Mechanisms of endotoxin tolerance in human intestinal microvascular endothelial cells

Lipopolysaccharide (endotoxin) tolerance is well described in monocytes and macrophages, but is less well characterized in endothelial cells. Because intestinal microvascular endothelial cells exhibit a strong immune response to LPS challenge and play a critical regulatory role in gut inflammation, we sought to characterize the activation response of these cells to repeated LPS exposure. Primary cultures of human intestinal microvascular endothelial cells (HIMEC) were stimulated with LPS over 6-60 h and activation was assessed using U937 leukocyte adhesion, expression of E-selectin, ICAM-1, VCAM-1, IL-6, IL-8, manganese superoxide dismutase, HLA-DR, and CD86. Effect of repeat LPS stimulation on HIMEC NF-kappaB and mitogen-activated protein kinase (MAPK) activation, generation of superoxide anion, and Toll-like receptor 4 expression was characterized. LPS pretreatment of HIMEC for 24-48 h significantly decreased leukocyte adhesion after subsequent LPS stimulation. LPS pretreatment inhibited expression of E-selectin, VCAM-1, IL-6, and CD86, while ICAM-1, IL-8, and HLA-DR were not altered. Manganese superoxide dismutase expression increased with repeated LPS stimulation, with a reduction in intracellular superoxide. NF-kappaB activation was transiently inhibited by LPS pretreatment for 6 h, but not at later time points. In contrast, p44/42 MAPK, p38 MAPK, and c-Jun N-terminal kinase activation demonstrated inhibition by LPS pretreatment 24 or 48 h prior. Toll-like receptor 4 expression on HIMEC was not altered by LPS. HIMEC exhibit endotoxin tolerance after repeat LPS exposure in vitro, characterized by diminished activation and intracellular superoxide anion concentration, and reduced leukocyte adhesion. HIMEC possess specific mechanisms of immunoregulatory hyporesponsiveness to repeated LPS exposure.     

Cytokine-induced upregulation of NF-kappaB, IL-8, and ICAM-1 is dependent on colonic cell polarity: implication for PKCdelta

As described for a long time, carcinoma-derived Caco-2 cells form a polarized epithelium in culture, whereas HT29-D4 cells are nonpolarized and undifferentiated but can form a polarized monolayer when cultured in a galactose-supplemented medium. Using NF-kappaB translocation and IL-8 and ICAM-1 gene activation as an index, we have studied the relationship between the differentiation state and the cell response to cytokines. We found that differentiated Caco-2 and HT29-D4 cells were responsive to both cytokines TNFalpha- and IL-1beta-mediated activation of NF-kappaB but that undifferentiated HT29-D4 cells were unresponsive to IL-1beta. However, the expression of endogenous ICAM-1 and IL-8 genes was upregulated by these cytokines in either cell lines differentiated or not. Upregulation of ICAM-1 gene occurred when IL-1beta or TNFalpha was added to the basal, but not apical surface of the differentiated epithelia. Finally, it appeared that in polarized HT29-D4 cells, the IL-1beta-induced translocation of NF-kappaB was connected to PKCdelta translocation.     

Syntenin negatively regulates TRAF6-mediated IL-1R/TLR4 signaling

Toll-like receptors are involved in host defense against invading pathogens. The two members of this superfamily, IL-1R and TLR4, activate overlapping NF-kappaB activate signaling pathway mediated by TRAF6. In this study, we identified syntenin as a negative regulator of IL-1R and TLR4 mediated NF-kappaB activation. Overexpressed syntenin inhibited IL-1- or LPS-, but not TNF- induced NF-kappaB activation and IL-8 mRNA expression in a dose dependent manner. Syntenin specifically interacted with TRAF6 in human 293 cells, and inhibited TRAF6 induced NF-kappaB and AP-1 activation. Syntenin also associated with TRAF6 under physiological condition, and dissociated from TRAF6 upon IL-1 stimulation. This might be due to a competition between syntenin and IRAK1, as overexpression of IRAK1 disrupted the interaction of syntenin with TRAF6, and rescued syntenin induced reduction of TRAF6 ubiquitination. Moreover, knockdown of syntenin potentiated IL-1- or LPS- triggered NF-kappaB activation and IL-8 mRNA expression. These findings suggest that syntenin is a physiological suppressor of TRAF6 and plays an inhibitory role in IL-1R- and TLR4- mediated NF-kappaB activation pathways.     

