Chinese herbal medicinal ingredients inhibit secretion of IL-6, IL-8, E-selectin and TXB2 in LPS-induced rat intestinal microvascular endothelial cells

The aim of the research was to investigate the anti-inflammatory mechanism of Pulsatillae Decoction (PD), the levels of interleukin (IL)-6, IL-8, E-selectin, and thromboxane B₂ (TXB₂) secreted by cultured rat intestinal microvascular endothelial cells (RIMECs) were determined after treatment with its active ingredients, namely anemoside B4, anemonin, berberine, jatrorrhizine, palmatine, aesculin, and esculetin. RIMECs were challenged with 1 μg/mL lipopolysaccharide (LPS) for 3?h, and then treated with each of the seven ingredients at three concentrations (1, 5 and 10 μg/mL) for 24?h. The results revealed that anemonin, aesculin and esculetin inhibited the production of IL-6, aesculin and esculetin inhibited the secretion of IL-8, anemoside B4, berberine and jatrorrhizine downregulated E-selectin expression, anemonin, berberine, jatrorrhizine and palmatine decreased the content of TXB₂. All these changes were significant. Taken together, the data suggest that all seven active ingredients of PD can effectively reduce inflammatory response, thus relieving intestinal dysfunction via multiple pathways.     

Production of soluble ICAM-1 from human endothelial cells induced by IL-1β and TNF-α

The present study was designed to establish the effects of cytokines on soluble ICAM-1 (sICAM-1) production by human endothelial cells (EC) and ICAM-1 expression on these cells and the effects of purified sICAM-1 on lympho-cyte-EC adhesion. Expression of ICAM-1 and production of sICAM-1 were measured by a specific ELISA method. ICAM-1 expression was enhanced by IL-1β, TNF-α, and most effectively by IFN-γ. IL-4, IL-6, M-CSF, or GM-CSF showed no effects on ICAM-1 expression. IL-4 (100 units/ml) or IL-6(100 units/ml) abolished the enhancing effect of IL-1β, while TNF-α (1, 10, 100 units/ml) synergized with IL-1β to promote ICAM-1 expression in EC. In contrast with the transient increase of cell-associated ICAM-1 expression after activiation by IL-1β, which peaked 40 h poststimulation and declined thereafter, sICAM-1 continued to accumulate in culture supernatants even after 48 h poststimulation in IL-1β-stimulated EC. IL-1β treatment resulted in an increase in adhesion. sICAM-1, purified from cell-free supernatants obtained after a 48-h culture of EC in IL-1β by affinity chromatography using monoclonal ICAM-1 antibody coupled to Sepharose beads, significantly inhibited lymphocyte EC adhesion. Preincubation of lymphocytes with conditioned medium of EC cultured with 100 units/ml IL-1β for 48 h, which contained a considerable amount of sICAM-1, resulted in a significant inhibition of lymphocyte adhesion to IL-1β-stimulated EC. These results suggest that there is a cumulative increase in sICAM-1 concentration in the vicinity of cytokine-stimulated EC and that this sICAM-1 modulates ICAM-1-mediated cell to cell interaction.     

Role of Chinese herbal medicinal ingredients in secretion of cytokines by PCV2-induced endothelial cells

While T-lymphocytes are the major cell type responsible for host responses to a virus (including induction of inflammatory responses to aid in ultimate removal of virus), other cells, including macrophages, epithelial and dendritic cells also have key roles. Endothelial cells also play important roles in physiologic/pathologic processes, like inflammation, during viral infections. As endothelial cells can be activated to release various endogenous compounds, including some cytokines, ex vivo measures of cytokine formation by the cells can be used to indirectly assess any potential endothelial dysfunction in situ. The research presented here sought to investigate potential immunolomodulatory effects of five saponins on endothelial cells: Saikosaponins A (SSA) and D (SSD), Panax Notoginseng Saponin (PNS) and Notoginsenoside R1 (SR1) and Anemoside B4 (AB4). For this, cells (porcine iliac artery endothelial line) were challenged with a virus isolate PCV2-AH for 24?h and then treated with the test saponin (at 1, 5 or 10?μg/ml) for an additional 24?h at 37?°C. The culture supernatants were then collected and analyzed for interleukin (IL)-2, -4 and -10, as well as interferon (IFN)-γ by ELISA. The results revealed that PNS and SR1 inhibited the production of IL-4; PNS, SR1 and AB4 inhibited the secretion of IL-10; SSA, SSD and PNS up-regulated IL-2 expression; SSA and SSD increased the level of IFNγ. All these changes were significant. Taken together, the data suggested these saponins might potentially have a capacity to regulate immune responses in vivo via changes in production of these select cytokines by infected endothelial cells. Nevertheless, the impact of these agents on other key cell types involved in anti-viral responses, including T-lymphocytes, remains to be determined.     

