Neisseria meningitidis can induce pro-inflammatory cytokine production via pathways independent from CD14 and toll-like receptor 4

Fulminant meningococcal sepsis (FMS) is considered the prototypical Gram-negative sepsis. Lipopolysaccharide (LPS) is thought to be the main toxic element that induces pro-inflammatory cytokine production after interaction with CD14 and toll-like receptor 4 (TLR4). However, there is increasing evidence that LPS is not the sole toxic element of meningococci. The aim of the present study was to determine the role of CD14 and TLR4 in pro-inflammatory cytokine induction by meningococci. To this end, cytokine induction by isolated meningoccal LPS, wild-type N. meningitidis H44/76 (LPS+-meningococci) matched for concentrations of LPS and LPS-deficient N. meningitidis H44/76lpxA (LPS - -meningococci) was studied in human PBMCs and murine peritoneal macrophages (PMs). Pre-incubation of PBMCs with WT14, a monoclonal antibody against CD14, abolished TNF-alpha and IL-1beta induction by E. coli LPS, while cytokine induction by meningococcal LPS was only partially inhibited. When LPS+- and LPS - -meningococci at higher concentrations were used as stimuli, anti-CD14 had a minimal effect. In C3H/HeJ murine PMs, devoid of a functional TLR4, minimal IL-1alpha, IL-6 and TNF-alpha production was seen after stimulation with 10 ng/mL E. coli or meningococcal LPS. However, at higher concentrations (1000 ng LPS/mL) the production of TNF-alpha, but not IL-1alpha or IL-6, occurred also independently of TLR4. The expression of a functional TLR4 in murine PMs had no effect on the cytokine induction by LPS+- or LPS - -meningococci. It is concluded that pro-inflammatory cytokine induction by N. meningitidis can occur independently of CD14 and TLR4.     

Activation of human meningeal cells is modulated by lipopolysaccharide (LPS) and non-LPS components of Neisseria meningitidis and is independent of Toll-like receptor (TLR)4 and TLR2 signalling

The interactions of Neisseria meningitidis with cells of the meninges are critical to progression of the acute, compartmentalized intracranial inflammatory response that is characteristic of meningococcal meningitis. An important virulence mechanism of the bacteria is the ability to shed outer membrane (OM) blebs containing lipopolysaccharide (LPS), which has been assumed to be the major pro-inflammatory molecule produced during meningitis. Comparison of cytokine induction by human meningeal cells following infection with wild-type meningococci, LPS-deficient meningococci or after treatment with OM isolated from both organisms, demonstrated the involvement of non-LPS bacterial components in cell activation. Significantly, recognition of LPS-replete OM did not depend on host cell expression of Toll-like receptor (TLR)4, the accessory protein MD-2 or CD14, or the recruitment of LPS-accessory surface proteins heat shock protein (HSP)70, HSP90alpha, chemokine receptor CXCR4 and growth differentiation factor (GDF)5. In addition, recognition of LPS-deficient OM was not associated with the expression of TLR2 or any of these other molecules. These data suggest that during meningococcal meningitis innate recognition of both LPS and non-LPS modulins is dependent on the expression of as yet uncharacterized pattern recognition receptors on cells of the meninges. Moreover, the biological consequences of cellular activation by non-LPS modulins suggest that clinical intervention strategies based solely on abrogating the effects of LPS are likely to be only partially effective.     

Neisseria meningitidis lipid A mutant LPSs function as LPS antagonists in humans by inhibiting TLR 4-dependent cytokine production

Lipopolysaccharide is a major constituent of the outer membrane of Gram-negative bacteria and important in the induction of pro-inflammatory responses. Recently, novel LPS species derived from Neisseria meningitidis H44/76 by insertional inactivation of the lpxL1 and lpxL2 genes have been created with a lipid A portion consisting of five (penta-acylated lpxL1) or four (tetra-acylated lpxL2) fatty acids connected to the glucosamine backbone instead of six fatty acids in the wild-type LPS. We show that these mutant LPS-types are poor inducers of cytokines (tumor-necrosis factor-α, IL-1β, IL-10, IL-RA) in human mononuclear cells. Both penta- and tetra-acylated meningococcal LPSs were able to inhibit cytokine production by wild-type Escherichia coli or meningococcal LPS. Binding of FITC-labelled E. coli LPS TLR4 transfected Chinese hamster ovary (CHO) cells was inhibited by both mutant LPS-types. Experiments with CHO fibroblasts transfected with human CD14 and TLR4 showed that the antagonizing effect was dependent on the expression of human TLR4. In contrast to the situation in humans, lpxL1 LPS has agonistic activity for cytokine production in peritoneal macrophages of DBA mice, and exacerbated arthritis in murine collagen induced arthritis model. N. meningitidis lipid A mutant LPSs lpxL1 and lpxL2 function as LPS antagonists in humans by inhibiting TLR4-dependent cytokine production but have agonistic activity in mice.     

