Effect of lisofylline and pentoxifylline on the bacterial-stimulated production of TNF-alpha, IL-1 beta IL-10 by human leucocytes.

The present study concerns the effect of the xanthine derivates lisofylline (LSF) and pentoxifylline (PTX) on the production of pro-inflammatory cytokines tumour-necrosis factor-alpha (TNF-alpha) and interleukin-1 beta (IL-1 beta) and the de-activating cytokine interleukin-10 (IL-10) by human leucocytes during stimulation with lipopolysaccharide (LPS), heat-killed Gram-negative bacteria (GNB) or Gram-positive bacteria (GPB). The production of TNF-alpha and IL-1 beta by leucocytes stimulated with LPS, Haemophilus influenzae type b (Hib) or Streptococcus pneumoniae was inhibited by both drugs. The production of IL-10 by leucocytes stimulated with LPS and Hib was inhibited by both xanthine derivates only at 48 hr. However, incubation of leucocytes with S. pneumoniae in the presence of LSF or PTX stimulated the production of IL-10 about four- and twofold at 24 hr and 48 hr, respectively. In all instances, the extent of inhibition or enhancement of cytokine production by LSF or PTX was equal. The divergent effects of xanthine derivates on the IL-10 production indicate the existence of distinct intracellular pathways depending on whether leucocytes are stimulated by GPB or GNB.     

Capsular polysaccharide of Cryptococcus neoformans induces proinflammatory cytokine release by human neutrophils.

Human polymorphonuclear leukocytes from normal subjects produced proinflammatory cytokines in response to stimulation with Cryptococcus neoformans yeast cells. The cytokines released after stimulation of neutrophils included interleukin-1beta (IL-1beta), IL-6, IL-8 and tumor necrosis factor alpha. The magnitude of the cytokine response was related to the yeast capsule size. Cells of a large-capsule isolate stimulated release of greater amounts of cytokine than did a thinly encapsulated isolate, which, in turn, stimulated release of greater amounts of cytokine than an acapsular isolate. Cytokine release was also stimulated by supernatant fluids from cryptococcal cells that were preincubated with 10% human serum, suggesting the generation of a soluble mediator. The major capsular polysaccharide, glucuronoxylomannan, stimulated release of tumor necrosis factor alpha, IL-1beta, and IL-8 in a dose-dependent fashion. These results differ from previous studies of cytokine secretion by human monocytes in several important respects, including the importance of encapsulation in stimulation of cytokine secretion and the ability of purified glucuronoxylomannan to induce cytokine secretion.     

Temporal sequence and kinetics of proinflammatory and anti-inflammatory cytokine secretion induced by toxic shock syndrome toxin 1 in human peripheral blood mononuclear cells.

The staphylococcal superantigen toxic shock syndrome toxin 1 (TSST-1) induces massive cytokine production, which is believed to be the key factor in the pathogenesis of TSS. The temporal sequence and kinetics of both proinflammatory and anti-inflammatory cytokines induced by TSST-1 in human peripheral blood mononuclear cells were investigated. A panel of loss-of-function single-amino-acid-substitution mutants of TSST-1, previously demonstrated to be defective in either major histocompatibility complex (MHC) class II binding (G31R) or T-cell receptor (TCR) interaction (H135A, S14N), was studied in parallel to further elucidate the mechanisms of cytokine secretion. Wild-type recombinant (WT r) TSST-1 induced a biphasic pattern of cytokine secretion: an early phase with rapid release of proinflammatory cytokines (especially gamma interferon, interleukin-2 [IL-2], and tumor necrosis factor alpha [TNF-alpha]) within 3 to 4 h poststimulation, and a later phase with more gradual production of both proinflammatory (IL-1beta, IL-12, and TNF-beta) and anti-inflammatory (IL-6, IL-10) cytokines within 16 to 72 h poststimulation. G31R, which is defective in MHC class II binding, induced a cytokine profile similar to that of WT rTSST-1, except that secretion of the early-phase proinflammatory cytokines was delayed and production of IL-1beta and IL-12 was markedly reduced. In contrast, mutant toxins defective in TCR interaction either demonstrated complete absence of any cytokine secretion during the entire observation period (H135A) or resulted in complete abolishment of IL-2 and other early-phase proinflammatory cytokines, while secretion of IL-10 appeared unaffected (S14N). Neither WT rTSST-1 nor the mutant toxins induced IL-4 or transforming growth factor beta. Our data indicate that effective TCR interaction is critical for the induction of the early-phase proinflammatory cytokine response, thus underscoring the importance of T-cell signaling in TSS.     

