IL-6 Receptor mRNA Expression Research Articles

Absence of IL-6 receptor expression in fresh childhood Burkitt's lymphoma cells and induction of IL-6 receptors by Epstein-Barr virus in vitro.

Interleukin-6 (IL-6) is a cytokine which is necessary for the differentiation of activated B cells and growth of early haemopoietic progenitors. It is used for ex-vivo expansion of myeloid progenitors in the setting of high-dose chemotherapy with autologous bone marrow transplantation (BMT). Expression of the IL-6 receptor (IL-6R) was examined in six fresh Burkitt's lymphoma (BL) cell preparations and 12 BL cell lines by reverse transcriptase polymerase chain reaction (RT-PCR) and flow cytometry (FCM). Inducibility of IL-6R mRNA expression by Epstein-Barr virus (EBV) was studied by comparing two uninfected cell lines with the same cell lines infected by EBV The phenotype of the BL cells lines was analysed by FCM and by proliferation assays in the presence of anti-IgM antibodies. None of the fresh BL cells expressed the IL-6 receptor. The BL cell lines expressed varying degrees of IL-6R mRNA and protein. In vitro infection of EBV-negative BL cell lines resulted in up-regulation of IL-6R mRNA. There was no proliferative response of the BL cell lines to IL-6, including the cells that expressed the receptor. Compared to uninfected BL cell lines, EBV-infected cell lines and lymphoblastoid cell lines (LCLs) showed a weaker or no response to anti-IgM antibodies, indicating a more mature phenotype of these cells. We conclude that the IL-6 receptor is not expressed in fresh childhood BLs, but only in BL cell lines. EBV infection in vitro leads to an up-regulation of IL-6R mRNA but not to increased proliferation. This makes growth stimulation of contaminating BL cells in the setting of autologous BMT unlikely.

P.6.012 - Expression of IL-6 receptor mRNA in the hypothalamus of restrained rat

P.6.012 - Expression of IL-6 receptor mRNA in the hypothalamus of restrained rat

Expression of interleukin-6 receptor (IL-6R) and gp130 mRNA in PC12 cells and sympathetic neurons: modulation by tumor necrosis factor α (TNF-α)

Recent findings indicate that IL-6, besides its various biological effects, also exerts neurotrophic and neuroprotective functions. Using the pheochromocytoma cell line PC12 and cultured primary sympathetic neurons, we investigated whether neurons express the IL-6 receptors, IL-6R and gp130, and how they might be regulated. For these studies we used RT-PCR and in situ hybridization. We provide here evidence for the expression of functional IL-6Rs in peripheral sympathetic neurons and PC12 cells. Furthermore we demonstrate that cytokines modulate the expression of IL-6R and gp130 mRNA. This modulation is much more pronounced in neuronally-differentiated PC12 cells than in undifferentiated cells. Among various cytokines tested, tumor necrosis factor α (TNF-α) turned out to be a major regulator of the IL-6R and gp130 mRNA expression. The induction was time- and dose-dependent for both genes. Maximal induction was reached within 16 h at a concentration of 0.1 nM TNF-α. The stimulatory effect of TNF-α on the IL-6R system was completely inhibited by the simultaneous addition of the glucocorticoid dexamethasone. In summary, our results show that sympathetic neurons and neuron-like differentiated PC12 cells express functional IL-6R and gp130, and that the expression of their mRNAs is modulated by cytokines. We suggest that cytokines such as IL-6 can modulate sympathetic neuron function.

Interleukin (IL)-6 induction of osteoclast differentiation depends on IL-6 receptors expressed on osteoblastic cells but not on osteoclast progenitors.

