Lester T. May

Role of interleukin-6 in regulating synthesis of C-reactive protein and serum amyloid A in human hepatoma cell lines.

Previous studies have demonstrated that synthesis of the two major human acute phase proteins, C-reactive protein (CRP) and serum amyloid A (SAA), by human hepatoma cell lines was not affected by preparations of interleukin-1 (IL-1) or tumor necrosis factor α but was increased after exposure to conditioned medium from lipopolysaccharide-activated monocytes. We report that neutralizing antibodies raised against E. coli-derived recombinant human interleukin-6 (IL-6) were capable of inhibiting induction of CRP and SAA by monocyte conditioned medium in both NPLC/PRF/5 and Hep 3B cell lines. Partially purified IL-6 from lipopolysaccharide-stimulated human fibroblasts and recombinant derived IL-6 induced both CRP and SAA synthesis in a concentration dependent manner in the NPLC/PRF/5 cell line. In contrast, in Hep 3B cells, IL-6 alone had no discernable effect on the induction of either CRP or SAA, but was capable of causing increased synthesis of α 1 -protease inhibitor and fibrinogen and reduced synthesis of albumin. The addition of IL-1 to IL-6 led to induction of both CRP and SAA in Hep 3B cells, but did not augment the response of either CRP or SAA in NPLC/PRF/5 cells. These studies indicate that IL-6 plays a central role in induction of both CRP and SAA synthesis in the two human hepatoma cell lines. The different observations in the two hepatoma lines we studied likely reflect differences between these transformed cell lines in genetic regulatory mechanisms or other intracellular mechanisms by which extracellular signals affect expression of the CRP and SAA genes. Furthermore, our findings suggest that the mechanisms by which IL-6 regulates synthesis of CRP and SAA in Hep 3B cells differ from those involved in induction of α 1 -protease inhibitor and fibrinogen and in inhibition of albumin synthesis.

Interleukin-6 gene expression in human endothelial cells: RNA start sites, multiple IL-6 proteins and inhibition of proliferation

Interleukin-6 (IL-6) is a cytokine which is not only produced by a wide variety of different cells but one which also affects the function of diverse tissues. We have studied the expression of the IL-6 gene in freshly explanted human umbilical vein endothelial cells (HUVEC) and have also evaluated the effect of IL-6 on HUVEC proliferation. Cytokines like interleukin-1 alpha (IL-1 alpha) and tumor necrosis factor (TNF) as well as bacterial products such as the lipopolysaccharide (LPS) rapidly enhance production of biologically active IL-6 by HUVEC (IL-6 bioassay: increase in alpha 1-antichymotrypsin secretion by Hep3B2 cells and its neutralization by antiserum to E. coli-derived human IL-6). The two inducible RNA start sites in the IL-6 gene that are used in cytokine-induced fibroblasts (at +1 and -21) are also used in the same relative proportion (+1 greater than -21) in cytokine or LPS-induced HUVEC as determined by S1-nuclease protection assays for IL-6 transcripts. Immunoaffinity chromatography followed by Western blotting shows that IL-6 species secreted by IL-1 alpha-induced HUVEC are of molecular mass 23-25, 27-30 and 45 kDa as judged by SDS-PAGE under reducing conditions. Finally, rIL-6 inhibits [3H]-thymidine incorporation by HUVEC in a dose-dependent manner. Thus IL-6 is not only produced by HUVEC but may also affect its proliferation. The ability of the vascular endothelium to rapidly secrete IL-6 in response to inflammation-associated cytokines is of strategic value since it generates a circulatory signal which helps mobilize the acute phase plasma protein response and enlists the immune system in host defence.

Regulation of Expression of Interleukin‐6

The human genome contains a single interleukin-6 (IL-6) gene located at 7p21.’-’ To a first approximation, the expression of 1L-6 is enhanced in almost every human tissue in response to “damage” of almost every kind (TABLES 1 and 2). Furthermore, all of the numerous effects of IL-6 on different tissue and organ systems are such that they appear to help restrict tissue damage and to allow the host to recover from injury (TABLE 3) . The regulation of expression of the IL-6 gene appears to be particularly adapted to the key function of this cytokine, namely, that of an alarm signal which recruits diverse nonspecific and specific host defense mechanisms in an attempt to limit tissue damage. Thus, every inflammation-associated cytokine, bacterial products such as endotoxin, and acute virus infections enhance IL-6 gene expression. Activation of all three of the major signal transduction pathways (protein kinase C-, CAMP-, and Ca’+-activated pathways) singly or in combination turns on IL-6 gene transcription. Perhaps the most remarkable adaptation is the paradoxical enhancement (superinduction) of IL-6 secretion by tissues as macromolecular synthesis (e.g., protein synthesis) is compromised. Therefore, even in the face of imminent cell death, this ensures that there is a net increase in the secretion of IL-6 by the damaged cell. The cell types that produce high levels of IL-6 in response to noxious stimuli are distributed all over the body. These include fibroblasts (fibrocytes), endothelial cells, keratinocytes, other epithelial cells, all cells of the monocytic series, and endometrial stromal cells to name only a few. A further principle to emerge is that not all stimuli work equally well in inducing IL-6 gene expression in every tissue. For example, interleukin-la (IL-la) and tumor necrosis factor (TNF) do not stimulate IL-6 gene expression in human peripheral blood monocytes; bacterial lipopolysaccharide (LPS) is the strongest stimulus in monocytes. In contrast, IL-la is the strongest stimulus in fibroblasts and endothelial cells. Anti-CD3 antibody thus far has been shown to induce IL-6 only in human mononuclear cell preparations. Elucidation of the biochemical basis for these tissue-specific differences in IL-6 gene expression and the

