Smad3 is acetylated by p300/CBP to regulate its transactivation activity.

Smad proteins are crucial for the intracellular signaling of transforming growth factor-beta (TGF-beta). Upon their receptor-induced activation, Smad proteins are phosphorylated and translocated to the nucleus to activate the transcription of a select set of target genes. Here, we show that the co-activator p300/CBP bound and acetylated Smad3 as well as Smad2 in vivo, and that the acetylation was stimulated by TGF-beta. A major acetylation site of Smad3 by p300/CBP is Lys-378 in the MH2 domain (Smad3C) known to be critical for the regulation of transcriptional activity. Replacement of Lys-378 with Arg decreased the transcriptional activity of GAL4-Smad3C in a luciferase assay. Moreover, p300/CBP potentiated the transcriptional activity of GAL4-Smad3C, but not the acetylation-resistant GAL4-Smad3C(K378R) mutant. These results suggest that acetylation of Smad3 by p300/CBP regulates positively its transcriptional activity.

Growth inhibition and augmentation of mouse myeloid leukemic cell line differentiation by interleukin 1.

The effect of interleukin 1 (IL 1) on the growth and differentiation of mouse myeloid leukemic cell line (M1) cells into macrophages has been studied. Purified human IL 1 beta appeared to be growth inhibitory (maximum, 50%) for M1 cells based on cell counts and [3H]thymidine incorporation. The replication of M1 cells was also inhibited by lipopolysaccharide (LPS), and as little as 1 unit/ml IL 1 augmented the growth inhibition by LPS. Although IL 1 inhibited M1 cell growth, it did not induce cell differentiation by the criteria of either effect on expression of Fc receptors or on phagocytic ability. However, IL 1 augmented M1 cell differentiation in conjunction with LPS. At low doses of LPS, addition of IL 1 induced differentiation even though LPS and IL 1 by themselves did not induce differentiation. Cells treated with IL 1 for 1 day and then with LPS for an additional 2 days showed considerable augmentation of Fc receptor expression, while cells treated with the same stimuli in the reverse sequence exhibited only a low level of differentiation. Cells treated with medium alone followed by LPS showed moderate increase in Fc receptor expression. In addition, exposure of cells to IL 1 for at least 16 h was required for IL 1 augmenting effect. Therefore, IL 1 appeared to primarily influence M1 cells to become more sensitive to LPS. Treatment with both of IL 1 and LPS induced differentiation of a LPS-resistant clone of M1 cells, and IL 1 pretreatment rendered the resistant clone to become responsive to the differentiation inducing effect of LPS. Culture supernatants of M1 cells after stimulation with LPS contained IL 1-like activity by thymocyte comitogenic assays. In addition, mouse recombinant IL 1 alpha appeared to have the same activity as purified human IL 1 beta on the growth and differentiation of M1 cells. These results suggest that IL 1 may play an important role in mouse myeloid leukemic cell differentiation by acting as an autostimulating factor. IL 1 has been shown to be growth inhibitory and cytocidal for several tumor cell lines. Our results therefore suggest that the effects of IL 1 may result in the induction of terminal differentiation of some tumor cells.

Human myelomonocytic cell line THP-1 produces a novel growth-promoting factor with a wide target cell spectrum.

Conditioned medium from a human myelomonocytic cell line THP-1 promoted the growth of a wide variety of cell types, i.e., human and mouse myeloid cells (HL-60, U937, K562, and M1), mouse T-cells (EL-4), human B cells (Daudi and Raji), mouse mastocytoma cells (IC-2), human melanoma cells (A375-C6), mouse transformed fibroblast cells (L929), human lung fibroblast cells (TIG-1), and mouse bone marrow fibroblast/stromal-like cells. The growth-promoting activity was acid-labile. The activity was resistant to 50 degrees C for 5 min but completely lost in 5 min at 70 degrees C. The activity was resistant to treatment with trypsin but sensitive to chymotrypsin alpha, Pronase E, and proteinase K, indicating the proteinous nature of this activity. The activity was lost by dithiothreitol and 2-mercaptoethanol. Molecular weight (M(r) 50,000-70,000) was estimated by gel filtration-high performance liquid chromatography. After the sequential anion exchange, hydrophobic, and hydroxylapatite high performance liquid chromatography, the partially purified factor exhibited the same target cell spectrum as the conditioned medium.

