell lines were assessed for the cell surface expression of IL-4R by radio-ligand receptor binding and for IL-4R gene expression by Northern blot analysis. Primary cultures of mesothelioma and neurofibrosarcoma cells were similarly investigated. Human melanoma, ovarian carcinoma and breast carcinoma cell lines expressed IL-4R on their cell surface with a dissociation constant (K_d) of 140-549 pM. These tumour lines expressed a single 4 kb species of mRNA for IL-4R. Similarly, primary cultures of mesothelioma and neurofibrosarcoma cells were positive for the IL-4R mRNA by Northern blot analysis. Fresh, non-cultured mesothelioma and neurofibrosarcoma tumour sections were also positive for the presence of IL-4R as determined by immunohistochemistry of frozen sections using anti-IL-4R antibody. In order to study possible functions of IL-4R, we evaluated the effects of 11.-4 on cell growth and its effect on MHC antigen expression in the presence or absence of interferon-gamma (IFN-γ)- In tissue culture, IL-4 reduced the growth of tumour cell lines and primary cell cultures studied. IL-4 had very title effect on MHC class I antigen expression on ovarian, breast and melanoma cell lines; however. MHC class I (HLA-DR) expression was enhanced on melanoma and breast carcinoma cells. IL-4 also enhanced the IFN-γ-induced class II expression on melanoma and breast carcinoma cells. Taken together, our observations indicate that IL-4R are expressed on a variety of human solid tumours and these receptors may be functional. IL-4 alone and in combination with IFN-γ may play a role in host immune response against cancers.     

The two-chain structure of high-affinity IL-2 receptors

Binding studies with radiolabeled interleukin 2 (IL-2) have suggested that T cells possess two classes of IL-2 binding site with different affinities but a shared epitope named Tac. The gene for a 55kDa Tac-positive protein has been cloned but on transfection induced only the expression of low-affinity IL-2 binding sites. The structure of the receptor has therefore been perplexing. Here Kendall Smith discusses recent studies which disclose the existence of a new Tac-negative 75kDa IL-2 binding protein and he suggests that a high affinity IL-2 receptor consists of an α(p75) chain non-covalently linked to a β(p55) chain.     

Expression of IgA and IgM Fc receptors on murine T lymphocytes

Fc receptors are induced on T cells following activation via the TCR. T cells that express Fc receptors transiently have the ability to use two different cognate systems: the TCR and immunoglobulins bound to the Fc receptors. The studies discussed in this article are focused on the Fc alpha and Fc mu receptors that can be induced on certain subsets of murine T lymphocytes. The article emphasizes the role of the T cell receptor for antigen in the expression of Fc alpha and Fc mu receptors on murine T cells and reviews experimental observations that suggest significant molecular heterogeneity of these Fc receptors. The finding that regulation of expression of Fc alpha receptors and Fc mu receptors on T lymphocytes is linked to cellular activation via the CD3/TCR complex implies that these Fc receptors might mediate important functions in the biology and pathology of T cells.     

Intermediate- and high-affinity interleukin-2 receptors expressed in an IL-4-dependent T-cell line induce different signals.

We have previously described a synergism between the two physiological hormones, retinoic acid (RA) and 1 alpha,25-dihydroxyvitamin D3 (VD) in the induction of U937 cell differentiation towards a more mature state. Herein, we investigated the regulation of cytokine production during RA and/or VD treatment of U937 cells. Cell differentiation was followed by measurement of their capacity to give oxidative responses, and interleukin-1 beta (IL-1 beta), tumour necrosis factor-alpha (TNF-alpha) and IL-6 gene and protein expression were determined in RA/VD-treated cells, activated or not with lipopolysaccharide (LPS). The undifferentiated and RA-treated U937 cells were unable to produce monokines even when they were stimulated by LPS. VD induced the monokine mRNA expression in U937 cells but failed to induce protein release. However, unlike RA, it primed the cells to secrete monokines upon endotoxin stimulation. A large enhancement of the production of the monokines both at mRNA and protein levels was observed in the U937 cells exposed to the combination of RA + VD. Nevertheless, protein release required a further step of activation of the RA + VD-primed cells. The co-inducer effect of RA and VD was not observed in HL-60 or THP-1 cells and seems to be restricted to U937 cells. These results on cytokine expression support our previous finding that a combination of RA and VD brings the U937 cells to a high stage of myeloid differentiation with major characteristics of monocytes/macrophages.     

