Co-Activation of Human Whole Blood Cells with Lipopolysaccharides and an Allergen.

The investigation of common inflammation mechanisms caused by exogenic compounds of microbial origin and allergens is one of the most important tasks in current biomedical science. The main manifestations of immune cell activation caused by pro-inflammatory agents are changes in receptor quantity on the surface of immune cells and the production of cytokines and chemokines by blood cells. The levels of expression of TLR4, CD14, and CD11b in the monocytes and neutrophils of human whole blood in response to LPS E. coli, Der p 2 allergen, or their combination reflect different functional activities in these cells, while the composition and amount of produced cytokines reflect the biological activity of the studied agonists. The activity of Der p 2 allergen in ex vivo experiments on whole blood samples is significantly lower compared with its activity in vitro in isolated PBMC cells, which should be taken into account when transferring the results obtained for isolated cells to whole blood cells. LPS R. capsulatus PG significantly decreases the synthesis of MyD88-dependent NF-κB-regulated cytokines activated by LPS E. coli, Der p 2, or their combination. This indirectly indicates the general mechanisms of cell activation caused by these structures and the unified mechanism of the protective action of LPS R. capsulatus PG against both endotoxin and a combination of endotoxin and the allergen.

Effect of lipopolysaccharide structure on functional response of whole blood cells.

Lipopolysaccharides (LPSs) induce a wide spectrum of functional activities after interaction with blood cells. Effect of structure of toxic LPS from S- and Re-chemotypes of E. coli and/or non-toxic LPS of Rhodobacter capsulatus PG (R. caps.) on activation of neutrophils and monocytes of human whole blood were studied, particularly, expression of TLR4, CD14 and CD11b receptors, phagocytosis of BioParticles Alexa Fluor 488, synthesis of cytokines and chemokines. A leading role of CD11b receptor in phagocytic activity of neutrophils primed by LPS from various E. coli chemotypes was shown. The non-toxic LPS of R. caps. does not affect the efficiency of phagocytosis activity of the neutrophils. The LPS of R. caps. was shown to induce production of TRIF-dependent cytokine IFN-ß in human whole blood leukocytes selectively, without activating MyD88-dependent pathway of pro-inflammatory cytokine synthesis, displaying properties of patrial agonist of TLR4. Structure and biological activity of LPS R. caps. allows considering it as a promising immunity stimulating pharmacological agent.

Monoclonal Antibody to CD14, TLR4, or CD11b: Impact of Epitope and Isotype Specificity on ROS Generation by Human Granulocytes and Monocytes.

Lipopolysaccharides (LPSs or endotoxins) from Gram-negative bacteria represent pathogen-associated molecular patterns (PAMPs) that are recognized by CD14 and Toll-like receptor 4 (TLR4). Lipopolysaccharides prime polymorphonuclear leukocytes (PMNs) for substantial production of reactive oxygen species (ROS) during its response to secondary stimuli such as chemoattractants or pathogens. The excessive ROS production can damage surrounding host tissues, thereby amplifying the inflammatory reaction caused by pathogens. Today, specific antibodies against CD14, TLR4, and CD11b are being used as the essential tools to elucidate the role of these receptors in acute inflammation and some of these antibodies have advised as therapeutic agents for clinical use. Because each antibody has two antigen-binding arms [F(ab')2] and one Fc arm, its effect on cellular response is much more complicated rather than simple blockage of target receptor. In fact, IgG antibody, once bound to target receptor, engages Fc receptors γ (FcγRs) and thereby is able to activate the adaptive immune system. The consequences of antibody-dependent binary heterotypic association of CD14, TLR4, or CD11b with FcγRs as well as homotypic one on ROS production are not well elucidated. Moreover, the consequences of antigenic recognition of CD14, TLR4, or CD11b by specific F(ab')2 fragments are not always investigated. In this review, we will discuss known mechanisms underlying the therapeutic efficiency of CD14, TLR4, and CD11b/CD18 antibodies with a focus on LPS-dependent ROS or cytokine production by PMNs or monocytes. The impacts of F(ab')2 as well as antibody IgG subclasses (isotypes) in therapeutic efficiency or agonistic potency of known antibodies against abovementioned receptors are presented. We also pay attention to how the efficiency of different IgG antibody subclasses is modulated during LPS-induced inflammation and by production of priming agents such as interferon γ (IFN-γ). Our review reinforces the molecular targets and therapeutic approaches to amelioration of harmful consequences of excessive activation of human pattern recognition receptors.

Impact of CD14 on Reactive Oxygen Species Production from Human Leukocytes Primed by Escherichia coli Lipopolysaccharides.

Lipopolysaccharides (LPS) from Gram-negative bacteria prime human polymorphonuclear neutrophils (PMNs) via multicomponent receptor cluster including CD14 and MD-2·TLR4 for the enhanced release of reactive oxygen species (ROS) were triggered by bacterial derived peptide N-formyl-methionyl-leucyl-phenylalanine (fMLP). In this study, we investigated the impact of CD14 on LPS-induced priming of human PMNs for fMLP-triggered ROS generation (respiratory or oxidative) burst. Monoclonal antibodies against human CD14 (mAbs) as well as isotype-matched IgG2a did not influence significantly fMLP-triggered ROS production from LPS-unprimed PMNs. Anti-CD14 mAbs (clone UCHM-1) attenuated LPS-induced priming of PMNs as it had been mirrored by fMLP-triggered decrease of ROS production. Similar priming activity of S-LPS or Re-LPS from Escherichia coli for fMLP-triggered ROS release from PMNs was found. Obtained results suggest that glycosylphosphatidylinositol-anchored CD14 is the key player in LPS-induced PMN priming for fMLP-triggered ROS production. We believe that blockade of CD14 on the cell surface and clinical use of anti-CD14 mAbs or their Fab fragments may diminish the production of ROS and improve outcomes during cardiovascular diseases manifested by LPS-induced inflammation.

Interaction of Gram-negative bacteria with cationic proteins: Dependence on the surface characteristics of the bacterial cell.

Gram-negative bacteria can enter the bloodstream and interact with serum cationic proteins. The character of interaction will depend on the surface characteristics of bacterial cells, which are determined by bacterial chemotype and density of lipopolysaccharide (LPS) packing in the cell wall. It was shown that the lysozyme treatment resulted in the increase sensitivity to hypotonic shock. Significant differences to this effect were found between Escherichia coli strain D21 and D21f2 under treatment with physiological protein concentration. On the basis of electrokinetic measurements and studies of the interaction of cells with lysozyme, the hypothesis was formed that the cell wall of the E. coli strain D21f2 contains more LPS and has a higher density of their packing than the cell wall of the E. coli D21 cells. The effect of lysozyme and lactoferrin on the viability of E. coli cells of two different strains was examined. Lysozyme was found to more effectively inhibit the growth of the E. coli D21 bacteria, and lactoferrin suppressed mainly the growth of the E. coli D21f2 bacteria. These results indicate that the differences in LPS core structure of bacterial R-chemotype, which determines surface charge and density of LPS packing, plays an essential role in the mechanisms of interaction of the cationic proteins with the cell wall.