Synergistic regulatory effects of interleukin 6 and interleukin 1 on the growth and differentiation of human and mouse myeloid leukemic cell lines.

We analyzed the effect of recombinant human interleukin 6 (IL-6), in combination with human recombinant interleukin 1 alpha (IL-1 alpha), on the growth and differentiation of several human and mouse myeloid leukemic cell lines, specifically U937, HL-60, M1, and its subclone M1-3b-N, into macrophage-like cells. IL-6 and IL-1 inhibited the growth of U937, M1, and M1-3b-N in a dose-dependent manner. Treatment of these cells with both IL-6 and IL-1 resulted in either an additive or a synergistic growth inhibition. IL-6 alone induced moderate differentiation of U937 and M1-3b-N, but the combination of IL-6 and IL-1 synergistically augmented this differentiation. In M1, only the combination of IL-1 and IL-6 resulted in differentiation. These two cytokines, whether alone or in combination, did not influence the growth and differentiation of HL-60. Therefore IL-6 in conjunction with IL-1 can induce differentiation in several human and mouse myeloid leukemic cell lines, although this effect varies with cell type. IL-6 did not stimulate the expression of IL-1 mRNA or IL-1 activity in U937 cells. IL-1 also failed to stimulate IL-6 production. Furthermore, the differentiation of U937 cells induced by IL-6 was not neutralized by antibody against either IL-1 alpha or IL-1 beta. The minimal differentiative effect of IL-1 was not affected by anti-IL-6 antibody. Therefore IL-6 and IL-1 appear to provide distinct signals for differentiation.

A human erythrocyte-derived growth-promoting factor with a wide target cell spectrum: identification as catalase.

We have reported previously that a factor with a molecular weight of 53,000 under SDS-polyacrylamide gel electrophoresis purified from human erythrocyte extracts promoted the growth of a wide variety of cell types from different species, including T cells, B cells, myeloid leukemia cells, melanoma cells, and mastocytoma cells, as well as normal and transformed fibroblast cells. In the present study, amino acid sequence analysis revealed that this factor has homology with human catalase. The purified factor exhibited catalase activity. Catalases derived from human erythrocytes, bovine liver, Aspergillus niger, and recombinant rat liver catalase are all able to promote the growth of cells. Antibody against human catalase absorbed both the growth-promoting activity and the enzyme activity of the purified factor. In addition, treatment of the factor with an irreversible enzyme inhibitor, aminotriazole, resulted in abrogation of both the growth-promoting activity and enzyme activity. These results indicate that the growth-promoting factor is catalase, and its activity is associated with the decomposition of hydrogen peroxide.

Inhibition of protein synthesis in mouse myeloma cells by Ricinus communis toxin.

The mechanism of inhibition of protein synthesis in mouse myeloma cells by Ricinus communis toxin was studied. No significant disaggregation of polysomes into monosomes was detected in the toxin-treated cells. The activity of the polysomes isolated from the cells treated with the toxin in protein synthesis was remarkably lower than that of the untreated cells, while the activity of the supernatant enzyme fraction was retained. The ribosomes derived from the polysomes of the toxin-treated cells were inactive in poly(U)-dependent polyphenylalanine synthesis. The activity of ribosomes reconstituted by hybridizing subunits derived from the ribosomes of normal and toxin-treated cells were measured in poly(U)-dependent polyphenylalanine synthesis, and the 60 S subunit was revealed to be inactive. These results indicate that the target of action of the toxin towards intact cells is the 60 S ribosomal subunit.