Detection of interleukin 2 receptors on murine lymphocytes using fluorescent interleukin 2.

Murine interleukin 2 receptors found on freshly isolated and on in vitro activated lymphocytes were identified using a fluorescent interleukin 2 (IL2F). Three percent of freshly isolated small thymocytes bound the IL2F; these cells appeared to be dual CD4 and CD8 positive cells. Ten percent of the larger thymocytes also bound the IL2F; phenotypically, these cells were more heterogenous in their CD4/CD8 composition than the small IL2F+ thymocytes. Freshly isolated splenocytes bound more IL2F than did the thymocytes. Twenty-four percent of the small splenocytes were IL2F+ and they were mostly B220+ cells. Half of the larger splenocytes were IL2 receptor positive and these cells consisted of B and T cells. Using mitogen stimulated splenocytes, three times as many LPS stimulated B220+ blasts bound the fluorescent IL2 than freshly isolated large B220+ cells; this level of IL2F binding was maintained for four days. Of the Con A blasts, more CD8+ cells (30%) bound IL2F than did CD4+ blasts (19%); these cells maintained this level of IL2F binding for only three days. The IL2F binding could be completely inhibited by excess unlabeled IL2 and could be inhibited by 92% using a monoclonal antibody directed against the IL2 binding region of the IL2 alpha receptor, indicating that IL2F can bind to both IL2 alpha and IL2 beta receptors.     

Tunicamycin inhibits function and expression of the high-affinity IL-2 receptor in a murine IL-2-dependent cell line.

Murine interleukin-2-dependent T-lymphocytes (CT6) were treated with tunicamycin, an inhibitor of both glycoprotein and ganglioside synthesis, to study the involvement of glycosylation in the IL-2 proliferative response. Tunicamycin inhibited proliferation in a dose-dependent manner at concentrations which did not inhibit protein synthesis (10-50 ng/ml). Swainsonine, a glycoprotein processing inhibitor, had no effect on proliferation. Inhibition of proliferation by tunicamycin was accompanied by an inhibition of binding of 125I-IL-2 to its high-affinity receptor. Scatchard analysis showed that receptor number was decreased by tunicamycin treatment. On the other hand, tunicamycin did not affect either the binding of the monoclonal antibody 7D4, specific for the 55 kDa low-affinity protein subunit of the IL-2 receptor, or the recycling of the IL-2 receptor. To determine the specific effects of tunicamycin on the biosynthesis of particular CT6 glycoconjugates, cells were radiolabeled with 3H-glucosamine and incorporation into ganglioside, neutral glycolipid and glycoprotein fractions was measured. Low doses of tunicamycin inhibited ganglioside synthesis and glycoprotein glycosylation to the same extent, whereas no effect on neutral glycolipid synthesis was observed. These results suggest that glycosylation of glycoprotein and/or gangliosides might play an important role in the formation of a functional high-affinity IL-2 receptor complex in CT6 cells.     

Recombinant interferon-gamma inhibits the expression of IL-4 receptors on human lymphocytes.

Interferon gamma (IFN-gamma) has been shown to inhibit many of the activities of IL-4, including the induction of IgE synthesis and the proliferation of T cell clones. Here we demonstrate that IFN-gamma is able to inhibit the expression of IL-4 receptors on peripheral blood lymphocytes from both normal healthy donors and from patients with chronic lymphocytic leukaemia. Inhibition was shown to be dose-dependent and did not affect the binding affinity of the receptor as shown by Scatchard analysis. IFN-gamma was unable to displace labelled IL-4 from its membrane receptor, which demonstrates that IFN-gamma and IL-4 do not compete for the same membrane binding protein. The ability of IFN-gamma to down-regulate IL-4 receptors may be important in controlling certain immune responses.     

