Cytokine Inhibition by A Novel Steroid,Mometasone Furoate

Abstract

Mometasone furoate (9α, 21 dichloro-11β, 17α dihydroxy-16α methyl-1, 4 pregnadiene-3, 20 dione-17–[2′] furoate) was an unexpectedly potent inhibitor of the in vitro production of three inflammatory cytokines, IL-11, IL-6, and TNF-α. the potency of mometasone furoate in inhibiting cytokine production was compared to that of hydrocortisone, betamethasone, dexamethasone, and beclomethasone.

IL-6 and TNF-α were both produced by WEHI-265.1 (murine myelomonocytic leukemia) cells following stimulation by lipopolysaccharide (LPS). Twenty-four hours after stimulation by LPS, the cell-free supernatant fluids were removed. Their cytokine content was analyzed using ELISAs specific for each cytokine.

IL-1 synthesis was induced in the harvested peritoneal macrophages of BALB/c mice by incubation with LPS for twenty-four hours. the IL-1 content in the cell-free supernatant fluids was determined by the thymocyte-costimulator bioassay.

Using these systems, mometasone furoate was found to be the most potent steroid tested for inhibiting the production of the three cytokines. the IC50′s were 0.05 nM (IL-1), 0.15 nM (IL-6), and 0.25 nM (TNF-α). the inhibition of the production of proinflammatory mediators by extremely low concentrations of mometasone furoate suggests that this steroid should be highly effective in various disorders.     

Enhanced interleukin-1beta, interleukin-6 and tumor necrosis factor-alpha production by LPS stimulated human monocytes isolated from HIV+ patients

Periodontal disease and tooth loss is a common finding among advanced HIV+ patients. In addition to local oral lipopolysaccharide (LPS) stimulation, systemic up-regulation of monocyte pro-inflammatory cytokine secretion may also be involved in the pathogenesis of HIV disease. A study was undertaken to investigate IL-1beta, IL-6 and TNF-alpha production by resting and LPS stimulated monocytes isolated from HIV+ patients and also to investigate the relationship of the patient's HIV viral load status to the cytokine production. Whole blood samples in EDTA were collected from 39 HIV-1 infected patients and 20 age and sex matched uninfected controls. Plasma was separated by centrifugation. Viral load was determined using a quantitative RT-PCR. Monocytes were isolated by Ficoll-hypaque gradient separation followed by overnight plastic adherence. Cultured monocytes (1x10(6)/ml) were stimulated with LPS (1 microg/ml) of either P. gingivalis or F. nucleatum for 2, 8, 24 and 48 h and supernatant fluids were collected. IL-1beta, IL-6, and TNF-alpha levels in supernatant fluids were estimated by ELISA. Increased overall production of IL-1beta, IL-6 and TNF-alpha by LPS stimulated monocytes isolated from HIV-1 infected patients was observed when compared to HIV-1 uninfected controls. LPS stimulated monocytes from HIV-1 infected patients with high viral load (HVL) produced significant (p<0.05) elevations in these pro-inflammatory cytokines when compared to HIV-1 uninfected controls. Both LPS of P. gingivalis and F. nucleatum produced a comparable cytokine production by monocytes after 8 h of stimulation. These data suggest that enhanced IL-1beta, IL-6 and TNF-alpha is produced by monocytes/macrophages isolated from HVL HIV+ patients and may be involved in the overall pathogenesis of HIV-1 infection.     

GM-CSF, IL-1 alpha, IL-1 beta, IL-6, IL-8, IL-10, ICAM-1 and VCAM-1 gene expression and cytokine production in human duodenal fibroblasts stimulated with lipopolysaccharide, IL-1 alpha and TNF-alpha.