Mannose-binding lectin binds to two major outer membrane proteins, opacity protein and porin, of Neisseria meningitidis

Human mannose-binding lectin (MBL) provides a first line of defense against microorganisms by complement activation and/or opsonization in the absence of specific Ab. This serum collectin has been shown to activate complement when bound to repeating sugar moieties on several microorganisms, including encapsulated serogroup B and C meningococci, which leads to increased bacterial killing. In the present study, we sought to identify the meningococcal cell surface components to which MBL bound and to characterize such binding. Outer membrane complex containing both lipooligosaccharide (LOS) and proteins and LOS from Neisseria meningitidis were examined for MBL binding by dot blot and ELISA. MBL bound outer membrane complex but not LOS. The binding to bacteria by whole-cell ELISA did not require calcium and was not inhibited by N-acetyl-glucosamine or mannose. With the use of SDS-PAGE, immunoblot analysis, and mAbs specific for meningococcal opacity (Opa) proteins and porin proteins, we determined that MBL bound to Opa and porin protein B (porB). The N-terminal amino acid sequences of the two MBL binding proteins confirmed Opa and PorB. Purified PorB inhibited the binding of MBL to meningococci. Escherichia coli with surface-expressed gonococcal Opa bound significantly more MBL than did the control strain. The binding of human factor H to purified PorB was markedly inhibited by MBL in a dose-dependent manner. Meningococci incubated with human serum bound MBL as detected by ELISA. We conclude that MBL binds to meningococci by a novel target recognition of two nonglycosylated outer membrane proteins, Opa and PorB.     

Human lipoproteins have divergent neutralizing effects on E. coli LPS, N. meningitidis LPS, and complete Gram-negative bacteria

The use of lipoproteins has been suggested as a treatment for Gram-negative sepsis because they inhibit lipopolysaccharide (LPS)-mediated cytokine production. However, little is known about the neutralizing effects of lipoproteins on cytokine production by meningococcal LPS or whole Gram-negative bacteria. We assessed the neutralizing effect of LDLs, HDLs, and VLDLs on LPS- or whole bacteria-induced cytokines in human mononuclear cells. A strong inhibition of Escherichia coli LPS-induced interleukin-1beta (IL-1beta), tumor necrosis factor-alpha, and IL-10 by LDL and HDL was seen, whereas VLDL had a less pronounced effect. In contrast, Neisseria meningitidis LPS, in similar concentrations, was neutralized much less effectively than E. coli LPS. Effective neutralization of meningococcal LPS required a longer interaction time, a lower concentration of LPS, or higher concentrations of lipoproteins. The difference in neutralization was independent of the saccharide tail, suggesting that the lipid A moiety accounted for the difference. Minimal neutralizing effects of the lipoproteins were observed on whole E. coli or N. meningitidis bacteria under all conditions tested. These results indicate that efficient neutralization of LPS depends on the type of LPS, but a sufficiently long interaction time, a low LPS concentration, or high lipoprotein concentration also inhibited cytokines by the less efficiently neutralized N. meningitidis LPS. Irrespective of these differences, whole bacteria showed no neutralization by lipoproteins.     

