Innate Immunity

L-Glycero-D- manno-heptopyranose is a characteristic compound of many lipopolysaccharide (LPS) core structures of Gram-negative bacteria. In Escherichia coli two heptosyltransferases, namely WaaC and WaaF, are known to transfer L- glycero-D-manno-heptopyranose to Re-LPS and Rd ₂-LPS, respectively. It had been proposed that both reactions involve ADPL- glycero-D-manno-heptose as a sugar donor; however, the structure of this nucleotide sugar had never been completely elucidated. In the present study, ADPL-glycero-D-manno-heptose was isolated from a heptosyltransferase-deficient E. coli mutant, and its structure was determined by nuclear magnetic resonance spectroscopy and matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry as ADPL-glycero-β-D-manno-heptopyranose. This compound represented the sole constituent of the bacterial extract that was accepted as a sugar donor by heptosyltransferases I and II in vitro .

The bacterial endotoxin LPS is a potent stimulator of monocyte and macrophage activation and has been shown to protect differentiated macrophages from de novo infection by HIV-1. However, the mechanisms of this inhibitory activity of LPS are not fully understood. We investigated the effect of LPS on the early post-binding steps of HIV-1 replication in primary macrophages. Paradoxically, when applied together with the virus, LPS stimulated entry of HIV-1 into macrophages, as judged by the amount of internalized HIV-1 RNA and p24. This stimulatory activity did not depend on receptors used for entry and did not require new protein synthesis. However, internalized viral RNA and p24 were rapidly degraded in LPS-stimulated macrophages. Surprisingly, while degradation of HIV-1 p24 in LPS-treated cells was inhibited by bafilomycin A1, HIV-1 RNA was not protected by this agent, suggesting that viral RNA is degraded by a pH-independent mechanism. These results indicate that LPS stimulates both virus uptake and virus degradation in macrophages.

Bacterial endotoxin (lipopolysaccharide, LPS), a glycolipid found in the outer membrane of Gram-negative bacteria, induces the secretion of pro-inflammatory cytokines such as tumor necrosis factor alpha (TNF-α), interleukin (IL)-1, and IL-6 by monocytes/macrophages. The secretion of these biologically active compounds leads to multiple pathological conditions, such as septic shock. There is substantial evidence that chronic exposure to LPS in periodontal diseases mediates, at least in part, the tissue destruction associated with the Gram-negative infection. LPS receptor has been shown to be CD14, a 55 kDa protein. LPS—CD14 interactions mediate many monocyte/macrophage functions in the inflammatory response. However, CD14 lacks a cytoplasmic domain, or any known signal transduction sequence motif, suggesting the existence of another cell surface domain capable of transducing signals. More recently, significant work has implicated Toll proteins in LPS-mediated signaling. The purpose of the present work was to investigate, identify, and characterize secondary LPS binding cell surface domain(s) on monocytes/macrophages. Initial experiments with anti-CD14 blocking antibody revealed only partial blocking of the LPS induced TNF-α response. The kinetics of these experiments suggested a second, low-affinity receptor. Cross-linking experiments were performed to identify LPS binding sites. Two domains were identified: a 55 kDa protein which was inhibited by anti-CD14 (presumably the CD14 receptor) and a second 78 kDa domain. Partial protein sequencing of the 78 kDa domain using mass spectroscopic analysis ascribed this domain to Moesin (membrane organizing extension spike protein). Preliminary experiments using anti-Moesin monoclonal antibody revealed a dose-dependent blocking of LPS induced TNF-α response with a total blocking at 50 µg/ml. Irrelevant isotype controls had no effect. Additional experiments were performed to evaluate the specificity of the anti-Moesin blocking. Separate experiments evaluated anti-Moesin effects on monocyte chemotaxis, IL-1 production in response to IL-1 stimulation, and TNF-α secretion in response to Staphylococcus aureus stimulation. Anti-Moesin antibody only blocked LPS-mediated events. Histological analysis of tissue sections harvested from LPS-induced skin lesions exhibited a 3-fold reduction of the polymorphonuclear neutrophil infiltrate in Moesin-deficient mice compared to wild type mice. The data suggest that Moesin functions as an independent LPS receptor on human monocytes.

