Estradiol Down-Regulates LPS-Induced Cytokine Production and NFkB Activation in Murine Macrophages

PROBLEM: In vivo and in vitro studies have indicated that estradiol can affect cytokine production in different cell types. This study examines whether estradiol affects inflammatory cytokine production by murine splenic macrophages. METHODS: Mouse splenic macrophages were first treated with 17β-estradiol, followed by lipopolysaccharide (LPS) stimulation. The production of cytokines by macrophages with or without estradiol treatment was determined at both the protein and mRNA levels. The nuclear factor-kB (NFkB) activity of activated mouse splenic macrophages was also evaluated by electrophoretic mobility shift assay. RESULT: Our results show that 17β-estradiol decreases LPS-induced IL-1α, IL-6, and TNF-α production but not IL-10, IL-12, and macrophage inflammatory protein (MIP) production by splenic macrophages. Furthermore, inhibition of cytokine production by 17β-estradiol was associated with a decreased LPS-induced NFkB-binding activity. CONCLUSION: Because cytokines are important mediators of immune function, the alteration of cytokine production by 17β-estradiol may thus have a profound effect on the outcome of immune response during inflammation.     

IL-32 up-regulation is associated with inflammatory cytokine production in allergic rhinitis

IL-32 is a described pro-inflammatory cytokine produced by T lymphocytes, natural killer cells, monocytes, and epithelial cells. However, the specific mechanism of IL-32 on allergic rhinitis (AR) has not been elucidated. Here, we report a significant increase of IL-32 protein and mRNA in the nasal mucosa of AR patients. In addition, in nasal mucosa tissue from AR patients, the level of IL-32 production correlated with inflammation, IL-1β, IL-18, and granulocyte-macrophage colony-stimulating factor (GM-CSF). In an AR animal model, IL-32 significantly increased IgE and inflammatory cytokine levels. IL-32 expression was induced by recombinant human GM-CSF via activation of caspase-1 in eosinophils. In addition, depletion of IL-32 prevents the production of inflammatory cytokines in eosinophils. In conclusion, IL-32 is an important cytokine involved in the inflammation of AR. The regulation of IL-32 expression may form the basis of a new strategy for the treatment of AR.     

Increased interleukin-10 production and Th2 skewing in the absence of 5-lipoxygenase

Eicosanoids (prostaglandins and leukotrienes) are important mediators of inflammatory responses. These lipid mediators may also regulate the production of peptide mediators of the immune system. In this study, we investigated the effect of the absence of 5-lipoxygenase (5-LO)-derived leukotrienes on interleukin (IL)-10 production. IL-10 is a key regulator of immune and inflammatory responses, and previous studies have suggested that prostaglandins effect their immunosuppressive functions in part by stimulation of IL-10 production. We therefore investigated whether leukotriene production would have a similar role in regulation of IL-10 production. We have made the striking observation that absence of 5-LO-derived leukotrienes results in increased IL-10 production with a concomitant decrease in the production of pro-inflammatory cytokines, including tumour necrosis factor (TNF)-alpha and IL-12. Moreover, T-cell cytokine production in the absence of 5-LO-derived leukotrienes results in increased IL-4 production and decreased interferon (IFN)-gamma production. This may be in part secondary to increased IL-10 production and its effects on dendritic cell function resulting in altered T-cell differentiation. These findings indicate that, in addition to the central role leukotrienes play in the acute inflammatory response, endogenous leukotrienes are also important regulators of inflammatory cytokine production, via regulation of IL-10 production and in vivo differentiation of T cells.     

