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.     

Toll-like receptor-4 mediates lipopolysaccharide-induced signal transduction

TLR4 is a member of the recently identified Toll-like receptor family of proteins and has been putatively identified as Lps, the gene necessary for potent responses to lipopolysaccharide in mammals. In order to determine whether TLR4 is involved in lipopolysaccharide-induced activation of the nuclear factor-kappaB (NF-kappaB) pathway, HEK 293 cells were transiently transfected with human TLR4 cDNA and an NF-kappaB-dependent luciferase reporter plasmid followed by stimulation with lipopolysaccharide/CD14 complexes. The results demonstrate that lipopolysaccharide stimulates NF-kappaB-mediated gene expression in cells transfected with the TLR4 gene in a dose- and time-dependent fashion. Furthermore, E5531, a lipopolysaccharide antagonist, blocked TLR4-mediated transgene activation in a dose-dependent manner (IC50 approximately 30 nM). These data demonstrate that TLR4 is involved in lipopolysaccharide signaling and serves as a cell-surface co-receptor for CD14, leading to lipopolysaccharide-mediated NF-kappaB activation and subsequent cellular events.     

Toll-like receptor-4 (TLR4) mediates human β-defensin-2 (HBD-2) induction in response to Chlamydia pneumoniae in mononuclear cells

Monocytes are pivotal effector cells of the innate immune system that are vital for recognizing and eliminating invasive microbial pathogens. When microbial products bind to pathogen-recognition receptors, monocytes are activated and release a broad array of cytokines and defensins that orchestrate the host innate and adaptive immune responses. The aim of the present study is to investigate whether Toll-like receptor-4 (TLR4) mediates human β-defensin-2 (HBD-2) induction in response to Chlamydia pneumoniae in mononuclear cells. We showed that TLR4 is expressed in U937 cells and monocytes infected with viable microorganisms in a time-dependent fashion, while heat-inactivated microorganisms induced a lesser expression, albeit still significant, of TLR4 compared with viable organisms; flow cytometric analysis, in particular, revealed a higher level of TLR4 expression at 48 and 72 h postinfection. In addition, U937 cells and monocytes responded to C. pneumoniae in a TLR4-dependent manner with induction of mRNA and protein of the antimicrobial peptide HBD-2. The treatment of cells with TLR4-neutralizing antibody resulted in a decrease in C. pneumoniae- induced HBD-2 production. This study reveals that TLRs not only recognize ligands but also the types of effector molecules induced, namely, antimicrobial peptides. An understanding of the importance of the TLR-mediated antimicrobial mechanisms may provide new avenues for the development of therapeutic regimens aimed at activating the body's own defenses by stimulating TLR-dependent pathways.     

2B4/CD48-mediated regulation of lymphocyte activation and function

2B4 (CD244) is a member of the CD2 subset of the Ig superfamily. This molecule is expressed on innate immune cells, including NK cells, and on subsets of T cells. The 2B4 molecule interacts with CD48, which is widely expressed on hemopoietic cells. Although earlier reports demonstrated a role for 2B4 as an activating receptor in both mice and humans, recent studies of 2B4-deficient mice have suggested that 2B4 functions predominantly as an inhibitory receptor in mice. In addition, 2B4 may also act as a costimulatory ligand for cells expressing CD48. Thus, the 2B4 molecule is more multifunctional than previously understood. In this study, we delineate the current view of 2B4-CD48 interactions among lymphocytes and other cells.     

