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...