Regulation by dietary essential fatty acid balance of tumor necrosis factor production in mouse macrophages.

Increasing the dietary alpha-linolenate (18:3n - 3)/linoleate (18:2n - 6) ratio results in an increase in lipopolysaccharide (LPS)-stimulated tumor necrosis factor (TNF) production in mouse resident and casein-induced peritoneal macrophages [3]. We found that prostaglandin E2 (PGE2) production is inversely related to TNF production and that indomethacin abolishes the effect of changing the essential fatty acid balance in resident macrophages. The resident macrophages enriched in n - 3 did not produce a significant amount of PGE3. Accordingly, the decreased production of PGE2 appears to be a major negative regulatory factor for enhancement of TNF production in the n - 3 enriched resident macrophages. In casein-induced macrophages the situation is more complex. Indomethacin decreased PGE2 production and increased TNF production; however, the differences in TNF production between the n - 6 enriched and n - 3 enriched macrophages were not completely abolished by indomethacin treatment. Lysosomal acid phosphatase activity, a marker of activation/maturation stages, was elevated in the n - 3 enriched compared to the n - 6 enriched casein-induced macrophages but was similar in the resident macrophages of the two dietary groups. Expression of CD14, which is a receptor for LPS, was not different in casein-induced macrophages of the two dietary groups. Thus, the differences in production of TNF between the n - 3 and n - 6 enriched resident macrophages can be accounted for mostly by a difference in the production of a negative feedback effector, PGE2. However, a significant portion of the TNF production in casein-induced macrophages is regulated by a factor(s) other than PGE2 and LPS receptor; advanced activation/maturation stages induced by the diet high in alpha-linolenate may underlie the enhanced TNF production in casein-induced macrophages.

Recombinant human interleukin 6 (B-cell stimulatory factor 2) is a potent inducer of differentiation of mouse myeloid leukemia cells (M1).

Recombinant human interleukin 6 (IL-6), a lymphokine involved in the final differentiation of activated B-cells into antibody-forming cells, greatly suppressed proliferation and induced differentiation of murine myeloid leukemia cells (M1) into mature macrophage-like cells. When M1 cells were treated with IL-6, their growth was completely arrested as early as on day 2, and they were induced to differentiate morphologically into macrophage-like cells. Differentiation-associated properties such as phagocytic activity, adherence to the dish surface, Fc and C3 receptors, were also induced within 24 h by IL-6, and they reached their respective maximal levels on day 2 or 3. The potency of IL-6 in suppressing proliferation and inducing differentiation was much greater than that of 1 alpha,25-dihydroxyvitamin D3 one of the most potent inducers of M1 cells. The present report indicates that IL-6 is involved in the differentiation of not only B-cells but also myeloid leukemia cells.

Molecular cloning of a novel gene involved in serotonin receptor-mediated signal transduction in rat stomach.

In Xenopus oocytes injected with small size mRNAs (500-700 b), obtained from rat stomach by fractionation, application of 10 microM 5-HT induced a substantial Ca2+-activated Cl- current (I(Cl-Ca)). I(Cl-Ca) was not elicited by 5-HT in native oocytes. Consistent results from this assay in the oocyte expression system motivated cDNA cloning experiments. A novel cDNA (named rat stomach serotonin receptor-related cDNA: RSS cDNA) which encodes a small protein involved in specific 5-HT receptor-mediated I(Cl-Ca) activation was identified. The molecular weight of RSS protein in the reticulocyte lysate translation system (approximately 10 kDa) is identical to that calculated from the amino acid sequence. Computer-aided analysis of the predicted protein does not show any obvious sequence homologies (< 18%) to any other proteins including G protein-coupled receptors. Northern analysis revealed that RSS mRNA is ubiquitously expressed at varying levels in a number of different tissues. Furthermore, the binding of [3H]spiperone, a 5-HT2 receptor antagonist, was examined in CHO cells, which highly expressed RSS transcripts (named CHO-RSS). Specific binding of [3H]spiperone was not clearly observed in native CHO but was detected in CHO-RSS. The dissociation constant was 10.3 nM in CHO-RSS. These results suggest that RSS protein may be a factor which facilitates 5-HT receptor expression or, alternatively, an enhancer of the affinity of native 5-HT receptor to 5-HT.