Toll-like receptor-2, but not Toll-like receptor-4, is essential for development of oviduct pathology in chlamydial genital tract infection

The roles of Toll-like receptor (TLR) 2 and TLR4 in the host inflammatory response to infection caused by Chlamydia trachomatis have not been elucidated. We examined production of TNF-alpha and IL-6 in wild-type TLR2 knockout (KO), and TLR4 KO murine peritoneal macrophages infected with the mouse pneumonitis strain of C. trachomatis. Furthermore, we compared the outcomes of genital tract infection in control, TLR2 KO, and TLR4 KO mice. Macrophages lacking TLR2 produced significantly less TNF-alpha and IL6 in response to active infection. In contrast, macrophages from TLR4 KO mice consistently produced higher TNF-alpha and IL-6 responses than those from normal mice on in vitro infection. Infected TLR2-deficient fibroblasts had less mRNA for IL-1, IL-6, and macrophage-inflammatory protein-2, but TLR4-deficient cells had increased mRNA levels for these cytokines compared with controls, suggesting that ligation of TLR4 by whole chlamydiae may down-modulate signaling by other TLRs. In TLR2 KO mice, although the course of genital tract infection was not different from that of controls, significantly lower levels of TNF-alpha and macrophage-inflammatory protein-2 were detected in genital tract secretions during the first week of infection, and there was a significant reduction in oviduct and mesosalpinx pathology at late time points. TLR4 KO mice responded to in vivo infection similarly to wild-type controls and developed similar pathology. TLR2 is an important mediator in the innate immune response to C. trachomatis infection and appears to play a role in both early production of inflammatory mediators and development of chronic inflammatory pathology.     

Toll-like receptor-4 modulates survival by induction of left ventricular remodeling after myocardial infarction in mice

Left ventricular (LV) remodeling is known to contribute to morbidity and mortality after myocardial infarction (MI). Because LV remodeling is strongly associated with an inflammatory response, we investigated whether or not TLR-4 influences LV remodeling and survival in a mice model of MI. Six days after MI induction, TLR4 knockout (KO)-MI mice showed improved LV function 32 and reduced LV remodeling as indexed by reduced levels of atrial natriuretic factor and total collagen as well as by a reduced heart weight to body weight ratio when compared with WT-MI mice. This was associated with a reduction of protein levels of the intracellular TLR4 adapter protein MyD88 and enhanced protein expression of the anti-hypertrophic JNK in KO-MI mice when compared with wild-type (WT)-MI mice. In contrast, protein activation of the pro-hypertrophic kinases protein kinase Cdelta and p42/44 were not regulated in KO-MI mice when compared with WT-MI mice. Improved LV function, reduced cardiac remodeling, and suppressed intracellular TLR4 signaling in KO-MI mice were associated with significantly improved survival compared with WT-MI mice (62 vs 23%; p < 0.0001). TLR4 deficiency led to improved survival after MI mediated by attenuated left ventricular remodeling.     

Transmembrane ion conductance in human B lymphocyte activation.

Human B lymphocytes were examined to determine whether transmembrane ion conductance plays a role in cell activation. Mitogens (anti-human IgM F(ab')2 fragment (anti-mu) and PMA) were used to stimulate B lymphocytes. Mitogen-induced DNA synthesis was inhibited by tetraethylammonium-Cl (TEA), 4-aminopyridine (4AP), verapamil, and diltiazem in a dose-dependent manner. This inhibition was not due to reduction in cell viability as determined by trypan blue exclusion. Mitogen-induced increases in RNA synthesis were partially inhibited by TEA and 4AP and were more completely inhibited by verapamil and diltiazem. Mitogen-induced cell volume increases were not affected by TEA or 4AP but were completely inhibited by verapamil and diltiazem. B lymphocytes stimulated with anti-mu expressed G1 phase cell surface antigens in the presence of TEA or 4AP, but failed to do so in the presence of verapamil or diltiazem. Substitution of PMA for anti-mu as the mitogen did not alter the effects of TEA or 4AP. However, verapamil inhibited PMA-induced expression of G1 phase cell surface markers although diltiazem did not. The patch clamp technique was used to directly examine plasma membrane ionic currents in whole-cell, cell-attached, and inside-out patch configurations. Activation of B lymphocytes with either anti-mu or the Ca2+ ionophore, A23187, inhibited opening of one type of channel in cell-attached patches. In inside-out patches, this channel type conducted current when the bath [Ca2+] was low (6 X 10(-8) M) but failed to conduct current when the bath [Ca2+] was increased above 1 X 10(-6) M. The results of these experiments are consistent with the hypothesis that activation of B lymphocytes induces alterations in plasma membrane ion conductance. Single channel studies suggest that activation induced increases in [Ca2+]i may directly inhibit a specific set of plasma membrane ion channels as one mechanism by which transmembrane ion flux is altered.     