TLR4 on follicular dendritic cells: an activation pathway that promotes accessory activity

Microbial molecular patterns engage TLRs and activate dendritic cells and other accessory cells. Follicular dendritic cells (FDCs) exist in resting and activated states, but are activated in germinal centers, where they provide accessory function. We reasoned that FDCs might express TLRs and that engagement might activate FDCs by up-regulating molecules important for accessory activity. To test this hypothesis, TLR4 expression on FDCs was studied in situ with immunohistochemistry, followed by flow cytometry and RT-PCR analysis. TLR4 was expressed on FDC reticula in situ, and flow cytometry indicated that TLR4 was expressed on surface membranes and TLR4 message was readily apparent in FDCs by RT-PCR. Injecting mice or treating purified FDCs with LPS up-regulated molecules important for accessory activity including, FDC-Fc gammaRIIB, FDC-ICAM-1, and FDC-VCAM-1. Treatment of purified FDCs with LPS also induced intracellular phospho-IkappaB-alpha, indicating NF-kappaB activation, and that correlated with increased Fc gammaRIIB, ICAM-1, and VCAM-1. FDCs in C3H/HeJ mice were not activated with LPS even when mice were reconstituted with C3H/HeN leukocytes, suggesting that engagement of FDC-TLR4 is necessary for activation. Moreover, activated FDCs exhibited increased accessory activity in anti-OVA recall responses in vitro, and the FDC number could be reduced 4-fold if they were activated. In short, we report expression of TLR4 on FDCs for the first time and that engagement of FDC-TLR4 activated NF-kappaB, up-regulated expression of molecules important in FDC accessory function, including Fc gammaRIIB, ICAM-1, and VCAM-1, as well as FDC accessory activity in promoting recall IgG responses.     

Neutralization of IL-18 attenuates lipopolysaccharide-induced myocardial dysfunction

Tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta) have been implicated in cardiac dysfunction during endotoxemia. Because IL-18 is a proinflammatory cytokine known to mediate the production of TNF-alpha and IL-1beta and to induce the expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1), we hypothesized that neutralization of IL-18 would attenuate lipopolysaccharide (LPS)-induced cardiac dysfunction. Mice (C57BL/6) were injected with LPS (0.5 mg/kg ip) or vehicle (normal saline), and left ventricular developed pressure (LVDP) was determined by the Langendorff technique. LVDP was depressed by 38% at 6 h after LPS. LPS-induced myocardial dysfunction was associated with increased myocardial levels of TNF-alpha and IL-1beta as well as increased expression of ICAM-1/VCAM-1. Pretreatment with neutralizing anti-mouse IL-18 antibody attenuated LPS-induced myocardial dysfunction (by 92%) and was associated with reduced myocardial IL-1beta production (65% reduction) and ICAM-1/VCAM-1 expression (50% and 35% reduction, respectively). However, myocardial TNF-alpha levels were not influenced by neutralization of IL-18. In conclusion, neutralization of IL-18 protects against LPS-induced myocardial dysfunction. IL-18 may mediate endotoxemic myocardial dysfunction through induction of and/or synergy with IL-1beta, ICAM-1, and VCAM-1.     

TLR4-independent and PKR-dependent interleukin 1 receptor antagonist expression upon LPS stimulation

Dendritic cells (DCs) induce innate immune responses by recognizing bacterial LPS through TLR4 receptor complexes. In this study, we compared gene expression profiles of TLR4 knockout (TLR4^neg) DCs and wild type (TLR4^pos) DCs after stimulating with LPS. We found that the expression of various inflammatory genes by LPS were TLR4-independent. Among them, interleukin 1 receptor antagonist (IL-1rn) was of particular interest since IL-1rn is a potent natural inhibitor of proinflammatory IL-1. Using RT-PCR, real-time PCR, immunoblotting and ELISA, we demonstrated that IL-1rn was induced by DCs stimulated with LPS in the absence of TLR4. 2-Aminopurine, a pharmacological PKR inhibitor, completely abrogated LPS-induced expression of IL-1rn in TLR4^neg DCs, suggesting that LPS-induced TLR4-independent expression of IL-1rn might be mediated by PKR pathways. Considering that IL-1rn is a physiological inhibitor of IL-1, TLR4-independent and PKR-dependent pathways might be crucial in counter-balancing proinflammatory effector functions of DCs resulted from TLR4-dependent activation by LPS.     

