Combinatory responses of proinflamamtory cytokines on nitric oxide-mediated function in mouse calvarial osteoblasts

Combinatory responses of proinflamamtory cytokines have been examined on the nitric oxide-mediated function in cultured mouse calvarial osteoblasts. Interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) induced iNOS gene expression and NO production, although these actions were inhibited by L-NG-monomethylarginine (L-NMMA) and decreased alkaline phosphatase (ALPase) activity. Furthermore, NO donors, sodium nitroprusside (SNP) and NONOate dose-dependently elevated ALPase activity. In contrast, transforming-growth factor-β (TGF-β) decreased NO production stimulated by IL-1β, TNF-α and interferon-γ (IFN-γ). iNOS was expressed by mouse calvarial osteoblast cells after stimulation with IL-1β, TNF-α, and IFN-γ. Incubation of mouse calvarial osteoblast cells with the cytokines inhibited growth and ALPase activity. However, TGF-β-treatment abolished these effects of IL-1β, TNF-α and IFN-γ on growth inhibition and stimulation of ALPase in mouse calvarial osteoblast cells. In contrast, IL-1β, TNF-α, and IFN-γ exerted growth-inhibiting effects on mouse calvarial osteoblast cells which were partly NO-dependent. The results suggest that NO may act predominantly as a modulator of cytokine-induced effects on mouse calvarial osteoblast cells and TGF-β is a negative regulator of the NO production stimulated by IL-1β, TNF-α and IFN-γ.     

Gender difference in tumor necrosis factor-α production in human neutrophils stimulated by lipopolysaccharide and interferon-γ

The gender difference in tumor necrosis factor-α (TNF-α) production in human neutrophils stimulated by lipopolysaccharide (LPS) and interferon-γ (IFN-γ) was explored by using peripheral blood neutrophils from young men and women. As compared with female neutrophils, male neutrophils released greater amounts of TNF-α, and exhibited stronger activation of mitogen-activated protein kinases and phosphatidylinositol 3-kinase in response to LPS stimulation. LPS-induced TNF-α production was markedly enhanced by pretreatment of cells with IFN-γ, and IFN-γ-mediated priming in male neutrophils was significantly greater than that in female neutrophils. Male neutrophils showed higher expression of TLR4, but not IFN-γ receptors, than female neutrophils, and its expression was increased by stimulation with IFN-γ or IFN-γ plus LPS. These findings indicate that male neutrophils show higher responsiveness to stimulation with LPS and IFN-γ than female neutrophils, and suggest that the gender difference in neutrophil responsiveness to LPS and IFN-γ is partly responsible for that in the outcome of sepsis, in which premenopausal women show a favorable prognosis as compared with men.     

Cytokine physiognomies of MSCs from varied sources confirm the regenerative commitment post-coculture with activated neutrophils

The interaction of mesenchymal stromal cells (MSCs) with paracrine signals and immunological cells, and their responses and regenerative commitment thereafter, is understudied. In the current investigation, we compared MSCs from the umbilical cord blood (UCB), dental pulp (DP), and liposuction material (LS) on their ability to respond to activated neutrophils. Cytokine profiling (interleukin-1α [IL-1α], IL-2, IL-4, IL-6, IL-8, tumor necrosis factor-α [TNF-α], interferon-γ [IFN-γ], transforming growth factor-β [TGF-β]), cellular proliferation and osteogenic differentiation patterns were assessed. The results showed largely comparable cytokine profiles with higher TNF-α and IFN-γ levels in LSMSCs owing to their mature cellular phenotype. The viability and proliferation between LS/DP/UCB MSCs were comparable in the coculture group, while direct activation of MSCs with lipopolysaccharide (LPS) showed comparable proliferation with significant cell death in UCB MSCs and slightly higher cell death in the other two types of MSC. Furthermore, when MSCs post-neutrophil exposure were induced for osteogenic differentiation, though all the MSCs devoid of the sources differentiated, we observed rapid and significant turnover of DPMSCs positive of osteogenic markers rather than LS and UCB MSCs. We further observed a significant turnover of IL-1α and TGF-β at mRNA and cytokine levels, indicating the commitment of MSCs to differentiate through interacting with immunological cells or bacterial products like neutrophils or LPS, respectively. Taken together, these results suggest that MSCs have more or less similar cytokine responses devoid of their anatomical niche. They readily switch over from the cytokine responsive cell phenotype at the immunological microenvironment to differentiate and regenerate tissue in response to cellular signals.     

