Human granulocytic ehrlichiosis agent inhibits superoxide anion generation by human neutrophils

Abstract

The human granulocytic ehrlichiosis (HGE) agent, which replicates in neutrophils, was found not to induce superoxide anion (O(2)(-)) generation or extracellular release by human peripheral blood neutrophils, as measured by a luminol-dependent chemiluminescence assay or a cytochrome c reduction assay, respectively. Furthermore, the HGE agent completely prevented O(2-) release by neutrophils upon stimulation with phorbol myristate acetate (PMA), formylmethionyl-leucyl-phenylalanine, or Escherichia coli. The inhibition was HGE agent dose dependent, required ehrlichial contact with the host cells, and was reversible upon removal of the extracellular HGE agent bound to the host cells prior to PMA stimulation. Structural integrity of or new protein synthesis by the HGE agent was not required for the inhibition; carbohydrate but not surface protein of the HGE agent was required. The HGE agent did not prevent O(2-) generation in human peripheral blood monocytes derived from the same individual. This neutrophil-specific prevention of O(2-) generation by the HGE agent would be critical in survival of the HGE agent. This is the first demonstration of the rapid inhibition of preexisting NADPH oxidase in human neutrophils by the HGE agent.

FIG. 1

The HGE agent blocks O₂⁻ release induced by PMA, E. coli, or fMLP. Neutrophils were incubated with or without PMA, E. coli, or fMLP in the presence or absence of the HGE agent. O₂⁻release was determined by measuring the reduction of ferricytochrome c after 2 h of stimulation. Bars represent means ± standard deviations (n = 3). ∗, P < 0.01 compared to neutrophil-alone control. (Student's t test).

FIG. 2

Time course of inhibition of total (extra- and intracellular) O₂⁻generation induced by PMA or E. coli by the HGE agent as measured by LDCL assay. Neutrophils were stimulated with PMA (A) or E. coli (B) in the presence or absence of the HGE agent. Results are representative of three independent experiments. CL, chemiluminescence.

FIG. 3

Inhibition of O₂⁻ release by the HGE agent is dose dependent. Neutrophils were incubated with decreasing numbers of host cell-free HGE agent with or without PMA. O₂⁻ secretion was determined by measuring the reduction of ferricytochrome c. Bars represent means ± standard deviations (n = 3). Results are representative of three independent experiments. ∗, P < 0.01 compared to neutrophil-alone (no PMA) control (Student's t test).

FIG. 4

HGE agent inhibition of superoxide release in response to PMA is not abrogated by pretreatment of neutrophils with neuraminidase type X. Neutrophils were treated with neuraminidase type X for 2 h prior to incubation with host cell-free HGE agent. PMA was then added to stimulate release of O₂⁻. O₂⁻ production was determined by measuring the reduction of ferricytochrome c. Bars represent means ± standard deviations (n = 3). Results are representative of three independent experiments. ∗, P < 0.01 compared to neutrophil-alone control (Student's t test).

FIG. 5

HGE agent inhibition of O₂⁻ secretion in response to PMA is partially blocked by cycloheximide but not oxytetracycline treatment. Neutrophils were incubated with cycloheximide (CHI) 30 min prior to addition of the HGE agent, or the HGE agent was incubated with oxytetracycline (OTC) for 30 min prior to addition of neutrophils. Mixtures were incubated with or without PMA stimulation in the presence of cycloheximide or oxytetracycline, and O₂⁻ release was determined by measuring the reduction of ferricytochrome c after 2 h of PMA stimulation. Bars represent means ± standard deviations (n = 3). Results are representative of three independent experiments. ∗, P < 0.01 compared to neutrophil-alone control (Student's t test).

FIG. 6

Lack of inhibition of PMA-induced total (extra- and intracellular) O₂⁻ generation in mononuclear cells by the HGE agent as measured by LDCL. Human peripheral blood mononuclear cells were stimulated with PMA in the presence or absence of the HGE agent. The neutrophil response from the same donor to the same stimulus is shown in the inset. Results are representative of three independent experiments. CL, chemiluminescence.