LPS from Neisseria meningitidis is crucial for inducing monocyte- and microparticle-associated tissue factor activity but not for tissue factor expression

Neisseria meningitidis causes sepsis with coagulopathy. The present study evaluated the tissue factor (TF)-inducing capacity of bacterial LPS in different presentation forms, i.e. membrane-bound LPS versus purified LPS, and of non-LPS components of N. meningitidis. By using a wild-type N. meningitidis, a mutant N. meningitidis lacking LPS (LPS-deficient N. meningitidis), purified LPS from N. meningitidis and Escherichia coli, we measured TF-expression and TF-activity on human monocytes and microparticles (MPs). The effect of TF-modulators, such as phosphatidylserine (PS), tissue factor pathway inhibitor (TFPI) and recombinant IL-10 (rhIL-10) was investigated. In plasmas from meningococcal patients, fibrinopeptide A (FPA), LPS and IL-10 were quantified. Monocytes and MPs exposed to purified LPS or wild-type N. meningitidis had much higher TF-activity than monocytes and MPs exposed to LPS-deficient N. meningitidis (clot formation assay). Incubation with wild-type N. meningitidis, but also LPS-deficient N. meningitidis, resulted in TF-expression on monocytes (flow cytometry, qRT-PCR). Increased cellular TF-activity is associated with coincident surface-exposure of PS and the number of monocytes positive for both PS and TF was significantly higher for monocytes exposed to wild-type N. meningitidis (7.6%) compared with monocytes exposed to LPS-deficient N. meningitidis (1.8%). Treatment with rhIL-10 reduced monocyte- and MP-associated TF-activity, the number of monocytes positive for both TF and PS, and microvesiculation. Patients with meningococcal septicemia had significantly higher levels of LPS, FPA and IL-10 than patients with distinct meningitis. Our results indicate that LPS from N. meningitidis is crucial for inducing TF-activity, but not for monocyte- and MP-associated TF-expression. TF-activity seems to require coincident expression of TF and PS on monocytes, and LPS induces such double-positive monocytes.     

Meningococcal lipopolysaccharides: virulence factor and potential vaccine component.

Lipopolysaccharides (LPS) are surface components of the outer membrane of Neisseria meningitidis. Today, 12 different types of meningococcal LPS (immunotypes) are known, of which 3 are prevalent in the western world. The differences between these immunotypes are in the oligosaccharide part of the LPS molecule and consist of small differences in the oligosaccharide structure, the amount and location of phosphoethanolamine groups, and the degree of O acetylation of individual monosaccharides. Although the differences between the various immunotypes are small, they have a profound influence on the immunochemical and immunological properties of these molecules. Furthermore, each individual strain synthesizes a number of different LPS molecules. The expression of the various components (protective epitopes) is influenced by growth conditions and growth phase. Meningococci can endogenously sialyate their LPS, which constitutes one of the mechanisms by which N. meningitidis can evade the response of the human host. Meningococcal LPS play a key role in the induction of septic shock and can probably enhance the invasiveness of meningococcal strains and shield protective epitopes. Therefore, incorporation of (detoxified) LPS or oligosaccharide components derived therefrom might be very beneficial for the efficacy of a vaccine against group B meningococci. An overview of the development of vaccines against group B meningococci is given, and the status and potential of meningococcal LPS-derived (synthetic) oligosaccharide-protein conjugate vaccines are discussed.     

Macrophage inflammatory protein-3 beta enhances IL-10 production by activated human peripheral blood monocytes and T cells.

We report that the addition of human macrophage inflammatory protein-3 beta (MIP-3 beta) to cultures of human PBMCs that have been activated with LPS or PHA results in a significant enhancement of IL-10 production. This effect was concentration-dependent, with optimal MIP-3 beta concentrations inducing more than a 5-fold induction of IL-10 from LPS-stimulated PBMCs and a 2- to 3-fold induction of IL-10 from PHA-stimulated PBMCs. In contrast, no significant effect on IL-10 production was observed when 6Ckine, the other reported ligand for human CCR7, or other CC chemokines such as monocyte chemoattractant protein-1, RANTES, MIP-1 alpha, and MIP-1 beta were added to LPS- or PHA-stimulated PBMCs. Similar results were observed using activated purified human peripheral blood monocytes or T cells. Addition of MIP-3 beta to nonactivated PBMCs had no effect on cytokine production. Enhancement of IL-10 production by MIP-3beta correlated with the inhibition of IL-12 p40 and TNF-alpha production by monocytes and with the impairment of IFN-gamma production by T cells, which was reversed by addition of anti-IL-10 Abs to the cultures. The ability of MIP-3 beta to augment IL-10 production correlated with CCR7 mRNA expression and stimulation of intracellular calcium mobilization in both monocytes and T cells. These data indicate that MIP-3 beta acts directly on human monocytes and T cells and suggest that this chemokine is unique among ligands binding to CC receptors due to its ability to modulate inflammatory activity via the enhanced production of the anti-inflammatory cytokine IL-10.     