IL-6 acts on endothelial cells to preferentially increase their adherence for lymphocytes

Using a quantitative monolayer adhesion assay, the current report shows that treatment of human umbilical vein endothelial cells (HUVEC) with IL-6 increases their adhesiveness for blood lymphocytes, particularly CD4⁺ cells, but not for polymorphonuclear cells and monocytes. This effect, which was most pronounced when using low concentrations of the cytokine (0.1–1.0 U/ml) and a short incubation period (4 h), was also apparent with microvascular endothelial cells and a hybrid endothelial cell line. Skin lesions from patients with mycosis fungoides contain high levels of IL-6, and blood lymphocytes from patients with this disorder also exhibited an enhanced adhesion to IL-6-treated HUVEC. The cytokine enhanced intercellular adhesion molecule-1 (ICAM-1) expression and induced the expression of vascular cell adhesion molecule-1 (VCAM-1) and E-selectin on endothelial cells. Antibody blocking studies demonstrated that the vascular adhesion molecules ICAM-1, VCAM-1 and E-selectin and the leucocyte integrin LFA-1 all contributed to lymphocyte binding to endothelium activated by IL-6. It is proposed that IL-6 may be involved in the recruitment of lymphocytes into non-lymphoid tissue.     

IL-6-deficient mice resist myelin oligodendrocyte glycoprotein-induced autoimmune encephalomyelitis

Experimental autoimmune encephalomyelitis (EAE) is induced by immunization with myelin components including myelin oligodendrocyte glycoprotein (MOG). Myelin-specific Th1 cells enter the central nervous system (CNS) via binding of very late antigen 4 (VLA-4) to the endothelial vascular cell adhesion molecule 1 (VCAM-1). In the present study, mice with a homologous disruption of the gene encoding IL-6 are found to be resistant to MOG-induced EAE as evidenced by absence of clinical symptoms, minimal infiltration of CD3⁺ T cells and monocytes into the CNS and lack of demyelination. The failure to induce EAE in IL-6^{− / −} mice is not due to the absence of priming, since lymphocytes of immunized IL-6^{− / −} mice proliferate in response to MOG and produce pro-inflammatory cytokines including IL-2 and IFN-γ. However, in MOG-immunized IL-6^{− / −} mice, serum anti-MOG antibody titers were found to be drastically reduced. This observation is unlikely to be responsible for resistance to EAE, because B cell-deficient (μMT) mice proved to be fully susceptible to the disease. A striking difference between MOG-immunized wild-type (wt) and IL-6^{− / −} mice was the expression of endothelial VCAM-1 and ICAM-1, which were dramatically up-regulated in the CNS in wt but not in IL-6^{− / −} mice. Taking into account recent studies on the role of VCAM-1 in the entry of Th1 cells into the CNS, the absence of VCAM-1 on endothelial cells in IL-6^{− / −} mice may explain their resistance to EAE.     

A synthetic hydroxypropenone inhibits nitric oxide,prostaglandin E2, and proinflammatory cytokine synthesis

HMP [3-(2-hydroxyphenyl)-1-(5-methyl-furan-2-y-l) propenone] was evaluated for its ability to inhibit the synthesis of major proinflammatory mediators and cytokines in interferon-γ (IFN-γ)- and lipopolysaccharide (LPS)-induced RAW 264.7 cells and phorbol myristate acetate (PMA)-differentiated/LPS-induced U937 cells. HMP suppressed the production of nitric oxide (NO) with significant inhibitory effects at doses as low as 0.78?μM (P?<?0.05). Prostaglandin E2 (PGE2) secretion was also inhibited at doses of 12.5?μM and above (P?<?0.01). The secretion of both TNF-α and IL-6 were only inhibited at the highest dose used (25?μM; P?<?0.001). IL-1β secretion was also inhibited from 12.5?μM onwards (P?<?0.01). This inhibition was demonstrated to be caused by down-regulation of inducible enzymes, inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2), without direct effect upon iNOS or COX-2 enzyme activity. HMP only inhibited iNOS (P?<?0.001) and IL-1β (P?<?0.05) gene expression at the highest tested concentration. HMP did not affect the secretion of chemokines IL-8 and monocyte chemotactic protein-1 (MCP-1) and the anti-inflammatory cytokine IL-10. The most striking effect of HMP was its NO inhibitory activity and therefore we conclude that HMP is a selective inhibitor of iNOS.     