The role of mucosal fibroblasts in intestinal inflammatory reactions is not known. In this study, we demonstrate that fibroblasts grown from histologically normal human duodenal biopsy tissues expressed mRNA genes for granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-1 alpha, IL-1 beta, IL-6, IL-8, IL-10, intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) when stimulated with lipopolysaccharide (LPS) or IL-1 alpha. The increased mRNA expression of GM-CSF, IL-1 alpha, IL-1 beta, IL-6 and IL-8 in response to IL-1 alpha and LPS stimulation was time- and dose-dependent. In contrast, IL-10 was weakly expressed when fibroblasts were stimulated with LPS, IL-1 alpha or tumour necrosis factor-alpha (TNF-alpha), but the expression was enhanced in the presence of cycloheximide combined with optimal concentrations of LPS, IL-1 alpha or TNF-alpha, IL-1 alpha was a more potent stimulator than LPS for GM-CSF, IL-6, IL-8 and IL-10 expression, but not for IL-1 alpha and IL-1 beta. Increased GM-CSF, IL-6 and IL-8 gene expression was associated with the production of cytokine proteins in culture supernatant, but IL-1 alpha and IL-1 beta remained undetectable. Dexamethasone suppressed both gene expression and protein production of GM-CSF, IL-6 and IL-8 when fibroblasts were exposed to IL-1 alpha. TNF-alpha stimulated the release of GM-CSF, IL-6 and IL-8 and, combined with IL-1 alpha, cytokine production was enhanced synergistically. Finally, both LPS and IL-1 alpha up-regulated ICAM-1 and VCAM-1 gene expression. These findings implicate duodenal fibroblasts in the initiation and/or regulation of intestinal inflammation.     

Comparative study of cytokine release by human peripheral blood mononuclear cells stimulated with Streptococcus pyogenes superantigenic erythrogenic toxins, heat-killed streptococci, and lipopolysaccharide.

The differences between toxic or septic shocks in humans during infections by streptococci and gram-negative bacteria remain to be fully characterized. For this purpose, a quantitative study of the cytokine-inducing capacity of Streptococcus pyogenes erythrogenic (pyrogenic) exotoxins (ETs) A and C, heat-killed S. pyogenes bacteria, and Neisseria meningitidis endotoxin (lipopolysaccharide [LPS]) on human peripheral blood mononuclear cells (PBMC) and monocytes has been undertaken. The levels of interleukin-1 alpha (IL-1 alpha), IL-1 beta, IL-6, IL-8, tumor necrosis factor alpha (TNF-alpha), and TNF-beta induced by these bacterial products and bacteria were determined by using cell supernatants. The capacity of ETs to elicit the monocyte-derived cytokines IL-1 alpha, IL-1 beta, IL-6, and TNF-alpha was found to depend on the presence of T lymphocytes, because of the failure of purified monocytes to produce significant amounts of these cytokines in response to ETs. PMBC elicited large amounts of these cytokines, as well as IL-8 and TNF-beta, with an optimal release after 48 to 96 h. The most abundant cytokine produced in response to ETA was IL-8. In contrast to the superantigens ETA and ETC, LPS and heat-killed streptococci stimulated the production of significant amounts of IL-1 alpha, IL-1 beta, IL-6, and TNF-alpha, with optimal production after 24 to 48 h in monocytes, indicating no significant involvement of T cells in the process. ETs, but neither LPS nor streptococci, were potent inducers of TNF-beta in PBMC. This study outlines the differences in the pathophysiological features of shock evoked by endotoxins and superantigens during infection by gram-negative bacteria and group A streptococci, respectively. The production of TNF-alpha was a common pathway for LPS, streptococcal cells, and ETs, although cell requirements and kinetics of cytokine release were different.     