The relative roles of factor H binding protein, neisserial surface protein A, and lipooligosaccharide sialylation in regulation of the alternative pathway of complement on meningococci

Neisseria meningitidis inhibits the alternative pathway (AP) of complement using diverse mechanisms, including expression of capsule (select serogroups), Neisserial surface protein A (NspA), factor H (fH) binding protein (fHbp), and lipooligosaccharide (LOS) sialylation. The contribution of the latter three molecules in AP regulation in encapsulated meningococci was studied using isogenic mutants. When LOS was unsialylated, deleting NspA alone from group A strain A2594 (low fHbp/high NspA) significantly increased AP-mediated C3 deposition. C3 deposition further increased ～2-fold in a ΔfHbpΔNspA double mutant, indicating cooperative fHbp function. LOS sialylation of A2594 ΔfHbpΔNspA decreased the rate of C3 deposition, revealing AP inhibition by LOS sialic acid. Maximal C3 deposition on group B strain H44/76 (high fHbp/low NspA) occurred when all three molecules were absent; again, LOS sialylation attenuated the AP in the absence of both fHbp and NspA. When H44/76 LOS was unsialylated, both fHbp and NspA independently inhibited the AP. LOS sialylation enhanced binding of fH C-terminal domains 18-20 to C3 fragments deposited on bacteria. Interaction of meningococci with nonhuman complement is relevant for animal models and vaccine evaluation studies that use nonhuman complement. Consistent with their human-specific fH binding, neither fHbp nor NspA regulated the rat AP. However, LOS sialylation inhibited the rat AP and, as with human serum, enhanced binding of rat fH to surface-bound C3. These data highlight the cooperative roles of meningococcal NspA and fHbp in regulating the human AP and broaden the molecular basis for LOS sialylation in AP regulation on meningococci in more than one animal species.     

Human MD-2 discrimination of meningococcal lipid A structures and activation of TLR4

MD-2, a eukaryotic accessory protein, is an essential component for the molecular pattern recognition of bacterial endotoxins. MD-2 interacts with lipid A of endotoxins [lipopolysaccharide (LPS) or lipooligosaccharide (LOS)] to activate human toll-like receptor (TLR) 4. The structure of lipid A influences the subsequent activation of human TLR4 and the immune response, but the basis for the discrimination of lipid A structures is unclear. A recombinant human MD-2 (rMD-2) protein was produced in the Pichia pastoris yeast expression system. Human embryonic kidney (HEK293) cells were transfected with human TLR4 and were stimulated with highly purified LOS (0.56 pmol) from Neisseria meningitidis or LPS from other structurally defined bacterial endotoxins in the presence or absence of human rMD-2. Human rMD-2 restored, in a dose-dependent manner, interleukin (IL-8) responsiveness to LOS or LPS in TLR4-transfected HEK293 cells. The interaction of endotoxin with human rMD-2 was then assessed by enzyme-linked immunosorbent assays. Wild-type meningococcal LOS (Wt m LOS) bound human rMD-2, and binding was inhibited by an anti-MD-2 antibody to MD-2 dose-dependently (P < 0.005). Wt m LOS or meningococcal KDO(2)-lipid A had the highest binding affinity for human rMD-2; unglycosylated meningococcal lipid A produced by meningococci with defects in the 3-deoxy-d-manno-2-octulosonic acid (KDO) biosynthesis pathway did not appear to bind human rMD-2 (P < 0.005). The affinity of meningococcal LOS with a penta-acylated lipid A for human rMD-2 was significantly less than that for hexa-acylated LOS (P < 0.05). The hierarchy in the binding affinity of different lipid A structures for human rMD-2 was directly correlated with differences in TLR4 pathway activation and cytokine production by human macrophages.     