Silica nanoparticles as an enhancer in the IL-1β-induced inflammation cycle of A549 cells

Objective: The industrial production and combustion of coal can produce silica nanoparticles (nano-SiO₂). It enters the human body mainly through the respiratory tract and exerts a toxic effect. However, whether nano-SiO₂ can increase the IL-1β-induced inflammatory expression in A549 cells has not been tested. Therefore, the synergistic toxicity of nano-SiO₂ and IL-1β to A549 was observed in our study.

Materials and methods: We exposed A549 cells to nano-SiO₂ (0, 100, 500, and 1000?μg/ml) for 12 and 24?h. The effect of nano-SiO₂ on the viability of A549 cells was observed by the CCK-8 method. The A549 cells were exposed to nano-SiO₂ (1?mg/mL) and cytokine IL-1β (10?ng/mL) for 4?h, and we detected the expression of IL-1β and IL-6 cytokines by real time quantitative polymerase chain (RT-qPCR) and enzyme linked immunosorbent assay (ELISA). The expression of β-Actin, I-κB, phospho-ERK_1/2 (P-ERK_1/2), total-ERK_1/2 (T-ERK_1/2), phospho-JNK (P-JNK), total-JNK (T-JNK), phospho-P38 (P-P38), and total-P38 (T-P38) in A549 cells was detected by the Western Blot method.

Results: The nano-SiO₂ treatment resulted in a time-dependent decrease in the viability of A549 cells. The synergistic effect of nano-SiO₂ and IL-1β was observed on the new production of IL-1β and IL-6 in A549 cells. The Western blot results showed that nano-SiO₂ can increase the expression of IL-1β and IL-6 by promoting the phosphorylation of ERK_1/2 and elevating the phosphorylation of I-κB by IL-1β. IL-1β and IL-6 were induced by nano-SiO₂, and the IL-1β treatment with 20?μM of I-κBα phosphorylation inhibitor (PD98059) and 20?μM of ERK_1/2 inhibitor (BAY11-7082) for 1?h was significantly lower than that of the control group in A549 cells.

Discussion and conclusion: These results indicated that nano-SiO₂ had a toxic effect on A549 cells, and this effect could increase IL-1β on the A549 cell-induced inflammatory response. The results suggested that the release of IL-1β and IL-6 in A549 was enhanced by the synergistic IL-1β-induced phosphorylation of ERK_1/2 and I-κB. This process is similar to a snowball, and it is possible that IL-1β is continuously produced and repeatedly superimposed in A549 cells to produce an inflammatory effect; then, a vicious circle occurs, and an inflammatory storm is accelerated.     

Marine peptides as immunomodulators: Californiconus californicus-derived synthetic conotoxins induce IL-10 production by regulatory T cells (CD4+Foxp3+)

Abstract

Context: CD4⁺ T lymphocytes are able to differentiate into distinct subtypes according to several immunological scenarios, including T helper (Th)1, Th2, Th17 and regulatory T (Treg) cells. CD4⁺ T cells are phenotypically flexible and have specific ion channels, such as the nicotinic acetylcholine receptors (nAChR) that could be modulated by peptides produced by marine snails, known as conotoxins. Their effect on T lymphocytes has not been explored and emerging evidence suggests that these peptides may have immunomodulatory activities.

Objective: This study investigated the effect of two Californiconus californicus-derived synthetic conotoxins on the proliferation and differentiation of T lymphocyte subpopulations Th1, Th2, Th17 and Treg.

Methods: Cells from lymph nodes of BALB/c mice were cultured in the presence of conotoxins cal14.1b and cal14.2c (5.5?μM), during 96?h. Cell proliferation and intracellular cytokine production (IFN-γ, IL-4, IL-17 and IL-10) were analyzed by flow cytometry.

