leukotriene-b4 and pepstatin

Eotaxin is a potent eosinophil chemoattractant that acts selectively through CCR3, which is expressed on eosinophils, basophils, mast cells, and Th2-type T cells. This arm of the immune system is believed to have evolved to control helminthic parasites. We hypothesized that helminths may employ mechanisms to inhibit eosinophil recruitment, to prolong worm survival in the host. We observed that the excretory/secretory products of the hookworm Necator americanus inhibited eosinophil recruitment in vivo in response to eotaxin, but not leukotriene B(4), a phenomenon that could be prevented by the addition of protease inhibitors. Using Western blotting, N. americanus supernatant was shown to cause rapid proteolysis of eotaxin, but not IL-8 or eotaxin-2. N. americanus homogenate was fractionated by gel filtration chromatography, and a FACS-based bioassay measured the ability of each fraction to inhibit the activity of a variety of chemokines. This resulted in two peaks of eotaxin-degrading activity, corresponding to approximately 15 and 50 kDa molecular mass. This activity was specific for eotaxin, as responses to other agonists tested were unaffected. Proteolysis of eotaxin was prevented by EDTA and phenanthroline, indicating that metalloprotease activity was involved. Production of enzymes inactivating eotaxin may be a strategy employed by helminths to prevent recruitment and activation of eosinophils at the site of infection. As such this represents a novel mechanism of regulation of chemokine function in vivo. The existence of CCR3 ligands other than eotaxin (e.g., eotaxin-2) may reflect the evolution of host counter measures to parasite defense systems.

Topics: Animals; Antigen-Antibody Reactions; Biological Assay; Cell Migration Inhibition; Chemokine CCL11; Chemokines, CC; Chemotactic Factors, Eosinophil; Cytokines; Enzyme Activation; Enzyme-Linked Immunosorbent Assay; Eosinophils; Guinea Pigs; Helminth Proteins; Humans; Hydrolysis; Injections, Intradermal; Kinetics; Leukotriene B4; Metalloendopeptidases; Necator americanus; Pepstatins; Phenanthrolines; Protease Inhibitors; Substrate Specificity

Exposure of polymorphonuclear neutrophils (PMNs) to phorbol 12-myristate 13-acetate (PMA) resulted in a concentration-dependent (1-10 ng/ml) inhibition of granule exocytosis induced with the receptor-specific ligands, N-formyl-methionyl-leucyl-phenylalanine (FMLP), pepstatin A, 5(S),12(R)-dihydroxy-6,14-cis-8,10-trans-eicosatetraenoic acid (LTB4), and acetyl-sn-glyceryl-3-phosphorylcholine (AGEPC). PMA exerted a marginal inhibitory effect on calcium ionophore A23187-induced PMN degranulation, and the PMA analog, 4 alpha-phorbol 12,13-didecanoate (4 alpha-PDD), was inactive. However, PMA potentiated AGEPC, pepstatin A, FMLP, LTB4, and A23187-stimulated superoxide anion (O2-) production. The mobilization of intracellular sequestered calcium (Ca2+) by the receptor-specific ligands, as reflected by a rise in the cytosolic-free Ca2+ concentration ([Ca2+]i) in PMNs loaded with the Ca2+-sensitive dye, Fura-2, was suppressed by PMA. A protein kinase C (PKC) inhibitor, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7) reversed the PMA-mediated inhibition of PMN degranulation and intracellular CA2+ mobilization. However, another, but less potent PKC inhibitor, N-(2-guanidino-ethyl)-5-isoquinolinesulfonamide (HA1004), had no effect on the inhibition of PMN activation by PMA.

Topics: Exocytosis; Humans; Leukotriene B4; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils; Pepstatins; Platelet Activating Factor; Protein Kinase C; Signal Transduction; Superoxides; Tetradecanoylphorbol Acetate