The green tea polyphenol (-)-epigallocatechin-3-gallate inhibits leukocyte activation by bacterial formylpeptide through the receptor FPR.

Although green tea polyphenol catechin is considered as a potential anti-inflammatory agent, its effect on bacterial component-induced inflammation has been poorly investigated. We examined the capacity of epigallocatechin gallate (EGCG) to regulate leukocyte responses to bacterial chemotactic peptide N-formylmethionyl-leucyl-phenylalanine (fMLF), which is recognized by a human G protein-coupled receptor FPR on phagocytic leukocytes. Pretreatment of human monocytic cells or FPR-transfected rat basophilic leukemia cells (ETFR cells) with EGCG significantly inhibited fMLF-induced chemotaxis. Intraperitoneal administration of EGCG in mice suppressed fMLF-induced leukocyte infiltration into the air pouch created in the skin. Mechanistic studies revealed that EGCG dose-dependently suppressed fMLF-induced calcium flux in monocytic cells and ETFR cells. fMLF-induced ETFR cell migration was significantly inhibited by a specific MEK1/2 inhibitor, PD98059, which was associated with reduction in fMLF-induced ERK1/2 phosphorylation. These results suggest that EGCG inhibits FPR-mediated leukocyte activation thus is a promising anti-inflammatory compound.

Sequential chemotactic and phagocytic activation of human polymorphonuclear neutrophils.

Human polymorphonuclear neutrophils (PMN) chemotax to a foreign entity. When the chemoattractants' origins are reached, specific receptors bind to the invader's surface, initiating phagocytosis, phagosome formation, and fusion with granule membranes, generating the bactericidal oxidative burst, and releasing lytic enzymes, specific peptides, and proteins. We explored the initial signaling involved in these functions by observing naïve, unprimed PMN in suspension using fluorescent indicators of cytoplasmic signals (Delta[Ca(2+)](i) and DeltapH(i)) and of bactericidal entities (oxidative species and elastase) exposed to N-formyl-methionyl-leucyl-phenylalanine (fMLP) and/or multivalent immune complexes (IC). fMLP and IC each initiate a rapid transient rise in [Ca(2+)](i), mostly from intracellular stores, simultaneously with a drop in pH(i); these are followed by a drop in [Ca(2+)](i) and a rise in pH(i), with the latter being due to a Na(+)/H(+) antiport. The impact of a second stimulation depends on the order in which stimuli are applied, on their dose, and on their nature. Provided that [Ca(2+)](i) is restored, 10(-7) M fMLP, previously shown to elicit maximal Delta[Ca(2+)](i) but no bactericidal functions, did not prevent the cells' responses with Delta[Ca(2+)](i) to a subsequent high dose of fMLP or IC; conversely, cells first exposed to 120 mug/ml IC, previously shown to elicit maximal Delta[Ca(2+)](i) and bactericidal functions, exhibited no subsequent Delta[Ca(2+)](i) or DeltapH(i) to either stimulus. While exposure to 10(-7) M fMLP, which saturates the PMN high-affinity receptor, did not elicit bactericidal release from these naïve unprimed PMN in suspension, 10(-5) M fMLP did, presumably via the low-affinity receptor, using a different Ca(2+) source.

Calcitonin gene related peptide and N-procalcitonin modulate CD11b upregulation in lipopolysaccharide activated monocytes and neutrophils.

OBJECTIVE: Circulating levels of calcitonin gene related peptide (CGRP) and calcitonin precursors, including procalcitonin (PCT) and its free aminopeptide N-procalcitonin (N-PCT), have been found dramatically increased in septic patients. PCT is known to attenuate the chemotaxis of monocytes in response to chemoattractants. This study examined whether CGRP and N-PCT modulate the LPS-induced expression of CD11b, which is one of the major integrins involved in monocyte and neutrophil chemotaxis during a response to microbial infections. DESIGN AND SETTING: In vitro cell culture study in the immunology laboratory of a university hospital. PARTICIPANTS: Healthy volunteers. MEASUREMENTS AND RESULTS: We assessed the effects of N-PCT and CGRP on CD11b expression on monocytes and neutrophils after LPS (2 ng/ml) or fMLP (10(-8) M) challenges. We used a human whole blood model, and measurements were made by flow cytometry. Both peptides in a dose-dependent manner decreased the LPS- and fMLP-induced rise in CD11b in monocytes and neutrophils. As these peptides are thought to act by raising cAMP, we also mimicked their effects with the use of rolipram and forskolin and found similar results. CONCLUSIONS: These findings are in line with recent studies demonstrating anti-inflammatory properties for this family of peptides. CGRP and calcitonin precursors may function as factors suppressing the propagation of inflammation through the inhibition of several processes involved during a response to a bacterial stimulus.