Results and Discussion: cal14.1b and cal14.2c increased intracellular IL-10 production in Treg (CD3⁺CD4⁺Foxp3⁺) cells and decreased intracellular IL-17 production (CD3⁺CD4⁺) after 72?h of culture. Conotoxins did not show any effect on T cell proliferation nor Th1/Th2 balance.

Conclusion: These results suggest that synthetic conotoxins exert immunomodulatory activity, especially by regulating specific functions on T lymphocytes.     

IL-10 up-regulates nitric oxide (NO) synthesis by lipopolysaccharide (LPS)-activated macrophages: improved control of Trypanosoma cruzi infection

We examined the effects of IL-10 on tumour necrosis factor-alpha (TNF-α) and NO production by LPS-activated macrophages and on the ability of these cells to control Trypanosoma cruzi infection. We first observed that the addition of rIL-10 to macrophages of the J774 cell line decreased their synthesis of TNF-α but increased their release of NO in a dose-dependent manner. In parallel, treatment of J774 cells with rIL-10 resulted in a better control of T. cruzi infection involving up-regulation of NO synthesis, as it was not observed in presence of N-nitro- L -arginine methyl ester ( L -NAME), a competitive inhibitor of NO synthase. The enhancing effect of rIL-10 on NO production was not observed on peritoneal macrophages from wild-type C57Bl/6 mice, but well on macrophages from IL-10 knock-out mice. The control of NO production by endogenous IL-10 was confirmed by the demonstration that neutralization of IL-10 secreted by LPS-activated macrophages from wild-type mice inhibited their production of NO and, in parallel, their ability to control T. cruzi infection. Taken together, these data demonstrate that both exogenous and endogenous IL-10 up-regulate the production of NO by LPS-activated macrophages and improve thereby their ability to clear T. cruzi infection.     

Increased adhesion of human monocytes to IL-4-stimulated human venous endothelial cells via CD11/CD18, and very late antigen-4 (VLA-4)/vascular cell adhesion molecule-1 (VCAM-1)-dependent mechanisms.

Expression of adhesion molecules on endothelial cells (EC) can be up-regulated or induced by cytokines. The aim of the present study was to investigate the effect of IL-4 on both the expression of adhesion molecules on EC and monocyte adhesion to EC. Flow cytometric analysis showed that VCAM-1 expression on EC was up-regulated after stimulation with IL-4 for 24 h, whereas the expression of E-selectin (formerly called endothelial leucocyte adhesion molecule-1 (ELAM-1)) was not enhanced, and that of intercellular adhesion molecule-1 (ICAM-1) only slightly. The adhesion of monocytes to EC increased to maximum values upon stimulation of EC with IL-4 for 24 h. Coating of monocytes with MoAb against the integrin beta 2-subunit (CD18) significantly inhibited their adhesion to IL-4-stimulated EC; maximal inhibition was found when monocytes were coated with anti-CD18 MoAb in combination with MoAb against CD49d (the alpha-chain of VLA-4), whereas no inhibition was found when monocytes were coated only with MoAb against CD49d. Monocyte adhesion was not significantly inhibited when IL-4-stimulated EC were coated with MoAbs against ICAM-1 or VCAM-1 alone or in combination. Adhesion of monocytes was inhibited to a greater extent when in addition to coating of monocytes with MoAb against CD18 the EC were coated with MoAb against VCAM-1. From these results we conclude that monocytes bind to IL-4-stimulated EC via interaction of CD11/CD18 molecules on the monocytes with an as yet unknown endothelial ligand, and interaction of VLA-4 on monocytes with VCAM-1 on EC.     

Histamine induces IL-6 production by human endothelial cells.

Histamine is one of the major mediators implicated in the physiopathology of allergy. On vascular endothelium, histamine mainly induces early effects: an increase in vasopermeability leading to oedema, a release of lipid mediators and a transient expression of P-selectin. The aim of this study was to evaluate the effects of histamine on adhesion molecule expression and IL-6 production by human endothelial cells. Histamine did not modulate the expression of intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) and E-selectin, but induced a transient expression of P-selectin as previously reported. In addition, histamine increased in a dose- (from 10(-5) to 10(-3) M) and time- (from 4 h to 24 h) dependent fashion the IL-6 synthesis by endothelial cells. Tumour necrosis factor-alpha (TNF-alpha)-induced IL-6 production was also potentiated in a dose-dependent manner by histamine, without modification of the time course of IL-6 secretion. Moreover, this increase of IL-6 production induced by histamine was inhibited in a dose-dependent manner by H1 and H2 histamine receptor antagonists (50% inhibition of IL-6 production at 5 x 10(-4) M and 4 x 10(-5) M, respectively). So, histamine induces, besides already well known effects, a late stimulation of endothelial cells, i.e. the production of IL-6.     

