Mitogen-activated protein kinases and NF-kappa B are involved in TNF-alpha responses to group B streptococci

TNF-alpha is a mediator of lethality in experimental infections by group B streptococcus (GBS), an important human pathogen. Little is known of signal transduction pathways involved in GBS-induced TNF-alpha production. Here we investigate the role of mitogen-activated protein kinases (MAPKs) and NF-kappa B in TNF-alpha production by human monocytes stimulated with GBS or LPS, used as a positive control. Western blot analysis of cell lysates indicates that extracellular signal-regulated kinase 1/2 (ERK 1/2), p38, and c-Jun N-terminal kinase MAPKs, as well as I kappa B alpha, became phosphorylated, and hence activated, in both LPS- and GBS-stimulated monocytes. The kinetics of these phosphorylation events, as well as those of TNF-alpha production, were delayed by 30-60 min in GBS-stimulated, relative to LPS-stimulated, monocytes. Selective inhibitors of ERK 1/2 (PD98059 or U0126), p38 (SB203580), or NF-kappa B (caffeic acid phenetyl ester (CAPE)) could all significantly reduce TNF-alpha production, although none of the inhibitors used alone was able to completely prevent TNF-alpha release. However, this was completely blocked by combinations of the inhibitors, including PD98059-SB203580, PD98059-CAPE, or SB203580-CAPE combinations, in both LPS- and GBS-stimulated monocytes. In conclusion, our data indicate that the simultaneous activation of multiple pathways, including NF-kappa B, ERK 1/2, and p38 MAPKs, is required to induce maximal TNF-alpha production. Accordingly, in septic shock caused by either GBS or Gram-negative bacteria, complete inhibition of TNF-alpha release may require treatment with drugs or drug combinations capable of inhibiting multiple activation pathways.     

Bacterial lipopolysaccharide and IFN-gamma induce Toll-like receptor 2 and Toll-like receptor 4 expression in human endothelial cells: role of NF-kappa B activation

Toll-like receptor (TLR) 4 has been identified as the primary receptor for enteric LPS, whereas TLR2 has been implicated as the receptor for Gram-positive and fungal cell wall components and for bacterial, mycobacterial, and spirochetal lipoproteins. Vascular endothelial cell (EC) activation or injury by microbial cell wall components such as LPS is of critical importance in the development of sepsis and septic shock. We have previously shown that EC express predominantly TLR4, and have very little TLR2. These cells respond vigorously to LPS via TLR4, but are unresponsive to lipoproteins and other TLR2 ligands. Here we show that LPS, TNF-alpha, or IFN-gamma induce TLR2 expression in both human dermal microvessel EC and HUVEC. Furthermore, LPS and IFN-gamma act synergistically to induce TLR2 expression in EC, and LPS-induced TLR2 expression is NF-kappaB dependent. LPS and IFN-gamma also up-regulate TLR4 mRNA expression in EC. These data indicate that TLR2 and TLR4 expression in ECs is regulated by inflammatory molecules such as LPS, TNF-alpha, or IFN-gamma. TLR2 and TLR4 molecules may render EC responsive to TLR2 ligands and may help to explain the synergy between LPS and lipoproteins, and between LPS and IFN-gamma, in inducing shock associated with Gram-negative sepsis.     

Involvement of protein tyrosine kinase in Toll-like receptor 4-mediated NF-kappa B activation in human peripheral blood monocytes

Bacterial lipopolysaccharide (LPS) is a powerful activator of the innate immune system. Exposure to LPS induces an inflammatory reaction in the lung mediated primarily by human blood monocytes and alveolar macrophages, which release an array of inflammatory chemokines and cytokines including IL-8, TNF-alpha, IL-1beta, and IL-6. The signaling mechanisms utilized by LPS to stimulate the release of cytokines and chemokines are still incompletely understood. Pretreatment with the protein tyrosine kinase-specific inhibitors genistein and herbimycin A effectively blocked LPS-induced NF-kappaB activation as well as IL-8 gene expression in human peripheral blood monocytes. However, when genistein was added 2 min after the addition of LPS, no inhibition was observed. Utilizing a coimmunoprecipitation assay, we further showed that LPS-stimulated tyrosine phosphorylation of Toll-like receptor 4 (TLR4) may be involved in downstream signaling events induced by LPS. These findings provide evidence that LPS-induced NF-kappaB activation and IL-8 gene expression use a signaling pathway requiring protein tyrosine kinase and that such regulation may occur through tyrosine phosphorylation of TLR4.     