Interleukin-10 inhibits proinflammatory activation of endothelium in response to Borrelia burgdorferi or lipopolysaccharide but not interleukin-1beta or tumor necrosis factor alpha

Interleukin (IL)-10 is generally regarded as an anti-inflammatory cytokine, since it acts on a variety of cell types to suppress production of proinflammatory mediators. In inflammation, endothelial cells (EC) play a crucial role in recruiting leukocytes to sites of injury or infection. In this study, the actions of IL-10 on human umbilical vein EC were investigated. IL-10 reduced migration of monocytes and T lymphocytes across endothelium stimulated by lipopolysaccharide and decreased endothelial production of chemokines in response to lipopolysaccharide and Borrelia burgdorferi, the agent of Lyme disease. However, IL-10 did not affect these responses when EC were activated by the host proinflammatory cytokines IL-lbeta or tumor necrosis factor alpha. Moreover, IL-10 did not prevent up-regulation of the adhesion molecules E-selectin and intercellular adhesion molecule-1 by EC exposed to any of these activating agents. IL-10 therefore inhibits proinflammatory activation of EC in a manner that is selective with respect to stimulus and effector response.     

IL-1alpha,IL-1beta and TNF-alpha secretion during in vivo/ex vivo cellular interactions with titanium and copper

Titanium (Ti) and copper (Cu) were used to evaluate cytokine secretion around materials with different chemical properties. Ti disks were coated with Cu or left uncoated. The disks were inserted subcutaneously in rats for 1, 3, 12, 18, 24 and 48 h. Interleukin-1alpha (IL-1alpha), IL-1beta and tumor necrosis factor-alpha (TNF-alpha) concentrations were measured in vivo around the materials, in sham operated sites, and after ex vivo incubation of surface adherent cells. Ti and Cu revealed distinct cytokine expression patterns. Cu recruited cells showed higher and prolonged release of IL-1alpha than Ti at longer times (>24 h), whereas Ti exhibited a transient IL-1alpha response at earlier periods (<24 h). An early enhanced secretion of TNF-alpha characterized Ti. Low amounts of IL-1beta were found around both materials. Sham site recruited cells produced lower levels of cytokines. The results after ex vivo incubations were similar to those in vivo. This study shows that material chemical properties influence early cytokine production. The Ti-associated transient rise of IL-1alpha and TNF-alpha may be of importance for the early tissue response around biocompatible materials, while a delayed high IL-1alpha expression could be a marker of inflammation induced by toxic materials.     

Role of EspA and intimin in expression of proinflammatory cytokines from enterocytes and lymphocytes by rabbit enteropathogenic Escherichia coli-infected rabbits

Enteropathogenic Escherichia coli (EPEC) produces attaching and effacing (A/E) lesions and watery diarrhea, both of which are intimin and EspA dependent. In this work, we explored the mucosal immune response by detecting cytokine induction in rabbits with diarrhea caused by rabbit EPEC (REPEC). Orally inoculated rabbits exhibited weight loss and mucosal inflammation, developed watery diarrhea, and died (day 7). At day 6 postinoculation, animals were analyzed for the induction of proinflammatory cytokines in enterocytes. The role of lymphocyte-dependent immunity was determined through the expression of proinflammatory cytokines by lymphocytes from Peyer's patches (PP) and the spleen. EspA and intimin mutants were used to explore the role of A/E lesions in the expression of these cytokines. REPEC-infected rabbit enterocytes showed increased interleukin 1beta (IL-1beta), IL-6, IL-8, and tumor necrosis factor alpha (TNF-alpha) mRNA expression, but that of anti-inflammatory IL-10 was increased only slightly. In contrast, intimin mutant-infected rabbits were unable to produce this proinflammatory cytokine profile but did produce a remarkable increase in IL-10 expression. Bacteria lacking EspA increased the expression of IL-8 and TNF-alpha, but that of IL-10 was increased only slightly. PP lymphocytes also produced proinflammatory cytokines, which were dependent on EspA (except for TNF-alpha) and intimin, while IL-10 was induced by EspA and intimin mutants. In contrast, spleen lymphocytes (systemic compartment) were unable to produce IL-1beta and TNF-alpha. These data show the importance of the proinflammatory cytokines secreted by enterocytes and those expressed locally by PP lymphocytes, which can activate effector mechanisms at the epithelium. Furthermore, this cytokine profile, including IL-6 and IL-1beta, which may be involved in the diarrhea produced by EPEC, depends on intimin.     