Paeoniflorin suppresses IL-33 production by macrophages

Abstract

Objective: Interleukin (IL)-33 has been attracting more and more attention as a new member of theIL-1 cytokine family in recent years. However, the underlying mechanisms referred to the regulation of endogenous IL-33 production are not fully illustrated. Paeoniflorin (PF) has been reported to possess multiple pharmacological activities, including anti-inflammation and anti-allergy. In this study, we aimed to investigate the effect of PF on IL-33 production by macrophages and explore the underlying mechanisms.

Methods: In vivo, IL-33 production in mice after lipopolysaccharide (LPS) injection together with PF application was detected by enzyme-linked immunosorbent assay (ELISA). In vitro, MTT, Real-time PCR, ELISA, Calcium (Ca²⁺) imaging and Western blot were used to assess the cytotoxicity of PF, IL-33 expression at mRNA and protein levels, Ca²⁺ influx, protein kinase C (PKC) activity, nuclear factor-kappa B (NF-κB), and mitogen-activated protein kinase (MAPK) activation in LPS-stimulated RAW264.7 macrophages with PF administration.

Results: Our results indicated that PF (5 and 25?mg/kg) significantly reduced the production of TNF-a, IL-1β, and IL-33 in the peritoneal exudate of LPS-treated mice. In vitro assay, upregulation of PF concentration (≥ 20?μM) showed an increased cytotoxicity in RAW264.7 cells during the 24-h cell culture. PF (10?μM) inhibited IL-33 production, Ca²⁺ influx, PKC activity, NF-κB (p65) activation, and P38MAPK phosphorylation in LPS-treated macrophages. Notably, NF-κB inhibitor (BAY 11-7085), P38MAPK inhibitor (SB203580), and Ca²⁺ blocker (NiCl₂) also curbed LPS-induced IL-33 production, respectively.

Conclusions: PF suppresses IL-33 production by macrophages via inhibiting NF-κB and P38MAPK activation associated with the regulation of Ca²⁺ mobilization.     

Role of Tyrosine Kinase Enzymes in TNF-α and IL-1 Induced Expression of ICAM-1 and VCAM-1 on Human Umbilical Vein Endothelial Cells

The aim of this study was to analyse the potential roles of protein kinase enzymes in tumour necrosis factor-α (TNF-α) and interleukin-1 (IL-1) induced expression of the adhesion molecules intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) on human umbilical vein endothelial cells (HUVEC). The authors observed a marked increase in ICAM-1 and VCAM-1 expression on HUVEC stimulated for 24 h by TNF-α (10 ng/ml) or IL-1 (20 ng/ml). Pre-treatment of HUVEC for 30 min with protein tyrosine kinase (PTK) inhibitors genistein and herbimycin A (10 μg/ml and 0.5 μg/ml, respectively) before stimulation with IL-1 did not affect the expression of these molecules. Similar results were observed with respect to VCAM-1 expression on HUVEC stimulated by TNF-α. In contrast, pre-incubation of HUVEC with PTK inhibitors prior to the addition of TNF-α significantly enhanced subsequent expression of ICAM-1, although spontaneous expression of ICAM-1 on unstimulated HUVEC was unaffected. Western blot analysis demonstrated a significant increase in phosphorylated tyrosine protein levels in HUVEC stimulated by TNF-α , and significantly lower levels of these proteins in TNF-α stimulated HUVEC pre-treated with PTK inhibitors. These results demonstrate that IL-1 induced ICAM-1 and VCAM-1 expression does not result from activation of PTK-dependent pathways. In the case of TNF-α induced responses, the selective co-stimulatory effect of this cytokine in combination with PTK inhibitors on ICAM-1 expression suggests a complicated intracellular pathway of TNF-α induced ICAM-1 expression, possibly involving down-modulation of increases in ICAM-1 by PTK enzymes.     