CBR2激活与小胶质细胞的活化和损伤的关系

目的：探讨大麻素CBR2受体激动剂AM1241预处理对脂多糖（LPS）和γ-干扰素（IFN-γ）所致炎症反应对小胶质细胞活化和损伤的影响。方法：联用LPS和IFN-γ作为小胶质细胞损伤模型,将细胞分为Control组、AM1241组、LPS/IFN-γ组和AM1241＋LPS/IFN-γ组;AM1241组和AM1241＋LPS/IFN-γ组经AM1241预处理2h,LPS/IFN-γ组和AM1241＋LPS/IFN-γ组用含LPS和IFN-γ的培养基培养24h。采用MTT法检测细胞代谢率,硝酸还原酶法检测细胞培养液中一氧化氮（NO）释放量,酶联免疫吸附剂测定细胞培养基中炎症因子释放量,倒置相差显微镜观察细胞形态。结果：与LPS/IFN-γ组相比,AM1241＋LPS/IFN-γ组细胞代谢率明显升高（P〈0.05）,NO、TNF-α、IL-1β和IL-10释放量明显减少（P〈0.05）,活化和损伤程度明显减轻。结论：大麻素CBR2受体激动剂AM1241预处理可减轻LPS和IFN-γ对小胶质细胞的活化和损伤。     

The role of inflammatory and anti-inflammatory cytokines in the pathogenesis of human tegumentary leishmaniasis

In tegumentary leishmaniasis caused by Leishmania braziliensis, there is evidence that increased production of IFN-γ, TNF-α and absence of IL-10 is associated with strong inflammatory reaction and with tissue destruction and development of the lesions observed in cutaneous leishmaniasis (CL) and mucosal leishmaniasis (ML). We evaluate the role of regulatory cytokines and cytokine antagonists in the downregulation of immune response in L. braziliensis infection. Peripheral blood mononuclear cells from CL and ML were stimulated with soluble Leishmania antigen in the presence or absence of regulatory cytokines (IL-10, IL-27 and TGF-β) or antagonists of cytokines (α-TNF-α and α-IFN-γ). Cytokines production (IL-10, IL-17, TNF-α and IFN-γ) was measured by ELISA. IL-10 and TGF-β downmodulate TNF-α and IL-17 production, whereas IL-27 had no effect in the production of TNF-α, IFN-γ and IL-17 in these patients. Neutralization of TNF-α decreased IFN-γ level and the neutralization of IFN-γ decreased TNF-α level and increased IL-10 production. This study demonstrate that IL-10 and TGF-β are cytokines that appear to be more involved in modulation of immune response in CL and ML patients. IL-10 might have a protective role, since the neutralization of IFN-γ decreases the production of TNF-α in an IL-10-dependent manner.     

Contribution of the exosome-associated form of secreted endoplasmic reticulum aminopeptidase 1 to exosome-mediated macrophage activation