Effects of tumor necrosis factor, lipopolysaccharide, and IL-4 on the expression of vascular cell adhesion molecule-1 in vivo. Correlation with CD3+ T cell infiltration.

We have injected human TNF, LPS, and IL-4 into the skin of baboons to examine regulation of endothelial leukocyte adhesion molecules (ELAM) in vivo and to determine which endothelial adhesion molecules correlate temporally and spatially with cytokine-induced T cell infiltration. The expression of adhesion molecules ELAM-1 (E-selectin), VCAM-1, and ICAM-1 (CD54) were quantified by immunocytochemical staining of frozen sections obtained from skin biopsies; T cell infiltration was measured by immunocytochemical staining of CD3+ T cells in serial sections. We found that injection of TNF causes late (24 to 48 h) T cell infiltration whereas injection of LPS, in doses that do not cause tissue necrosis, does not. The ability of TNF (but not LPS) to recruit T cells correlates with the ability of TNF to cause sustained endothelial cell adhesion molecule expression. Expression of VCAM-1 on post-capillary venules showed the highest degree of spatial localization with infiltrates. IL-4, although not proinflammatory by itself, can cause T cell infiltration in combination with an ineffective dose of TNF. The ability of IL-4 to augment TNF-induced inflammation best correlates with the ability of the combination of IL-4 and TNF to increase endothelial VCAM-1 expression. In contrast, IL-4 does not promote T cell infiltration or endothelial VCAM-1 expression in combination with LPS. In cytokine-injected tissues, VCAM-1 is also expressed on connective tissue cells other than endothelium, including smooth muscle and perineural cells, where it is induced by cytokines in parallel with endothelial VCAM-1. Overall, our data support the hypothesis that endothelial VCAM-1 expression contributes to T cell extravasation at sites of inflammation. Furthermore, we find that IL-4, a product a Ag-activated T cells, can interact with TNF to selectively promote VCAM-1 expression and the development of T cell-rich infiltrates, characteristic of Ag-induced inflammatory reactions.     

Bcl10 mediates LPS-induced activation of NF-kappaB and IL-8 in human intestinal epithelial cells

Lipopolysaccharide (LPS) is recognized as an inducer of the inflammatory response associated with gram-negative sepsis and systemic inflammatory response syndrome. LPS induction proceeds through Toll-like receptor (TLR) in immune cells and intestinal epithelial cells (IEC). This report presents the first identification of Bcl10 (B-cell CLL/lymphoma 10) as a mediator of the LPS-induced activation of IL-8 in human IEC. Bcl10 is a caspase-recruitment domain-containing protein, associated with constitutive activation of NF-kappaB in MALT (mucosa-associated lymphoid tissue) lymphomas. The normal human IEC line NCM460, normal primary human colonocytes, and ex vivo human colonic tissue were exposed to 10 ng/ml of LPS for 2-6 h. Effects on Bcl10, phospho-IkappaBalpha, NF-kappaB, and IL-8 were determined by Western blot, ELISA, immunohistochemistry, and confocal microscopy. Effects of Bcl10 silencing by small-interfering RNA (siRNA), TLR4 blocking antibody, TLR4 silencing by siRNA, and an IL-1 receptor-associated kinase (IRAK)-1/4 inhibitor on LPS-induced activation were examined. Following Bcl10 silencing, LPS-induced increases in NF-kappaB, IkappaBalpha, and IL-8 were significantly reduced (P < 0.001). Increasing concentrations of LPS were associated with higher concentrations of Bcl10 protein when quantified by ELISA, and the association between LPS exposure and increased Bcl10 was also demonstrated by Western blot, immunohistochemistry, and confocal microscopy. Exposure to TLR4 antibody, TLR4 siRNA, or an IRAK-1/4 inhibitor eliminated the LPS-induced increases in Bcl10, NF-kappaB, and IL-8. Identification of Bcl10 as a mediator of LPS-induced activation of NF-kappaB and IL-8 in normal human IEC provides new insight into mechanisms of epithelial inflammation and new opportunities for therapeutic intervention.     