Detection of IL-5 and IL-1 receptor antagonist in bronchoalveolar lavage fluid in acute eosinophilic pneumonia

BACKGROUND: Acute eosinophilic pneumonia is an idiopathic cause of respiratory failure, characterized by very high numbers of alveolar eosinophils without significant blood eosinophilia. OBJECTIVE: The purpose of this study was to determine which cytokines are associated with acute eosinophilic pneumonia. METHODS: Soluble IL-1 type II receptor and the cytokines IL-1β, IL-1ra, IL-3, IL-5, granulocyte-macrophage colony-stimulating factor, and tumor necrosis factor-α were measured in serum and in bronchoalveolar lavage fluid from two patients with acute eosinophilic pneumonia during both acute and convalescent phases. RESULTS: Compared with patients with adult respiratory distress syndrome, the patients with acute eosinophilic pneumonia had high bronchoalveolar lavage fluid levels of IL-5, IL-1ra, and soluble type II IL-1 receptor but not IL-1β, tumor necrosis factor-α>, IL-3, or granulocyte-macrophage colony-stimulating factor. Bronchoalveolar lavage fluid levels of IL-5 and IL-1ra fell after resolution of symptoms. In the serum of patients with acute eosinophilic pneumonia, IL-5 was not detectable, and IL-1ra was initially high but fell after corticosteroid treatment. CONCLUSION: Acute eosinophilic pneumonia is characterized by locally high levels of IL-5, IL-1ra, and soluble type II IL-1 receptor in the alveolar space. (J ALLERGY CLIN IMMUNOL 1996;97:1366-74.)     

生物素-链霉亲和素系统在检测人PBMC IL-2R_α中的应用

本文报道用自制生物素-链霉亲和素系统配以抗IL-2R_aMcAb,建立检测人PBMCIL-2R_a的方法,并对本法中的关键条件进行探索,确定生物素化二抗和链霉亲和素的最适浓度以及多种诱导物的最适诱导时间和浓度。用本法对比检测了健康献血者和肿瘤患者PBMC IL-2R_a的表达水平,表明未经诱导的肿瘤患者PBMC IL-2R_a百分率显著高于正常人(P<0.01)。     

Effects of combination of Cryptococcus gattii and IFN-γ, IL-4 or IL-27 on human bronchial epithelial cells

BackgroundAirway epithelial cells are crucial for the establishment of cryptococcosis. In experimental cryptococcosis, the Th2 immune response is associated with host susceptibility, while Th1 cells are associated with protection. The absence of IL-27 receptor alpha in mice favor the increase Cryptococcus neoformans burden in the lung. Here, we evaluated the effects of the combination of IL-4, IFN-γ or IL-27 with C. gattii on human bronchial epithelial cells (BEAS-2B).MethodsBEAS-2B were stimulated with IL-4, IFN-γ or IL-27 (100 ng/mL) and/or live yeast forms of C. gattii (multiplicities of infection (MOI) of 1–100) and vice-versa, as well as with heat-killed cells of C. gattii for 24 h.ResultsNone of the C. gattii MOIs had cytotoxic effects on BEAS-2B when compared to control. The cells stimulated by cytokines (IL-4, IFN-γ or IL-27) followed by live yeast forms of C. gattii (MOI of 100) infection and vice-versa demonstrated a reduction in IL-6, IL-8 and/or CCL2 production and activation of STAT6 (induced by IL-4) and STAT1 (induced by IL-27 or IFN-γ) when compared to cells stimulated with C. gattii, IL-4, IFN-γ or IL-27. In the combination of cytokines and heat-killed cells of C. gattii, no inhibition of these inflammatory parameters was observed. The growth of C. gattii was increased while the phagocytosis of live yeast forms of C. gattii in the BEAS-2B were reduced in the presence of IL-4, IFN-γ or IL-27.ConclusionThe association of live yeast forms, but not heat-killed yeast forms, of C. gattii with IL-4, IFN-γ or IL-27 induced an anti-inflammatory effect.     

