Measurement of interleukin 6.

Interleukin 6 (IL-6) is a pluripotent cytokine with multiple effects on many different cell types. It is produced by a variety of cells in response to immunological and other stimuli. This unit describes a simple and sensitive assay for human, rat, rabbit, pig, and mouse IL-6, based on IL-6-dependent proliferation of a murine B cell hybridoma cell line, B9.

NF-kappaB elements contribute to junB inducibility by lipopolysaccharide in the murine macrophage cell line RAW264.7.

Macrophages respond to bacterial lipopolysaccharide (LPS) by activating latent cis-acting factors that initiate transcription of immediate early genes. One such immediate early gene, junB, is induced by LPS in macrophages within 30 min. To identify elements that mediate the induction of junB by LPS, upstream and downstream sequences flanking the junB gene were examined by transient expression in the RAW264.

Constitutive activation of STAT3 is associated with the acquisition of an interleukin 6-independent phenotype by murine plasmacytomas and hybridomas.

Interleukin 6 (IL-6), the major growth factor for myeloma cells, signals through the activation of signal transducers and activators of transcription (STAT) proteins. An important step in the malignant progression of murine plasmacytomas is the transition from dependence on IL-6 to a state of IL-6 independence. To elucidate the mechanism whereby IL-6 independence occurs, intracellular signaling events elicited by IL-6 in both IL-6-dependent and -independent plasmacytomas and hybridomas were compared.

Evaluation of methods for transient transfection of a murine macrophage cell line, RAW 264.7.

Monocyte/macrophage cell lines are fastidious cells commonly used in transient transfection experiments. In the course of a study of gene regulation by lipopolysaccharide (LPS), we have compared several methods for DNA-mediated cell transfection to determine which would be optimally applicable to the macrophage line, RAW 264.7.

Regulation of actin cytoskeleton in lymphocytes: PKC-delta disrupts IL-3-induced membrane ruffles downstream of Rac1.

In the murine pre-B lymphoid cell line Baf3, the presence of IL-3 is required for the formation of membrane ruffles that intensely stain for actin and are responsible for the elongated cell phenotype. Withdrawal of IL-3 dissolves ruffled protrusions and converts the cell phenotype to round. Flow cytometric analysis of the cell shape showed that an inactive analog of Rac1 but not inactive RhoA or inactive cdc42 rounds the cells in the presence of IL-3.

Sodium butyrate suppresses apoptosis in human Burkitt lymphomas and murine plasmacytomas bearing c-myc translocations.

We report that sodium butyrate, a natural product of fiber degradation by colonic bacteria, markedly suppresses c-Myc-mediated apoptosis induced in murine plasmacytomas and human Burkitt lymphomas by growth factor deprivation, but not in cell lines devoid of c-myc translocations. Attenuation of cell death is associated with downregulation of the rearranged c-myc and activation of pRb via its dephosphorylation. We suggest that in vivo sodium butyrate may play an important role in plasmacytomagenesis by supporting the survival of cells with c-myc translocations, which otherwise would be eliminated by the lack of growth factors.

Cross-talk between protein kinase C-alpha (PKC-alpha) and -delta (PKC-delta): PKC-alpha elevates the PKC-delta protein level, altering its mRNA transcription and degradation.

Studies utilizing the overexpression of individual isoforms indicated that both PKC-alpha and -delta promote a number of biological effects, including inhibition of DNA synthesis associated with rearrangements of the actin cytoskeleton in the murine B-cell lymphoma (Baf3), differentiation of the murine promyelocyte line 32D, and activation of MAP kinase in CHO fibroblasts. We postulated that these results reflect some form of cross-regulation between PKC-alpha and -delta rather than their functional redundancy. In this report, we show that overexpression of PKC-alpha in Baf3 and 32D leads to an elevation of the endogenous PKC-delta mRNA and protein levels.

Mechanism of apoptosis suppression by phorbol ester in IL-6-starved murine plasmacytomas: role of PKC modulation and cell cycle.

We show here that the mode of cell death in IL-6-starved T1165 and T1198 plasmacytoma cell lines is apoptosis, and that it can be suppressed by phorbol ester (PMA) treatment in a protein kinase C (PKC)-mediated process that involves alpha and/or delta isozymes. PMA-induced PKC activation, but not the depletion that follows it, participates in the suppression of apoptosis. Extended PKC activation is necessary but not sufficient for the apoptosis suppression.

An acute phase response factor/NF-kappa B site downstream of the junB gene that mediates responsiveness to interleukin-6 in a murine plasmacytoma.