Contact allergic response to dinitrochlorobenzene (DNCB) in rats: insight from sensitization phase

Contact hypersensitivity (CHS) is a T-cell-mediated skin inflammatory reaction to cutaneous exposure to small sensitizing chemicals, haptens. Majority of CHS studies were conducted in mice and there is paucity of data in other experimental animals. In the present study, characteristics of contact hypersensitivity reaction to dinitrochlorobenzene (DNCB) were determined in Th1-prone Dark Agouti (DA) rats by evaluating sensitization phase as a function of time-dependent changes in draining lymph nodes (DLN). Apart from basic indices of DLN activity (cellularity and proliferation), the production of cytokines relevant for CHS induction, interleukin-6 (IL-6), interferon-γ (IFN-γ), interleukin-17 (IL-17) and interleukin-4 (IL-4) was analyzed. Anti-inflammatory cytokine interleukin-10 (IL-10) production by DLN cells was determined as well. Highest production of IL-6, IFN-γ and IL-17 in sensitized animals was observed at day 3 after DNCB application, with a decrease at day 5. Increased messages for IFN-γ and IL-17 were noted at this time point. In contrast to inflammatory cytokines, anti-inflammatory cytokine interleukin-4 (IL-4) was undetectable during the entire sensitization phase. Differential pattern (IL-6 and IFN-γ) and level (IFN-γ and IL-17) of inflammatory cytokine production was noted in sensitized Th2-prone Albino Oxford (AO) rats. Similarly to DA rats, no changes in IL-4 were noted in AO rats. Strain-dependent differences in inflammatory cytokine production seem to be based on anti-inflammatory cytokine interleukin-10 (IL-10). Production of IFN-γ concomitantly with undetectable IL-4 in both strains classify rat CHS to DNCB as Th1/type 1 reaction. Detection of IL-17 in sensitized DLN cells points to the involvement of T_IL-17 cells in rat contact hypersensitivity.     

Combined cell wall polysaccharide,mycotoxin and bacterial lipopolysaccharide exposure and inflammatory cytokine responses

Human exposure to environmental microbes occurs regularly. Microbial compounds may interact with each other to affect cellular responses. We hypothesized that interactions between microbial compounds could modulate inflammatory cytokine responses in vitro. We investigated monocyte production of the pro‐inflammatory cytokine tumour necrosis factor‐α (TNF‐α) and the regulatory cytokine interleukin‐10 (IL‐10) after combined exposure to the fungal cell wall polysaccharide mannan and to the β‐glucan laminarin, the mycotoxin citrinin and bacterial lipopolysaccharide (LPS). Interactions between the cell wall microbial compounds were estimated statistically in a general linear mixed model. We found that LPS (100 ng/ml) and the used β‐glucan (up to 1000 μg/ml) significantly interacted with each other to reduce TNF‐α production. Mannan (up to 100 μg/ml) did not interact with the β‐glucan, but interacted with LPS. IL‐10 production was induced by LPS only. The mycotoxin citrinin did not induce cytokine production, but was toxic to the cells in a dose‐ and time‐dependent manner. However, non‐toxic doses of citrinin reduced LPS‐induced IL‐10 production while LPS‐induced TNF‐α production was not similarly reduced by citrinin. In conclusion, interactions between microbial compounds can modulate cellular inflammatory cytokine production and experimental investigations of one compound at a time could give misleading conclusions about these combined effects.     

Insulin-like growth factor-I stimulates IL-10 production in human T cells

There is vast body of evidence that the insulin-like growth factor (IGF)-I exerts immunomodulatory effects in vitro and in vivo. In vitro studies indicate that stimulatory effects of IGF-I may be exerted through augmentation of inflammatory cytokine production. To further explore the immunomodulatory effects of IGF-I through regulation of cytokine production, we tested the in vitro effects of IGF-I on the secretion of inflammatory T helper cell type 1 (Th1) and Th2 cytokines by human peripheral blood mononuclear cells (PBMC). To this end, PBMC were stimulated with the T cell mitogen phytohemagglutinin (PHA), and cytokines in the culture media were assessed after 18, 42, 66, and 80 h of culture. We found that IGF-I stimulated the secretion of the Th2 cytokine interleukin (IL)-10 by 40-70% in PHA-stimulated PBMC. In addition, we observed a small stimulatory effect (15%) on the secretion of another Th2 cytokine IL-4. The secretion of IL-2, IL-5, IL-6, interferon-gamma, and the inflammatory cytokines IL-1beta, IL-8, and tumor necrosis factor alpha was not or was hardly affected. IL-10 secretion was also stimulated in purified T cells, and we established that IGF-I also stimulated IL-10 mRNA expression by 100-150%. The monocyte-activating bacterial cell-wall product lipopolysaccharide induced IL-10 production in PBMC, but this was not affected by IGF-I. As IL-10 predominantly exerts anti-inflammatory actions and suppresses Th1-dependent immune responses, our results indicate that IGF-I may exert inhibitory actions on inflammatory and Th1-mediated cellular immune responses through stimulation of IL-10 production in T cells.     