Endotoxin-induced upregulation of type I interleukin-1 receptor mRNA expression in hepatocytes of mice: role of cytokines.

Interleukin-1 (IL-1) signal is transduced through the type I IL-1 receptor (IL-1RI). Although regulation of IL-1R expression has been extensively studied in vitro, little is known about it in vivo. By using RT-PCR analysis, we investigated the regulation of the IL-1RI mRNA expression level in various organs of mice at 2, 6, and 24 h following lipopolysaccharide (LPS) administration. IL-1RI mRNA expression in response to LPS appeared to be different in various organs. As a marked and sustained increase of IL-1RI mRNA expression in the liver was observed, we investigated the mechanism of the upregulation. IL-1, IL-6, and tumor necrosis factor (TNF) all increased the mRNA expression in the liver when administrated in vivo. In situ hybridization revealed that upregulation of IL-1R mRNA was observed in parenchymal liver cells (hepatocytes) in response to LPS administration. When primary cultured hepatocytes were treated in vitro, IL-1, IL-6, conditioned medium from LPS-treated mouse macrophages, and serum from LPS-treated mouse upregulated IL-1RI mRNA expression, but LPS, TNF, and prostaglandin E2 failed to do so. Therefore, these results suggest that the upregulation of IL-1RI mRNA in the hepatocytes by LPS administration is mediated by cytokines, especially by IL-1 and IL-6. The results also indicate that the regulation is different in different organs, and microenvironmental factors may be important.

Role of glucocorticoid in the upregulation of type I interleukin-1 receptor mRNA expression in hepatocytes of endotoxin-administrated mice.

The interleukin-1 (IL-1) signal is transduced through type I IL-1 receptor (IL-1RI). We have recently reported that lipopolysaccharide (LPS) upregulated IL-1RI mRNA expression in mouse liver in vivo and that IL-1 and IL-6 directly upregulated IL-1RI mRNA expression in primary cultured mouse hepatocytes. Glucocorticoid (GC) has been reported to increase IL-1 binding to the cell surface and the expression level of IL-1R mRNA in a variety of cell types. As serum GC level is elevated in an inflammatory response, we evaluated the role of GC in LPS-induced upregulation of IL-1RI mRNA in the mouse liver. When LPS was administered to adrenalectomized (ADX) mice, IL-1RI mRNA was upregulated at a level comparable to those of untreated or sham-operated mice. A high dose of dexamethasone (Dex), however, caused upregulation of the mRNA. When primary cultured mouse hepatocytes were treated with Dex, only a weak upregulation of IL-1RI mRNA was observed. However, Dex in combination with IL-1 or IL-6 markedly enhanced the IL-1RI mRNA expression. A marked upregulation of the mRNA was also induced by treatment with a combination of IL-1 and IL-6 in the absence of Dex, reflecting the observation in ADX mice. These results suggest that the upregulation of IL-1RI mRNA in response to LPS is induced by the interaction of IL-1 and IL-6 and that GC augments their effect.

Endotoxin and cytokine regulation of toll-like receptor (TLR) 2 and TLR4 gene expression in murine liver and hepatocytes.