Regulation of lung surfactant secretion by phospholipase A2

Arachidonic acid has been shown to stimulate lung surfactant secretion from alveolar epithelial type II cells. To identify the (phospho)lipases responsible for generating arachidonic acid during lung surfactant secretion, the effects of various (phospho)lipase inhibitors on phosphatidylcholine (PC) secretion from rat alveolar type II cells were investigated. N-(p-amylcinnamoyl)anthranilic acid (ACA), a general inhibitor of phsopholipase A2 (PLA2), inhibited ATP-stimulated PC secretion in a dose-dependent manner. ACA also blocked PC secretion from type II cells stimulated by other secretagogues including phorbol 12-myristate 13-acetate, Ca2+ ionophore A23187 and terbutaline, indicating that PLA2 acts at a late step distal to the generation of second messengers. To determine which PLA2 isoform(s) is involved in lung surfactant secretion, selective inhibitors to different types of PLA2 were used to inhibit PLA2 activity in type II cells. The cytosolic PLA2 (cPLA2) inhibitor, arachidonyl trifluoromethyl ketone, was found to inhibit ATP-stimulated PC secretion, whereas the secretory PLA2 inhibitors, oleoyloxyethylphosphocholine, aristolochic acid, or p-bromophenacyl bromide, and the Ca2+-independent PLA2 inhibitors, palmitoyl trifluoromethyl ketone, or haloenol lactone suicide substrate, had no effect. In addition to PLA2, arachidonic acid is released from diacylglycerol (DAG) by DAG and monoacylglycerol lipases. The DAG lipase inhibitor, RHC-80267 also blocked ATP-stimulated PC secretion. The results suggest that both pathways for generating arachidonic acid via cPLA2 and DAG lipase may participate in lung surfactant secretion.     

Dermatan sulfate inhibits osteoclast formation by binding to receptor activator of NF-kappa B ligand

Dermatan sulfate (DS) is a major component of extracellular matrices in mammalian tissues. In the present study, DS demonstrated a high level of binding activity to receptor activator of NF-kappaB ligand (RANKL) and obstructed the binding of RANK to RANKL, determined using a quartz-crystal microbalance (QCM) technique. Further, when mouse bone marrow cells were cultured with RANKL and macrophage colony-stimulating factor, DS suppressed tartrate-resistant acid phosphatase-positive multinucleated cell formation in a dose-dependent manner. In addition, immunoblot analyses revealed that DS reduced the levels of phosphorylation of p38 mitogen-activated protein kinase and extracellular signal-regulated kinase protein in mouse osteoclast progenitor cells stimulated with RANKL. Together, these results indicate that DS regulates osteoclast formation through binding to RANKL and inhibition of signal transduction in osteoclast progenitor cells, suggesting that it has an important role in bone metabolism in pathological conditions.     

Mannose binding lectin and lung collectins interact with Toll-like receptor 4 and MD-2 by different mechanisms

Background

We have previously shown that lung collectins, surfactant protein A (SP-A) and surfactant protein D, interact with Toll-like receptor (TLR) 2, TLR4, or MD-2. Bindings of lung collectins to TLR2 and TLR4/MD-2 result in the alterations of signaling through these receptors, suggesting the immunomodulatory functions of lung collectins. Mannose binding lectin (MBL) is another collectin molecule which has structural homology to SP-A. The interaction between MBL and TLRs has not yet been determined.

Methods

We prepared recombinant MBL, and analyzed its bindings to recombinant soluble forms of TLR4 (sTLR4) and MD-2.

Results

MBL bound to sTLR4 and MD-2. The interactions were Ca²⁺-dependent and inhibited by mannose or monoclonal antibody against the carbohydrate-recognition domain of MBL. Treatment of sTLR4 or MD-2 by peptide N-glycosidase F significantly decreased the binding of MBL. SP-A bound to deglycosylated sTLR4, and this property did not change in chimeric molecules of SP-A/MBL in which Glu¹⁹⁵–Phe²²⁸ or Thr¹⁷⁴–Gly¹⁹⁴ of SP-A were replaced with the corresponding MBL sequences.

General Significance

These results suggested that MBL binds to TLR4 and MD-2 through the carbohydrate-recognition domain, and that oligosaccharide moieties of TLR4 and MD-2 are important for recognition by MBL. Since our previous studies indicated that lung collectins bind to the peptide portions of TLRs, MBL and lung collectins interact with TLRs by different mechanisms. These direct interactions between MBL and TLR4 or MD-2 suggest that MBL may modulate cellular responses by altering signals through TLRs.     

