Chris Galanos

Crucial role for human Toll-like receptor 4 in the development of contact allergy to nickel

Allergies to nickel (Ni2+) are the most frequent cause of contact hypersensitivity (CHS) in industrialized countries. The efficient development of CHS requires both a T lymphocyte-specific signal and a proinflammatory signal. Here we show that Ni2+ triggered an inflammatory response by directly activating human Toll-like receptor 4 (TLR4). Ni2+-induced TLR4 activation was species-specific, as mouse TLR4 could not generate this response. Studies with mutant TLR4 proteins revealed that the non-conserved histidines 456 and 458 of human TLR4 are required for activation by Ni2+ but not by the natural ligand lipopolysaccharide. Accordingly, transgenic expression of human TLR4 in TLR4-deficient mice allowed efficient sensitization to Ni2+ and elicitation of CHS. Our data implicate site-specific human TLR4 inhibition as a potential strategy for therapeutic intervention in CHS that would not affect vital immune responses.

Lipopolysaccharides of Gram-Negative Bacteria

Publisher Summary Lipopolysaccharides (LPS) form a large, unique class of macromolecules representing a characteristic attribute of gram-negative bacteria. Associated with proteins, they are located in the outer leaflet of the outer membrane of the bacterial cell. In this exposed position on the cell surface, lipopolysaccharides are involved in the interaction of the cell with the environment. Thus, contact of the bacterium with the immune system leads to the stimulation of specific antibodies directed predominantly against determinant structures of the lipopolysaccharide. Hence, lipopolysaccharides represent the main surface antigens of gram-negative bacteria. The chapter describes general aspects of the O-specific chains and the core, and principles of their biosynthesis. The O-specific chains of lipopolysaccharides are made up of repeating units of identical oligosaccharides. These units usually contain different constituents, thus the O chain represents a heteropolysaccharide. The chapter discusses the structure of Salmonella lipopolysaccharides (lipid A), and its biosynthesis. Lipid As of other gram-negative bacteria, recently investigated is also described. The smallest lipid A substructure exhibits antigenicity, mitogenicity, lethal toxicity, (weak) pyrogenicity, and (weak) complement reactivity, but strong Limulus lysate activity. Some aspects of the biological properties of lipopolysaccharides are also discussed. In Gram-positive and Gram-negative bacteria, cell wall components other than lipopolysaccharide may also be endowed with endotoxin-like activities. Like lipopolysaccharide, these constituents are amphipathic in nature (probably with the exception of murein, though it may contain lipoprotein). Thus, the studies indicate that at least some lipid A activities are not restricted to one specific structure, but are rather connected with general physicochemical properties.

Picogram-sensitive assay for endotoxin: Gelation of Limulus polyphemus blood cell lysate induced lipopolysaccharides and lipid A from gram-negative bacteria

Abstract The original endotoxin assay of Levin et al. ( N. Engl. J. Med. , 283 (1970) 1313) has been improved to allow detection of picogram quantities of lipopolysaccharides and free lipid A. The response of this assay to both alkali-treated and untreated lipopolysaccharides and their derivatives parallels the previously demonstrated effect of these substances on guinea pig complement fixation.

Toll-Like Receptor 4 Contributes to Efficient Control of Infection with the Protozoan Parasite Leishmania major

ABSTRACT The essential role of Toll-like receptors (TLR) in innate immune responses to bacterial pathogens is increasingly recognized, but very little is known about the role of TLRs in host defense against infections with eukaryotic pathogens. For the present study, we investigated whether TLRs contribute to the innate and acquired immune response to infection with the intracellular protozoan parasite Leishmania major. Our results show that TLR4 contributes to the control of parasite growth in both phases of the immune response. We also addressed the mechanism that results in killing or growth of the intracellular parasites. Control of parasite replication correlates with the early induction of inducible nitric oxide synthase in TLR4-competent mice, whereas increased parasite survival in host cells from TLR4-deficient mice correlates with a higher activity of arginase, an enzyme known to promote parasite growth. This is the first study showing that TLR4 contributes to the effective control of Leishmania infection in vivo.

Mechanisms of endotoxin shock and endotoxin hypersensitivity

Endotoxins (lipopolysaccharide, LPS) are biologically active substances present in Gram-negative bacteria. Injection of purified LPS into experimental animals leads to the development of many biological activities that can lead to shock with lethal outcome. The biological activities of LPS are not direct effects of the LPS molecule since LPS usually expresses no direct cytotoxic activity. The toxic and other biological properties of LPS are caused indirectly through the action of endogenous mediators that are formed following interaction of LPS with cellular targets, macrophages occupying a key position in the development of endotoxin shock. The interaction of LPS with macrophages may proceed directly leading to activation of these cells, with subsequent synthesis and secretion of a number of endogenous mediators which initiate the different biological activities of LPS. Tumor necrosis factor alpha (TNF-alpha), a macrophage derived cytokine, is a primary mediator of the lethal action of endotoxin. Sensitivity to LPS is genetically determined, varying considerably among different species. The sensitivity of normal animals (mice) to endotoxin may be enhanced considerably under different experimental conditions that include treatment with live (infection) or killed Gram-negative and -positive bacteria. Sensitization to endotoxin proceeds in all LPS-responder strains investigated and in the LPS-resistant mice of the strain C3H/HeJ. It does not proceed in a second LPS-resistant strain, C57BL/10ScCr. The absence of sensitization in the latter mice was found to be due to an impaired IFN-gamma production. IFN-gamma could be identified as the mediator of endotoxin hypersensitivity induced by bacteria.