The differential response of human dendritic cells to live and killed Neisseria meningitidis

There is currently no effective vaccine for Neisseria meningitidis (Nm) serogroup B. Generation of optimal immune responses to meningococci could be achieved by targeting meningococcal antigens to human dendritic cells (DCs). Recent studies have shown that diverse DC responses and subsequent generation of protective immunity can be observed if the microbes are viable or killed. This is important because the host is likely to be exposed to both live and killed bacteria during natural infection. There are currently few data on comparative DC responses to live and killed meningococci. We show here that exposure of human DC to live meningococci does not result in a typical maturation response, as determined by the failure to upregulate CD40, CD86, HLA-DR and HLA-Class I. Despite this, live meningococci were potent inducers of IL-12 and IL-10, although the ratios of these cytokines differed from those to killed organisms. Our data also suggest that enhanced phagocytosis of killed organisms compared with live may be responsible for the differential cytokine responses, involving an autocrine IL-10-dependent mechanism. The consequences of these findings upon the effectiveness of antigen presentation and T-cell responses are currently under investigation.     

Reversal of defective IL-6 production in lipopolysaccharide-tolerant mice by phorbol myristate acetate

The development of LPS tolerance has been suggested to be mediated by an inhibition of cytokine synthesis. Here we have studied serum IL-6 and TNF levels in mice after LPS administration. Repeated administration of LPS (35 micrograms daily for 4 days) to mice induced a refractoriness (tolerance) to subsequent administrations of LPS in terms of induction of circulating IL-6 and TNF. To investigate the mechanism by which LPS down-regulates its own induction of cytokine synthesis and the relationship between IL-6 and TNF production, we attempted to revert the inhibition of IL-6 and TNF production using agents like PMA or IFN-gamma, previously reported to activate macrophage production of cytokines. Pretreatment with PMA (4 micrograms, 10 min before LPS) partially restored IL-6 production in LPS-tolerant mice given 2 micrograms LPS. On the other hand, PMA did not restore TNF induction in LPS-tolerant mice, even when administered with high doses of LPS (up to 200 micrograms). A similar reversal of LPS resistance to IL-6, but not TNF, induction by PMA was observed in genetically LPS-resistant C3H/HeJ mice. IFN-gamma also restored, although to a lesser extent than PMA, IL-6 production. However, unlike PMA, IFN-gamma could also partially restore TNF production in LPS-tolerant mice, although only when LPS was administered at high doses. By contrast with PMA, IFN-gamma was clearly more active in restoring TNF synthesis than that of IL-6. Similar results were obtained in genetically LPS-unresponsive C3H/HeJ mice. These data suggest that different mechanisms are implicated in the inhibition of IL-6 and TNF synthesis in LPS-tolerant mice and that part of this inhibition can be overcome by PMA or IFN-gamma.     

Regulation of murine B cell Thy-1 expression by IL-4, IFN-gamma, and CD4+ T cell subsets

Interleukin 4 (IL-4) induces the expression of membrane Thy-1 on the vast majority of lipopolysaccharide (LPS)-stimulated normal murine B cells in vitro. This induction is inhibited by interferon-gamma (IFN-gamma). IL-4 and IFN-gamma are required late in culture to effect maximal induction and inhibition of Thy-1 expression by LPS- or LPS + IL-4-stimulated B cells, respectively. IFN-gamma suppresses IL-4-induced Thy-1 expression by inhibiting the induction of steady-state levels of Thy-1-specific mRNA. Three distinct CD4+ Th2 clones, through their release of IL-4, induce B cells to express high levels of Thy-1, by 24 hr, in striking contrast to the 3 days required to induce Thy-1 expression after stimulation with LPS and IL-4. This induction is abrogated by the addition of IFN-gamma. B cells stimulated with three distinct Th1 clones (IFN-gamma- and IL-2-producing) exhibit a modest, non-IL-4-dependent, expression of Thy-1. In contrast to intrinsic expression of Thy-1 by Th2-stimulated B cells. Thy-1 expressed by Th1-stimulated B cells is acquired, having the allotype specificity of the stimulating T cell.     

Thyroid hormone enhances nitric oxide-mediated bacterial clearance and promotes survival after meningococcal infection

Euthyroid sick syndrome characterized by reduced levels of thyroid hormones (THs) is observed in patients with meningococcal shock. It has been found that the level of THs reflects disease severity and is predictive for mortality. The present study was conducted to investigate the impact of THs on host defense during meningococcal infection. We found that supplementation of thyroxine to mice infected with Neisseria meningitidis enhanced bacterial clearance, attenuated the inflammatory responses and promoted survival. In vitro studies with macrophages revealed that THs enhanced bacteria-cell interaction and intracellular killing of meningococci by stimulating inducible nitric oxide synthase (iNos)-mediated NO production. TH treatment did not activate expression of TH receptors in macrophages. Instead, the observed TH-directed actions were mediated through nongenomic pathways involving the protein kinases PI3K and ERK1/2 and initiated at the membrane receptor integrin αvβ3. Inhibition of nongenomic TH signaling prevented iNos induction, NO production and subsequent intracellular bacterial killing by macrophages. These data demonstrate a beneficial role of THs in macrophage-mediated N. meningitidis clearance. TH replacement might be a novel option to control meningococcal septicemia.     