Effects of TNF-alpha on leukocyte adhesion molecule expressions in cultured human lymphatic endothelium.

TNF-alpha alters leukocyte adhesion molecule expression of cultured endothelial cells like human umbilical vein endothelial cells (HUVEC). This study was designed to investigate the changes in vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), and platelet endothelial cell adhesion molecule-1 (PECAM-1) expression with TNF-alpha stimulation in cultured human neonatal dermal lymphatic endothelial cells (HNDLEC). The real-time quantitative PCR analysis on HNDLEC showed that TNF-alpha treatment leads to increases of VCAM-1 and ICAM-1 mRNAs to the 10.8- and 48.2-fold levels of untreated cells and leads to a reduction of PECAM-1 mRNA to the 0.42-fold level of untreated cells. Western blot and immunohistochemical analysis showed that TNF-alpha leads to VCAM-1 and ICAM-1 expressions that were inhibited by antiserum to human TNF receptor or by AP-1 inhibitor nobiletin. In flow cytometry analysis, the number of VCAM-1- and ICAM-1-positive cells increased, and PECAM-1-positive cells decreased with TNF-alpha treatment. Regarding protein amounts produced in cells and amounts expressed on the cell surface, VCAM-1 and ICAM-1 increased in HNDLEC and HUVEC, and PECAM-1 decreased in HNDLEC in a TNF-alpha concentration-dependent manner. VCAM-1, ICAM-1, and PECAM-1 protein amounts in TNF-alpha-stimulated cells were lower in HNDLEC than in HUVEC. This suggests that the lymphatic endothelium has the TNF-alpha-induced signaling pathway, resulting in increased VCAM-1 and ICAM-1 expression to a weaker extent than blood endothelium and PECAM-1 reduction to a stronger extent than blood endothelium.     

Tobacco smoking inhibits expression of proinflammatory cytokines and activation of IL-1R-associated kinase, p38, and NF-kappaB in alveolar macrophages stimulated with TLR2 and TLR4 agonists

Tobacco smoking has been associated with impaired pulmonary functions and increased incidence of infections; however, mechanisms that underlie these phenomena are poorly understood. In this study, we examined whether smokers' alveolar macrophages (AM) exhibit impaired sensing of bacterial components via TLR2 and TLR4 and determined the effect of smoking on expression levels of TLR2, TLR4 and coreceptors, and activation of signaling intermediates. Smokers' AMs exhibited reduced gene expression and secretion of proinflammatory cytokines (TNF-alpha, IL-1beta, IL-6) and chemokines (RANTES and IL-8) upon stimulation with TLR2 and TLR4 agonists, S-[2,3-bis(palmitoyloxy)-(2-RS)-propyl]-N-palmitoyl-(R)-Cys-(S)-Ser-Lys4-OH trihydrochloride (Pam(3)Cys), and LPS, whereas expression of anti-inflammatory cytokines (IL-10 and IL-1 receptor antagonist) was not affected. TLR3 activation with polyinosinic-polycytidylic acid led to comparable or even higher cytokine responses in smokers' AMs, indicating that smoking-induced suppression does not affect all TLRs. Comparable expression of cytokines and chemokines was detected in PBMC and purified monocytes obtained from smokers and nonsmokers, demonstrating that the suppressive effect of smoking is restricted to the lung. TLR2/4-inducible IL-1R-associated kinase-1 (IRAK-1) and p38 phosphorylation and NF-kappaB activation was suppressed in smokers' AMs, whereas TLR2, TLR4, CD14, MD-2 mRNA levels, and TLR4 protein expression were not altered. These data suggest that changes in expression and/or activities of signaling intermediates at the postreceptor level account for smoking-induced immunosuppression. Thus, exposure of AMs to tobacco smoke induces a hyporesponsive state similar to endotoxin tolerance as manifested by inhibited TLR2/4-induced expression of proinflammatory cytokines, chemokines, and impaired activation of IRAK-1, p38, and NF-kappaB, resulting in suppressed expression of proinflammatory mediators.     