Toll-like receptor (TLR) 2 and TLR4 are members of the interleukin-1 receptor (IL-1R) family and transduce similar signals as IL-1R in response to bacteria and bacterial components. In this study, we investigated the regulation of their gene expression in murine tissues, especially in the liver and hepatocytes. When mice were administered lipopolysaccharide (LPS), TLR2 mRNA was upregulated in the brain, heart, lung, liver, and kidney. In contrast, it was downregulated in the spleen. TLR4 mRNA was decreased in the brain. In the heart and lung, it increased, and it was not affected in the liver, kidney, and spleen. TLR mRNA was further analyzed in the liver and hepatocytes. Like LPS treatment, administration of IL-1, IL-6, or tumor necrosis factor (TNF) upregulated TLR2 mRNA. However, none of them affected the TLR4 mRNA level. In primary cultured hepatocytes, TLR2 mRNA was upregulated by LPS, IL-1, or TNF but not by IL-6 or dexamethasone. None of them affected TLR4 mRNA expression. Similar responses were observed in the murine hepatoma cell line Hepa 1-6. These results suggest that in infection with gram-negative bacteria, LPS and proinflammatory cytokines differentially regulate gene expression of TLR2 and TLR4 in murine hepatocytes, which may lead to pathologic and host defense reactions in the liver.

CHOP, a basic leucine zipper transcriptional factor, contributes to the antiproliferative effect of IL-1 on A375 human melanoma cells through augmenting transcription of IL-6.

Interleukin-1 (IL-1) inhibits the proliferation of A375 human melanoma cells. We have demonstrated previously that p38 mitrogen-activated protein kinase (MAPK) mediated the antiproliferative effect of IL-1 partially through the downregulation of activity and protein level of ornithine decarboxylase (ODC). In this study, we investigated the role of CCAAT/enhancer-binding protein (C/EBP) homologous protein (CHOP), one of the p38 MAPK target transcriptional factors. The mRNA level of CHOP was not affected by IL-1 treatment in A375-6 cells. Unexpectedly, CHOP was constitutively phosphorylated, and IL-1 or p38 MAPK inhibitor, SB203580, did not affect the phosphorylation level. However, A375-6 cells exhibited enhanced sensitivity to IL-1 by transfecting CHOP expression plasmid and reduced sensitivity to IL-1 by antisense CHOP mRNA expression plasmid. Furthermore, CHOP appeared to regulate positively IL-6 production at the transcriptional level. The experiments using CHOP muteins revealed that dimerization ability - but not p38 MAPK-dependent phosphorylation or DNA binding activity - is important for the IL-6 inducing activity of CHOP. These results indicate that CHOP contributes to the IL-1 growth-inhibitory signal through augmenting IL-6 production.

Human interleukin-1 receptor antagonist: large-scale expression in Bacillus brevis 47-5Q.

Interleukin-1 receptor antagonist (IL-1RA) has been used as a tool to study the biologic activity of IL-1 and as a possible therapeutic substance for inflammatory disease. To perform in vivo study, however, large quantities of IL-1RA are required. Bacillus brevis strains secrete large amounts of protein but little protease into the medium. Using B. brevis 47-5Q, we developed a large-scale expression system of human IL-1RA (HuIL-1RA). The bacteria secreted HuIL-1RA into the culture medium at very high levels, approximately 200 mg/L. The protein was isolated in one-step purification with monoclonal antibody (mAb) against HuIL-1RA. The IL-1RA molecule was determined to be functionally active by the inhibiting assay of HuIL-1-induced cell proliferation in a mouse T cell line, D10N4M.

D-mannose dimer introduced human recombinant interleukin- 1 alpha, NEO IL-1 alpha, exhibits altered tissue distribution in mice.