We reported that interleukin (IL) 6 alone cannot induce osteoclast formation in cocultures of mouse bone marrow and osteoblastic cells, but soluble IL-6 receptor (IL-6R) strikingly triggered osteoclast formation induced by IL-6. In this study, we examined the mechanism of osteoclast formation by IL-6 and related cytokines through the interaction between osteoblastic cells and osteoclast progenitors. When dexamethasone was added to the cocultures, IL-6 could stimulate osteoclast formation without the help of soluble IL-6R. Osteoblastic cells expressed a very low level of IL-6R mRNA, whereas fresh mouse spleen and bone marrow cells, both of which are considered to be osteoclast progenitors, constitutively expressed relatively high levels of IL-6R mRNA. Treatment of osteoblastic cells with dexamethasone induced a marked increase in the expression of IL-6R mRNA. By immunoblotting with antiphosphotyrosine antibody, IL-6 did not tyrosine-phosphorylate a protein with a molecular mass of 130 kD in osteoblastic cells but did so in dexamethasone-pretreated osteoblastic cells. Osteoblastic cells from transgenic mice constitutively expressing human IL-6R could support osteoclast development in the presence of human IL-6 alone in cocultures with normal spleen cells. In contrast, osteoclast progenitors in spleen cells from transgenic mice overexpressing human IL-6R were not able to differentiate into osteoclasts in response to IL-6 in cocultures with normal osteoblastic cells. These results clearly indicate that the ability of IL-6 to induce osteoclast differentiation depends on signal transduction mediated by IL-6R expressed on osteoblastic cells but not on osteoclast progenitors.

Investigative Urology: Inhibitory Effect of Dexamethasone and Progesterone in Vitro on Proliferation of Human Renal Cell Carcinomas and Effects on Expression of Interleukin-6 or Interleukin-6 Receptor

Interleukin-6 (IL-6) has been suggested as an autocrine growth factor of human renal cell carcinomas. Since steroids are known to inhibit IL-6 gene expression, we investigated their effects on the growth of renal cell carcinoma. Dexamethasone inhibited proliferation of 2 of 4 renal cell carcinoma cell lines in a dose-dependent manner. In one of these 2 cell lines, IL-6 gene expression was also inhibited, but not in the other. The inhibitory effect of dexamethasone on cell proliferation was not reversed by the exogenous IL-6. In 1 of the 2 remaining cell lines, the inhibition of IL-6 gene expression was observed, although there was no inhibition of cell proliferation. Thus, inhibition of growth by dexamethasone did not correlate with an inhibitory action of dexamethasone on IL-6 mRNA expression. Progesterone inhibited the growth of 1 cell line without concomitant inhibition of IL-6 gene expression. Expression of IL-6 receptor mRNA was not altered. A dose-dependent increase in mRNA expression of gp130, the transducer of IL-6 signal, was induced by dexamethasone and progesterone in 2 and 1 of the 4 cell lines, respectively. These data suggest that, in some renal cell carcinomas, steroids may inhibit cell proliferation by a mechanism independent of their effects on mRNA expression of IL-6 and IL-6 receptors. Dexamethasone may be useful, not only for palliation of paraneoplastic syndrome caused by overproduction of IL-6, but also for inhibition of growth of renal cell carcinomas.

Interleukin-6 and interleukin-6 receptor are expressed by cultured glomerular epithelial cells.

Interleukin-6 (IL-6) has been extensively studied in mesangial cells but little is known about the expression of this cytokine and its receptor in glomerular epithelial cells (GEC). IL-6 was detected in the culture supernatants of human GEC and its production was enhanced in time and dose dependent manner by lipopolysaccharide (LPS), interleukin-1 beta (IL-1 beta) and tumour necrosis alpha (TNF-alpha). Quiescent, serum-starved GEC did not express clearly IL-6 mRNA. Stimulation of cells with LPS, TNF-alpha or IL-1 beta resulted in an increase of detectable IL-6 mRNA. Interestingly, it was found that IL-6 induced its own mRNA attesting that this cytokine was secreted in autocrine fashion by GEC. GEC expressed IL-6 receptor (IL-6R) as demonstrated directly by the existence of IL-6R mRNA detected by northern blotting. Stimulation of GEC by pro-inflammatory mediators such as LPS increased the expression of IL-6R mRNA. The soluble form of IL-6 receptor (sIL-6R) was not detectable in the culture supernatants harvested from untreated or cytokine-treated cells. We investigated further, whether IL-6 may influence growth of cultured GEC. Incubation of GEC with recombinant (r) IL-6 resulted in a dose dependent increase in 3H thymidine incorporation indicating that IL-6 acts as an autocrine growth factor for GEC. We conclude that GEC are a potent source of IL-6, the local excessive expression of IL-6 and its receptor may play a substantive role in the regulation of processes which appear critical to the initiation of progressive glomerular disease such as cell proliferation.

