Leukocyte infection by the granulocytic ehrlichiosis agent is linked to expression of a selectin ligand

Abstract

Human granulocytic ehrlichiosis (HGE) is a newly recognized tickborne infection of humans (1-3). HGE is a febrile illness characterized by reductions in blood leukocyte and platelet counts and the presence of colonies of infecting organisms (morulae) proliferating within cytoplasmic vacuoles of circulating neutrophils. No other organism is known to replicate within human granulocytes. Our laboratory recently isolated and propagated the causative agent of this disease using the human promyelocytic leukemia cell line HL60 (4). In addition, we found that the agent can grow in primary bone marrow granulocytic and monocytic precursors, blood neutrophils, and nonactivated monocytes (5).The clinical and in vitro tropism of this obligate intracellular parasite for myelomonocytic cells suggested that specific cell-surface molecules are required for adhesion, cell signaling, and entry into a permissive intracellular compartment. Of interest, sialyl Lewis x ([NeuAcα(2− 3)GalΒ1−4(Fucα1-3)GlcNAc, or CD15s]), which serves as a ligand to initially tether leukocytes to endothelial cell Eselectin (6, 7), is richly expressed on both HL60 cells and on those bone marrow and blood cells permissive for the HGE agent (8-11), where it modifies a variety of lipid and protein molecules. We hypothesized that cell-surface sialylated Lewis x might be important for infection by HGE. Methods HGE infection, cultivation, and antibody blocking studies.A cell-free bacterial inoculum of HGE was prepared from fully infected HL60 cells, propagated as described (4). The infected cells, at a density of 10 6 per ml, were passed through a 25-gauge needle three times, and cellular debris were removed by centrifugation at 700 g for 5 min. The supernatant was centrifuged at 1,236 g for 5 min, and the resultant bacterial pellet was resuspended in 10 µl supernatant per ml of the original culture. Counting in a Petroff-Hausser chamber revealed ∼10 7 organisms per 10 µl. Infections were carried out by coincubating 10 µl of this cell-free HGE preparation with 10 6 cells for a multiplicity of infection of ∼10 organisms per cell used in all experiments. For studies to determine whether bacterial binding requires calcium, both HL60 cells and the bacterial inoculum were separately incubated in HBSS with or without calcium for 20-70 min before coincubation, as described later here. For blocking studies, 10 µg (or the quantity specified) of the monoclonal antibody (MAB) tested (or control diluent or murine IgM alone) was added to 1 ml of 10 6 HL60 cells (or bone marrow progenitors, peripheral blood monocytes, or granulocytes, prepared as described; ref. 5) in medium and incubated at room temperature for 1 h. Blocking studies were performed six times with similar results. As noted, antibody blocking studies were also performed at 4°C or 37°C. Cells were centrifuged at 200 g for 5 min, 900 µl of supernatant saved, the cells resuspended in the 100 µl of medium remaining, and 10 µl of the HGE bacteria added and then coincubated for 15 min at room temperature. The cells w...

Structurally Distinct Requirements for Binding of P-selectin Glycoprotein Ligand-1 and Sialyl Lewis x to Anaplasma phagocytophilum and P-selectin

“…2D). Chelation of divalent cations with EDTA had no effect on binding, as observed previously (25) for bacterial binding to HL-60 cells. This contrasts with the Ca 2ϩ -dependent binding of selectins to PSGL-1 and other sLe x -capped glycoconjugates (11,12).…”

“…Binding of A. phagocytophilum to human myeloid HL-60 cells requires cell surface sialylation and correlates with expression of FTVII (25). Moreover, mAbs to N-terminal epitopes on human PSGL-1 inhibit binding of A. phagocytophilum to human neutrophils or HL-60 cells (26).…”

Human Granulocytic Ehrlichiosis Agent Inhibits Superoxide Anion Generation by Human Neutrophils

“…The HGE agent completely inhibited O 2 Ϫ release by neuraminidasetreated neutrophils in the presence or absence of PMA. Unlike the case for HL-60 cells (10), treatment of neutrophils with neuraminidase did not inhibit infection by the HGE agent. Following 16 h of incubation, distinct morulae (microcolonies of ehrlichiae) were present in 6.2% Ϯ 0.5% and 4.1% Ϯ 0.5% (n ϭ 3) of nontreated and treated neutrophils, respectively.…”

“…A recent study by Goodman et al (10) has shown that treatment of HL-60 cells with neuraminidase prevents both binding and infection by the HGE agent. To examine whether an external sialic acid on the neutrophil surface is required for inhibition of O 2 Ϫ release by the HGE agent, neutrophils were pretreated with neuraminidase prior to incubation with the HGE agent.…”

Kinetics of CD11b/CD18 Up-Regulation During Infection with the Agent of Human Granulocytic Ehrlichiosis in Mice

“…One potential direct mechanism by which bacterial contact or infection may result in CD11b/CD18 up-regulation is via PSGL-1. It was recently demonstrated that the aoHGE directly infects human neutrophils via receptor-mediated endocytosis using PSGL-1 as a receptor (Goodman et al, 1999;Herron et al, 2000). As stimulation of PSGL-1 activates mouse neutrophils, it is indeed possible that binding of aoHGE is an initiating event that leads to cellular activation and increased expression of CD11b/CD18 on the host cell surface (Blanks et al, 1998).…”