Regulation of the production of soluble IL-4 receptors in murine cutaneous leishmaniasis. The roles of IL-12 and IL-4.

These studies were undertaken with the purpose of elucidating the key signals involved in the regulation of the production of soluble interleukin-4 receptors (sIL-4R) in mice during Th1 and Th2 responses to infection with the parasite Leishmania major. Our results showed that the production of sIL-4R was consistently higher in lymph node cell cultures from animals mounting a predominant Th2 response (BALB/c mice), and that sIL-4R production paralleled that of IL-4 in both mouse strains, even in the presence of a dominant Th1 response (C3H/FeJ mice). Consistently, administration of anti-IL-12 antibodies to infected C3H/ FeJ mice induced a switch from a Th1- to a Th2-type response and resulted in enhanced production of sIL-4R. Addition of rIL-12 to splenic cell cultures, however, was found not to have a direct effect on sIL-4R production induced by IL-4 or T cell mitogens. Moreover, the production of sIL-4R appears to be little influenced by Th1-produced cytokines, inasmuch as recombinant interferon-gamma or supernatants derived from antigen-stimulated Th1 clones did not affect the production of sIL-4R by activated splenic cultures. Despite its correlation with Th2 responses, the presence of IL-4 was not an absolute requirement for the up-regulation of the expression of sIL-4R because increased levels could be induced on cells obtained from IL-4-/- mice. These results indicate that, although enhanced sIL-4R production is a feature related to the activation and/or generation of Th2 responses, it is not absolutely dependent on IL-4 or directly inhibited by IL-12 or Th1 cytokines.     

Isolation of high-affinity murine interleukin 2 receptors as detergent-resistant membrane complexes

Murine T cells and T cell lines bearing high- and low-affinity receptors for interleukin (IL) 2 were chemically cross-linked to radiolabeled IL 2 and subjected to differential detergent extractions to evaluate the extent of IL 2 receptor association with the nonionic detergent-resistant framework of the plasma membrane. Low-affinity receptors were readily solubilized by nonionic detergent extraction of whole cross-linked cells, while solubilization of high-affinity receptors required a stronger ionic detergent suggesting their association with a membrane structure that is resistant to nonionic detergents. To achieve physical separation of low- and high-affinity receptors, cells cross-linked to 125I-labeled IL 2 were centrifuged through a sucrose barrier containing Triton X-100. Alternatively, Triton X-114 extracts of plasma membrane fractions were partitioned into aqueous and detergent phases. By either approach, high-affinity receptors differed from low-affinity ones by their increased density and consisted of detergent-resistant complexes containing p55-p75 heterodimers. The low-affinity receptors, on the contrary, were of low density and consisted exclusively of detergent-soluble p55 subunits. High density and resistance to nonionic detergent extraction of high-affinity IL 2 receptors suggest their integration into lateral microdomains of the detergent-resistant framework of the plasma membrane.     

PGE2 receptors on murine splenic lymphocytes: effects of tumor bearing.

