Inhibition of neutrophil oxidative metabolism by lysosomotropic weak bases

Abstract

Maintenance of an acidic intralysosomal compartment may be relevant to multiple aspects of neutrophil function. The effect of lysosomal alkalinization on the neutrophil respiratory burst was studied by measuring cytochrome c reduction in response to soluble stimuli in the presence of lysosomotropic weak bases. The weak bases chloroquine, ammonium chloride, methylamine, and clindamycin all raised the intralysosomal pH and inhibited neutrophil oxidative metabolism at concentrations ranging from 0.1 to 100 mmol/L. Inhibition was dose dependent for each base and correlated significantly with the degree of lysosomal alkalinization. Concentrations that did not alkalinize the lysosome did not inhibit the respiratory burst. Inhibition by weak bases was seen when oxidative metabolism was stimulated by phorbol myristate acetate, calcium ionophore A23187, formyl-methionyl-leucyl- phenylalanine, opsonized zymosan, or sodium fluoride. Increasing the stimulus concentration (from 5 ng/mL to 5 micrograms/mL phorbol myristate acetate and from 0.5 to 1 mumol/L A23187) diminished or abolished inhibition by weak bases. Washing the cells after incubation with bases and before stimulation substantially reversed the inhibition. None of the bases impaired detection of superoxide in a cell-free xanthine-xanthine oxidase assay. Other indexes of oxidative metabolism, including oxygen consumption and hydrogen peroxide release, were also inhibited by weak bases. Analysis of particulate NADPH oxidase activity from neutrophils stimulated in the presence of bases suggested that these cells assemble a subnormal amount of an enzyme complex with normal kinetic characteristics. Lysosomotropic weak bases alkalinized the neutrophil lysosome and produced inhibition of oxidative metabolism that was dose related, was not stimulus specific, and was largely reversed by washing the cells before stimulation. A possible explanation would be altered assembly of the enzyme complex involved in respiratory burst activation as a consequence of impaired granule/plasma membrane fusion in the presence of diminished transmembrane pH gradients.

A new mechanism for anti‐inflammatory actions of proton pump inhibitors – inhibitory effects on neutrophil–endothelial cell interactions

“…demonstrated that alkalinization of lysosomes interfered with the fusion of cytoplasmic vesicles, which subsequently inhibited NADPH oxidase activation. 28 As upregulation of CD11b/CD18 also requires translocation to the cell surface by granule fusion, the inhibitory effect by proton pump inhibitors in the present study may partially depend on the prevention of lysosomal acidification. While the exact molecular mechanisms by which proton pump inhibitors inhibited the expression of CD11b/CD18 on PMN are unclear, the present results indicate that proton pump inhibitors may protect against gastroduodenal inflammation by inhibiting the infiltration of neutrophils into the extravascular space elicited by infection with H. pylori .…”

Interactions between HMR 3647, a New Ketolide, and Human Polymorphonuclear Neutrophils

“…Only L-cladinose-bearing macrolides both triggered granule exocytosis and impaired the oxidative response of PMNs. However, we have observed that HMR 3004 impairs oxidant production by PMNs (2), an effect probably related to the presence of a quinoline ring, a structure also present in various antimalarials which inhibit oxidant production by PMNs (20,22,33) and also induce PMN exocytosis (12). Unpublished observations by our group show that HMR 3004 is also able to promote PMN degranulation.…”

Cellular accumulation of the new ketolide RU 64004 by human neutrophils: comparison with that of azithromycin and roxithromycin

“…This has also been advocated as a mechanism for the concentration of some quinolone-containing antimalarial drugs inside the digestive vacuole of Plasmodium spp. (3) and in the azurophilic granules of neutrophils (33). A passive and slow egress of such protonated drugs from loaded cells was suggested to be the main (or exclusive) mechanism of efflux.…”