Pathogen Recognition and Innate Immunity

At high concentrations, free radicals and radical-derived, nonradical reactive species are hazardous for living organisms and damage all major cellular constituents. At moderate concentrations, how...

/pdf/free-radicals-in-the-physiological-control-of-cell-function-g3w3iyr0ph.pdf

Free Radicals in the Physiological Control of Cell Function

▪ Abstract The innate immune system is a universal and ancient form of host defense against infection. Innate immune recognition relies on a limited number of germline-encoded receptors. These receptors evolved to recognize conserved products of microbial metabolism produced by microbial pathogens, but not by the host. Recognition of these molecular structures allows the immune system to distinguish infectious nonself from noninfectious self. Toll-like receptors play a major role in pathogen recognition and initiation of inflammatory and immune responses. Stimulation of Toll-like receptors by microbial products leads to the activation of signaling pathways that result in the induction of antimicrobial genes and inflammatory cytokines. In addition, stimulation of Toll-like receptors triggers dendritic cell maturation and results in the induction of costimulatory molecules and increased antigen-presenting capacity. Thus, microbial recognition by Toll-like receptors helps to direct adaptive immune responses ...

/pdf/innate-immune-recognition-3eedeqyq83.pdf

Innate Immune Recognition

One of the mechanisms by which the innate immune system senses the invasion of pathogenic microorganisms is through the Toll-like receptors (TLRs), which recognize specific molecular patterns that are present in microbial components. Stimulation of different TLRs induces distinct patterns of gene expression, which not only leads to the activation of innate immunity but also instructs the development of antigen-specific acquired immunity. Here, we review the rapid progress that has recently improved our understanding of the molecular mechanisms that mediate TLR signalling.

Toll-like receptor signalling

ObjectiveTo determine the incidence, cost, and outcome of severe sepsis in the United States.DesignObservational cohort study.SettingAll nonfederal hospitals (n = 847) in seven U.S. states.PatientsAll patients (n = 192,980) meeting criteria for severe sepsis based on the International Classification

Epidemiology of severe sepsis in the United States: analysis of incidence, outcome, and associated costs of care.

The serum protein lipopolysaccharide (LPS)-binding protein (LBP) seems to play an important role in regulating host responses to LPS. Complexes of LPS and LBP form in serum and stimulate monocytes, macrophages, or polymorphonuclear leukocytes after binding to CD14. Previous reports have described the structure and properties of LBP from human and rabbit sera. Since mice are used in some experimental models of endotoxemia or gram-negative bacterial infections, information is needed about the properties of murine LBP. Murine LBP was purified by ion-exchange chromatography and high-pressure liquid chromatography; its NH2-terminal sequence (TNPGLVTRIT) was very similar to those of human and rabbit LBPs (80 to 90% amino acid identity). Murine LBP resembled LBPs from other species in that it promoted the binding of LPS to monocytes and enhanced the sensitivity of monocytes to LPS at least 100-fold. Mouse LBP, like rabbit and human LBPs, was found to be an acute-phase protein. Further in vivo studies with mice and anti-CD14 or anti-LBP reagents should help determine the role of LBP in response to LPS challenges.

https://iai.asm.org/content/iai/61/2/378.full.pdf

Purification and characterization of murine lipopolysaccharide-binding protein.

Optimal activation of endothelial cells by nanomolar quantities of endotoxin (lipopolysaccharide, LPS) requires the presence of plasma or serum We and others have demonstrated that soluble CD14 (sCD14) and LPS binding protein (LBP) were the key plasma proteins mediating endothelial cell responses to LPS The role of LBP is to transfer LPS to sCD14 and newly formed LPS-sCD14 will in turn activate endothelial cells via an as yet unknown surface receptor This plasma-dependent pathway of endothelial cells activation is referred as to the direct pathway However, endothelial cells are in constant contact with whole blood and not only with plasma In experiments where whole blood was substituted for plasma, we showed that endothelial cells became sensitive to picomolar, rather than nanomolar quantities of LPS The fact that endothelial cell responses were amplified by the presence of whole blood prompted us to search for the responsible blood cell(s) and mediator(s) for this effect Blood cell fractionation experiments, experiments with blood from PNH patients and the use of anti-CD14 antibodies pointed to the monocyte as the blood cell responsible for the amplification effect Moreover, the blood effect could be entirely reproduced by cells from a CD14-expressing cell line, such as calcitriol-differentiated HL-60 cells Inhibitors to TNF and to IL-1 blocked LPS-induced activation of endothelial cells partially when added separately to whole blood, but abrogated endothelial cell responses when added together Thus, the whole blood effect begins with LPS activation of monocytes via cell membrane CD14 and results in endothelial cell activation by the effects of TNF and IL-1 The monokine-mediated endothelial cell activation is referred as to the indirect pathway

Activation of endothelial cells by endotoxin: direct versus indirect pathways and the role of CD14.

