Bactericidal activity of human eosinophilic granulocytes against Escherichia coli

Eosinophils participate in allergic inflammation and may have roles in the bodys defense against helminthic infestation. Even under noninflammatory conditions, eosinophils are present in the mucosa of the large intestine, where large numbers of gram-negative bacteria reside. Therefore, roles for eosinophils in host defenses against bacterial invasion are possible. In a system for bacterial viable counts, the bactericidal activity of eosinophils and the contribution of different cellular antibacterial systems against Escherichia coli were investigated. Eosinophils showed a rapid and efficient killing of E. coli under aerobic conditions, whereas under anaerobic conditions bacterial killing decreased dramatically. In addition, diphenylene iodonium chloride (DPI), an inhibitor of the NADPH oxidase and thereby of superoxide production, also significantly inhibited bacterial killing. The inhibitor of nitric oxide (NO) production L-N5-(1-iminoethyl)-ornithine dihydrochloride did not affect the killing efficiency, suggesting that NO or derivatives thereof are of minor importance under the experimental conditions used. To investigate the involvement of superoxide and eosinophil peroxidase (EPO) in bacterial killing, EPO was blocked by azide. The rate of E. coli killing decreased significantly in the presence of azide, whereas addition of DPI did not further decrease the killing, suggesting that superoxide acts in conjunction with EPO. Bactericidal activity was seen in eosinophil extracts containing granule proteins, indicating that oxygen-independent killing may be of importance as well. The findings suggest that eosinophils can participate in host defense against gram-negative bacterial invasion and that oxygen-dependent killing, i.e., superoxide acting in conjunction with EPO, may be the most important bactericidal effector function of these cells

Chemiluminescence in activated human neutrophils

Human neutrophils (PMNs) suspended in Hanks' balanced salt solution (HBSS), which are stimulated either by polycation-opsonized streptococci or by phorbol myristate acetate (PMA), generate nonamplified (CL), luminol-dependent (LDCL), and lucigenin-dependent chemiluminescence (LUCDCL). Treatment of activated PMNs with azide yielded a very intense CL response, but only a small LDCL or LUCDCL responses, when horse radish peroxidase (HRP) was added. Both CL and LDCL depend on the generation of Superoxide and on myeloperoxidase (MPO). Treatment of PMNs with azide followed either by dimethylthiourea (DMTU), deferoxamine, EDTA, or detapac generated very little CL upon addition of HRP, suggesting that CL is the: result of the interaction among H 2 O 2 , a peroxidase, and trace metals. In a cell-free system practically no CL was generated when H 2 O 2 was mixed with HRP in distilled water (DW). On the other hand significant CL was generated when either HBSS or RPMI media was employed. In both cases CL was markedly depressed either by deferoxamine or by EDTA, suggesting that these media might be contaminated by trace metals, which catalyzed a Fenton-driven reaction. Both HEPES and Tris buffers, when added to DW, failed to support significant HRP-induced CL. Nitrilotriacetate (NTA) chelates of Mn 2+ , Fe 2+ , Cu 2+ , and Co 2+ very markedly enhanced CL induced by mixtures of H 2 O 2 and HRP when distilled water was the supporting medium. Both HEPES and Tris buffer when added to DW strongly quenced NTA-metal-catalyzed CL. None of the NTA-metal chelates could boost CL generation by activated PMNs, because the salts in HBSS and RPMI interfered with the activity of the added metals. CL and LDCL of activated PMNs was enhanced by aminotriazole, but strongly inhibited by diphenylene iodonium (an inhibitor of NADPH oxidase) by azide, sodium cyanide (CN), cimetidine, histidine, benzoate, DMTU and moderately by Superoxide dismutase (SOD) and by deferoxamine. LUCDCL was markedly inhibited only by SOD but was boosted by CN. Taken together, it is suggested that CL generated by stimulated PMNs might be the result of the interactions among, NADPH oxidase, (inhibitable by diphenylene iodonium), MPO (inhibitable by sodium azide), H 2 O 2 probably of intracellular origin (inhibitable by DMTU but not by catalase), and trace metals that contaminate salt solutions. The nature of the salt solutions employed to measure CL in activated PMNs is critical.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/44510/1/10753_2004_Article_BF00918987.pd