Macrophages secrete endoplasmic reticulum aminopeptidase 1 (ERAP1) in response to lipopolysaccharide (LPS) and interferon (IFN)-γ to enhance their phagocytic and nitric oxide (NO) synthetic activities. In this study, we found that a subset of secreted ERAP1 bound to exosomes released from LPS/IFN-γ-treated murine RAW264.7 macrophages compared to untreated cells. ERAP1-bound exosomes enhanced phagocytic and NO synthetic activities of macrophages more efficiently than free ERAP1 and exosomes derived from untreated cells. Deletion of the exon 10 coding sequence in ERAP1 gene resulted in loss of binding to exosomes. By comparing the activities of exosomes derived from wild-type and ERAP1 gene-deficient RAW264.7 cells, we observed that ERAP1 contributed to the exosome-dependent phagocytosis and NO synthesis of the cells. Upon stimulation of RAW264.7 cells with LPS/IFN-γ, TNF-α, IFN-γ, and CCL3 were also associated with the released exosomes. Analyses of cytokine function revealed that while CCL3 in the exosomes was crucial to the phagocytic activity of RAW264.7 cells, TNF-α and IFN-γ primarily contributed to the enhancement of NO synthesis. These results suggest that treatment with LPS/IFN-γ alters the physicochemical properties of exosomes released from macrophages in order to facilitate association with ERAP1 and several cytokines/chemokines. This leads to exosome-mediated enhancement of macrophage functions. It is possible that packaging effector molecules into exosomes upon inflammatory stimuli, facilitates the exertion of effective pathophysiological functions on macrophages. Our data provide the first evidence that ERAP1 associated with exosomes plays important roles in inflammatory processes via activation of macrophages.     

Promoting effect of IFN-γ on the expression of LPS-induced IL-12 p40 and p35 mRNA in murine suppressor macrophages

We detected the expression of IL-12 p40/p35 mRNA by semi-quantitative RT-PCR and silver staining, and studied the molecular interaction between the IL-12 expression and the NF-kB activation induced by LPS and IFN-γ/LPS in murine peritoneal suppressor macrophages (MPSMs). It was found that IFN-γ strongly enhanced the LPS-induced IL-12 p40 and p35 mRNA expression. Both p40 and p35 mRNA levels were approximately equal. IFN-γ also greatly promoted the LPS-induced secretion of IL-12 p70 in MPSMs. The Proteasome Inhibitor I (PSI) could block the expres-sion of IL-12 p40 and p35 mRNA, and the degradation of IkBα induced by LPS or LPS/IFN-γ. EM-SA showed that LPS could augment the NF-kB binding activity to p40 promoter DNA. However, IFN-γ could neither enhance the LPS-induced NF-kB activity nor promote the degradation of IkBα. Taken together, the data suggest: (i) IFN-γ/LPS could strongly induce the expression of IL-12 p40 and p35 mRNA; both the expression levels were equal; this phenomenon coincided with the high-level secretion of IL-12 p70 induced by IFN-γ/LPS; (ii) NF-kB signal pathway is essential for IFN-γ/LPS to induce IL-12 mRNA expression; (iii) by blocking the degradation of IkB, the PSI sup-presses the IL-12 p40/p35 mRNA expression induced by LPS and IFN-γ/LPS; (iv) NF-kB signal may not be involved in the mechanism by which IFN-γ enhanced the expression of the LPS-induced IL-12 p40/p35 mRNA.     

Characterization of L1210 S Cells with Low Sensitivity to Mouse Interferon-γ

A mouse leukemic cell line L1210 Sg with a low sensitivity to interferon-γ (IFN-γ) was described. On the nature of the antiviral action and binding of IFN, L1210 Sg cells were compared with L1210m cell line which is sensitive to IFN-γ. For a half reduction of the vesicular stomatitis virus-RNA synthesis, L1210 Sg cells required 500–fold more IFN-γ than L1210m cells did. However, both cell lines were induced to the antiviral state to the same extent with IFN-α or -β. L1210 Sg and L1210m cells were sensitive to the anti-proliferative action of IFN-α and -β, but insensitive to IFN-γ. (2′-5′)Oligoadenylate synthetase was induced in these cell lines by IFN-β, but not by IFN-γ, which suggests that the induction of this synthetase may not be responsible for the antiviral action of IFN-γ. No substantial difference between L1210 Sg and L1210m cells was found in IFN receptors for IFN-γ and IFN-β either in number per cell or in their affinity to corresponding IFN type. However, differences were noted in time course profiles of cell-associated IFN-γ at 37 C: in L1210m cells, a rise-and-decay profile of IFN-γ bound to cells was observed at 37 C, but in L1210 Sg cells, rise and slight decay was observed. On the other hand, a similar rise-and-decay curve of IFN-β bound to these cells was observed. These results indicated that the low sensitivity of L1210 Sg cells to IFN-γ may be due to this slight decay of receptor-bound IFN-γ.     