Blocking NF-kappaB activation may be an effective strategy in the fever therapy

Lipopolysaccharide (LPS) stimulates peripheral mononuclear cells (PBMC) to synthesize or release pyrogenic cytokines, including interleukin-1beta (IL-1beta), IL-6, and tumor necrosis factor-alpha (TNF-alpha). Nuclear factor-kappa B (NF-kappaB) influences inflammatory responses through the regulation of genes encoding cytokines. In the present study, experiments were carried out to determine whether an inhibition of NF-kappaB mechanisms causes an inhibition of pyrogenic cytokine synthesis or release from PBMC and results in antipyresis. Intravenous administration of the supernatant fluids obtained from the human PBMC incubated with LPS caused feverlike hyperthermia in rabbits. The febrile responses were in parallel with the levels of IL-1beta, IL-6, and TNF-alpha in supernatant fluids. Both the fever and the increased levels of these cytokines in supernatant fluids were decreased by incubating LPS-PBMC with NF-kappaB inhibitors, including pyrrolidine dithiocarbamate, sodium pyrithione, N-acetyl-cysteine, and curcumin. Moreover, an intravenous administration of LPS (0.5-2 microg/kg) produced dose-dependent fever in the rabbits. The fevers were in parallel with the levels of IL-1beta, IL-6, and TNF-alpha in rabbit serum. A pretreatment of rabbits with an intravenous injection of pyrrolidine dithiocarbamate, sodium pryithione, N-acetyl-cysteine, or curcumin 1 h before the intravenous administration of LPS significantly attenuated the LPS-induced fever and/or increased levels of these cytokines in the serum of rabbits. Furthermore, pretreatment with an intravenous dose of anti-IL-1beta, anti-IL-6, or anti-TNF-alpha monoclonal antibody significantly attenuated the fever induced by the intravenous injection of LPS in rabbits. The antipyretic effects exerted by anti-L-1beta monoclonal antibody were greater than those exerted by anti-L-6 or anti-NF-alpha monoclonal antibody. The data indicate that NF-kappaB activation correlates with an LPS-induced synthesis or a release of cytokines (in particular, IL-1beta) from PBMC and triggers fever. Blocking NF-kappaB mechanisms in the PBMC with NF-kappaB inhibitors may be an effective strategy in the fever therapy.     

Dendritic cell activation and cytokine production induced by group B Neisseria meningitidis: interleukin-12 production depends on lipopolysaccharide expression in intact bacteria

Interactions between dendritic cells (DCs) and microbial pathogens are fundamental to the generation of innate and adaptive immune responses. Upon stimulation with bacteria or bacterial components such as lipopolysaccharide (LPS), immature DCs undergo a maturation process that involves expression of costimulatory molecules, HLA molecules, and cytokines and chemokines, thus providing critical signals for lymphocyte development and differentiation. In this study, we investigated the response of in vitro-generated human DCs to a serogroup B strain of Neisseria meningitidis compared to an isogenic mutant lpxA strain totally deficient in LPS and purified LPS from the same strain. We show that the parent strain, lpxA mutant, and meningococcal LPS all induce DC maturation as measured by increased surface expression of costimulatory molecules and HLA class I and II molecules. Both the parent and lpxA strains induced production of tumor necrosis factor alpha (TNF-alpha), interleukin-1alpha (IL-1alpha), and IL-6 in DCs, although the parent was the more potent stimulus. In contrast, high-level IL-12 production was only seen with the parent strain. Compared to intact bacteria, purified LPS was a very poor inducer of IL-1alpha, IL-6, and TNF-alpha production and induced no detectable IL-12. Addition of exogenous LPS to the lpxA strain only partially restored cytokine production and did not restore IL-12 production. These data show that non-LPS components of N. meningitidis induce DC maturation, but that LPS in the context of the intact bacterium is required for high-level cytokine production, especially that of IL-12. These findings may be useful in assessing components of N. meningitidis as potential vaccine candidates.     