Regional localization of the interferon-β2B-cell stimulatory factor 2/hepatocyte stimulating factor gene to human chromosome 7p15-p21

The human interferon-beta 2 gene (IFNB2) is identical to the genes encoding the B-cell stimulatory factor (BSF-2), the hybridoma growth factor (HGF), and the hepatocyte stimulating factor (HSF). This protein mediates major alterations in the secretion of a wide spectrum of plasma proteins by the liver in response to tissue injury (the acute-phase response). We have used a cDNA probe specific to the human IFNB2 gene in DNA hybridization experiments and report the regional localization of this gene to human chromosome 7p15-p21. Southern blot analyses of DNA extracted from a panel of mouse X human somatic cell hybrids localized this gene to human chromosome 7p. In situ hybridization of the IFNB2 cDNA probe to prebanded human metaphase chromosome spreads allowed the further localization of this gene to 7p15-p21.

Expression of Retrovirally Transduced IL-1 α in IL-6-Dependent B Cells: A Murine Model of Aggressive Multiple Myeloma

Retroviral-mediated gene transfer was employed to introduce an IL-1 alpha cDNA into an IL-6-dependent murine B-cell line. Bone marrow metastases and bone lesions were frequently observed following intravenous injection of these B cells into syngeneic mice. Because the retroviral vector also contained the neomycin phosphotransferase gene, metastatic cells could be easily recovered from bone marrow by addition of G418 to the culture medium. Interestingly, the metastatic B cells were found to retain their IL-6 dependency through several transplant generations. By comparison, intravenous injection of autonomously-growing B-cell lines generated in vitro by retroviral introduction of an IL-6 cDNA rarely resulted in bone marrow metastases. These results demonstrate that abrogation of growth factor dependency is neither necessary nor sufficient for the in vivo growth and dissemination of tumor cells in this experimental system. It is proposed that the increased metastasis of the IL-1 alpha-producing B-cells to bone marrow is due to alterations in cell adhesion molecules. The B-cell bone marrow metastasis model described here may be useful for studies of bone marrow homing and for evaluation of therapeutic regimens for multiple myeloma.

Interleukin-1 and Interleukin-6 Synergize to Increase Plasma Amyloid P and C3 Concentrations in the Mousea

Following inflammation, trauma, or infection, significant changes occur in the pattern of protein synthesis by the liver. Increased synthesis of a variety of hepatic “acute phase reactant proteins” is presumed to play an integral role in how the host responds to tissue damage or invading pathogen.’ The increased hepatic protein synthesis seen during inflammation is presumed to be regulated by several cytokines, including interleukin1, interferon-beta, /interleukin6, and tumor necrosis factor-alpha (cachectin). However, controversy exists regarding the quantitative and qualitative importance of these three cytokines in inducing the individual plasma protein responses. Although initial studies emphasized the importance of interleukin-1 as the primary inducer of the hepatic acute phase response,’ more recent studies have emphasized interleukin-6’ and, to a lesser extent, tumor necrosis fa~tor -a lpha .~ .~ Therefore, the present study was undertaken to evaluate the induction of two murine acute phase proteins-amyloid P and the third component

Regulation of Human Fibroblast Growth by Autocrine Interferon-Beta

Several studies suggested a role for autocrine IFN-beta in the regulation of cell cycle progression and cellular differentiation. Spontaneous production of a beta-type IFN was shown to occur in growth-arrested leukemic cells during the process of terminal differentiation (1,2), in density-arrested, quiescent human melanoma cells (3), or during the progression of synchronized murine fibroblasts through the cell cycle (3). In addition, secretion of a beta-type IFN was demonstrated in cultures of murine bone marrow cells stimulated to differentiate by the addition of colony stimulating factor-1 (2,5). The role of endogenously produced beta-type IFN in these earlier studies was documented by the demonstration that the addition of neutralizing antibodies specific for IFN-beta either delayed growth arrest or inhibited terminal differentiation of the cells in culture.

Structure, Genetics and Function of Human β2 Interferon

It was reported in 1980 that polyadenylated cytoplasmic RNA extracted from human diploid fibroblasts (FS-4 strain) induced with poly(I).poly(C) and cycloheximide could be resolved by electrophoresis through an agarose-methylmercury hydroxide gel into two translationally active (in the Xenopus laevis oocyte assay) mRNA species that coded for human interferon (IFN) of the β serotype (1). The translation product of the 0.9 kb mRNA species, which hybridized a cDNA probe corresponding to the IFN-β gene on chromosome 9, was designated as IFN-β1 while that of the 1.3 kb mRNA species, which did not cross-hybridize the IFN-β1- cDNA probe, was designated IFN-B2 (1). The two species of translationally active human IFN-β mRNA were also resolved by sucrose-gradient sedimentation of polyadenylated RNA extracted from poly(I).poly(C) and cycloheximide-induced SV40-transformed human fibroblasts (SV80 cell line) and partial cDNA clones corresponding to the 1.3 kb IFN-β2 mRNA were described (2). IFN-β2 cDNA probes did not cross-hybridize the 0.9 kb IFN-β1 mRNA (2). Translationally active 1.3 kb long human IFN-β mRNA was detected in appropriately induced human-rodent somatic cell hybrids that lacked human chromosome 9 strongly suggesting that the IFN-β2 gene was not syntenic with the IFN-β1 gene (3).