SIGIRR promotes resistance against Pseudomonas aeruginosa keratitis by down-regulating type-1 immunity and IL-1R1 and TLR4 signaling

Pseudomonas aeruginosa keratitis destroys the cornea in susceptible Th1 responder C57BL/6 (B6), but not resistant Th2 responder (BALB/c) mice. To determine whether single Ig IL-1R-related molecule (SIGIRR) played a role in resistance, mRNA and protein expression levels were tested. Both were constitutively expressed in the cornea of the two mouse groups. A disparate mRNA and protein expression pattern was detected in the cornea of BALB/c vs B6 mice after infection. SIGIRR protein decreased significantly in BALB/c over B6 mice at 1 day postinfection. Thus, BALB/c mice were injected with an anti-SIGIRR Ab or IgG control. Anti-SIGIRR Ab over control-treated mice showed increased corneal opacity, stromal damage, and bacterial load. Corneal mRNA levels for IL-1beta, MIP-2, IL-1R1, TLR4, IL-18, and IFN-gamma and protein levels for IL-1beta and MIP-2 also were significantly up-regulated in anti-SIGIRR Ab over control mice, while no changes in polymorphonuclear cell number, IL-4, or IL-10 mRNA expression were detected. To further define the role of SIGIRR, RAW264.7 macrophage-like cells were transiently transfected with SIGIRR and stimulated with heat-killed P. aeruginosa or LPS. SIGIRR transfection significantly decreased mRNA levels for IL-1R1, TLR4, and type 1 immune response-associated cytokines (IL-12, IL-18, and IFN-gamma) as well as proinflammatory cytokines IL-1beta and MIP-2 protein expression. SIGIRR also negatively regulated IL-1 and LPS, but not poly(I:C)-mediated signaling and NF-kappaB activation. These data provide evidence that SIGIRR is critical in resistance to P. aeruginosa corneal infection by down-regulating type 1 immunity, and that it negatively regulates IL-1 and TLR4 signaling.     

Nuclear translocation of p65 NF-kappaB is sufficient for VCAM-1, but not ICAM-1, expression in TNF-stimulated smooth muscle cells: Differential requirement for PARP-1 expression and interaction

Although nuclear translocation of NF-kappaB and subsequent binding to promoters of ICAM-1 and VCAM-1 have been shown to be decisive for their expression, a number of discrepancies in the expression patterns of these adhesion molecules have been reported in both cell culture systems and disease settings, including atherosclerosis, asthma, and autoimmune diseases. Here we show that while p65 NF-kappaB nuclear translocation in TNF-treated smooth muscle cells (SMCs) was sufficient for the expression of VCAM-1, expression of ICAM-1 showed a critical requirement for PARP-1. I-kappaBalpha phosphorylation and subsequent degradation were virtually identical in both TNF-treated wild-type and PARP-1-/- SMCs. VCAM-1 expression in TNF-treated PARP-1-/- SMCs was completely inhibited by the NF-kappaB inhibitor, pyrrolidine dithiocarbamate, confirming that VCAM-1 expression was indeed NF-kappaB-dependent. The expression of both VCAM-1 and ICAM-1 was associated with a transient interaction between PARP-1 and p65 NF-kappaB when examined in the fibroblastic cell line, COS-7, and in the airway epithelial cell line, A549. Such interactions were confirmed using florescence resonance energy transfer analysis. Protein acetylation activity, mediated by p300/CBP, was required for both VCAM-1 and ICAM-1 expression in TNF-treated SMCs; however, the interaction of PARP-1 with p300/CBP was dispensable for VCAM-1 expression. These findings indicate that p65 NF-kappaB nuclear translocation may be sufficient for certain genes (e.g., VCAM-1) while insufficient for others (e.g., ICAM-1), thus providing a novel insight into the role of NF-kappaB in driving target gene expression. Furthermore, the data suggest a differential requirement for PARP-1 expression in inflammatory processes.     