Toll-like receptor 2 and Toll-like receptor 4 expression in human adrenals.

Toll-like receptors (TLRs) are key elements in the innate immune response, functioning as pattern-recognition receptors for the detection and response to endotoxins and other microbial ligands. Inflammatory cytokines play an important role in the activation of the hypothalamic-pituitary-adrenal HPA axis during inflammation and sepsis. The newly recognized major role of TLR2 and TLR4 and the adrenal stress response during critical illnesses such as inflammation and sepsis demand comprehensive analysis of their interactions. Therefore, we analyzed TLR2 and TLR4 expression in human adrenal glands. Western blot analysis demonstrated the expression of TLR2 and TLR4 in the human adrenocortical cell line NCI-H295. Immunohistochemical analysis of normal human adrenal glands revealed TLR2 and TLR4 expression in the adrenal cortex, but not in the adrenal medulla. Considering the crucial role of the HPA axis and the innate immune response during acute sepsis or septic shock, elucidating the functional interaction of these systems should be of great clinical relevance.     

Spinal cord injury triggers systemic autoimmunity: evidence for chronic B lymphocyte activation and lupus-like autoantibody synthesis

Clinical and experimental data indicate that spinal cord injury (SCI) elicits pathological T-cell responses. Implicit in these data, but poorly understood, is that B lymphocytes (B cells) also contribute to the delayed pathophysiology of spinal trauma. Here, for the first time, we show that experimental spinal contusion injury elicits chronic systemic and intraspinal B cell activation with the emergence of a B cell-dependent organ-specific and systemic autoimmune response. Specifically, using sera from spinal cord injured mice, immunoblots reveal oligoclonal IgG reactivity against multiple CNS proteins. We also show SCI-induced synthesis of autoantibodies that bind nuclear antigens including DNA and RNA. Elevated levels of anti-DNA antibodies are a distinguishing feature of systemic lupus erythematosus and, via their ability to cross-react with neuronal antigens, can cause neuropathology. We show a similar pathologic potential for the autoantibodies produced after SCI. Thus, mammalian SCI produces marked dysregulation of B cell function (i.e. autoimmunity) with pathological potential.     

Transient P2X7 receptor activation triggers macrophage death independent of Toll-like receptors 2 and 4, caspase-1, and pannexin-1 proteins

The function of P2X(7) receptors (ATP-gated ion channels) in innate immune cells is unclear. In the setting of Toll-like receptor (TLR) stimulation, secondary activation of P2X(7) ion channels has been linked to pro-caspase-1 cleavage and cell death. Here we show that cell death is a surprisingly early triggered event. We show using live-cell imaging that transient (1-4 min) stimulation of mouse macrophages with high extracellular ATP ([ATP]e) triggers delayed (hours) cell death, indexed as DEVDase (caspase-3 and caspase-7) activity. Continuous or transient high [ATP]e did not induce cell death in P2X(7)-deficient (P2X(7)(-/-)) macrophages or neutrophils (in which P2X(7) could not be detected). Blocking sustained Ca(2+) influx, a signature of P2X(7) ligation, was highly protective, whereas no protection was conferred in macrophages lacking caspase-1 or TLR2 and TLR4. Furthermore, pannexin-1 (Panx1) deficiency had no effect on transient ATP-induced delayed cell death or ATP-induced Yo-Pro-1 uptake (an index of large pore pathway formation). Thus, "transient" P2X(7) receptor activation and Ca(2+) overload act as a death trigger for native mouse macrophages independent of Panx1 and pro-inflammatory caspase-1 and TLR signaling.     