Molsidomine Ameliorates Experimental Allergic Encephalomyelitis in Lewis Rats

Experimental allergic encephalomyelitis (EAE) is an autoimmune CD4⁺ T cell-mediated disease of the central nervous system (CNS). Nitric oxide (NO) plays an important role in preventing the development of EAE. Molsidomine (Mol) is a drug used for the treatment of coronary artery disease. Its therapeutic effects are the consequences of NO formation. In this study, we investigated the effects of Mol on EAE development in myelin basic protein (MBP)-immunized Lewis rats. All rats immunized with MBP developed typical clinical signs of acute EAE. In the EAE rats receiving Mol, the severity of clinical signs and the infiltration of inflammatory cells in CNS were clearly reduced. Furthermore, Mol administration significantly reduced the production of interferon-γ, a Th1 inflammatory cytokine, but increased the production of interleukin-10, a Th2 anti-inflammatory cytokine. Our findings suggest that the administration of the exogenous NO donor Mol is of considerable benefit in limiting the development of EAE and other Th1 cell-mediated inflammatory diseases.     

Molsidomine ameliorates experimental allergic encephalomyelitis in Lewis rats

Experimental allergic encephalomyelitis (EAE) is an autoimmune CD4⁺ T cell-mediated disease of the central nervous system (CNS). Nitric oxide (NO) plays an important role in preventing the development of EAE. Molsidomine (Mol) is a drug used for the treatment of coronary artery disease. Its therapeutic effects are the consequences of NO formation. In this study, we investigated the effects of Mol on EAE development in myelin basic protein (MBP)-immunized Lewis rats. All rats immunized with MBP developed typical clinical signs of acute EAE. In the EAE rats receiving Mol, the severity of clinical signs and the infiltration of inflammatory cells in CNS were clearly reduced. Furthermore, Mol administration significantly reduced the production of interferon-γ, a Th1 inflammatory cytokine, but increased the production of interleukin-10, a Th2 anti-inflammatory cytokine. Our findings suggest that the administration of the exogenous NO donor Mol is of considerable benefit in limiting the development of EAE and other Th1 cell-mediated inflammatory diseases.     

Short-chain fatty acids induce pro-inflammatory cytokine production alone and in combination with toll-like receptor ligands

Citation Mirmonsef P, Zariffard MR, Gilbert D, Makinde H, Landay, AL, Spear GT. Short‐chain fatty acids induce pro‐inflammatory cytokine production alone and in combination with Toll‐like receptor ligands. Am J Reprod Immunol 2012; 67: 391–400 Problem Short‐chain fatty acids (SCFAs), produced at relatively high levels by anaerobic bacteria in bacterial vaginosis (BV), are believed to be anti‐inflammatory. BV, a common alteration in the genital microbiota associated with increased susceptibility to HIV infection, is characterized by increased levels of both pro‐inflammatory cytokines and SCFAs. We investigated how SCFAs alone or together with Toll‐like receptor (TLR) ligands affected pro‐inflammatory cytokine secretion. Method of study Cytokines were measured by ELISA. Flow was used for phenotyping and reactive oxygen species (ROS) measurement. Results Short‐chain fatty acids, at 20 mm , induced interleukin (IL)‐8, IL‐6, and IL‐1β release, while lower levels (0.02–2 mm ) did not induce cytokine secretion. Levels >20 mm were toxic to cells. Interestingly, lower levels of SCFAs significantly enhanced TLR2 ligand‐ and TLR7 ligand‐induced production of IL‐8 and TNFα in a time‐ and dose‐dependent manner, but had little effect on lipopolysaccharide‐induced cytokine release. SCFAs mediated their effects on pro‐inflammatory cytokine production at least in part by inducing the generation of ROS. Conclusion Our data suggest that SCFAs, especially when combined with specific TLR ligands, contribute to a pro‐inflammatory milieu in the lower genital tract and help further our understanding of how BV affects susceptibility to microbial infections.     