Effect of Danchunwhangagam on LPS or DFX-induced cytokine production in peripheral mononuclear cells of cerebral infarction patients

The Danchunwhangagam (DCWGG) has long been used for various cerebrovascular diseases. However, little scientific investigation has been carried out. Cytokines involved in the regulation of inflammatory reactions and immune responses may play a role in the pathogenesis of cerebral infarction (CI). The aim of the present study is to investigate the effect of DCWGG on the production of proinflammatory cytokines in peripheral blood mononuclear cells (PBMCs) from CI patients. The amount of tumor necrosis factor (TNF)-alpha, interleukin (IL)-1alpha, IL-1beta, IL-6, and IL-8 in PBMC culture supernatant was significantly increased in the lipopolysaccaride (LPS)- or desferrioxamine (DFX)-treated cells compared with unstimulated cells. We showed that DCWGG inhibited the production of TNF-alpha, IL-1alpha, IL-1beta IL-6, and IL-8 induced by LPS in a dose-dependent manner. Also, DCWGG inhibited TNF-alpha, IL-1alpha, IL-beta, IL-6, and IL-8 production-induced DFX in dose-dependent manner. These results suggest that DCWGG might have regulatory effects on LPS- or DFX-induced cytokine production, which might explain its beneficial effect in the treatment of CI.     

The phosphatidylinositol 3-kinase/protein kinase B signaling pathway is activated by lipoteichoic acid and plays a role in Kupffer cell production of interleukin-6 (IL-6) and IL-10

Sepsis caused by gram-positive bacteria lacking lipopolysaccharide (LPS) has become a major and increasing cause of mortality in intensive-care units. We have recently demonstrated that the gram-positive-specific bacterial cell wall component lipoteichoic acid (LTA) stimulates the release of the proinflammatory cytokines in Kupffer cells in culture. In the present study, we have started to assess the signal transduction events by which LTA induces the production of tumor necrosis factor alpha (TNF-alpha), interleukin-6 (IL-6), and the anti-inflammatory cytokine IL-10 in rat Kupffer cells. LTA was found to trigger phosphorylation of mitogen-activated protein kinases (MAPK) (p38 MAPK and ERK 1/2) and protein kinase B (PKB). Compared to LPS, LTA was more potent in inducing PKB phosphorylation after 40 min, although we found that the cytokine responses were similar. For both bacterial molecules, blocking phosphatidylinositol 3-kinase (PI3-K; Ly294002) or Janus kinase 2 (JAK-2; AG490) particularly affected the induction of IL-6 and IL-10 release, whereas TNF-alpha levels were strongly reduced by inhibition of Src family tyrosine kinases (PP2). All three cytokines were reduced by inhibition of p38 MAPK (SB202190) or the broad-range tyrosine kinase inhibitor genistein, whereas IL-6 release was particularly blocked by inhibition of ERK 1/2 (PD98059). Divergences in the regulatory pathways controlling TNF-alpha, IL-10, and IL-6 production in Kupffer cells following LPS or LTA stimulation may create a basis for understanding how the balance between pro- and anti-inflammatory cytokines is regulated in the liver following infections by gram-positive or gram-negative bacteria.     

Tumor necrosis factor-alpha and interleukin-1 alpha synergistically enhance phorbol myristate acetate-induced superoxide production by rat bone marrow-derived macrophages.

Tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 alpha (IL-1 alpha) are secreted by macrophages in response to endotoxin challenge. In addition, macrophages express receptors for both of these cytokines. Macrophage function can therefore be modulated by regulation of both cytokine production and receptor levels. We have initiated studies to investigate the effects of TNF-alpha and IL-1 alpha on macrophage function. Macrophages were obtained by in vitro differentiation of rat bone marrow cells. The biologic response to TNF-alpha and IL-1 alpha was assessed by measurement of superoxide production quantitated by the reduction of cytochrome c in response to phorbol myristate acetate. Macrophages were treated with endotoxin (LPS), TNF-alpha, and IL-1 alpha, alone and in combination. None of these agents was a primary stimulus for superoxide production. However, after treatment with endotoxin or TNF-alpha for 24 h, macrophages were primed for enhanced production of superoxide. The priming effect of LPS was due, at least in part, to endogenously produced TNF-alpha, since anti-murine TNF-alpha antibodies blocked the LPS-mediated priming by approximately 30%. IL-1 alpha did not prime macrophages, but treatment with IL-1 alpha followed by TNF-alpha or LPS resulted in enhanced superoxide production. IL-1 alpha treatment of macrophages resulted in an increase in TNF-alpha receptors, which might explain the synergistic priming of TNF-alpha and IL-1 alpha.     