Previous studies have demonstrated that the carbohydrate-introduced recombinant human IL-l alpha exhibited impairment in both biologic activities in all the experiments in vitro and receptor binding capacity compared with intact IL-l alpha. However, the glycosylated IL-l alpha exhibited selective activities in vivo. In this study, we compared the tissue distribution of IL-l alpha and IL-l alpha coupled with D-Mana (l-6)Man [Man2 alpha) (l-6)IL-l alpha] in mice. Mice were injected by intravenous and intraperitoneal routes with 2.0 mu g radiolabeled IL-l alpha. At 1 and 2 h after IP injection, the level of Man2 alpha) (l-6)IL-l alpha decreased twofold compared with that of IL-l alpha in kidney. In contrast, at 1 hour after administration, Man2 alpha) (l-6)IL-l alpha exhibited higher levels than IL-l alpha in blood, heart, and liver. No significant difference was observed in brain at each time point. IV injection demonstrated that Man2 alpha)(l-6)IL-l alpha decreased to approximately one-half the level of rhIL-l alpha in kidney. In contrast, Man(2 alpha) (l-6)IL-l alpha increased twofold over that of IL-l alpha in liver at 1 h after dosing. These findings are consistent with the result of IP injection. There was no significant difference between IL-l alpha and glycosylated IL-l alpha at 4 h after IV administration. These differences in tissue distribution may contribute to the selective activities of glycosylated IL-l alpha in vivo. The results also suggest that by coupling with mannose dimer, it is possible to develop neocytokines prone to liver distribution.

Interleukin-1 or tumor necrosis factor-alpha augmented the cytotoxic effect of mycobacteria on human fibroblasts: application to evaluation of pathogenesis of clinical isolates of Mycobacterium tuberculosis and M. avium complex.

Mycobacteria-induced in vitro events reflecting human tuberculosis can contribute to the evaluation of the pathogenesis of Mycobacterium tuberculosis (MTB). In this study, we propose such an in vitro method based on live mycobacteria-induced cytotoxicity to human cell lines. When human lung-derived normal fibroblast cell line MRC-5 was infected with various strains of mycobacteria (M. tuberculosis H(37)Rv and H(37) Ra, Mycobacterium avium 427S and 2151SmO, and Mycobacterium bovis BCG Pasteur and Tokyo), the fibroblasts were killed by mycobacteria according to the degree of virulence. Other human originated macrophage (U-937, THP-1), myeloid (HL-60), and epithelial carcinoma (A549) cell lines exhibited a similar cytotoxic response to virulent mycobacteria. MRC-5 was most susceptible to virulent mycobacteria among various human cell lines examined. The cytotoxicity was enhanced by the proinflammatory cytokines, interleukin-1 (IL-1) and tumor necrosis factor-a (TNF-alpha), which in the absence of mycobacteria stimulate the growth of normal human fibroblasts. This in vitro evaluation system was applied to clinical isolates of drug-sensitive MTB (DS-MTB), drug-resistant MTB (DR-MTB) including multidrug-resistant (MDR-MTB), and M. avium complex (MAC). MTB strains (n = 24) exhibited strong cytotoxic activity, but MAC strains (n = 5) had only weak activity. Furthermore, there was no significant difference in cytotoxicity between DS-MTB (n = 11) and DR-MTB (n = 13). Collectively, these results suggest that this new in vitro system is useful for evaluating the pathogenesis of mycobacteria and that there was no difference in the pathogenesis between drug-susceptible and drug-resistant clinical isolates.

Glycosylated human recombinant interleukin-1 alpha, neo interleukin-1 alpha, with D-mannose dimer exhibits selective activities in vivo.

To investigate the effect of carbohydrate-introduction on IL-1 activity, especially in vivo, and to develop IL-1 with less deleterious effects, recombinant human IL-1 alpha was coupled with mannose dimer, alpha-D-Man-1-6-D-Man [Man2 alpha(1-6)] by an acyl azide method. Previous studies demonstrated that the glycosylated IL-1 exhibited reduced activities compared with original IL-1 in all the experiments performed in vitro. In this study, we investigated the in vivo activities of Man2 alpha(1-6)-conjugated IL-1 alpha. The glycosylated IL-1 alpha exhibited very low pyrogenic activity and alpha 1-acid glycoprotein induction compared with untreated IL-1 alpha. Untreated IL-1 alpha increased the serum level of IL-6, but the glycosylated IL-1 alpha did not. However, the glycosylated IL-1 alpha possessed the same potency as untreated IL-1 alpha in reduction of serum levels of glucose and triglyceride and in recovery of peripheral white blood cells in 5-fluorouracil-treated mice. Therefore, glycosylation of IL-1 appeared to be useful for the development of neoIL-1 with selective activity in vivo.