Hypophysectomy enhances interleukin-1beta, tumor necrosis factor-alpha, and interleukin-10 mRNA expression in the rat brain.

Although the effects of various cytokines as regulators of hormone synthesis and production are well documented, the role for pituitary hormones as modulators of cytokine synthesis is not fully understood. In this study, we investigated the effect of pituitary hormones' depletion on cytokine synthesis after short- (21 days) and long- (35 days) term hypophysectomy (ST-HX and LT-HX, respectively). The expresssion of the proinflammatory cytokine interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha) and the anti-inflammatory cytokines IL-10 and transforming growth factor-beta (TGF-beta) in the rat brain was studied using in situ hybridization. Our results indicate that IL-1beta mRNA-expressing cells were significantly upregulated at day 21 in hypophysectomized rats compared to sham-operated controls. This enhanced expression was also detected later at day 35 post hypophysectomy. However, TNF-alpha mRNA expression was significantly increased only at the later sampling interval. IL-10 mRNA-expressing cells were increased after long-term hypophysectomy compared to controls. TGF-beta mRNA-expressing cells were not increased after hypophysectomy. In conclusion, these results suggest a role for pituitary hormones in IL-1beta, TNF-alpha, and IL-10 synthesis.     

Interleukin-1 beta (IL-1 beta), IL-1 receptor antagonist, and TNF alpha production in whole blood.

The ability of an individual to mount defense responses to infection depend in part on the capacity to produce cytokines such as interleukin 1 (IL-1) and tumor necrosis factor (TNF). The specialized equipment, labor intensity, and sterile practice required for the standard in vitro evaluation of cytokine production can make such evaluation impractical in some clinical situations. We report a method for stimulating whole blood to produce cytokines that can be implemented in laboratories without tissue culture facilities and requires minimal sample preparation. IL-1 beta and TNF alpha production in whole blood samples was stimulated with endotoxin and/or phytohemagglutinin in standard EDTA-containing vacuum collection tubes. After incubation, plasma was removed and frozen for later assay. Comparison of this whole blood method with isolated mononuclear cell cultures indicated a significant correlation for IL-1 beta production (r = 0.746, P = 0.005). This technique also produced the newly described cytokine, IL-1 receptor antagonist. We conclude that the whole blood method is an acceptable alternative to isolated cell culture methods for measuring IL-1 beta in situations that preclude the standard in vitro approach.     

Induction of cytokines and chemokines in human monocytes by Mycoplasma fermentans-derived lipoprotein MALP-2

Bacterial infections are characterized by strong inflammatory reactions. The responsible mediators are often bacterially derived cell wall molecules, such as lipopolysaccharide or lipoteichoic acids, which typically stimulate monocytes and macrophages to release a wide variety of inflammatory cytokines and chemokines. Mycoplasmas, which lack a cell wall, may also stimulate monocytes very efficiently. This study was performed to identify mycoplasma-induced mediators. We investigated the induction of cytokines and chemokines in human monocytes exposed to the Mycoplasma fermentans-derived membrane component MALP-2 (macrophage-activating lipopeptide 2) by dose response and kinetic analysis. We found a rapid and strong MALP-2-inducible chemokine and cytokine gene expression which was followed by the release of chemokines and cytokines with peak levels after 12 to 20 h. MALP-2 induced the neutrophil-attracting CXC chemokines interleukin-8 (IL-8) and GRO-alpha as well as the mononuclear leukocyte-attracting CC chemokines MCP-1, MIP-1alpha, and MIP-1beta. Production of the proinflammatory cytokines tumor necrosis factor alpha and IL-6 started at the same time as chemokine release but required 10- to 100-fold-higher MALP-2 doses. The data show that the mycoplasma-derived lipopeptide MALP-2 represents a potent inducer of chemokines and cytokines which may, by the attraction and activation of neutrophils and mononuclear leukocytes, significantly contribute to the inflammatory response during mycoplasma infection.     