IFN-γ、IL-1α、NGF-β和TNF-α在皖西白鹅小脑皮质发育中的表达

用免疫组织化学strept actividin-biotin complex(SABC)法,以干扰素-γ(IFN-γ)、白介素-1α(IL-1α)、神经生长因子-β(NGF-β)和肿瘤坏死因子-α(TNF-α)对胚龄13d、19d、24d、28d(E13、E19、E24、E28)和日龄7d、15d(P7、15)的皖西白鹅(WesternAnhuiwhitegoose)小脑皮质中的阳性细胞进行定位和半定量检测,探讨IFN-γ、IL-1α、NGF-β和TNF-α在小脑皮质发育中的作用。研究表明,外颗粒层细胞在E13、E19、E24、E28、P7有IFN-γ和TNF-α阳性表达;在E13、E19、E24、E28有IL-1α阳性表达;在E13、E19、E24有NGF-β阳性表达;且在所检测的6个时期中,4种细胞因子均在E19表达最强。Purkinje细胞层在E13、E19、E24、E28、P7、P15均有IFN-γ、IL-1α、TNF-α阳性表达;在E13、E19、E24、E28、P7有NGF-β阳性表达;内颗粒层细胞在E13、E19、E24、E28、P7、P15有IFN-γ阳性表达;在E13、E19、E24、E28、P7有IL-1α、TNF-α阳性表达;在E13、E19、E24、E28有NGF-β阳性表达。结果表明,E19可能为小脑皮质发育的"关键期";IFN-γ、IL-1α和TNF-α可能由小脑皮质自身合成;NGF-β可能由投射到Purkinje细胞的区域转运而来,且可能在Purkinje细胞生长发育过程中起营养作用;IFN-γ可能在颗粒细胞迁移过程中起干扰作用。     

Blockade of hypoxia-inducible factor-1α by YC-1 attenuates interferon-γ and tumor necrosis factor-α-induced intestinal epithelial barrier dysfunction

Proinflammatory cytokines play vital roles in intestinal barrier function disruption. YC-1 has been reported to have potent anti-inflammatory properties, and to be a potential agent for sepsis treatment. Here, we investigated the protective effect of YC-1 against intestinal barrier dysfunction caused by interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α). To assess the protective effect of YC-1 on intestinal barrier function, Caco-2 monolayers treated with simultaneous IFN-γ and TNF-α were used to measure transepithelial electrical resistance (TER) and paracellular permeability. To determine the mechanisms involved in the protective action of YC-1, expression and distribution of tight junction proteins ZO-1 and occludin in Caco-2 monolayers challenged with simultaneous IFN-γ and TNF-α were analyzed by Western blot and immunofluorescence, respectively. Expressions of phosphorylated myosin light chain (MLC), MLC kinase (MLCK) and hypoxia-inducible factor-1α (HIF-1α) were analyzed by Western blot in IFN-γ and TNF-α-treated Caco-2 monolayers. It was found that YC-1 attenuated barrier dysfunction caused by IFN-γ and TNF-α, and also prevented IFN-γ and TNF-α-induced morphological redistribution of tight junction proteins ZO-1 and occludin in Caco-2 monolayers. In addition, YC-1 suppressed IFN-γ and TNF-α-induced upregulation of MLC phosphorylation and MLCK protein expression. Furthermore, enhanced expression of HIF-1α in Caco-2 monolayers treated with IFN-γ and TNF-α was also suppressed by YC-1. It is suggested that YC-1, by downregulating MLCK expression, attenuates intestinal barrier dysfunction induced by IFN-γ and TNF-α, in which HIF-1α inhibition, at least in part, might by involved. YC-1 may be a potential agent for treatment of intestinal barrier disruption in inflammation.     