Up-regulation of IL-6-receptors by IL-3 on a plasma cell leukaemia cell line which proliferates dependently on both IL-3 and IL-6.

In this report we show that IL-3 up-regulates the expression of IL-6-receptors on a plasma cell leukaemia cell line which proliferates in response to both IL-3 and IL-6. Quantitative binding studies and Scatchard analysis revealed that HSM-2.3 has a single class of high-affinity IL-6 binding sites (200/cell, dissociation constant at the equilibrium 1.43 x 10(-11) M). After stimulation with IL-3, HSM-2.3 has 1200 binding sites. IL-6 receptor mRNA expression was detected in HSM-2.3 only after stimulation with IL-3.

Regulation of IL-6 and the hepatic IL-6 receptor in acute inflammation in vivo

The serum levels of interleukin 6 (IL-6) and the expression (mRNA) of the 80 kDA IL-6 receptor (IL-6R) were examined in three different models of acute inflammation. Rats were treated with either Freund's complete adjuvant (FA) via intraperitoneal injection, LPS via intravenous injection, or turpentine via subcutaneous injection. Using bio- and specific immunoassays, rat serum levels of IL-6, corticosterone, and acute phase proteins were quantified. LPS treatment induced the quickest and greatest serum IL-6 response (>100 ng/ml within 3 h). In comparison, sera from turpentine and FA-treated rats contained much lower levels of IL-6 activity (<10 ng/ml). Serum corticosterone levels increased by 3 h after injection in all three models, and equivalent raised serum levels of acute phase proteins were detected within 12–24 h. The expression of IL-6 receptor mRNA in hepatocytes increased markedly as early as 3 h after treatment and message levels began to decline by 6–12 h in all three models. To analyze the individual effects of raised corticosterone and IL-6 on the expression of hepatic IL-6R mRNA, rats were injected with either dexamethasone (Dex) or purified recombinant rat IL-6 (rIL-6) via intraperitoneal injection. Rats injected with rIL-6 showed highly induced IL-6R mRNA levels as early as 1 h after injection, and Dex-injected rats showed a significant but less dramatic rise in IL-6R message levels. Dex- or rIL-6-injected rats demonstrated a distinct profile of acute phase protein response different from that seen in the three experimental models. Regulation of IL-6R gene expression in the liver in vivo depends on a complex interaction between the hepatocyte and a combination of cytokines and other hormones.

Retinole Acid Inhibits Interleukin-6–Induced Macrophage Differentiation and Apoptosis in a Murine Hematopoietic Cell Line, Y6

We established a radiation-induced murine hematopoietic cell line, Y6, that could be induced to differentiate into macrophages by interleukin-6 (IL-6). IL-6 also induced growth inhibition and apoptosis in Y6 cells. Retinoic acid (RA) inhibited such effects of IL-6 on Y6 cells. The inhibitory effect of RA on the effects of IL-6 was not caused by the downregulation of the IL-6 receptor, because RA neither affected the expression of IL-6 receptor mRNA nor the expression of IL-6 receptor molecule on the cell surface. Furthermore, RA did not inhibit the IL-6-induced expression of junB mRNA, indicating that the expression of functionally active IL-6 receptor and the signal transduction pathway activating the junB gene are not inhibited by RA. IL-6-induced macrophage differentiation of Y6 cells was preceded by the downregulation of the c-myc gene, which was also prevented by RA. Because the inhibitory effect of RA on Y6 cells was reversible and seemed not to require de novo protein synthesis, the RA receptor by itself might be directly involved in the inhibition of the IL-6 signal transduction pathway. The results indicated that the IL-6 signal transduction pathways leading to the induction of macrophage differentiation and junB gene expression can be dissected by RA.