Earlier studies from this laboratory revealed that killer lymphocyte lineages are inactivated in the tumor-bearing host by macrophage-derived PGE2. In this study, we examined whether tumor bearing causes a change in the density or affinity of PGE2 receptors on lymphocytes, making them more vulnerable to PGE2 action, and whether it enhances PGE2 production by host macrophages. PGE2 receptors were examined on both unfractionated and monocyte-macrophage-depleted splenocytes of normal or tumor-bearing C3H/HeJ mice (at 25 days following s.c. transplantation of 10(6) C3 mammary adenocarcinoma cells) using a [3H]PGE2 binding assay in the presence of increasing (up to 10(4)-fold) concentrations of unlabeled PGE2 (or PGA or PGF2 alpha as specificity controls) followed by a Scatchard analysis. PGE2 production by splenic macrophages was measured with a radio-immunoassay. Results revealed that splenocytes in normal and tumor-bearing mice bear specific receptors for PGE2, since splenocyte binding of [3H]PGE2 (10(-9) M) was inhibited in the presence of excess unlabeled PGE2, but not 10(-4)-fold excess PGA or PGF2 alpha. Tumor-bearing did not appear to cause an appreciable change in the affinity or density of these receptors, since Kd and Bmax values for PGE2 binding were similar for normal and tumor-bearing mice. However, specific PGE2-binding by splenocytes in vitro was reduced in tumor-bearing mice. Three types of evidence indicate that this resulted from a partial occupation of PGE2 receptors on lymphocytes with PGE2 produced by splenic macrophages of tumor-bearing hosts. First, depletion of monocyte-macrophages from the splenocyte population improved this binding in tumor-bearing but not normal mice.(ABSTRACT TRUNCATED AT 250 WORDS)     

Human IFN-gamma up-regulates IL-2 receptors in mitogen-activated T lymphocytes.

This study examined the role of human recombinant interferon-gamma (rIFN-gamma) in the expression of interleukin-2 receptors (IL-2R) by human T lymphocytes. rIFN-gamma enhanced total numbers of IL-2R in mitogen-activated but not resting T cells. Scatchard plot analysis indicated that rIFN-gamma increased both high- and low-affinity receptors, with a predominant effect on the latter. Phytohaemagglutinin (PHA)-activated T cells treated with IFN-gamma showed higher IL-2 binding and greater IL-2 internalization and degradation than cells treated with PHA alone. There was a corresponding increase of mitogen-driven proliferative responses, indicating an increase of functional receptors in IFN-treated cultures. IFN-gamma may influence T-cell activation and proliferation by enhancing expression of IL-2R and promoting IL-2 uptake by mitogen-activated lymphocytes.     

Analysis of clonality of tumour infiltrating lymphocytes in breast cancer and uveal melanoma.

Fresh tumour infiltrating lymphocytes (TILs) from 6 uveal melanomas and 4 breast cancers were analysed by flow cytometry with a panel of 6 monoclonal antibodies to V beta regions of the T cell receptor (V betas 5a, 5b, 5c, 6, 8a and 12a). With a single exception where one TIL sample lacked V beta 12a, lymphocytes from both tumour and blood contained cells reactive with all 6 probes, and no probe was highly dominant or missing. The proportions of reactive cells differed between tumour and blood within each patient. The data indicate that while tumour infiltrating lymphocytes have a capacity to locate selectively within the tumour they nonetheless comprise a population expressing a diversity of TCR V beta genes.     

Role of IL-2 and IL-4 in exacerbations of murine antigen-induced arthritis.

In this study the roles of different T-cell subsets, and produced cytokines, were investigated in an animal model for acute exacerbations. Flare-up reactions are inducible in the chronic phase of a smouldering antigen-induced inflammation by injection of a small amount of an antigen into a hyper-reactive knee joint. In vivo treatment with anti-CD4 monoclonal antibodies (mAb) almost totally blocked the flare reaction, whereas anti-CD8 treatment did not exert any effect. The role of T-helper 1 (Th1) cells in delayed-type hypersensitivity-resembling diseases is generally entitled proinflammatory, whereas Th2 cells act in an anti-inflammatory manner. To investigate the role of these T-cell subsets in flare-up reactions, anti-interleukin-2 (IL-2) and anti-IL-4 mAb treatments were performed. Anti-IL-2 treatment partly blocked the flare reaction, and anti-IL-4 treatment, although the result was unexpected, blocked the flare more efficiently. Furthermore, when human recombinant IL-2 (hrIL-2) and murine recombinant IL-4 (mrIL-4) were co-injected with the antigen to test their ability respectively to potentiate or down-regulate the flare reaction, both cytokines demonstrated additional pro-inflammatory effects, although hrIL-2 was more potent than mrIL-4. The mere effect of hrIL-2 and mrIL-4 was studied by direct injection into a hyperreactive joint. No flare-up reaction or cell-influx could be induced, suggesting that other mediators are needed to exert pro-inflammatory effects of IL-2 or IL-4. We conclude that not only Th1 cells, but also Th2 lymphocytes (at least regarding IL-4 production) may play a pro-inflammatory role in flare-up reactions of chronic arthritis. Considering therapeutic application of Th2 cell-derived cytokines, one should be aware of the possible pro-inflammatory potential of IL-4.     