The innate immune system coordinates the inflammatory response to pathogens. To do so, cells of the innate immune system must rapidly discriminate between self and non-self. All bacteria express membrane-associated lipoproteins. These molecules activate cells of the innate immune system to initiate host defense mechanisms. However, it is currently unknown how the innate immune system recognizes bacterial lipoproteins. Here, we describe that in response to bacterial lipoprotein, human Toll-like receptor-2 activates three different cellular responses: nuclear factor-κB dependent transcription, programmed cell death and reactive oxygen species production. We propose that Toll-like receptor-2 fulfils multiple roles in the genesis of the immune response to bacterial pathogens.

Even though the pan-caspase inhibitor zVAD has been widely used as an anti-apoptotic agent, inefficient prevention or even enhancement of cell death has been reported in certain cells. To further investigate its effects on cell death, three different cell types were exposed to various apoptotic stimuli in the presence or absence of zVAD. In Jurkat cells, zVAD protected against cell death induced by tumor necrosis factor (TNF), sodium nitroprusside (SNP) and etoposide, whereas in L929 cells cell death was increased. In RAW246.7 macrophages, zVAD showed similar effects as in L929 cells. However, unlike L929 cells, in which the cell death by TNF is known to be necrosis, RAW246.7 cells manifested features of apoptosis such as chromatin condensation and nuclear fragmentation. Induction of cell death by zVAD in lipopolysaccharide (LPS)-activated RAW246.7 cells also showed the same features as those observed in SNP- and etoposide-treated cells. Initiation of an apoptotic process by zVAD not only disputes the sole role of caspases in apoptosis but also suggests an anti-apoptotic function of certain caspase(s). Death of LPS-activated macrophages may be controlled by an anti-apoptotic caspase.

Toll is a Drosophila gene essential for ontogenesis and antimicrobial resistance. Several homologues of Toll have been identified and cloned in vertebrates, namely Toll-like receptors (TLRs). TLRs are structurally characterized by a cytoplasmic Toll/interleukin-1R (TIR) domain and by extracellular leucine rich repeats. TLRs characterized so far activate the MyD88/IRAK signalling cascade which activates NF-κB transcription factor. Genetic, gene transfer, and dominant negative approaches have involved TLR family members (TLR2 and TLR4) in distinct bacterial components for recognition and signalling. However, very little information is available regarding other TLRs. Here we propose to classify TLRs based on their expression pattern, in ubiquitous (TLR1), restricted (TLR2, 4, 5) and specific (TLR3) TLRs. Differential expression and regulation as well as distinct, though overlapping, ligand recognition patterns may underlie the existence of a such a large, seemingly redundant, TLR family.

Severe infection induces both activation of the coagulation system and multiple other inflammatory mediator cascades. This concise review summarizes the current knowledge of mechanisms that are considered to contribute to the procoagulant response to sepsis. Furthermore, evidence is discussed that mediators traditionally involved in the regulation of the hemostatic balance may also influence other inflammatory pathways.

Septic shock is one of the leading causes of death in intensive care units world-wide. Scientists have made great improvements in understanding mechanisms of inflammation, and the sequence of activation of the various pro- and anti-inflammatory markers is now well known. In contrast, physicians have failed to improve survival from septic shock despite the development of specific targets at various points in the cytokine cascade considered to have a key role in host survival in sepsis. Corticosteroids were among the first anti-inflammatory drugs to be tested in large randomized controlled trials. These trials showed that patients with septic shock did not benefit from a short course of large doses of steroids. More recent findings highlighting the role of the integrity of the hypothalamic—pituitary—adrenal axis to respond appropriately to a septic insult, have led to a re-appraisal of the use of steroids in septic shock. Several randomized controlled trials have evaluated the efficacy of a replacement therapy with hydrocortisone in severe sepsis. These trials strongly suggest that this replacement therapy reduces the morbidity of septic shock and may favorably affect survival from septic shock.