Retinoic acid inhibits interleukin-6-induced macrophage differentiation and apoptosis in a murine hematopoietic cell line, Y6

We established a radiation-induced murine hematopoietic cell line, Y6, that could be induced to differentiate into macrophages by interleukin- 6 (IL-6). IL-6 also induced growth inhibition and apoptosis in Y6 cells. Retinoic acid (RA) inhibited such effects of IL-6 on Y6 cells. The inhibitory effect of RA on the effects of IL-6 was not caused by the downregulation of the IL-6 receptor, because RA neither affected the expression of IL-6 receptor mRNA nor the expression of IL-6 receptor molecule on the cell surface. Furthermore, RA did not inhibit the IL-6-induced expression of junB mRNA, indicating that the expression of functionally active IL-6 receptor and the signal transduction pathway activating the junB gene are not inhibited by RA. IL-6-induced macrophage differentiation of Y6 cells was preceded by the downregulation of the c-myc gene, which was also prevented by RA. Because the inhibitory effect of RA on Y6 cells was reversible and seemed not to require de novo protein synthesis, the RA receptor by itself might be directly involved in the inhibition of the IL-6 signal transduction pathway. The results indicated that the IL-6 signal transduction pathways leading to the induction of macrophage differentiation and junB gene expression can be dissected by RA.

The human hepatoma Hep3B cell line as an experimental model in the study of the long-term regulation of acute-phase proteins by cytokines.

The regulation of the synthesis by the cytokines interleukin-1 (IL-1) and IL-6 of the positive acute-phase protein alpha 1-acid glycoprotein (AGP) and of the negative acute-phase protein alpha 2-HS glycoprotein (AHSG) has been studied in a long-term culture system of the human hepatoma cell line Hep3B. The culture system contained 30 nM-sodium selenite as the only supplement. This allowed maintenance of the synthesis of the proteins under study at a near steady state for over 3 months. An increase in AGP mRNA and a decrease in AHSG mRNA were observed when cells were treated for two successive 48 h-periods with monocyte-conditioned medium. A return to basal levels was obtained after cessation of the cytokine addition. Two further additions of cytokines led to alterations in mRNA levels similar to those observed following the first cytokine treatment. The amounts of AGP and AHSG secreted were altered in accordance with the mRNA modifications. These results suggest that new cytokine receptors were being constantly synthesized during cell culture. When cytokines were present in the culture medium for 10 days, maximum alterations in AGP and AHSG synthesis were obtained following 2 and 4 days of treatment respectively, but further alterations in protein levels could not be observed afterwards. Expression of IL-6 receptor mRNA was not up-regulated by cytokines, but only by 1 microM-dexamethasone. Our results show that, in this long-term culture system, cytokines induce a response in hepatoma cells similar to that observed in vivo during human inflammatory states. This model could be used to evaluate the effects of agonists or antagonists of cytokines responsible for the hepatic acute-phase protein response.

Modulation of GM-CSF receptor beta-subunit and interleukin-6 receptor mRNA expression in a human megakaryocytic leukemia cell line.

Our present study was designed to clarify the mechanism by which the same megakaryocyte progenitor cells respond to various cytokines at different stages of megakaryocyte development. We examined the changes in mRNA expression of granulocyte macrophage colony-stimulating factor receptor beta-subunit (GM-CSFR beta-subunit), which was a common subunit of a high-affinity interleukin-3 receptor (IL-3R) and a high-affinity GM-CSFR, and interleukin-6 receptor (IL-6R) during megakaryocyte development in a human megakaryocytic leukemia cell line (CMK) which could proliferate and/or differentiate in the presence of 12-O-tetradecanoylphorbol 13-acetate (TPA), IL-3, GM-CSF, and IL-6. We found that GM-CSFR beta-subunit mRNA was expressed constitutively in CMK cells and was transiently down-regulated by TPA and IL-6, while the expression of IL-6R mRNA was increased by TPA in association with the differentiation of megakaryocytes. Furthermore, the TPA-induced down-regulation of GM-CSFR beta-subunit mRNA expression and its recovery were blocked by cycloheximide (CHX), a protein synthesis inhibitor, suggesting that these modulations required de novo protein synthesis. These findings imply that multi-lineage cytokines such as GM-CSF and IL-3 may contribute preferentially to the regulation of the earlier development of megakaryocyte progenitor cells with high densities of multi-lineage cytokine receptors, while IL-6 may be limited in its action to supporting the maturation of more differentiated megakaryocyte progenitor cells.(ABSTRACT TRUNCATED AT 250 WORDS)