Involvement of p42/p44 MAPK, p38 MAPK, JNK, and NF-kappaB in IL-1beta-induced VCAM-1 expression in human tracheal smooth muscle cells

Interleukin-1beta (IL-1beta) has been shown to induce the expression of adhesion molecules on airway epithelial and smooth cells and contributes to inflammatory responses. Here, the roles of mitogen-activated protein kinases (MAPKs) and nuclear factor-kappaB (NF-kappaB) pathways for IL-1beta-induced vascular cell adhesion molecule (VCAM)-1 expression were investigated in human tracheal smooth muscle cells (HTSMC). IL-1beta induced expression of VCAM-1 protein and mRNA in a time-dependent manner, which was significantly inhibited by inhibitors of MEK1/2 (U0126 and PD-98059), p38 (SB-202190), and c-Jun NH(2)-terminal kinase (JNK; SP-600125). Consistently, IL-1beta-stimulated phosphorylation of p42/p44 MAPK, p38, and JNK was attenuated by pretreatment with U0126, SB-202190, or SP-600125, respectively. IL-1beta-induced VCAM-1 expression was significantly blocked by the specific NF-kappaB inhibitors helenalin and pyrrolidine dithiocarbamate. As expected, IL-1beta-stimulated translocation of NF-kappaB into the nucleus and degradation of IkappaB-alpha were blocked by helenalin but not by U0126, SB-202190, or SP-600125. Moreover, the resultant enhancement of VCAM-1 expression increased the adhesion of polymorphonuclear cells to a monolayer of HTSMC, which was blocked by pretreatment with helenalin, U0126, SB-202190, or SP-600125 before IL-1beta exposure or by anti-VCAM-1 antibody. Together, these results suggest that in HTSMC, activation of p42/p44 MAPK, p38, JNK, and NF-kappaB pathways is essential for IL-1beta-induced VCAM-1 gene expression. These results provide new insight into the mechanisms of IL-1beta action that cytokines may promote inflammatory responses in airway disease.     

Involvement of p42/p44 MAPK, JNK, and NF-kappaB in IL-1beta-induced ICAM-1 expression in human pulmonary epithelial cells

Interleukin-1beta (IL-1beta) has been shown to induce the expression of intercellular adhesion molecule-1 (ICAM-1) on airway epithelial cells and contributes to inflammatory responses. However, the mechanisms regulating ICAM-1 expression by IL-1beta in human A549 cells was not completely understood. Here, the roles of mitogen-activated protein kinases (MAPKs) and NF-kappaB pathways for IL-1beta-induced ICAM-1 expression were investigated in A549 cells. IL-1beta induced expression of ICAM-1 protein and mRNA in a time- and concentration-dependent manner. The IL-1beta induction of ICAM-1 mRNA and protein were partially inhibited by U0126 and PD98059 (specific inhibitors of MEK1/2) and SP600125 [a specific inhibitor of c-Jun-N-terminal kinase (JNK)]. U0126 was more potent than other inhibitors to attenuate IL-1beta-induced ICAM-1 expression. Consistently, IL-1beta stimulated phosphorylation of p42/p44 MAPK and JNK which was attenuated by pretreatment with U0126 or SP600125, respectively. Moreover, transfection with dominant negative mutants of MEK1/2 (MEK K97R) or ERK2 (ERK2 K52R) also attenuated IL-1beta-induced ICAM-1 expression. The combination of PD98059 and SP600125 displayed an additive effect on IL-1beta-induced ICAM-1 gene expression. IL-1beta-induced ICAM-1 expression was almost completely blocked by a specific NF-kappaB inhibitor helenalin. Consistently, IL-1beta stimulated translocation of NF-kappaB into the nucleus and degradation of IkappaB-alpha which was blocked by helenalin, U0126, or SP600125. Taken together, these results suggest that activation of p42/p44 MAPK and JNK cascades, at least in part, mediated through NF-kappaB pathway is essential for IL-1beta-induced ICAM-1 gene expression in A549 cells. These results provide new insight into the mechanisms of IL-1beta action that cytokines may promote inflammatory responses in the airway disease.