The evolution of three types of indoleamine 2,3 dioxygenases in fungi with distinct molecular and biochemical characteristics

Indoleamine 2,3-dioxygenase (IDO) is a tryptophan-degrading enzyme and known as a mammalian immunosuppressive molecule. In fungi, the primary role of IDO is to supply nicotinamide adenine dinucleotide (NAD(+)) via the kynurenine pathway. We previously reported that the koji-mold, Aspergillus oryzae has two IDO genes, IDOα and IDOβ. In the present study, we found that A. oryzae also has the third IDO, IDOγ. These three-types of IDOs are widely distributed among the Pezizomycotina fungi, although the black truffle, Tuber melanosporum has only one corresponding gene to IDOα/IDOβ. The yeast, Saccharomyces cerevisiae has a single IDO gene. Generally, Pezizomycotina IDOα showed similar enzymatic properties to the yeast IDO, suggesting that the IDOα is a functional homologue of the S. cerevisiae IDO. In contrast to IDOα, the K(m) value of IDOβ is higher. However, the reaction velocity of IDOβ is very fast, resulting in comparable or higher catalytic efficiency than IDOα. Thus IDOβ may functionally substitute for IDOα in fungal L-Trp metabolism. The enzymatic activity of IDOγ was comparatively very low with the values of enzymatic parameters comparable to vertebrate IDO2 enzymes. IDOα and IDOβ have similar gene structures, suggesting that they were generated by gene duplication which occurred rather early in Pezizomycotina evolution, although the timing of the duplication remains debatable. In contrast, the phylogenetic trees suggest that IDOγs form an evolutionarily distinct group of IDO enzymes, with a closer relationship to group I bacterial IDOs than other fungal IDOs. The ancestor of the IDOγ family is likely to have diverged from other eukaryotic IDOs at a very early stage of eukaryotic evolution.     

Functional role of endogenous CD14 in lipopolysaccharide-stimulated bone resorption

Lipopolysaccharide (LPS) is a bacterial cell component that plays multifunctional roles in inflammatory reactions, and one of these roles is that of a powerful stimulator of bone resorption. However, the mechanism by which LPS stimulates bone resorption is not yet understood. In the present study, we show, by using mouse embryonic calvarial cells, that endogenous CD14 and interleukin-1β (IL-1β) play an important role in the LPS-mediated bone resorption and that interferon-γ (IFN-γ)functions as a strong inhibitor of this resorption by suppressing LPS-stimulated expression of CD14 and IL-1β genes in the calvarial cells. We observed that LPS-stimulated differentiation of osteoclastic cells and bone and resorption were markedly neutralized by anti-mouse CD14 antibody were clearly inhibited by anti-sense CD14 oligonucleotide treatment. In addition, because LPS stimulated CD14 gene expression in the calvarial cells, these observations demonstrate the precise role of endogenous CD14 in LPS-stimulated differentiation of osteoclastic cells and boneresorption. However, the stimulation of the differentiation of osteoclastic cells and bone resorption was also inhibited by anti-mouse IL-1β antibody. Interestingly, anti-sense CD14 oligonucleotide inhibited LPS-stimulated expression of the IL-1β gene in the calvarial cells. These observations suggest a functional role of endogenous CD14 in LPS-stimulated expression of the IL-1β gene in the cells. Because IFN-γ is a potent inhibitor of bone resorption stimulated by IL-1, in additional experiments, we examined whether IFN-γ is able to inhibit LPS-stimulated differentiation of osteoclastic cells and bone resorption. We found that IFN-γ inhibited these stimulations by suppressing CD14 and IL-1β genes in the calvarial cells. The present study thus clearly demonstrates a functional role of endogenous CD14 in LPS-stimulated bone resorption. J. Cell. Physiol. 173:301–309, 1997. © 1997 Wiley-Liss, Inc.     