The immediate early gene, junB, is induced by interleukin-6 (IL-6) in plasmacytomas. In order to identify enhancers that mediate this effect, we cloned upstream and downstream sequences flanking the gene into a luciferase reporter gene vector containing the junB promoter and evaluated the IL-6 inducibility of these sequences by transient expression in an IL-6-dependent plasmacytoma cell line. Although a 6.

Suramin blocks the binding of interleukin-1 to its receptor and neutralizes IL-1 biological activities.

This report demonstrates the ability of the anti-cancer drug suramin to interfere with the binding of interleukin (IL)-1 to its receptor and to inhibit IL-1-induced biological activities. In a radioreceptor cell based assay, suramin inhibits the binding of IL-1 alpha to several murine cell lines expressing predominantly type I and type II IL-1 receptors. Affinity cross-linking experiments using IL-1 alpha and EL-4.

Suramin interferes with interleukin-6 receptor binding in vitro and inhibits colon-26-mediated experimental cancer cachexia in vivo.

Neoplastic diseases are frequently associated with metabolic changes collectively known as cancer cachexia. The presence of cachexia complicates therapeutic intervention and is an important cause of death in cancer patients. At present there is no effective treatment for cachexia.

Direct association of interleukin-6 with a 130-kDa component of the interleukin-6 receptor system.

Affinity cross-linking of membrane bound 125I-interleukin-6 (IL-6) on several cell lines revealed a three-band pattern of IL-6-containing cross-linked complexes with molecular masses of 100, 120, and 150 kDa. To identify the membrane components that were associated with IL-6 in the three complexes, we employed the Denny-Jaffe reagent, a heterobifunctional, cleavable cross-linker that allows the transfer of 125I from the ligand to its receptor. Samples cross-linked with Denny-Jaffe reagent were analyzed by two-dimensional SDS-polyacrylamide gel electrophoresis in which the cross-linker was cleaved prior to the second dimension.

Interleukin 6 reduces lipoprotein lipase activity in adipose tissue of mice in vivo and in 3T3-L1 adipocytes: a possible role for interleukin 6 in cancer cachexia.

To investigate whether interleukin 6 (IL-6) might be a potential mediator of the depleted fat reserves observed in malignancy-associated cachexia, we measured lipoprotein lipase (LPL) activity in adipose tissue of mice after administration of IL-6 or tumor necrosis factor and in cultured adipocytes after addition of these cytokines. Injection of IL-6 i.p.

Multichain structure of the IFN-alpha receptor on hematopoietic cells.

The structure of IFN-alpha receptor was studied by 1) developing antibodies against the receptor, and 2) screening a number of cell lines by affinity cross-linking to identify cells that express different IFN-alpha 2 receptor structures. We report that two different patterns of IFN-alpha 2 receptor are observed in human cells of hematopoietic origin. The predominant form of the IFN-alpha receptor is a multichain structure in which IFN-alpha 2 forms complexes of 110 and 130 kDa (alpha-subunit).

Detection of functional interferon alpha receptors in human neuroendocrine tumor cell lines using a new monoclonal antibody.

While first described as antiviral agents, interferons (IFNs) exhibit significant antiproliferative and antitumor effects as well. IFN alpha has been successfully used in clinical trials to treat several malignancies, including leukemias and certain solid tumors. While many cell types have been studied for IFN alpha receptor expression, very little is known about receptor expression on human neuroendocrine cells.

IL-6 and tumor necrosis factor-alpha. Autocrine and paracrine cytokines involved in B cell function.

IL-6 and TNF-alpha are synthesized and secreted by normal tonsillar B cells after stimulation with the polyclonal B cell activator Staphylococcus aureus Cowan strain 1 (SAC) and IL-2 as well as spontaneously by in vivo activated B cells from patients with hypergammaglobulinemia. Using specific neutralizing antibodies, both factors were shown to be involved in autocrine and/or paracrine regulation of B cell differentiation. IgG induced by SAC/IL-2 stimulation was reduced 73% with an anti-IL-6 antibody and 40% with an anti-TNF-alpha antibody.

Characterization of an interleukin-6-mediated autocrine growth loop in the human multiple myeloma cell line, U266.

It has been reported recently that freshly isolated human myeloma cell cultures proliferate in response to added interleukin-6 (IL-6). Endogenous levels of IL-6 found in the same cultures suggested that an autocrine growth loop may contribute to cell growth. However, the lack of homogenous cell populations in primary myeloma cultures has made it difficult to distinguish between paracrine and autocrine growth mechanisms.