骨髓间充质干细胞体外对T淋巴细胞分泌IL-2、IL-4功能的影响

目的探讨间充质干细胞(Mesenchymal stem cells,MSCs)对T淋巴细胞分泌功能的调节作用.方法体外分离培养、扩增人骨髓MSCs,并通过形态学特征及流式细胞术检测其表面标志加以鉴定.将不同数量的MSCs(5×103、1×104、5×104个细胞/孔)分别与PHA激活的T细胞和混合淋巴细胞反应(MLR)体系共培养,并将不同浓度(25%、50%、75%)的MSCs培养上清和MLR体系共培养.应用ELISA分别检测各培养上清液中T细胞分泌IL-2、IL-4的水平.结果骨髓MSCs能抑制PHA作用下的T细胞分泌IL-2、IL-4(P＜0.05),并呈MSCs数量相关性(P＜0.05),且对IL-2的抑制作用更显著;也可抑制MLC体系中T细胞分泌IL-2、IL-4(P＜0.05),但各数量组的MSCs的抑制作用无显著性差异(P＞0.05).不同浓度的MSCs培养上清均可抑制MLC体系中T细胞分泌IL-2、IL-4(P＜0.05),但不同浓度的MSCs培养上清组对IL-4抑制作用无显著性差异(P＞0.05).结论骨髓MSCs及其培养上清均可抑制PHA或异体抗原作用下的T细胞分泌IL-2、IL-4,提示MSCs可能是直接作用于T细胞或通过分泌可溶性因子调节 Th1/Th2反应平衡而发挥免疫调节作用的.     

Expression of IL-10 receptors on epithelial cells from the murine small and large intestine

The appearance of chronic intestinal inflammation in IL-10 knockout mice suggests IL-10 may inhibit adverse responses to luminal antigen. Moreover, this inflammation is associated with an increase in class II MHC molecule expression on intestinal epithelial cells. Thus, the role of IL-10 regulation in epithelial cell function was investigated. Using RT-PCR, it was shown that intestinal epithelial cells express mRNA for both subunits of the IL-10 receptor-signaling complex. In addition, biotinylated IL-10 was shown to bind to both cultured and freshly isolated intestinal epithelial cells prepared from the small or large intestine. This binding appeared specific as it was blocked by neutralizing antibodies to IL-10 but not the isotype control. Moreover, an excess of native IL-10 also inhibited the binding of radiolabeled IL-10. To evaluate whether IL-10 mediated any functions through this receptor, epithelial cells were cultured with IL-10 alone or with IFN-gamma plus IL-10. IL-10 alone had no detectable effects on epithelial cell growth or their expression of class II MHC molecules but it did antagonize the effect of IFN-gamma on the viability of cultured cells. In addition, IL-10 blocked the IFN-gamma-induced expression of class II MHC molecules on cultured epithelial cells. These results suggest that IL-10 binds to a specific receptor on intestinal epithelial cells and may regulate the contribution of epithelial cells to the inflammatory and immune response in the digestive tract.     

IL-2 receptors on rabbit T-cell lines and their transfectants expressing the human IL-2 receptor alpha chain.