In vitro activation of murine peritoneal macrophages by recombinant YopJ: production of nitric oxide, proinflammatory cytokines and chemokines

Recently it was reported that 3 μg/ml of recombinant YopJ induced apoptosis in murine peritoneal macrophages in vitro. However, in this study, we report the activation of murine peritoneal macrophages in vitro on treatment with sub-apoptotic dose of recombinant YopJ protein (1 μg/ml). The activation involves enhanced production of nitric oxide (NO), tumor necrosis factor-α (TNF-α), IL-12, and IL-6. Production of NO and IL-6 was found to peak at 24 h of rYopJ treatment, whereas IL-12 and IFN-γ production peaked at 18 h of rYopJ treatment. Increased mRNAs expression of nitric oxide, IL-12, IL-6 and IFN-γ molecules, was also observed in rYopJ-treated macrophages by RT-PCR. rYopJ induced the enhanced activity of protein tyrosine kinases which was inhibited by pharmacological inhibitor genestein, wortmanin and H-7 suggesting the role of tyrosine kinases, PI3K and PKC in the above process. rYopJ also induced increased enhanced production chemokines MIP-1α, MCP-1, and RANTES in macrophages. Significantly, increased expression of TLR-2, TLR-6, MyD 88 and IRAK-1 was also observed by immunoblotting in rYopJ-treated macrophages. rYopJ induced production of NO, TNF-α and IL-6 was significantly inhibited in macrophages pretreated with pharmacological inhibitor wortmanin, genestein and H-7 demonstrating the probable involvement of protein tyrosine kinases in the above process.     

Carcinoembryonic antigen promotes tumor cell survival in liver through an IL-10-dependent pathway

Most circulating tumor cells die within 24 h of entering the hepatic microvasculature because their arrest initiates an ischemia-reperfusion (I/R) injury that is cytotoxic. Human colorectal carcinomas (CRC) produce the glycoprotein Carcinoembryonic Antigen (CEA) that increases experimental liver metastasis in nude mice. Since CEA induces release of IL-6 and IL-10, we hypothesized that CEA inhibits the I/R injury through a Kupffer cell-mediated cytokine-dependent pathway. We assessed cytokine effects in CRC co-cultured with liver and in vivo. Human CRC prelabeled with fluorescent dyes were incubated with a reoxygenated suspension of ischemic nude mouse liver fragments in a bioreactor. CEA, rhIL-6 or rhIL-10 were either administered to the donor mice prior to hepatic ischemia or during co-culture. Liver donors were athymic nude or iNOS, IL-6 or IL-10 knock out mice. Ischemic-reoxygenated liver kills Clone A CRC through production of nitric oxide (NO) and superoxide anion. Treatment of liver donors with CEA prior to hepatic ischemia inhibited this in vitro cytotoxicity through an IL-10 and Kupffer cell dependent pathway that inhibited NF-κB activation, NO production and iNOS upregulation. IL-10 but not IL-6 enhanced CRC survival in nude mouse liver in vivo. Thus, CEA enhanced metastasis by inducing IL-10 to inhibit iNOS upregulation in host liver.This revised version was published online in August 2005 with a corrected cover date.     

Orientia tsutsugamushi induced endothelial cell activation via the NOD1-IL-32 pathway

Orientia tsutsugamushi (OT), the causative agent of scrub typhus, is an obligate intracellular bacterium. In order to verify the inflammatory responses involved in the pathogenesis of scrub typhus, we assessed the cytokine profile of the human endothelial cell line, ECV304, after OT infection. We noted that CCL5, CCL17, IL-1α, IL-6, IL-8, IL-10, IL-15, TNF-α and TNF-β were strongly induced in response to OT. Additionally, IL-32, the candidate modulator for the induction of IL-6 and IL-8, was increased significantly with OT infection and these increases coincided with NOD1 pathway activation. Thus, we hypothesized that NOD1 pathway and IL-32 might act on cytokine release in endothelial cells as a modulator of the inflammation caused by OT infection. NOD1 siRNA resulted in a reduction in IL-32 levels, and also reduced IL-1β, IL-6, IL-8, and ICAM-1 expression in OT-infected ECV304 cells. These changes in IL-1β, IL-6, IL-8, and ICAM-1 induced by NOD1 knockdown were reversed as the result of IL-32 treatment. This indicated that OT infection activated the NOD1 pathway followed by IL-32 secretion, thus resulting in the production and expression of IL-1β, IL-6, IL-8, and ICAM-1. Therefore, IL-32 might perform a role upstream of the inflammatory reaction in endothelial cells of OT infection.