Expression of the interleukin 6 receptor and interleukin 6 in prostate carcinoma cells.

We have probed for the presence of interleukin 6 (IL6) receptors in prostatic carcinoma cell lines (LNCaP, DU 145, and PC3) by examining their sensitivity to the cytotoxic effects of a chimeric toxin composed of IL6 and Pseudomonas exotoxin (PE). All three cell lines were killed by IL6-PE66(4)Glu, a version of IL6-PE in which the binding domain of native PE has been mutated to debilitate PE binding to its own receptor. This cytotoxic activity confirmed the presence of IL6 receptors on prostatic carcinoma cells.

Characterization of three monoclonal antibodies that recognize the interferon alpha 2 receptor.

The interferon system plays an important role in the control of viral infections and cell proliferation. These effects are mediated through the interaction of interferons with specific cell surface receptors. We report here the development of monoclonal antibodies against one of the subunits of the interferon alpha receptor.

Cell-specific toxicity of a chimeric protein composed of interleukin-6 and Pseudomonas exotoxin (IL6-PE40) on tumor cells.

IL6-PE40 is a chimeric toxin composed of human interleukin-6 (IL6) linked by a peptide bond to PE40, a form of Pseudomonas exotoxin (PE) devoid of its cell recognition domain. To identify cancer cell lines with high numbers of IL6 receptors and to assess the usefulness of IL6-PE40 as a possible anticancer agent, we evaluated the toxicity of IL6-PE40 on a variety of tumor cell lines and demonstrated that certain human myeloma and hepatoma cell lines were particularly sensitive. IL6 binding to selected hepatoma and myeloma cell lines were determined by using [125I]IL6.

T cell derived IL-6 is differentially required for antigen-specific antibody secretion by primary and secondary B cells.

IL-6 (formerly PCTGF, HP-1, BSF-2, HGF, IFN-beta 2, 26 kDa) is a recently defined lymphokine demonstrating activity on multiple cell types, including hepatocytes, thymocytes, T cells, plasmacytomas, and B cells. The biologic effects of IL-6 on lymphocytes, particularly B cells, suggest this factor may be involved in the regulation of normal immune responses. Accordingly, we have investigated the role of IL-6 in Ag-specific responses of B cells from both naive and Ag-primed mice.

Interleukin 6 (interferon beta 2) and interferon alpha/beta present in postendotoxin serum induce differentiation of murine M1 myeloid leukemia cells.

Serum from lipopolysaccharide-treated mice (postendotoxin serum, PES) induces the differentiation of M1 myeloid leukemia cells into mature macrophages, as well as supporting the proliferation of the interleukin 6 (IL6)-dependent B9 hybridoma cells. The kinetics of appearance of these two activities in PES were identical. To determine whether these two activities are due to the presence of the same substance, we tested whether anti-IL6 antibodies could neutralize the differentiation-inducing activity of PES.

IL-6 is a potent cofactor of IL-1 in IgM synthesis and of IL-5 in IgA synthesis.

In these studies we determined the capacity of IL-6 to act as a differentiation cofactor for murine Peyer's patch B cells producing different Ig classes and subclasses. In preliminary studies we determined that sufficient endogenous IL-6 was produced in LPS-induced cell systems to obscure responses to exogenous IL-6. We therefore studied IL-6 effects on Peyer's patch B cells (T cell-depleted cell populations) in the absence of LPS, relying on responses of in vivo-activated cells.

Murine thymocytes proliferate in direct response to interleukin-7.

The ability of interleukin-7 (IL-7) to stimulate murine thymocyte proliferation was investigated. IL-7, either alone or in concert with lectin, induced proliferation of adult thymocytes as well as day 13 fetal and adult CD4-/CD8-thymocytes. The IL-7-induced proliferative response of unfractionated thymocytes could not be inhibited by antibodies to IL-2, or IL-4, IL-6, or the IL-2 receptor.

In vivo induction of IL-6 by administration of exogenous cytokines and detection of de novo serum levels of IL-6 in tumor-bearing mice.

We investigated the capacity of several recombinant cytokines to induce IL-6 in vivo in both normal and tumor-bearing (TB) mice. Intravenous administration of human rhTNF-alpha, rhIL-1, rhIL-2, rhIFN-alpha A/D, and rmIFN-gamma were all capable of inducing circulating IL-6. rhTNF-alpha administration caused the greatest induction of IL-6.