In the present study we demonstrate that human monocytes activated by lipopolysaccharides (LPS) were able to produce high levels of interleukin 10 (IL-10), previously designated cytokine synthesis inhibitory factor (CSIF), in a dose dependent fashion. IL-10 was detectable 7 h after activation of the monocytes and maximal levels of IL-10 production were observed after 24-48 h. These kinetics indicated that the production of IL-10 by human monocytes was relatively late as compared to the production of IL-1 alpha, IL-1 beta, IL-6, IL-8, tumor necrosis factor alpha (TNF alpha), and granulocyte colony-stimulating factor (G-CSF), which were all secreted at high levels 4-8 h after activation. The production of IL-10 by LPS activated monocytes was, similar to that of IL-1 alpha, IL-1 beta, IL-6, IL-8, TNF alpha, granulocyte-macrophage colony-stimulating factor (GM-CSF), and G-CSF, inhibited by IL-4. Furthermore we demonstrate here that IL-10, added to monocytes, activated by interferon gamma (IFN-gamma), LPS, or combinations of LPS and IFN-gamma at the onset of the cultures, strongly inhibited the production of IL-1 alpha, IL-1 beta, IL-6, IL-8, TNF alpha, GM-CSF, and G-CSF at the transcriptional level. Viral-IL-10, which has similar biological activities on human cells, also inhibited the production of TNF alpha and GM-CSF by monocytes following LPS activation. Activation of monocytes by LPS in the presence of neutralizing anti-IL-10 monoclonal antibodies resulted in the production of higher amounts of cytokines relative to LPS treatment alone, indicating that endogenously produced IL-10 inhibited the production of IL-1 alpha, IL-1 beta, IL-6, IL-8, TNF alpha, GM-CSF, and G-CSF. In addition, IL-10 had autoregulatory effects since it strongly inhibited IL-10 mRNA synthesis in LPS activated monocytes. Furthermore, endogenously produced IL-10 was found to be responsible for the reduction in class II major histocompatibility complex (MHC) expression following activation of monocytes with LPS. Taken together our results indicate that IL-10 has important regulatory effects on immunological and inflammatory responses because of its capacity to downregulate class II MHC expression and to inhibit the production of proinflammatory cytokines by monocytes.

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Interleukin 10(IL-10) inhibits cytokine synthesis by human monocytes: an autoregulatory role of IL-10 produced by monocytes.

systemic reaction characterized by fever, leukocytosis, increase in erythrocyte sedimentation rate, increases in Leucocytosis secretion of ACTH and glucocorticoids, activation of Complement activat complement and clotting cascades, decreases in serum levels of iron and zinc, a negative nitrogen balance, and by dramatic changes in the concentration of some plasma ,l' proteins. These proteins are named acute phase proteins. i

Interleukin-6 and the acute phase response

Interleukin 6 (IL-6), promptly and transiently produced in response to infections and tissue injuries, contributes to host defense through the stimulation of acute phase responses, hematopoiesis, and immune reactions. Although its expression is strictly controlled by transcriptional and posttranscriptional mechanisms, dysregulated continual synthesis of IL-6 plays a pathological effect on chronic inflammation and autoimmunity. For this reason, tocilizumab, a humanized anti-IL-6 receptor antibody was developed. Various clinical trials have since shown the exceptional efficacy of tocilizumab, which resulted in its approval for the treatment of rheumatoid arthritis and juvenile idiopathic arthritis. Moreover, tocilizumab is expected to be effective for other intractable immune-mediated diseases. In this context, the mechanism for the continual synthesis of IL-6 needs to be elucidated to facilitate the development of more specific therapeutic approaches and analysis of the pathogenesis of specific diseases.

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IL-6 in Inflammation, Immunity, and Disease

The biology of interleukin-6.

Interleukin 6 (IL-6) has a broad effect on cells of the immune system and those not of the immune system and often displays hormone-like characteristics that affect homeostatic processes. IL-6 has context-dependent pro- and anti-inflammatory properties and is now regarded as a prominent target for clinical intervention. However, the signaling cassette that controls the activity of IL-6 is complicated, and distinct intervention strategies can inhibit this pathway. Clinical experience with antagonists of IL-6 has raised new questions about how and when to block this cytokine to improve disease outcome and patient wellbeing. Here we discuss the effect of IL-6 on innate and adaptive immunity and the possible advantages of various antagonists of IL-6 and consider how the immunobiology of IL-6 may inform clinical decisions.

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IL-6 as a keystone cytokine in health and disease

When stimulated with antigen, B cells are influenced by T cells to proliferate and differentiate into antibody-forming cells. Since it was reported that soluble factors could replace certain functions of helper T cells in the antibody response, several different kinds of lymphokines and monokines have been reported in B-cell growth and differentiation. Among these, human B-cell differentiation factor (BCDF or BSF-2) has been shown to induce the final maturation of B cells into immunoglobulin-secreting cells. BSF-2 was purified to homogeneity and its partial NH2-terminal amino-acid sequence was determined. These studies indicated that BSF-2 is functionally and structurally unlike other known proteins. Here, we report the molecular cloning, structural analysis and functional expression of the cDNA encoding human BSF-2. The primary sequence of BSF-2 deduced from the cDNA reveals that BSF-2 is a novel interleukin consisting of 184 amino acids.

Complementary DNA for a novel human interleukin (BSF-2) that induces B lymphocytes to produce immunoglobulin

Further studies on the site-specific protein modification by microbial transglutaminase.

A process for modifying a physiologically active peptide or a physiologically active protein which comprises reacting a physiologically active peptide or a physiologically active protein having at least a glutamine residue with a substance having an amino donor in the presence of a transglutaminase originating in a microorganism to thereby form an acid amide bond at the γ-position acid amide group of the glutamine residue with the amino group of the amino donor; and the product of modification obtained thereby.

Modification of peptide and protein

Recombinant human interleukin-6 (hIL-6), a pleiotropic cytokine containing two intramolecular disulfide bonds, was expressed in Escherichia coli as an insoluble inclusion body, before being refolded and purified in high yield providing sufficient qualities for clinical use. Quantitative reconstitution of the native disulfide bonds of hIL-6 from the fully denatured E. coli extracts could be performed by glutathione-assisted oxidation in a completely denaturating condition (6M guanidinium chloride) at protein concentrations higher than 1 mg/mL, preventing aggregation of reduced hIL-6. Oxidation in 6M guanidinium chloride (GdnHCl) required remarkably low concentrations of glutathione (reduced form, 0.01 mM; oxidized form, 0.002 mM) to be added to the solubilized hIL-6 before the incubation at pH 8.5, and 22°C for 16 h. After completion of refolding by rapid transfer of oxidized hIL-6 into acetate buffer by gel filtration chromatography, residual contaminants including endotoxin and E. coli proteins were efficiently removed by successive steps of chromatography. The amount of dimeric hIL-6s, thought to be purification artifacts, was decreased by optimizing the salt concentrations of the loading materials in the ion-exchange chromatography, and gradually removing organic solvents from the collected fractions of the preparative reverse-phase HPLC. These refolding and purification processes, which give an overall yield as high as 17%, seem to be appropriate for the commercial scale production of hIL-6 for therapeutic use. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 62: 301–310, 1999.

Yoshiyuki Takahara

Papers

Complementary DNA for a novel human interleukin (BSF-2) that induces B lymphocytes to produce immunoglobulin

Further studies on the site-specific protein modification by microbial transglutaminase.

Modification of peptide and protein

High yield refolding and purification process for recombinant human interleukin‐6 expressed in Escherichia coli

Gene transfer into hepatoma cells mediated by galactose-modified α-helical peptides