Mixing lipopolysaccharide (LPS, Salmonella minnesota R595) with acute-phase rabbit serum (APRS) results in the formation of two types of complexes. The two complexes are separable from each other and from LPS by CsCl density gradient ultracentrifugation. LPS alone had density 1.38 g/cm3, complex 1.3 had density 1.3 +/- 0.02 g/cm3, and complex 1.2 had density less than 1.20 g/cm3. At 1 to 10 micrograms LPS/ml APRS, up to 23 micrograms of LPS could be found in complex 1.3, with the remainder of the LPS in complex 1.2. The capacity of complex 1.2 for LPS was 500 to 600 micrograms LPS/ml APRS. The ability of rabbit serum to form complex 1.3 rose to a maximum in 24 hr post-acute-phase induction. The two complexes were purified by equilibrium density gradient ultracentrifugation and immunoaffinity chromatography using antibodies to LPS and subjected to SDS-PAGE. Complex 1.3 had major peptides after reduction of 91, 64, 61 and 50 kilodaltons. The 61-kilodalton peptide is probably rabbit serum albumin; the others are unidentified. Complex 1.2 had major peptides after reduction whose mobility corresponded to apolipoprotein A-I and serum amyloid A. Complex 1.2 thus seems to be analogous to the LPS-high-density-lipoprotein complexes that form in normal serum except that the latter complexes do not contain serum amyloid A. Rabbit serum amyloid A (SAA) was purified by CsCl equilibrium density gradient ultracentrifugation, gel filtration, and ion-exchange chromatography into two forms, SAA-1 and SAA-2. The two forms of SAA have very similar amino acid compositions and m.w. (SAA-1, 11,526 daltons; SAA-2, 11,469 daltons). They differ primarily in their isoelectric points, 6.57 and 6.27 for SAA-1 and SAA-2, respectively. The complexes 1.2 from several individual rabbits after acute-phase induction were studied by isoelectric focusing. Seven of 10 rabbits showed both forms of SAA; three showed a single form. Two rabbits showing SAA-1 on the first acute-phase induction were reinduced; one rabbit again gave SAA-1 and the other gave SAA-2. These results suggest that SAA and perhaps other acute-phase reactants may modulate the biologic activity of LPS.

Interactions of bacterial lipopolysaccharide with acute phase rabbit serum

Interactions of bacterial lipopolysaccharide with acute-phase rabbit serum and isolation of two forms of rabbit serum amyloid A.

Objective: This review describes efforts to define new therapeutic targets by utilizing information derived from studies of the innate immune response to infection. These targets will provide new means to design novel therapeutics for the treatment of human disease caused by dysregulation of the innate immune response. Data Sources and Analysis: The results were derived from model systems that reflect the cellular changes accompanying activation of the innate immune system. These model systems include the use of a rabbit model of endotoxin shock that requires multiple exposures to lipopolysaccharide to induce severe pathophysiological changes that result in death. The cellular systems range from primary explants of myeloid lineage cells to stably transfected lines bearing key receptors of the innate immune system, the latter of which includes the Toll-like receptors and CD14. Studies in animal models include blocking monoclonal antibodies to rabbit CD14 and rabbit tumor necrosis factor-α. Cellular studies use measurements of cell activation as defined by activation of nuclear factor-κB, MAP kinase, and gene expression. In some studies, direct measurements of secreted cytokine levels were performed. Standard approaches to data analysis have been used. Conclusions: Evidence demonstrates the utility of anti-CD14 monoclonal antibody therapy in septic shock and the potential value of targeting intracellular kinases to modulate harmful cellular responses during sepsis. The key element to each of these approaches is to blunt, but not eliminate, the dysregulated inflammatory response that may occur in sepsis.

Richard J. Ulevitch

Papers

Purification and characterization of murine lipopolysaccharide-binding protein.

Activation of endothelial cells by endotoxin: direct versus indirect pathways and the role of CD14.

Interactions of bacterial lipopolysaccharide with acute phase rabbit serum

Interactions of bacterial lipopolysaccharide with acute-phase rabbit serum and isolation of two forms of rabbit serum amyloid A.

New therapeutic targets revealed through investigations of innate immunity.