Type I and type II interferons upregulate functional type I interleukin-1 receptor in a human fibroblast cell line TIG-1.

The regulation of type I interleukin-1 receptor (IL-1R) expression by type I, interferon (IFN)-alpha A/D, and type II IFN, IFN-gamma, in a human fibroblast cell line TIG-1 was investigated. After 2 h stimulation with human IFN-alpha A/D or IFN-gamma, the levels of type I IL-1R mRNA increased. We previously reported that IL-1 upregulates transcription and cell surface molecules of type I IL-1R in TIG-1 cells through induction of prostaglandin (PG) E2 and cAMP accumulation. However, indomethacin was unable to inhibit the effect of IFNs, indicating that IFNs augment IL-1R expression through a pathway distinct from that of IL-1. The augmentation was also observed in other fibroblast cell lines. Nuclear run-on assays and studies of the stability of mRNA suggested that the increase in IL-1R mRNA was a result of the enhanced transcription of IL-1R gene. Binding studies using 125I-IL-1 alpha revealed that the number of cell surface IL-1R increased with no change in binding affinity by treatment with these IFNs. Pretreatment of the cells with IFNs enhanced IL-1-induced IL-6 production, indicating that IFNs upregulate functional IL-1R. IL-1 and IFNs are produced by the same cell types, as well as by the adjacent different cell types, and are concomitantly present in lesions of immune and inflammatory reactions. These results therefore suggest that IFNs exhibit synergistic effects with IL-1 through upregulation of IL-1R. Augmented production of IL-6 may also contribute to the reactions.

Serotonin derivative, N-(p-coumaroyl) serotonin, inhibits the production of TNF-alpha, IL-1alpha, IL-1beta, and IL-6 by endotoxin-stimulated human blood monocytes.

We have reported that N-(p-coumaroyl) serotonin (CS) and its derivatives with antioxidative activity are present in safflower seeds. As reactive oxygen species (ROS) are implicated in the signaling of lipopolysaccharide (LPS), we examined whether CS has a suppressive effect on inflammatory cytokine generation from human monocytes in vitro. CS at 50-200 microM reduced tumor necrosis factor (TNF), interleukin-1 (IL-1), and IL-6 activities in the culture supernatants from LPS-stimulated human blood monocytes without cytotoxicity. ELISA assay revealed that the production of TNF-alpha, IL-1alpha, IL-1beta, and IL-6 was inhibited by CS. Northern blot analysis showed that LPS-induced expression of these cytokine mRNA in monocytes was suppressed by CS. NF-kappaB activation was also inhibited by CS. These findings indicate that CS has a suppressive effect on proinflammatory cytokine production from monocytes, and this effect is based in part on the suppression of cytokine mRNA expression through inhibition of NF-kappaB activation.

Cytokines produced in lymph follicles.

The events occurring inside lymph follicles during a germinal center reaction are poorly understood. Using B and T lymphoid cell populations prepared from human tonsillar lymph follicles, and enriched or not in macrophages or in follicular dendritic cells, we examined the production of cytokines by these cells in vitro. Interleukin 6 (IL-6) and tumor necrosis factor (TNF) were found in the supernatants of cultures stimulated with phytohemagglutinin or pokeweed mitogen. IL-1 beta was occasionally detected; its secretion apparently depends on the origin of the tonsils, the stimulation, and the cell populations. IFN-gamma and IL-2 were not produced in significant amounts by these lymph follicle cells. IL-4 was only found in very low concentrations in the supernatant of the different cell cultures. The cell populations containing follicular dendritic cells produced more IL-6 and TNF than the others, especially than those composed of only B and T cells.