Inflammatory cell numbers and cytokine expression in atopic dermatitis after topical pimecrolimus treatment

BACKGROUND: In several clinical trials the topical application of pimecrolimus was shown to be effective in the treatment of atopic dermatitis (AD). By targeting calcineurin-dependent signaling pathways, pimecrolimus controls cytokine gene expression. The purpose of this study was to investigate the effect of pimecrolimus on the inflammatory infiltrate and cytokine expression pattern in AD upon topical therapy. METHODS: From 10 patients with acute AD, skin biopsies as well as immunophenotype and cytokine production of peripheral blood mononuclear cells (PBMC) were examined before and 3 weeks after therapy. RESULTS: The clinical improvement was associated with a marked regression of histopathological features. In particular, the density of the inflammatory infiltrate mostly containing lymphocytes and eosinophils declined. By double immunofluorescent staining, a reduced expression of the T helper (Th) 2 cytokines interleukin (IL)-5, IL-10, and IL-13 in both CD4+ and CD8+ T cells was demonstrated after therapy. Pimecrolimus therapy was also associated with a reduced expression of the Th1 cytokine interferon (IFN)-gamma. Interestingly, the numbers of epidermal CD1a+ dendritic cells increased following treatment. In the peripheral blood, a decrease of lymphocytes and eosinophils was noticed, but the distribution of lymphocyte subpopulations and their capacity of cytokine production did not change. CONCLUSIONS: Topical pimecrolimus exhibits anti-inflammatory effects in AD by reducing the inflammatory cell infiltrate and cytokine expression in the dermis.     

Toll-like receptor-mediated inhibition of Gas6 and ProS expression facilitates inflammatory cytokine production in mouse macrophages

Activation of Toll-like receptors (TLRs) triggers rapid inflammatory cytokine production in various cell types. The exogenous product of growth-arrest-specific gene 6 (Gas6) and Protein S (ProS) inhibit the TLR-triggered inflammatory responses through the activation of Tyro3, Axl and Mer (TAM) receptors. However, regulation of the Gas6/ProS-TAM system remains largely unknown. In the current study, mouse macrophages are shown to constitutively express Gas6 and ProS, which synergistically suppress the basal and TLR-triggered production of inflammatory cytokines, including those of tumour necrosis factor-α, interleukin-6 and interleukin-1β, by the macrophages in an autocrine manner. Notably, TLR signalling markedly decreases Gas6 and ProS expression in macrophages through the activation of the nuclear factor-κB. Further, the down-regulation of Gas6 and ProS by TLR signalling facilitates the TLR-mediated inflammatory cytokine production in mouse macrophages. These results describe a self-regulatory mechanism of TLR signalling through the suppression of Gas6 and ProS expression.     

Histamine H1-receptor antagonists with immunomodulating activities: potential use for modulating T helper type 1 (Th1)/Th2 cytokine imbalance and inflammatory responses in allergic diseases