The role of IL-1beta in stress-induced sensitization of proinflammatory cytokine and corticosterone responses

Proinflammatory cytokines often sensitize neuronal, hormonal, and behavioral responses to subsequent challenge. Recently, it was observed that exposure to inescapable tailshock enhances peripheral and central proinflammatory cytokine and corticosterone (CORT) responses to subsequent immune challenge up to 4 days later. Thus, we examined the role of central interleukin-1beta (IL-1beta) in stress-induced sensitization of proinflammatory cytokine and CORT responses to a subsequent immune challenge. Rats were administered IL-1 receptor antagonist (IL-1ra) or vehicle into the intra-cisterna magna 1 h prior to tailshock (100, 1.6 mA 5 s shocks) exposure. Twenty-four hours later, rats were challenged i.p. with 10 microg/kg lipopolysaccharide (LPS) and killed 1 h later. IL-1ra had no effect on basal proinflammatory cytokines, but completely blocked the stress-induced enhancement in central and pituitary IL-1beta and plasma IL-6 release following LPS challenge. IL-1ra had no effect on stress-induced enhancement in CORT responses following LPS challenge. Additional rats were administered i.c.v. hrIL-1beta or vehicle and returned to their home cage. Twenty-four hours later, rats were challenged i.p. with either saline or 10 microg/kg LPS and killed 1 h later. Central hrIL-1beta administration significantly elevated central IL-1beta levels and plasma CORT following LPS challenge compared with vehicle-injected controls. These data demonstrate that elevations in central IL-1beta, whether stress-induced or exogenously administered, are sufficient for sensitizing central IL-1beta and CORT responses to subsequent immune challenge. However, during times of stress, exogenous central IL-1ra administration only blocked sensitization of subsequent central IL-1beta responses, not CORT responses, suggesting other factors during the stress response can sensitize CORT responses.     

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.     

The Type II heat-labile enterotoxins LT-IIa and LT-IIb and their respective B pentamers differentially induce and regulate cytokine production in human monocytic cells

The type II heat-labile enterotoxins, LT-IIa and LT-IIb, exhibit potent adjuvant properties. However, little is known about their immunomodulatory activities upon interaction with innate immune cells, unlike the widely studied type I enterotoxins that include cholera toxin (CT). We therefore investigated interactions of LT-IIa and LT-IIb with human monocytic THP-1 cells. We found that LT-II enterotoxins were inactive in stimulating cytokine release, whereas CT induced low levels of interleukin-1beta (IL-1beta) and IL-8. However, all three enterotoxins potently regulated cytokine induction in cells activated by bacterial lipopolysaccharide or fimbriae. Induction of proinflammatory (tumor necrosis factor alpha [TNF-alpha]) or chemotactic (IL-8) cytokines was downregulated, whereas induction of cytokines with anti-inflammatory (IL-10) or mucosal adjuvant properties (IL-1beta) was upregulated by the enterotoxins. These effects appeared to depend on their A subunits, because isolated B-pentameric subunits lacked regulatory activity. Enterotoxin-mediated inhibition of proinflammatory cytokine induction in activated cells was partially attributable to synergism for endogenous production of IL-10 and to an IL-10-independent inhibition of nuclear factor kappaB (NF-kappaB) activation. In sharp contrast to the holotoxins, the B pentamers (LT-IIaB and, to a greater extent, LT-IIbB) stimulated cytokine production, suggesting a link between the absence of the A subunit and increased proinflammatory properties. In this regard, the ability of LT-IIbB to activate NF-kappaB and induce TNF-alpha and IL-8 was antagonized by the LT-IIb holotoxin. These findings support distinct immunomodulatory roles for the LT-II holotoxins and their respective B pentamers. Moreover, the anti-inflammatory properties of the holotoxins may serve to suppress innate immunity and promote the survival of the pathogen.     

Protective role of interleukin-1 in mycobacterial infection in IL-1 alpha/beta double-knockout mice

To understand the role of the proinflammatory cytokine interleukin-1 (IL-1) in mycobacterial inflammation, IL-1 alpha/beta double-knockout (KO) mice were produced. These mice were infected with either Mycobacterium tuberculosis H37Rv by the airborne route using an airborne infection apparatus, and their capacities to control mycobacterial growth, granuloma formation, cytokine, and nitric oxide (NO) production were examined. The IL-1 alpha/beta mice developed significantly larger (p < 0.01) granulomatous, but not necrotic, lesions in their lungs than wild-type (WT) mice after infection with H37Rv. Inflammatory lesions, but not granulomas, were observed in spleen and liver tissues from both IL-1 alpha/beta KO and wild-type mice. Granulomatous lesion development in IL-1 alpha/beta KO mice was not significantly inhibited by treatment with exogenous recombinant IL-1 alpha/beta. Compared with wild-type mice, splenic IFN-gamma and IL-12 levels were within the normal range. NO production by cultured alveolar macrophages from IL-1 alpha/beta KO mice was lower than in wild-type mice but were increased by the addition of recombinant IL-1 alpha/beta. Our data clearly indicate that IL-1 is important for the generation of early-phase protective immunity against mycobacterial infection.     