Interleukin-10 modulates proinflammatory cytokines in the human monocytic cell line THP-1 stimulated with Borrelia burgdorferi lipoproteins

We determined previously that lipoproteins of Borrelia burgdorferi stimulate inflammatory and anti-inflammatory cytokines (interleukin-10 [IL-10]) in monocytes. IL-10 could have an effect on innate and acquired immune responses to B. burgdorferi and influence the magnitude of the infectious inoculum and disease outcome. To understand the mechanism(s) of IL-10 action during early infection, when innate immunity expressed chiefly by skin macrophages is key, we investigated the effect of exogenous and endogenous IL-10 on the production of the macrophage-derived cytokines IL-6, IL-1beta, IL-12, and tumor necrosis factor alpha (TNF-alpha). We used the THP-1 human monocytic cell line and recombinant lipidated OspA (L-OspA) as the model target cell and stimulant, respectively. To determine the kinetics of cytokine production by THP-1 cells, we stimulated them with L-OspA and also with heat-killed B. burgdorferi cells (HBb) and lipopolysaccharide (LPS). Exogenous IL-10 dampened production of inflammatory cytokines, as elicited by lipoproteins. The inhibition of endogenous IL-10 function by anti-IL-10 antibody reduced the production of IL-12 and IL-6 but not that of IL-1beta and TNF-alpha. An inspection of the kinetics of cytokine production clarified this finding. TNF-alpha was produced prior to, and IL-beta was produced at the same time as, IL-10, whereas IL-6 and IL-12 were produced later. HBb, LPS, and L-OspA yielded similar kinetics of cytokine production. This result reinforces the notion that lipoproteins are the functional molecules in HBb and perhaps in vivo. It indicates also that signaling pathways utilized by LPS and lipoproteins may be extensively shared. The results are consistent with a major role played by IL-10 in controlling the initial phase of infection with this spirochete.     

Interaction between porcine reproductive-respiratory syndrome virus and bacterial endotoxin in the lungs of pigs: potentiation of cytokine production and respiratory disease

Porcine reproductive-respiratory syndrome virus (PRRSV) is a key agent in multifactorial respiratory disease of swine. Intratracheal administration of bacterial lipopolysaccharides (LPSs) to PRRSV-infected pigs results in markedly enhanced respiratory disease, whereas the inoculation of each component alone results in largely subclinical disease. This study examines whether PRRSV-LPS-induced respiratory disease is associated with the excessive production of proinflammatory cytokines in the lungs. Gnotobiotic pigs were inoculated intratracheally with PRRSV and then with LPS at 3, 5, 7, 10, or 14 days of infection and euthanatized 6 h after LPS inoculation. Controls were inoculated with PRRSV or LPS only or with phosphate-buffered saline. Virus titers, (histo)pathological changes in the lungs, numbers of inflammatory cells, and bioactive tumor necrosis factor alpha (TNF-alpha), interleukin-1 (IL-1), and IL-6 levels in bronchoalveolar lavage fluids were examined. All pigs inoculated with PRRSV-LPS developed severe respiratory disease, whereas the controls that were inoculated with PRRSV or LPS alone did not. PRRSV infection significantly enhanced cytokine production in response to LPS. Peak TNF-alpha, IL-1, and IL-6 titers were 10 to 100 times higher in the PRRSV-LPS-inoculated pigs than in the pigs inoculated with PRRSV or LPS alone; and the titers correlated with the respiratory signs. The levels of neutrophil infiltration and the pathological changes detected in the lungs of PRRSV-LPS-inoculated pigs resembled those detected when the effects of PRRSV and LPS inoculated alone are combined, but with no synergistic effects between PRRSV and LPS. These data demonstrate a synergism between PRRSV and LPS in the induction of proinflammatory cytokines and an association between induction of these cytokines and disease.     