An IFN-gamma-dependent pathway controls stimulation of memory phenotype CD8+ T cell turnover in vivo by IL-12, IL-18, and IFN-gamma

Unlike naive T cells, memory phenotype (CD44(high)) T cells exhibit a high background rate of turnover in vivo. Previous studies showed that the turnover of memory phenotype CD8(+) (but not CD4(+)) cells in vivo can be considerably enhanced by products of infectious agents such as LPS. Such stimulation is TCR independent and hinges on the release of type I IFNs (IFN-I) which leads to the production of an effector cytokine, probably IL-15. In this study, we describe a second pathway of CD44(high) CD8(+) stimulation in vivo. This pathway is IFN-gamma rather than IFN-I dependent and is mediated by at least three cytokines, IL-12, IL-18, and IFN-gamma. As for IFN-I, these three cytokines are nonstimulatory for purified T cells and under in vivo conditions probably act via production of IL-15.     

IL-10 inhibits cytokine production by activated macrophages

IL-10 inhibits the ability of macrophage but not B cell APC to stimulate cytokine synthesis by Th1 T cell clones. In this study we have examined the direct effects of IL-10 on both macrophage cell lines and normal peritoneal macrophages. LPS (or LPS and IFN-gamma)-induced production of IL-1, IL-6, and TNF-alpha proteins was significantly inhibited by IL-10 in two macrophage cell lines. Furthermore, IL-10 appears to be a more potent inhibitor of monokine synthesis than IL-4 when added at similar concentrations. LPS or LPS- and IFN-gamma-induced expression of IL-1 alpha, IL-6, or TNF-alpha mRNA was also inhibited by IL-10 as shown by semiquantitative polymerase chain reaction or Northern blot analysis. Inhibition of LPS-induced IL-6 secretion by IL-10 was less marked in FACS-purified peritoneal macrophages than in the macrophage cell lines. However, IL-6 production by peritoneal macrophages was enhanced by addition of anti-IL-10 antibodies, implying the presence in these cultures of endogenous IL-10, which results in an intrinsic reduction of monokine synthesis after LPS activation. Consistent with this proposal, LPS-stimulated peritoneal macrophages were shown to directly produce IL-10 detectable by ELISA. Furthermore, IFN-gamma was found to enhance IL-6 production by LPS-stimulated peritoneal macrophages, and this could be explained by its suppression of IL-10 production by this same population of cells. In addition to its effects on monokine synthesis, IL-10 also induces a significant change in morphology in IFN-gamma-stimulated peritoneal macrophages. The potent action of IL-10 on the macrophage, particularly at the level of monokine production, supports an important role for this cytokine not only in the regulation of T cell responses but also in acute inflammatory responses.     

Human dendritic cell activation by Neisseria meningitidis: phagocytosis depends on expression of lipooligosaccharide (LOS) by the bacteria and is required for optimal cytokine production

Group B Neisseria meningitidis is a human pathogen, for which a universally effective vaccine is still not available. Immune responses to bacteria are initiated by dendritic cells (DC), which internalize and process bacterial antigens for presentation to T cells. We show here that optimal IL-12 and TNF-alpha production by human monocyte derived DC in response to killed serogroup B N. meningitidis depends on physical contact and internalization of the bacteria by DC. The majority of DC producing cytokines had internalized N. meningitidis while inhibition of bacterial internalization markedly impaired IL-12 and TNF-alpha, but not IL-6 production. Internalization of N. meningitidis was shown to depend on lipooligosaccharide (LOS) expressed by the bacteria with poor internalization of LOS deficient bacteria compared to wild-type bacteria. Restoration of LOS biosynthesis in a LOS regulatory strain also restored both internalization and cytokine production and was enhanced in the presence of LPS binding protein (LBP). These results suggest that DC phagocytosis depends on expression of LOS within the bacteria and that optimal cytokine production, particularly IL-12, requires internalization of the bacteria. These findings have important implications for designing vaccines that will induce protective immune responses to group B N. meningitidis.     