ICAM-1 and alpha3beta1 expression by bronchial epithelial cells and their in vitro modulation by inflammatory and anti-inflammatory mediators

BACKGROUND: Adhesion molecules are involved in inflammatory and repair processes of the bronchial epithelium. ICAM-1 is mainly involved in inflammatory reactions, whereas integrins, such as alpha3beta1, are mainly involved in repair processes. METHODS: Using bronchial biopsies from 10 asthmatics and eight controls, we first evaluated by immunohistochemistry expression of alpha3beta1 and ICAM-1 in intact and damaged epithelium. Then, using the human pulmonary epithelial cell line WI-26 VA, we studied, by flow-cytometry, the modulation of ICAM-1 and alpha3beta1 expression, and, by ELISA, the release of fibronectin by proinflammatory cytokines, such as IL-5, and anti-inflammatory cytokines, such as IL-4, TGF-beta, and EGF. RESULTS: alpha3beta1 expression was slightly higher in asthma than in controls, as well as in damaged epithelium than in undamaged epithelium. ICAM-1 expression was higher in asthma than in controls, and similarly distributed in intact or damaged epithelium. In vitro, alpha3beta1 was significantly increased by TGF-beta, EGF, and IL-4, and significantly decreased by IL-5. Fibronectin release was significantly increased by TGF-beta and IL-4, unchanged by EGF, and slightly but significantly decreased by IL-5. ICAM-1 expression was significantly decreased by TGF-beta and IL-4, unchanged by EGF, and significantly increased by IL-5. CONCLUSIONS: These differences in adhesion molecule expression and fibronectin release may be important in epithelial cell inflammation and repair.     

Swertia chirayita mediated modulation of interleukin-1beta, interleukin-6, interleukin-10, interferon-gamma, and tumor necrosis factor-alpha in arthritic mice

The effect of aqueous extract of Swertia chirayita stem on the pro- and anti-inflammatory cytokines balance in primary joint synovium of adjuvant-induced arthritic mice has been studied. The level of pro-inflammatory cytokines was found elevated in the joint synovium of arthritic mice in comparison to normal joints. Administration of S. chirayita extract in varying doses through the oral route did not modulate the proinflammatory cytokines on day 2. In contrast, by day 12, a dose dependent (0, 11.86 and 23.72 mg/kg body weight) reduction of tumor necrosis factor-alpha (INF-alpha) interleukin-1beta, (IL-beta) and interferon-gamma, (IFN-gamma) and elevation of Interleukin-10 (IL-10) was observed in the joint homogenates of arthritic mice. Interleukin-6 (IL-6) was not down regulated in joint homogenate of arthritic mice at the dose 11.86 mg/kg but at higher doses (23.72 and 35.58 mg/kg) significant reduction was observed. The aqueous extract was found to possess two polar compounds, amerogentin and mangiferin but was devoid of swerchirin, chiratol, methyl swetianin, and swertanone. Mangiferin has been reported to possess potent anti-inflammatory property and we presume its presence in the aqueous extract of S. chirayita is responsible for reducing TNF-alpha, IL-1beta, IL-6, and IFN-gamma and/or elevating IL-10 in the joint homogenates of arthritic mice on day 12. This study will help in our understanding of the mechanism of anti-inflammatory action of S. chirayita in the light of proinflammatory and anti-inflammatory cytokine balance.     

Mycobacterial purified protein derivatives stimulate innate immunity: Malawians show enhanced tumor necrosis factor alpha, interleukin-1beta (IL-1beta), and IL-10 responses compared to those of adolescents in the United Kingdom

To investigate the role of innate immunity in variable efficacy of Mycobacterium bovis BCG vaccination in Malawi and the United Kingdom, we examined 24-h tumor necrosis factor alpha, interleukin-1beta (IL-1beta), and IL-10 responses to mycobacterial purified protein derivatives (PPDs). The rank order in stimulatory potency for different PPDs was the same for all three cytokines. Before vaccination Malawians made higher pro- and anti-inflammatory responses than did United Kingdom subjects. Fewer than 5% of United Kingdom subjects made IL-10 in response to any PPD, compared to 19 to 57% responders among Malawians. Priming for regulatory IL-10 may contribute to the smaller increase in gamma interferon responses in Malawians compared to United Kingdom subjects following BCG vaccination.