Chemokine gene expression and secretion by cytokine-activated human microvascular endothelial cells. Differential regulation of monocyte chemoattractant protein-1 and interleukin-8 in response to interferon-gamma.

The elicitation of leukocytes from the circulation to inflamed tissue depends on the activation of both the leukocyte and endothelial cell. In this study we determined the gene expression and secretion patterns for the chemokines interleukin-8 (IL-8) and monocyte chemoattractant protein-1 (MCP-1) in cytokine- and lipopolysaccharide (LPS)-treated cultured human lung microvascular endothelial cells (HLE). HLE constitutively expressed low levels of MCP-1 and IL-8. Treatment of HLE with a variety of cytokines and LPS up-regulated both IL-8 mRNA expression and release of immunoreactive IL-8 with an order of potency tumor necrosis factor-alpha (TNF-alpha) >> IL-1 alpha > LPS, whereas interferon-gamma (IFN-gamma) had no effect on IL-8 mRNA or antigenic levels. However, IFN-gamma, in combination with high doses of IL-1 alpha, resulted in a synergistic increase in IL-8 generation. MCP-1 gene expression and secretion was induced in a dose-dependent manner after IL-1 alpha, TNF-alpha, IFN-gamma, and LPS activation of HLE. IL-1 alpha was the most potent inducer of MCP-1 generation and LPS was relatively ineffective. IFN-gamma, in combination with low doses of IL-1 alpha, resulted in a synergistic increase in MCP-1 generation by HLE. These results demonstrate that although IL-8 and MCP-1 generation by HLE occurs on cytokine treatment, the relative ability of a given cytokine to elicit IL-8 generation is not directly parallel to effects on MCP-1 generation. These data suggest that the regulation of IL-8 and MCP-1 expression exhibit significant differences in their mechanisms. Such differences in the expression of specific chemokines may explain the specific appearance of various leukocytes at sites of inflammation and injury. These data also directly demonstrate that the lung microvascular endothelium contribute to the cytokine network of the lung, with the ability to respond to locally generated cytokines and to produce potent mediators of the local inflammatory response.     

The effects of dehydroepiandrosterone (DHEA) on in vitro spleen cell proliferation and cytokine production.

Dehydroepiandrosterone (DHEA), a weak androgenic steroid, has been associated with enhancing immune responses and upregulating resistance against viral, parasitic, and bacterial infections. The objective of this study was to assess the effects of DHEA on murine spleen cell viability, proliferation, and cytokine production following in vitro stimulation with the mitogens concanavalin A (ConA) and lipopolysaccharide (LPS). Results showed that exposure to 6 microM DHEA significantly decreased the viability and proliferation of murine spleen cells stimulated with LPS, whereas no effect was seen on murine spleen cells stimulated with ConA. DHEA did influence the production of both ConA-induced and LPS-induced cytokines. DHEA also significantly reduced the mitogen-induced production of the proinflammatory cytokine interleukin-1 (IL-1) as well as the Th1 cytokines IL-2 and interferon-gamma (IFN-gamma). Increasing concentrations of DHEA significantly increased the production of the Th2 cytokine IL-10 but had no effect on the production of the Th2 cytokine IL-4, the proinflammatory cytokine tumor necrosis factor-alpha (TNF-alpha), or IL-6. These results suggest that DHEA may be an important factor for increasing Th2 cytokine production and decreasing Th1 and proinflammatory cytokine production. This study provides a more comprehensive understanding of the effects of DHEA on the rates of cell proliferation, cell viability, and cytokine production.     