Low-affinity (dissociation constant: Kd = 7 nM) and high-affinity (Kd = 27 pM) interleukin-2 receptors (IL-2R) were detected on rabbit T-cell lines by IL-2 binding studies. Chemical cross-linking studies using 125I-labelled IL-2 showed that rabbit low-affinity IL-2R was singly expressed alpha-chain (MW 55,000) and that high-affinity IL-2R was composed of at least alpha- and beta- (MW 75,000) chains, similar to the human and murine counterparts. The existence of an additional chain (MW 25,000) was suggested in the rabbit IL-2R. Rabbit T-cell transfectant lines were established by human IL-2R alpha-chain (IL-2R alpha) cDNA transfection. These transfectant lines possessed not only extremely large numbers of human IL-2R alpha (over 10 times more than endogenous rabbit alpha-chain) but also twice as many high-affinity sites as their parental lines. The number of high-affinity sites on the transfectants significantly decreased when human alpha-chains were blocked, indicating that these transfectants expressed high-affinity receptor consisting of the exogenous human alpha-chain and rabbit beta-chain. This was confirmed by cross-linking experiments. The observation that expression of extremely large numbers of exogenous alpha-chains lead to an increase of the total number of high-affinity sites in the apparent absence of an increase of beta-chain expression raises the possibility that not only the beta-chain but also the alpha-chain may play an important role in regulating the number of high-affinity receptors.     

Isolated hapten-binding receptors of sensitized lymphocytes. IV. Expression of immunoglobulin variable regions in (4-hydroxy-3-nitrophenyl) acetyl (NP)-specific receptors isolated from murine B and T lymphocytes.

Primary anti-NP [(4-hydroxy-3-nitrophenyl)acetyl] antibodies and NP-specific receptors isolated from T cells of mice carrying the Ig-1^b allotype express the NP^b idio-type and a fine specificity marker (heteroclicity) which are both controlled by V genes in the heavy chain linkage group. While in antibodies both markers are invariably expressed in association with Ig light chains of the Λ type, antigen-binding T cell-derived receptors appear not to follow this rule in that they do not significantly bind to anti-Λ antisera. Mice of the SJL strain also carry the Ig-l^b allotype but express a defect in Λ chain synthesis, and their primary anti-NP response is thus almost completely devoid of Λ-bearing heteroclitic molecules of the NP^b idiotype. In contrast, both NP^b idiotype and heteroclicity are expressed in the majority of T cell-derived receptors. This result permits a positive discrimination between T cell-derived receptors and humoral antibodies in the SJL strain and shows that V gene expression follows different rules in the T and the B cell compartment. The expression of Ig V regions in isolated T cell-derived receptors was also analyzed with antisera binding to presumed “framework” determinants of the V_H and the V_Λdomains of Ig. A reaction of anti-V_H antiserum with isolated T cell receptors could clearly be demonstrated. The anti-V_Λ antiserum may bind to a minor fraction of the NP-specific molecules in our T cell receptor preparations.     

Expression of functional interleukin 2 receptors on chronic lymphocytic leukaemia B lymphocytes is modulated by recombinant interleukin 2.

In 16 of 18 chronic lymphocytic leukaemia (CLL) patients examined, a significant proportion of B cells in the leukaemic clone bound monoclonal antibodies specific for the interleukin 2 (IL-2) receptor site (CD25). B lymphocytes from patients tested showed a direct response to recombinant interleukin (rIL-2) during culture in vitro as shown by: (a) a ligand-mediated upregulation in the level of IL-2 receptor (IL-2R) expression (12 of 12 patients), (b) an increase in cell size (eight of nine patients), (c) an increase in 3H-thymidine uptake (four of six patients). Taken together, this evidence suggests that the majority of leukaemic B cells from all the CLL patients examined expressed functional IL-2 receptors in vitro. Intriguingly, maximal receptor upregulation or increase in cell size was achieved at a lower concentration (50 u/ml) of rIL-2 than was required to achieve maximal 3H-thymidine incorporation.