Tumor necrosis factor-alpha induces functionally active hyaluronan-adhesive CD44 by activating sialidase through p38 mitogen-activated protein kinase in lipopolysaccharide-stimulated human monocytic cells

Interaction of CD44, an adhesion molecule, with its ligand, hyaluronan (HA), in monocytic cells plays a critical role in cell migration, inflammation, and immune responses. Most cell types express CD44 but do not bind HA. The biological functions of CD44 have been attributed to the generation of the functionally active, HA-adhesive form of this molecule. Although lipopolysaccharide (LPS) and cytokines induce HA-adhesive CD44, the molecular mechanism underlying this process remains unknown. In this study, we show that LPS-induced CD44-mediated HA (CD44-HA) binding in monocytes is regulated by endogenously produced tumor necrosis factor (TNF)-alpha and IL-10. Furthermore, p38 mitogen-activated protein kinase (MAPK) activation was required for LPS- and TNF-alpha-induced, but not IL-10-induced, CD44-HA-binding in normal monocytes. To dissect the signaling pathways regulating CD44-HA binding independently of cross-regulatory IL-10-mediated effects, IL-10-refractory promonocytic THP-1 cells were employed. LPS-induced CD44-HA binding in THP-1 cells was regulated by endogenously produced TNF-alpha. Our results also suggest that lysosomal sialidase activation may be required for the acquisition of the HA-binding form of CD44 in LPS- and TNF-alpha-stimulated monocytic cells. Studies conducted to understand the role of MAPKs in the induction of sialidase activity revealed that LPS-induced sialidase activity was dependent on p42/44 MAPK-mediated TNF-alpha production. Blocking TNF-alpha production by PD98059, a p42/44 inhibitor, significantly reduced the LPS-induced sialidase activity and CD44-HA binding. Subsequently, TNF-alpha-mediated p38 MAPK activation induced sialidase activity and CD44-HA binding. Taken together, our results suggest that TNF-alpha-induced p38 MAPK activation may regulate the induction of functionally active HA-binding form of CD44 by activating sialidase in LPS-stimulated human monocytic cells.     

Interaction of Neisseria meningitidis with human brain microvascular endothelial cells: role of MAP- and tyrosine kinases in invasion and inflammatory cytokine release

Neisseria meningitidis traversal across the blood-cerebrospinal fluid barrier is an essential step in the pathogenesis of bacterial meningitis. We have previously shown that invasion of human brain microvascular endothelial cells (HBMEC) by meningococci is mediated by bacterial outer membrane protein Opc that binds fibronectin, thereby anchoring the bacterium to the integrin alpha 5 beta 1-receptor on the endothelial cell surface. However, subsequent signal transduction mechanisms essential for or regulated by N. meningitidis adhesion and invasion, or HBMEC responses to N. meningitidis are unknown. In this report we investigated the role of c-Jun N-terminal kinases 1 and 2 (JNK1 and JNK2), p38 mitogen-activated (MAP) kinase and protein tyrosine kinases in endothelial-N. meningitidis interaction. Binding of meningococci to HBMEC phosphorylated and activated JNK1 and JNK2 and p38 MAPK as well as their direct substrates c-Jun and MAP kinase activated kinase-2 (MAPKAPK-2), respectively. Non-invasive meningococcal strains lacking opc gene (opc mutants and sequence type 11 complex meningococci) still activated p38 MAPK, however, failed to activate JNK. Inhibition of JNK1 and JNK2 significantly reduced internalization of N. meningitidis by HBMEC without affecting its adherence. Blocking the endothelial integrin alpha 5 beta 1 also decreased N. meningitidis-induced JNK activation in HBMEC. These findings indicate the crucial role of JNK signalling pathway in N. meningitidis invasion in HBMEC. In contrast, p38 MAPK pathway was important for the control of interleukin-6 (IL-6) and IL-8 release by HBMEC. Genistein, a protein tyrosine kinase inhibitor, decreased both invasion of N. meningitidis into HBMEC and IL-6 and IL-8 release, indicating that protein tyrosine kinases, which link signals from integrins to intracellular signalling pathways are essential for both bacterial internalization and cytokine secretion by HBMEC.