Preexposure of murine macrophages to CpG oligonucleotide results in a biphasic tumor necrosis factor alpha response to subsequent lipopolysaccharide challenge

Bacterial DNA and synthetic oligonucleotides containing CpG sequences (CpG-DNA and CpG-ODN) provoke a proinflammatory cytokine response (tumor necrosis factor alpha [TNF-alpha], interleukin-12 [IL-12], and IL-6) and increased mortality in lipopolysaccharide (LPS)-challenged mice via a TNF-alpha-mediated mechanism. It was hypothesized that preexposure of macrophages to CpG-ODN would result in an increased TNF-alpha response to subsequent LPS challenge in vitro. Using the murine macrophage cell line RAW 264.7, we demonstrated both a rapid proinflammatory cytokine response (TNF-alpha) and a delayed inhibitory cytokine response (IL-10) with CpG-ODN. Preexposure of macrophages to CpG-ODN for brief periods (1 to 3 h) augmented TNF-alpha secretion and mRNA accumulation following subsequent LPS challenge (1 microg/ml). However, prolonged preexposure to CpG-ODN (6 to 9 h) resulted in suppression of the TNF-alpha protein and mRNA response to LPS. The addition of anti-IL-10 antibody to CpG-ODN during preexposure resulted in an increase in the LPS-induced TNF-alpha response over that induced by CpG-ODN preexposure alone. Thus, while brief preexposure of macrophages to CpG-ODN augments the proinflammatory cytokine response to subsequent LPS challenge, prolonged preexposure elicits IL-10 production, which inhibits the TNF-alpha response. Although the initial proinflammatory effects of CpG-DNA are well established, the immune response to CpG-DNA may also include autocrine or paracrine feedback mechanisms, leading to a complex interaction of proinflammatory and inhibitory cytokines.     

Downregulation by cryptococcal polysaccharide of tumor necrosis factor alpha and interleukin-1 beta secretion from human monocytes.

The regulation by Cryptococcus neoformans encapsulation of interleukin 1 beta (IL-1 beta) and tumor necrosis factor alpha (TNF-alpha) production by human monocytes was investigated. By using encapsulated and acapsular C. neoformans, we demonstrated that both strains induce cytokine production, although the acapsular strain was a better stimulator than the thinly encapsulated strain. The cytokine levels produced by cells stimulated by the two strains were lower and followed a different kinetic than those stimulated by lipopolysaccharide (LPS). Purified capsular polysaccharide inhibits TNF-alpha secretion induced by LPS or acapsular C. neoformans. In contrast, no regulator effect on IL-1 beta was observed when LPS was used. The secretory response of these cytokines follows different pathways of macrophage activation; in fact, complete inhibition of TNF-alpha does not affect IL-1 beta production and vice versa. These data indicate that purified capsular polysaccharide of C. neoformans could contribute to the in vivo progress of cryptococcosis by suppressing cytokine production of macrophages and suggest that a therapeutic approach to address the suppressive effect of cryptococal polysaccharide could be devised.     

Induction of interleukin-1 and -6 in human gingival fibroblast cultures stimulated with Bacteroides lipopolysaccharides.

Normal human gingival fibroblasts stimulated in vitro by lipopolysaccharides (LPS) from oral Bacteroides species produced cell-free and cell-associated thymocyte-activating factors (TAF). Neutralization assays using antisera to human interleukin-1 alpha (HuIL-1 alpha), HuIL-1 beta, and HuIL-6 revealed that cell-free TAF was attributable mainly to IL-1 beta and that IL-6 augmented the TAF activity of IL-1 beta in the culture supernatant. Another factor(s), however, may also be involved in cell-free TAF. By contrast, the active entity of cell-associated TAF was ascribed to IL-1 alpha alone. Furthermore, IL-6 was detected mainly in the supernatant of fibroblast cultures stimulated with Bacteroides LPS. Fibroblasts pretreated with natural human beta or gamma interferon, but not those pretreated with alpha interferon, synthesized higher levels of cell-associated IL-1 alpha in response to stimulation by Bacteroides LPS; however, no interferons exhibited direct IL-1-inducing activity or synergistic IL-1-inducing activity with LPS. Endogenously induced beta interferon was suggested to be necessary for fibroblasts to produce cell-associated IL-1 alpha in response to Bacteroides LPS.     