Pathophysiology of neonatal lung injury induced by monoclonal antibody to surfactant protein B

Grossmann, Gertie, Yasuhiro Suzuki, Bengt Robertson, TsutomuKobayashi, Per Berggren, Wen-Zhi Li, Guo-Wei Song, and Bo Sun.Pathophysiology of neonatal lung injury induced by monoclonal antibody to surfactant protein B. J. Appl.Physiol. 82(6): 2003-2010, 1997.Near-termnewborn rabbits were exposed via the airways to a monoclonal antibodyto surfactant protein B and ventilated for 0-120 min. Controlanimals received nonspecific rabbit or mouse immunoglobulin G, saline,or no material via the airways. Administration of the antibody at 40mg/kg elicited an immediate, significant fall in lung-thorax complianceassociated with progressive intra-alveolar edema and/oralveolar collapse and necrosis and desquamation of airway epithelium,and hyaline membranes. The vascular-to-alveolar leak of human albuminand human immunoglobulin G, injected intravenously at birth anddetermined in lung lavage fluid 60-120 min after instillation ofthe antibody, was 1.8% for the left lung, with no difference betweenthe markers. The average leak in control animals ventilated for 120 minwas <0.3% (P < 0.05). Cytospin preparations of lung lavage fluid from animals exposed to the antibodyshowed significantly increased recruitment of neutrophilic granulocytes. The pathology and pathophysiology of neonatal lung injuryinduced by the monoclonal antibody to surfactant protein B probablyreflect a combination of direct inactivation of surfactant and aninflammatory response triggered by the immune reaction.

     

Toll-like receptor activation suppresses ER stress factor CHOP and translation inhibition through activation of eIF2B

Activation of Toll-like receptors (TLRs) induces the endoplasmic reticulum (ER) unfolded protein response (UPR) to accommodate essential protein translation. However, despite increased levels of phosphorylated eIF2α (p-eIF2α), a TLR-TRIF-dependent pathway assures that the cells avoid CHOP induction, apoptosis and translational suppression of critical proteins. As p-eIF2α decreases the functional interaction of eIF2 with eIF2B, a guanine nucleotide exchange factor (GEF), we explored the hypothesis that TLR-TRIF signalling activates eIF2B GEF activity to counteract the effects of p-eIF2α. We now show that TLR-TRIF signalling activates eIF2B GEF through PP2A-mediated serine dephosphorylation of the eIF2B ?-subunit. PP2A itself is activated by decreased Src-family-kinase-induced tyrosine phosphorylation of its catalytic subunit. Each of these processes is required for TLR-TRIF-mediated CHOP suppression in ER-stressed cells in vitro and in vivo. Thus, in the setting of prolonged, physiologic ER stress, a unique TLR-TRIF-dependent translational control pathway enables cells to carry out essential protein synthesis and avoid CHOP-induced apoptosis while still benefiting from the protective arms of the UPR.     

Direct interaction of surfactant protein A with myometrial binding sites: signaling and modulation by bacterial lipopolysaccharide

Surfactant protein A (SFTPA1), a member of the collagenous lectin (collectin) family, was first described as a major constituent of lung surfactant, but has recently also been found in the female genital tract. Various microorganisms colonize this area and may cause intrauterine infection or trigger preterm labor. We found that SFTPA1 was not produced in the uterus. Instead, it was immunodetected transiently in rat myometrium at the end (Days 19 and 21) of gestation, but not postpartum, and in cultured myometrial cells. Fluorescence microscopy showed that Texas Red-labeled SFTPA1 bound to myometrial cells. This result was confirmed by biochemical approaches. [(125)I]-SFTPA1 bound to two myometrial cell proteins (55 and 210 kDa). This interaction was dependent on the integrity of the collagenlike domain of SFTPA1. SFTPA1 rapidly activated mitogen-activated protein kinase 1/3 (MAPK1/3) in myometrial cells. Bacterial lipopolysaccharide (LPS), an agent known to trigger uterine contractions and preterm birth, also activated MAPK1/3. The prolonged treatment of myometrial cells with LPS or SFTPA1 upregulated PTGS2 (COX2) protein levels. The addition of rough-type LPS to SFTPA1 blocked the interaction of SFTPA1 with its binding sites and the activation of MAPK1/3 and PTGS2 by SFTPA1. Our data provide the first demonstration of a direct effect of SFTPA1 on rat myometrial cells and inhibitory cross talk between SFTPA1 and LPS signals, providing new insight into the mechanisms of normal and preterm parturition.