Cefodizime enhances phagocytosis and intracellular killing of Staphylococcus aureus but does not influence polymorphonuclear leukocytes response to fMLP stimulation.

The influence of Cefodizime (CDZ) on in vitro activity of polymorphonuclear leukocytes (PMNL) from healthy subjects was assessed. Preincubation with CDZ enhanced phagocytosis and intracellular killing of Staphylococcus aureus by PMNL. Contrary to numerous clinical reports, no significant effect of CDZ preincubation on PMNL response to n-formyl-methionyl-leucyl-phenylalanine was found with respect to intracellular calcium changes, degranulation, hydrogen peroxide production, and chemiluminescence. These results suggest that augmented microbicidal activity of PMNL is not related to the enhanced production of reactive oxygen species in healthy subjects.

Monocyte chemotactic and activating factor is a potent histamine-releasing factor for human basophils.

Recombinant monocyte chemotactic-activating factor (MCAF) has been shown to induce histamine release from human basophils with a dose response between 10(-9) and 10(-6) M. The peak of activity was reached at 10(-7) M. Histamine release by MCAF was rapid with an initial rate comparable with histamine release by an optimal dose of anti-IgE. MCAF led to peak histamine release within 1 min. 80% of the subjects tested were responsive to MCAF or anti-IgE, while all were responsive to FMLP. The percentage histamine release by MCAF was, however, less than that seen with anti-IgE or FMLP, but this was attributable to a lesser percent release in nonatopic subjects; atopic subjects responded similarly to all three agonists. MCAF was also shown to activate highly purified human basophils more readily than mixed leukocytes, and its activity was inhibited by a polyclonal rabbit antibody. At a suboptimal concentration (2.5 x 10(-9) M), MCAF was unable to prime the basophil to histamine release by other secretagogues. However, interleukin 3 (IL-3) and IL-5 could each prime basophils for MCAF-induced secretion. Therefore, our results suggest that MCAF may be a major contributor to the histamine-releasing activity seen in peripheral blood mononuclear cell supernatants that has been designated histamine releasing factor(s).

Demonstration of human, rye grass pollen extract-specific, helper and suppressor stimulating activity of modified allergens by effects on in vitro hapten-specific antibody production.

An in vivo system is described in which penicilloyl antibody was produced from peripheral leucocytes of a grass pollen-sensitive patient who had received penicillin therapy, by challenge of the cells with penicilloyl-grass pollen extract conjugate. Incubation of these leucocytes with a number of modified preparations of grass pollen extract with various T-cell-stimulating properties was shown to affect penicilloyl antibody production. Both chymotryptically fragmented rye grass pollen extract and a conjugate of f met-leu-phe and rye grass pollen extract enhanced penicilloyl-specific antibody similarly to the enhancement induced by unmodified extract, though at high concentration some suppression was seen. A conjugate of polysarcosine and rye grass pollen extract, previously shown to cause antibody suppression in mice, was similarly suppressive for penicilloyl-specific antibody. The system therefore shows potential for the evaluation of the effects of modified allergen treatment on antibody levels via T-cell mechanisms.

Induction of allergen-specific T cells by conjugates of N-formyl-methionyl-leucyl-phenylalanine and rye grass pollen extract.

A conjugate of the biologically active peptide N-formyl-methionyl-leucyl-phenylalanine and rye grass pollen extract (F-MLP/rye), previously shown to react with rye grass pollen extract-specific T cells, induced the formation of allergen-specific T cells in mice. Lymph node cells prepared from mice immunized with either native extract or F-MLP/rye gave an enhanced response to unmodified rye grass pollen allergens in vitro. Syngeneic spleen macrophages were able to present the unmodified allergens to T cells obtained from both groups of mice causing their proliferation in vitro. Conjugation of the peptide into the extract brought about an extensive reduction in its reactivity with grass pollen-specific human IgE, and a loss of its ability to induce specific IgG antibody in guinea-pigs. A state of delayed hypersensitivity specific for rye grass pollen extract was produced in guinea-pigs by immunization with either the F-MLP/rye or unmodified extract. It is concluded that conjugates such as F-MLP/rye or other T' allergoids could be used as probes to investigate whether changes in T cell activity are important in immunotherapy.

T cell reactivity of conjugates of N-formyl-methionyl-leucyl-phenylalanine and rye-grass pollen allergens.

Conjugates of rye-grass pollen extract and N-formyl-methionyl-leucyl-phenylalanine were prepared using an activated form of the tripeptide. Introduction of the peptide into the extract brought about an extensive reduction of reactivity with grass-pollen-specific IgE, as measured by RAST inhibition. Despite this loss, guinea pig alveolar macrophages and murine splenic macrophages readily presented the conjugates to T lymphocytes specific for grass pollen allergens and caused their proliferation in vitro.