Effective phage therapy is associated with normalization of cytokine production by blood cell cultures

The aim of this study was to investigate the effect of phagotherapy on tumor necrosis factor alpha (TNF-alpha) and interleukin 6 (IL-6) serum levels and the ability of blood cells to produce these cytokines in culture. Fifty one patients with long-term, suppurative infections of various tissues and organs were enrolled. The ability of cells to secrete cytokines was tested using whole blood cell cultures, unstimulated or stimulated with lipopolysaccharide (LPS) from E. coli. In addition, cytokine serum levels were determined. Measurement of cytokine activity was performed using bioassays. We showed that TNF-alpha, but not IL-6 serum levels, were regulated upon division of patients into categories exhibiting initial: low, moderate and high cytokine levels. The low spontaneous production of IL-6 by blood cell cultures was elevated significantly on day 21 of phage therapy, whereas high release of this cytokine was inhibited. No such correlation was observed with LPS-induced IL-6 production in cell cultures when cells from low-, moderately- or highly-reactive patients were studied. Phage therapy modified TNF release according to the initial ability to produce that cytokine: it reduced TNF production in high responders and increased it in low responders. Patients infected only with Gram-positive bacteria demonstrated analogous changes in the spontaneous and LPS-induced TNF-alpha production as in the whole studied group. A similar kind of regulation was observed in TNF-alpha and LPS-induced production, i.e. low production was significantly elevated, high strongly inhibited, and moderate only slightly affected. In summary, we demonstrated for the first time that effective phage therapy can normalize TNF-alpha serum levels and the production of TNF-alpha and IL-6 by blood cell cultures.     

Cultured human monocytes secrete fibronectin in response to activation by proinflammatory cytokines

We studied the effects of the cytokines IL-1alpha, IL-6, tumour necrosis factor-alpha (TNF-alpha), IL-4, IL-10, IL-13 and transforming growth factor-beta (TGF-beta) on fibronectin (FN) production by cultured-human monocytes. IL-1alpha, IL-6 and TNF-alpha all increased FN production, an indicator of monocyte activation. These cytokines increased FN production in a dose-dependent fashion, with a 4-h treatment being sufficient to measure FN production by radioimmunoassay. Conversely, IL-4, IL-10 and IL-13 strongly inhibited cytokine-induced FN production, while TGF-beta only partially inhibited FN production. The combination of suboptimal doses of cytokines (IL-1alpha + IL-6, IL-1alpha + TNF-alpha, IL-6 + TNF-alpha), which could not singly induce substantial amounts of FN, were able to induce FN production by cultured monocytes. Northern blot analysis with a cDNA specific for FN confirmed the expression of FN mRNA in cultured monocytes stimulated with a single cytokine or a combination of cytokines. Our data demonstrate that monocytes may not always require high concentrations of cytokines for activation in vitro, and that the synergistic or additive action of low levels of cytokines on monocyte activation may be sufficient to promote immune or inflammatory reactions. Our data also suggest that certain T cell cytokines may regulate monocyte activation.     

Gram-negative bacteria induce proinflammatory cytokine production by monocytes in the absence of lipopolysaccharide (LPS)

Tumour necrosis factor-alpha (TNF-alpha), IL-1alpha and IL-6 production by human monocytes in response to a clinical strain of the Gram-negative encapsulated bacteria Neisseria meningitidis and an isogenic lpxA- strain deficient in LPS was investigated. Wild-type N. meningitidis at concentrations between 105 and 108 organisms/ml and purified LPS induced proinflammatory cytokine production. High levels of these cytokines were also produced in response to the lpxA- strain at 107 and 108 organisms/ml. The specific LPS antagonist bactericidal/permeability-increasing protein (rBPI21) inhibited cytokine production induced by LPS and wild-type bacteria at 105 organisms/ml but not at higher concentrations, and not by LPS-deficient bacteria at any concentration. These data show that proinflammatory cytokine production by monocytes in response to N. meningitidis does not require the presence of LPS. Therapeutic strategies designed to block LPS alone may not therefore be sufficient for interrupting the inflammatory response in severe meningococcal disease.