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.An emerging tick-borne zoonosis caused by the human granulocytic ehrlichiosis (HGE) agent was first described in 1994 (5) and has been increasingly recognized in the United States and several European countries. HGE is a disease characterized by systemic illness such as fever, chills, headache, malaise, and/or myalgia. Laboratory tests may reveal thrombocytopenia, leukopenia, elevated C-reactive protein levels, and elevated liver enzyme activities. The HGE agent is a unique obligatory intracellular bacterium that replicates in neutrophils. Neutrophils have a strong ability to kill invading microorganisms through activation of the NADPH oxidase, which rapidly generates superoxide anion (O 2 Ϫ ) and subsequent formation of other bactericidal reactive oxygen intermediates (hydrogen peroxide, myeloperoxide, hypochlorous acid, hydroxyl radical, or longer-lived N-chloroamines) (1, 7, 9). Recently Banerjee et al. (4) reported that after 5 days of infection with the HGE agent, HL-60 cells (a human promyelocytic leukemia cell line) exhibited a lack of luminol-dependent chemiluminescence (LDCL) response to phorbol myristate acetate (PMA) and down regulation of mRNA of gp91 phox , one component of the NADPH complex. However, for the HGE agent to survive in neutrophils, this inhibition must occur immediately rather than 5 days after establishment of infection. Although use of neutrophils is more difficult than use of cell lines, we determined whether human peripheral blood neutrophils produce O 2 Ϫ upon exposure to the HGE agent. Since we found that the HGE agent does not induce O 2 Ϫ generation, we determined whether this is a generalized and/or active inhibition and whether both intracellular and extracellular O 2 Ϫ generation is prevented. We further examined what type of ehrlichial factors and inter...

Insights into Pathogen Immune Evasion Mechanisms: Anaplasma phagocytophilum Fails to Induce an Apoptosis Differentiation Program in Human Neutrophils

“…1C). These findings are consistent with an earlier report that demonstrated that inhibition of neutrophil superoxide generation is not dependent on viable or structurally intact bacteria (10).…”

“…1C). As with previous studies (10,11,13), neither live nor heat-killed pathogens elicited significant PMN ROS production compared with either PMA or S. aureus (Fig. 1C).…”

“…This observation suggests that there is an A. phagocytophilum-specific response, very different from that in bacteria studied to date (e.g., S. aureus), which most likely involves in part a bacterial surface structure or protein. The observation that a bacterial surface structure is sufficient to alter PMN function has been reported by others (6,10). At 9 h, over one-half of the genes differentially regulated by live A. phagocytophilum were similarly altered by heat-killed pathogen.…”

Effects ofAnaplasma phagocytophilaon NADPH Oxidase Components in Human Neutrophils and HL-60 Cells

“…The inhibition requires the carbohydrate rather than the protein residue of A. phagocytophila as well as A. phagocytophila and host cell contact (22). The inhibition is specific to neutrophils and does not occur with human monocytes (only a few minutes' delay in O 2 Ϫ release), and it requires at least 30 min of preincubation of A. phagocytophila and neutrophils, as well as host cell protein synthesis, for the complete inhibition (22).…”

“…We have previously demonstrated that A. phagocytophila not only does not induce O 2 Ϫ generation by human peripheral blood neutrophils but also rapidly (within 30 min) deprives neutrophils of the ability to generate O 2 Ϫ in response to a variety of potent stimuli, such as phorbol myristic acetate (PMA), formylmethionyl-leucyl-phenylalanine (fMLP), or Escherichia coli as determined by both ferricytochrome c reduction and luminol-dependent chemiluminescent assays (22).…”