Toll-like receptor-3-induced mitochondrial dysfunction in cultured human hepatocytes

Several studies have shown the presence of liver mitochondrial dysfunction during sepsis. TLR3 recognizes viral double-stranded RNA and host endogenous cellular mRNA released from damaged cells. TLR3 ligand amplifies the systemic hyperinflammatory response observed during sepsis and in sepsis RNA escaping from damaged tissues/cells may serve as an endogenous ligand for TLR3 thereby modulating immune responses. This study addressed the hypothesis that TLR3 might regulate mitochondrial function in cultured human hepatocytes.HepG2 cells were exposed to TLR-3 ligand (dsRNA — polyinosine–polycytidylic acid; Poly I:C) and mitochondrial respiration was measured. Poly I:C induced a reduction in maximal mitochondrial respiration of human hepatocytes which was prevented partially by preincubation with cyclosporine A (a mitochondrial permeability transition pore-opening inhibitor). Poly-I:C induced activation of NF-κB, and the mitochondrial dysfunction was accompanied by caspase-8 but not caspase-3 activation and by no major alterations in cellular or mitochondrial ultrastructure.     

Isoaspartyl post-translational modification triggers anti-tumor T and B lymphocyte immunity

A hallmark of the immune system is the ability to ignore self-antigens. In attempts to bypass normal immune tolerance, a post-translational protein modification was introduced into self-antigens to break T and B cell tolerance. We demonstrate that immune tolerance is bypassed by immunization with a post-translationally modified melanoma antigen. In particular, the conversion of an aspartic acid to an isoaspartic acid within the melanoma antigen tyrosinase-related protein (TRP)-2 peptide-(181-188) makes the otherwise immunologically ignored TRP-2 antigen immunogenic. Tetramer analysis of iso-Asp TRP-2 peptide-immunized mice demonstrated that CD8+ T cells not only recognized the isoaspartyl TRP-2 peptide but also the native TRP-2 peptide. These CD8+ T cells functioned as cytotoxic T lymphocytes, as they effectively lysed TRP-2 peptide-pulsed targets both in vitro and in vivo. Potentially, post-translational protein modification can be utilized to trigger strong immune responses to either tumor proteins or potentially weakly immunogenic pathogens.     

The apoptotic signaling pathway activated by Toll-like receptor-2

The innate immune system uses Toll family receptors to signal for the presence of microbes and initiate host defense. Bacterial lipoproteins (BLPs), which are expressed by all bacteria, are potent activators of Toll-like receptor-2 (TLR2). Here we show that the adaptor molecule, myeloid differentiation factor 88 (MyD88), mediates both apoptosis and nuclear factor-kappaB (NF-kappaB) activation by BLP-stimulated TLR2. Inhibition of the NF-kappaB pathway downstream of MyD88 potentiates apoptosis, indicating that these two pathways bifurcate at the level of MyD88. TLR2 signals for apoptosis through MyD88 via a pathway involving Fas-associated death domain protein (FADD) and caspase 8. Moreover, MyD88 binds FADD and is sufficient to induce apoptosis. These data indicate that TLR2 is a novel 'death receptor' that engages the apoptotic machinery without a conventional cytoplasmic death domain. Through TLR2, BLP induces the synthesis of the precursor of the pro-inflammatory cytokine interleukin-1beta (IL-1beta). Interestingly, BLP also activates caspase 1 through TLR2, resulting in proteolysis and secretion of mature IL-1beta. These results indicate that caspase activation is an innate immune response to microbial pathogens, culminating in apoptosis and cytokine production.     

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.     

Innate immune responses in murine pleural mesothelial cells: Toll-like receptor-2 dependent induction of beta-defensin-2 by staphylococcal peptidoglycan