Toll-like receptor and IL-12 signaling control susceptibility to contact hypersensitivity

Allergic contact hypersensitivity (CHS) is a T cell–mediated inflammatory skin disease. Interleukin (IL)-12 is considered to be important in the generation of the allergen-specific T cell response. Loss of IL-12 function in IL-12Rβ2–deficient mice, however, did not ameliorate the allergic immune response, suggesting alternate IL-12–independent pathways in the induction of CHS. Because exposure to contact allergens always takes place in the presence of microbial skin flora, we investigated the potential role of Toll-like receptors (TLRs) in the induction of CHS. Using mice deficient in TLR4, the receptor for bacterial lipopolysaccharide (LPS), IL-12 receptor (R) β2, or both, we show that the concomitant absence of TLR4 and IL-12Rβ2, but not the absence of TLR4 or IL-12Rβ2 alone, prevented DC-mediated sensitization, generation of effector T cells, and the subsequent CHS response to 2,4,6-trinitro-1-chlorobenzene (TNCB), oxazolone, and fluorescein isothiocyanate. Introduction of the TLR4 transgene into the TLR4/IL-12Rβ2 mutant restored the CHS inducibility, showing a requirement for TLR4 in IL-12–independent CHS induction. Furthermore, the concomitant absence of TLR2 and TLR4 prevented the induction of CHS to TNCB in IL-12–competent mice. Finally, CHS was inducible in germ-free wild-type and IL-12Rβ2–deficient mice, but not in germ-free TLR4/IL-12Rβ2 double deficient mice, suggesting that the necessary TLR activation may proceed via endogenous ligands.

Bacterial sensing, cell signaling, and modulation of the immune response during sepsis.

ABSTRACT Since the definition of systemic inflammatory response syndrome/sepsis was originally proposed, a large amount of new information has been generated showing a much more complex scenario of inflammatory and counterinflammatory responses during sepsis. Moreover, some fundamental mechanisms of sensing and destroying invading microorganisms have been uncovered, which include the discovery of TLR4 as the lipopolysaccharide (LPS) gene, implications of innate immune cells as drivers of the adaptive response to infection, and the modulation of multiple accessory molecules that stimulate or inhibit monocyte/macrophage and lymphocyte interactions. The complexity of the infection/injury-induced immune response could be better appreciated with the application of genomics and proteomics studies, and LPS was a useful tool in many of these studies. In this review, we discuss aspects of bacterial recognition and induced cellular activation during sepsis. Because of the relevance of endotoxin (LPS) research in the field, we focus on LPS and host interactions as a clue to understand microorganisms sensing and cell signaling, then we discuss how this response is modulated in septic patients.

R-form LPS, the master key to the activation ofTLR4/MD-2-positive cells

Lipopolysaccharide (endotoxin, LPS) is a major recognition marker for the detection of gram‐negative bacteria by the host and a powerful initiator of the inflammatory response to infection. Using S‐ and R‐form LPS from wild‐type and R‐mutants of Salmonella and E. coli, we show that R‐form LPS readily activates mouse cells expressing the signaling receptor Toll‐like receptor 4/myeloid differentiation protein 2 (TLR4/MD‐2), while the S‐form requires further the help of the LPS‐binding proteins CD14 and LBP, which limits its activating capacity. Therefore, the R‐form LPS under physiological conditions recruits a larger spectrum of cells in endotoxic reactions than S‐form LPS. We also show that soluble CD14 at high concentrations enables CD14‐negative cells to respond to S‐form LPS. The presented in vitro data are corroborated by an in vivo study measuring TNF‐α levels in response to injection of R‐ and S‐form LPS in mice. Since the R‐form LPS constitutes ubiquitously part of the total LPS present in all wild‐type bacteria its contribution to the innate immune response and pathophysiology of infection is much higher than anticipated during the last half century.

Interaction of lipopolysaccharides with plasma high-density lipoprotein in rats.

The method of crossed immunoelectrophoresis was used to investigate early changes in plasma proteins of rats treated with lipopolysaccharide (LPS). Intravenous injection of a smooth (S)- and a rough (R)-form preparation led to alterations in the high-density lipoprotein (HDL) precipitation peak. The changes were dose dependent and characteristic for each LPS. The changes were identified as being due to the formation of a complex of LPS with HDL, the complex of the S-form LPS with HDL migrating slower and that of the R-form LPS with HDL migrating faster than free HDL. The fate of the complex was followed in the plasma of injected rats, and it was shown that the R-form LPS complex disappeared after several hours, whereas the S-form LPS complex was still partly present after 2 days. Plasma clearance studies, carried out with 14C-labeled LPS, revealed similar differences in the rate of elimination of the two LPSs. In both cases the time of clearance resembled that of the disappearance of LPS-HDL complex. These results may indicate that HDL represents a transport protein for LPS in plasma to organs of clearance or to other cellular targets.

Chemical structure and biological activities of lipid A's from various bacterial families

The endotoxic principle of lipopolysaccharides (LPS) is localized in their lipid A component. Biological effects of LPS on, for instance, body temperature, blood pressure, and blood picture, are also induced by free lipid A. In contrast to the great variability of the 0-specific chains, the chemical structure of lipid A is much more constant. It is common for Salmonella and similar for other genera of the Enterobacteriaceae. Recently, a number of lipid As have been recognized that exhibited distinct structural features compared with Enterobacteriaceae. These lipid As were found to be also distinct with regard to some of their biological properties.