Being a first-line treatment for hypersensitivity allergic disease, histamine H1-receptor antagonists possess anti-inflammatory activity in addition to being H1-receptor antagonists. While it is not purely a histamine-related condition, hypersensitivity allergic disease is associated with an increase in the number of T helper type 2 (Th2) cells and Th2 cytokines, and a decrease in the number of Th1 cells and Th1 cytokines. Suppression of Th2-type cytokine production in addition to H1-receptor blockade may therefore represent a successful therapeutic strategy for the treatment of hypersensitivity allergic diseases. H1-receptor antagonists have been reported to modulate immune cascade at various points by acting on T cell-related inflammatory molecules, including adhesion molecules, chemokines and inflammatory cytokines. These effects of H1-receptor antagonists may be optimized for the treatment of allergic diseases. Besides their ability to regulate inflammatory molecules, some H1-receptor antagonists have been reported to down-regulate Th2 cytokine production. In particular, it has been shown that several H1-receptor antagonists specifically inhibit the production of Th2, but not Th1, cytokines. Accumulating evidence indicates a crucial role for Th1/Th2 cytokine imbalance on the development of allergic diseases. Accordingly, the use of H1-receptor antagonist with Th2 cytokine inhibitory activity to modulate Th1/Th2 cytokine imbalance might be a favourable strategy for the treatment of hypersensitivity allergic diseases. Furthermore, the identification of H1-receptor antagonists which possess immunoregulatory activities in addition to their anti-histamine activity will provide an important insight into the development of novel immunoregulatory drugs.     

CD44 as a novel target for treatment of staphylococcal enterotoxin B-induced acute inflammatory lung injury

Exposure to bacterial superantigens, such as staphylococcal enterotoxin B (SEB), can lead to the induction of acute lung injury/acute respiratory distress syndrome (ALI/ARDS). In the current study, we investigated the role of CD44 in ALI/ARDS. Intranasal exposure of CD44 wild-type mice to SEB led to a significant increase in the expression of CD44 on lung mononuclear cells. CD44 knockout mice developed significantly reduced SEB-induced ALI/ARDS, through reduced inflammatory cytokine production and reduced lung inflammatory cells, compared to similarly treated CD44 wild-type mice. Mechanistically, deletion of CD44 altered SEB-induced cytokine production in the lungs and reduced the ability of SEB-exposed leukocytes to bind to lung epithelial cells. Finally, treatment of SEB-exposed mice with anti-CD44 mAbs led to significant reduction in vascular permeability, reduction in cytokine production, and prevented inflammatory cell infiltration in the lungs. Together, these results suggest the possibility of targeting CD44 for the treatment of SEB-induced ALI/ARDS.     

Intergenotypic variation of nitric oxide and inflammatory markers in preeclampsia: a pilot study in a North Indian population

Cytokines appeared to contribute to the development of pathologic condition and eNOS gene polymorphism may affect cytokine production. The aim of this study was to evaluate cytokines pattern in preeclampsia and whether there is any relationship between gene and cytokines production and cytokine with disease severity. This cross-sectional study included 100 women with preeclampsia and 100 healthy pregnant women. Their blood samples were analyzed for nitric oxide (NO), inflammatory cytokines, and eNOS gene polymorphism. Decreased NO and increased cytokine (tumor necrosis factor-α, interleukin-2, and interferon-γ) levels were found in preeclampsia (p < 0.001). Significant differences were found in genotype/allele distribution between the two groups. A significant negative correlation was observed between NO and cytokine levels (tumor necrosis factor-α and interferon-γ) in the preeclamptic group (p = 0.001). We conclude that preeclampsia is associated with decreased levels of NO and increased levels of circulating inflammatory cytokines due to single nucleotide polymorphisms, pointing toward the role of endothelial and inflammatory components.     

Pro-inflammatory signaling by Jun-N-terminal kinase in inflammatory bowel disease