The innate immune response is mediated in part by pattern recognition receptors including Toll-like receptors (TLRs). The pleural mesothelial cells (PMCs) that line the pleural surface are in direct contact with pleural fluid and accordingly carry the risk of exposure to infiltrating microorganisms or their components in an event of a complicated parapneumonic effusion. Here we show that murine primary PMCs constitutively express TLR-1 through TLR-9 and, upon activation with peptidoglycan (PGN), mouse PMC produce antimicrobial peptide beta-defensin-2 (mBD-2). Treatment of PMCs with staphylococcal PGN, a gram-positive bacterial cell wall component and a TLR-2 agonist, resulted in a significant increase in TLR-2 and mBD-2 expression. Silencing of TLR-2 expression by small interfering RNA led to the downregulation of PGN-induced mBD-2 expression, thereby establishing causal relationship between the activation of TLR-2 receptor and mBD-2 production. PMCs exposed to PGN showed increased p38 MAPK activity. In addition, PGN-induced mBD-2 expression was attenuated by SB203580, a p38 MAPK inhibitor, underlining the importance of p38 MAPK in mBD-2 induction. Inhibition of erk1/erk2 or phosphatidylinositol 3-kinase did not block PGN-induced mBD-2 expression in PMC. PGN-activated PMC-derived mBD-2 significantly killed Staphylococcus aureus, and mBD-2-neutralizing antibodies blunted this antimicrobial activity. Taken together, these data indicate that PMCs may contribute to host innate immune defense upon exposure to gram-positive bacteria or their products within the pleural space by upregulating TLR-2 and mBD-2 expression.     

The role of the transferrin receptor in human B lymphocyte activation

Transferrin receptors are expressed on proliferating cells and are required for their growth. Transferrin receptors can be detected after, but not before, mitogenic stimulation of normal peripheral blood T and B cells. T cells demonstrate a functional requirement for transferrin receptors in the activation process. These receptors, in turn, are induced to appear by T cell growth factor (interleukin 2). In the experiments reported here, we examined the regulation of transferrin receptor expression on activated human B cells and whether these receptors are necessary for activation to occur. Activation was assessed by studying both proliferation and immunoglobulin secretion. We determined that transferrin receptor expression on B cells is regulated by a factor contained in supernatants of mitogen-stimulated T cells (probably B cell growth factor). This expression is required for proliferation to occur, because antibody to transferrin receptor (42/6) blocks B cell proliferation. Induction of immunoglobulin secretion, however, although dependent on phytohemagglutinin-treated T cell supernatant, is not dependent on transferrin receptor expression and can occur in mitogen-stimulated cells whose proliferation has been blocked by anti-transferrin receptor antibody. These findings support a model for B cell activation in which mitogen (or antigen) delivers two concurrent but distinct signals to B cells: one, dependent on B cell growth factor and transferrin receptor expression, for proliferation; and a second, dependent on T cell-derived factors and not requiring transferrin receptors, which leads to immunoglobulin secretion.     

Regulation of ultraviolet radiation induced cutaneous photoimmunosuppression by Toll-like receptor-4

UVB radiation is a potent immunosuppressive agent that inhibits cell-mediated immune responses. The mechanisms by which UVB radiation influences cell-mediated immune responses have been the subject of extensive investigation. However, the role of innate immunity on photoimmunological processes has received little attention. The purpose of this study was to determine whether Toll-like receptor-4 (TLR4) contributed to UV-induced suppression of contact hypersensitivity (CHS) responses. TLR4^−/− and wild type C57BL/6 (TLR4^+/+) mice were subjected to a local UVB immunosuppression regimen consisting of 100 mJ/cm² UVB radiation followed by sensitization with the hapten DNFB. Wild type TLR4^+/+ mice exhibited significant suppression of contact hypersensitivity response, whereas TLR4^−/− developed significantly less suppression. The suppression in wild type TLR4^+/+ mice could be adoptively transferred to naïve syngeneic recipients. Moreover, there were significantly fewer Foxp3 expressing CD4⁺CD25⁺ regulatory T-cells in the draining lymph nodes of UV-irradiated TLR4^−/− mice than TLR4^+/+ mice. When cytokine levels were compared in these two strains after UVB exposure, T-cells from TLR4^+/+ mice produced higher levels of IL-10 and TGF-β and lower levels of IFN-γ and IL-17. Strategies to inhibit TLR4 may allow us to develop immunopreventive and immunotherapeutic approaches for management of UVB induced cutaneous immunosuppression.