CD1d-restricted IFN-γ-secreting NKT cells promote immune complex-induced acute lung injury by regulating macrophage-inflammatory protein-1α production and activation of macrophages and dendritic cells

Immune complex-induced acute lung injury (IC-ALI) has been implicated in various pulmonary disease states. However, the role of NKT cells in IC-ALI remains unknown. Therefore, we explored NKT cell functions in IC-ALI using chicken egg albumin and anti-chicken egg albumin IgG. The bronchoalveolar lavage fluid of CD1d(-/-) and Jα18(-/-) mice contained few Ly6G(+)CD11b(+) granulocytes, whereas levels in B6 mice were greater and were increased further by α-galactosyl ceramide. IFN-γ and MIP-1α production in the lungs was greater in B6 than CD1d(-/-) mice. Adoptive transfer of wild type (WT) but not IFN-γ-, MIP-1α-, or FcγR-deficient NKT cells into CD1d(-/-) mice caused recruitment of inflammatory cells to the lungs. Moreover, adoptive transfer of IFN-γR-deficient NKT cells enhanced MIP-1α production and cell recruitment in the lungs of CD1d(-/-) or CD1d(-/-)IFN-γ(-/-) mice, but to a lesser extent than WT NKT cells. This suggests that IFN-γ-producing NKT cells enhance MIP-1α production in both an autocrine and a paracrine manner. IFN-γ-deficient NKT cells induced less IL-1β and TNF-α production by alveolar macrophages and dendritic cells in CD1d(-/-) mice than did WT NKT cells. Taken together, these data suggest that CD1d-restricted IFN-γ-producing NKT cells promote IC-ALI by producing MIP-1α and enhancing proinflammatory cytokine production by alveolar macrophages and dendritic cells.     

MiR-375-3p alleviates the severity of inflammation through targeting YAP1/LEKTI pathway in HaCaT cells

ABSTRACT

Atopic dermatitis (AD) is a relapsing inflammatory skin disease with a complicated pathogenesis. This study aimed to investigate whether miR-375-3p could regulate AD through the Yes-associated protein 1 (YAP1) pathway. In this study, inflammatory response was induced by TNF-α and IFN-γ administration in HaCaT cells. We found that viability and inflammatory factor release, including interleukin-1β (IL-1β) and IL-6, were negatively related to miR-375-3p expression in HaCaT cells. We also found that YAP1 overexpression down-regulated lympho-epithelial Kazal type inhibitor (LEKTI) levels and aggravated viability and inflammation in TNF-α and IFN-γ-treated HaCaT cells. Dual-luciferase reporter assay proved the targeted binding of miR-375-3p and YAP1 3?-UTR. Additionally, the protective effect of miR-375-3p on inflammatory response in TNF-α and IFN-γ-treated HaCaT cells could be impeded by YAP1 overexpression. Collectively, our results suggested that miR-375-3p could modulate HaCaT cell viability and inflammation through the YAP1/LEKTI pathway.     

IL-11 expression in retinal and corneal cells is regulated by interferon-γ

Interleukin-11 (IL-11) is an anti-apoptotic, anti-inflammatory cytokine with hematopoietic potential. The expression and protective actions of IL-11 have not been explored in the eye. The expression of IL-11 in primary cultures of human retinal pigment epithelial (HRPE) and human corneal fibroblast (HCRF) cells were evaluated in these studies. Constitutive secretion of IL-11 was not observed in either HRPE or HCRF. TNF-α + IL-1 induced IL-11 secretion and this production was inhibited by NFκB pathway inhibitors. IFN-γ significantly inhibited TNF-α and IL-1 induced IL-11 secretion and inhibitors of JAK-STAT pathway reversed this inhibition. TGF-β induced IL-11 secretion that was blocked by TGF-β receptor 1 inhibitor but not by IFN-γ. RT-PCR analysis confirmed the effects of IL-1, TNF-α, IFN-γ and TGF-β on IL-11 secretion at mRNA levels. Our results demonstrate that IL-11 is dramatically up regulated in retina and cornea cells and that IFN-γ is a physiological inhibitor of IL-11 expression.