CD14 is a pattern recognition receptor for the bacterial cell wall components from Gram-positive and Gram-negative bacteria as well as mycobacteria. Binding of lipopolysaccharide (LPS) or other cell wall constituents to CD14 initiates signal transduction through the Toll-like receptors resulting in the release of pro-inflammatory cytokines and the initiation of the systemic inflammatory response. In rabbits and non-human primates, CD14 specific antibodies were shown to attenuate responses to LPS or Escherichia coli challenge including pro-inflammatory cytokine release, acute lung injury, hypotension and changes in lung, liver, spleen and adrenal gland morphology. In healthy human subjects, single doses of a chimeric CD14 antibody (IC14) have been shown to be well tolerated and result in a dose-related degree of saturation of CD14 receptors on monocytes and granulocytes. Pretreatment of healthy subjects with IC14 2 h prior to LPS resulted in an attenuation of the LPS-induced fever, clinical symptoms, and leukocyte activation and degranulation. IC14 inhibited the release of TNF-α, IL-6, and IL-10 and delayed the release of sTNFR_I and IL-1ra. Further studies are in progress to characterize the safety and clinical pharmacology of IC14 in patients with severe sepsis.

Effective therapies against overwhelming Gram-negative bacteremia, or sepsis, have eluded successful development. The discovery that tumor necrosis factor (TNF), a host-derived inflammatory mediator, was both necessary and sufficient to recapitulate Gram-negative sepsis raised cautious optimism for developing a targeted therapeutic. However, the rapid kinetics of the TNF response to infection defined an extremely narrow window of opportunity during which anti-TNF therapeutics could be successfully administered. HMGB1 was previously studied as a DNA-binding protein involved in DNA replication, repair, and transcription; and as a membrane-associated protein that mediates neurite outgrowth. A decade-long search has culminated in our identification of HMGB1 as a late mediator of endotoxemia. HMGB1 is released by macrophages upon exposure to endotoxin, activates many other proinflammatory mediators, and is lethal to otherwise healthy animals. Elevated levels of HMGB1 are observed in the serum of patients with sepsis, and the highest levels were found in those patients that died. The delayed kinetics of HMGB1 release indicate that it may be useful to target this toxic cytokine in the development of future therapies.

Some infectious agents may contribute to atherosclerosis by maintaining a heightened state of inflammatory response. Although the risk for atherosclerosis was associated with elevated plasma levels of endotoxin, it is difficult to firmly establish what place endotoxin assumes in the etiology of this disease. As the ability for endotoxin to promote disease may depend on its ability to initiate an inflammatory response, it may be controlled by additional regulatory factors. We measured plasma levels of endotoxin and serum levels of neopterin and soluble interleukin-2 receptor in a random population of 402 men and women, 50—79 years old at the 1990 baseline evaluation (Bruneck Study). End point of the prospective survey was incident (early) atherosclerosis in the carotid arteries as assessed with duplex ultrasound. Subjects with high endotoxin levels (90th percentile) in combination with low neopterin or soluble interleukin-2 receptor levels (below median) did not differ from those with low endotoxin in their risk of incident atherosclerosis. The risk associated with high endotoxin, however, was markedly elevated in subjects with high (above median) neopterin or soluble interleukin-2 receptor levels. The study provides epidemiological evidence that the atherogenic potential of endotoxemia is affected by concomitant immune activation.

We suggest that successful defense against microbial invasion requires both local inflammation and systemic anti-inflammation. The key systemic responses involve the hypothalamic-pituitary-adrenocortical axis, the sympathetic-adrenomedullary axis, acute phase protein production, thermoregulation and alterations in leukocyte responsiveness to agonists such as bacterial endotoxin. These integrated responses raise blood and tissue concentrations of several anti-infective molecules, mobilize leukocytes into the circulation, and increase blood flow to injured or infected sites. They also neutralize cytokines, proteases and oxidants that enter the bloodstream from inflamed local sites and forestall endothelial activation in distant vessels. Together, these forces help concentrate activated phagocytes at injured or infected local sites while preventing potentially damaging inflammation in uninvolved tissues.