Since Jun-N-terminal kinase participates in intracellular signaling cascades resulting in inflammatory responses, inhibiting this pathway may represent a new treatment for inflammatory bowel disease including ulcerative colitis and Crohn's disease. However, the functional significance of the activation of this kinase in inflammatory bowel disease remains unclear. We investigated whether Jun-N-terminal kinase activation is increased in inflammatory bowel disease and analyzed the effects of SP600125, which decreases inflammatory cytokine synthesis by inhibiting the phosphorylation of this kinase. Phosphorylation of the kinase was examined in affected human colon using an enzyme-linked immunosorbent assay and immunohistochemistry. The effect of SP600125 on cytokine production was examined in cultures of patients' leukocytes and colonic tissue. Finally, rats received injection of SP600125 (30 mg/kg, s.c.) or vehicle twice daily 2 h before the induction of colitis with dextran sulfate sodium. SP600125 effects were determined observationally and histologically. Colonic tissue contained increased phosphorylated kinase in patients with inflammatory bowel disease with expression localized to the nucleus of epithelial and lamina propria mononuclear cells in lesions. Culturing mononuclear cells or colonic tissue with SP600125 down-regulated inflammatory cytokine production. Prophylactic treatment with SP600125 significantly reduced clinical and pathological scores in dextran sulfate sodium-treated rats. This first demonstration of the pathogenetic role of Jun-N-terminal kinase in the development of intestinal inflammation suggests that inhibiting its phosphorylation could benefit patients with inflammatory bowel disease.     

Role of interleukin-18 in intrahepatic inflammatory cell recruitment in acute liver injury

Although the innate immune system has been demonstrated to play important roles as the first line of defense against various infections, little is known about the interactions between intrahepatic inflammatory cells and the cytokine network in the liver. Here, we examined the role of IL-18 in IHL recruitment in acute liver injury. C57BL/6 mice were injected with an αCD40 mAb, and their serum IL-18 levels were observed to increase, with subsequent recruitment of IHLs into the liver. NKT cells were involved in this liver injury, as the serum ALT levels were reduced in NKT KO mice through the suppression of macrophage and monocyte migration and cytokine production. In contrast, depletion of neutrophils exacerbated the liver injury associated with high levels of TNF-α and IL-18 and increased numbers of macrophages and monocytes. Treatment with a neutralizing antibody against IL-18 reduced the serum ALT levels and inflammatory cell accumulation in the liver. Finally, additional administration of rIL-18 with αCD40 injection caused severe liver injury with increased IFN-γ production by NK cells. In conclusion, these findings demonstrate that IL-18 modulates liver inflammation by the recruitment of inflammatory cells, including NKT cells, macrophages, monocytes, and neutrophils.     

The modulatory effects of prostaglandin-E on cytokine production by human peripheral blood mononuclear cells are independent of the prostaglandin subtype

The production of inflammatory mediators, relevant to (auto)immune diseases and chronic inflammatory conditions, can be modulated by dietary intake of n-3 and n-6 long chain polyunsaturated fatty acids (PUFAs). It was suggested that these effects are related to the formation of different series of eicosanoids, in particular prostaglandin-E (PGE). In this study we investigated whether prostaglandin subtypes metabolized from arachidonic acid (PGE2), dihomo-gamma-linolenic acid (PGE1) or eicosapentaenoic acid (PGE3) have different effects on T-cell proliferation and cytokine production in vitro. Freshly isolated human peripheral blood mononuclear cells (PBMC) were stimulated with concanavalin A (ConA) or lipopolysaccharide (LPS) in the presence or absence of exogenous PGE1, PGE2 or PGE3. We found that tumour necrosis factor-alpha (TNF-alpha), interferon-gamma (IFN-gamma) and to a lesser extent interleukin (IL)-10 production was inhibited by all PGE-subtypes in ConA-stimulated PBMC concomitant with unaffected IL-2 levels. The modulated cytokine production of ConA stimulated cells was independent of T-cell proliferation. PGE2 and PGE1 moderately stimulated proliferation, while PGE3 inhibited the proliferative response to some extent. In LPS-stimulated PBMC, TNF-alpha production was inhibited by all PGE-subtypes, whereas IL-6 remained unaffected and IL-10 production was increased. Time course experiments on the effects of PGE-subtypes on cytokine production after ConA or LPS stimulation showed these effects to be time dependent, but indifferent of the prostaglandin subtype added. Overall, the modulatory effects of PGE on cytokine production were irrespective of the subtype. This may implicate that the immunomodulatory effects of PUFAs, with respect to cytokine production, are not caused by a shift in the subtype of PGE.