Primary Bone Marrow Progenitors of Both Granulocytic and Monocytic Lineages Are Susceptible to Infection with the Agent of Human Granulocytic Ehrlichiosis

Klein, Marina B.; Miller, Jeffrey S.; Nelson, Curtis M.; Goodman, Jesse L.

doi:10.1086/517332

Cited by 71 publications

(55 citation statements)

References 14 publications

(7 reference statements)

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“…The VIM-2 MAB, directed against the closely related sialylated CDw65 carbohydrate moiety, had irregular blocking activity noted only at the highest concentration tested (40 µg/ml), with a maximal reduction in infection to 60% of the level seen in untreated controls, suggesting either nonspecific effects or biologic cross-reactivity of high concentrations of VIM-2 against CD15s (22). Anti-CD15s antibody was also tested for its ability to block infection of those nontransformed cells likely to be natural targets of the HGE agent: human marrow progenitors and peripheral blood monocytes and granulocytes (5). As observed in HL60 cells, 10 µg/ml of MAB CSLEX1 prevented infection of all of these cell types (P < 0.001 vs. untreated controls).…”

Section: Resultsmentioning

confidence: 99%

“…This procedure killed any remaining infectious extracellular organisms (data not shown), thus enabling assessment of the effects of antibody on intracellular organisms alone. Cells were incubated as described (4,5) and assessed for infection (see later here) daily.…”

Section: Hge Infection Cultivation and Antibody Blocking Studiesmentioning

confidence: 99%

“…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).…”

Section: Introductionmentioning

confidence: 99%

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

et al. 1999

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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...

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Section: Resultsmentioning

confidence: 99%

Section: Hge Infection Cultivation and Antibody Blocking Studiesmentioning

confidence: 99%

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

et al. 1999

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“…12 The finding of morulae within monocytes was interesting and was similar to some human cases of HGE (Dumler SJ, unpublished data) but infrequently reported in the literature. However, Klein and others 20 reported that bone marrow monocyte progenitor cells became infected with the HGE agent in vitro.…”

Section: Discussionmentioning

confidence: 99%

A simian model of human granulocytic ehrlichiosis.

Foley

Lerche²,

Dumler

et al. 1999

The American Journal of Tropical Medicine and Hygiene

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Abstract. Following intravenous inoculation with horse blood-infected with the agent of human granulocytic ehrlichiosis (HGE) from a human fatality, two rhesus macaques (Macaca mulatta) exhibited pyrexia and lethargy on days 4-12 postinfection (PI). Hematology revealed neutropenia, thrombocytopenia, and anemia, with ehrlichial morulae in monocytes and neutrophils on days 4-12. Blood was polymerase chain reaction (PCR)-positive on days 4-12 and bone marrow was PCR-positive on day 11. There was a minor increase in gamma-glutamyl transpeptidase on day 12 and serum interferon-␥ levels increased by day 18. Seroconversion occurred on day 20 PI to a titer of 100 by day 22. Western blot bands characteristic of HGE included 25-, 44-, 80-, 94-, 105-, and 125-kD 10 The development of clinical signs appears to be largely host-species dependent. An early attempt to infect non-human primates with Ehrlichia equi was successful based on visualization of ehrlichial inclusions within the cytoplasm of neutrophils.3 However, that study was hampered by the lack of some diagnostic procedures such as the polymerase chain reaction (PCR) and DNA sequencing, cytokine assays, and other immunologic assays. Moreover, the investigators did not know at that time that E. equi or its close relative, the agent of human granulocytic ehrlichiosis (HGE), was capable of producing life-threatening disease in humans. 1 In the present study, we report successful experimental infection of rhesus macaques with the HGE agent from a severely ill human case. This animal model of human disease is important for further studies of HGE diagnosis, management, and pathogenesis. MATERIALS AND METHODS Animals and inoculation.Three rhesus macaques were pre-screened for ehrlichiosis by serology and E. equi-specific PCR. Monkey 312 was a 16-year-old reproductively intact female who had been used previously for breeding and a teratogenesis study (zinc deprivation). Monkey 121 was a 17-year-old intact female with a history of use for breeding and anti-progestin drug studies. Monkey 324 was a 14-yearold intact female from the breeding colony. Monkeys 121 and 312 were inoculated intravenously with fresh horse blood containing 2 ϫ 10 6 neutrophils of which 15% contained HGE agent strain BDS inclusions (morulae); monkey 324 was control-inoculated with 2 ϫ 10 6 uninfected horse neutrophils. All monkeys were monitored twice a day for clinical signs of disease, including lethargy, inappetance, and depression. Rectal temperature was assessed at the time of each blood sample collection. Blood samples were collected every two days into tubes containing EDTA for Wright-Giemsa staining and PCR and without anticoagulant for serology and serum chemistry. Bone marrow aspirates were performed on days 3, 11, and 14 postinfection (PI) and submitted for cytology and PCR. On day 21 PI, monkeys 121 and 324 were killed with an intravenous overdose of barbiturate and necropsied. Samples from tissues were either snap-frozen in liquid nitrogen for PCR or fixed overnight in 10% buffered forma...

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“…In some assays, HL-60 cells were exposed to heat-killed HGE bacteria for 24 h or to supernatant (10 ml of supernatant from Ehrlichia-infected HL-60 cells) for 5 days. HL-60 cells (2 ϫ 10 5 /ml) were incubated at 37°C in 5% CO 2 with 1 M retinoic acid and cultured for 6 days for maximum differentiation (3,16,17). Superoxide anion was measured in both control and infected (both uninduced and retinoic acidinduced) HL-60 cells.…”

Section: Cultivation Of the Hge Agent And Superoxide Releasementioning

confidence: 99%

Cutting Edge: Infection by the Agent of Human Granulocytic Ehrlichiosis Prevents the Respiratory Burst by Down-Regulatinggp91phox

Banerjee

Anguíta

Roos

et al. 2000

The Journal of Immunology

113

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The agent of human granulocytic ehrlichiosis (HGE) is an emerging tick-borne pathogen that resides in neutrophils and can be cultured in a promyelocytic (HL-60) cell line. In response to microbes, polymorphonuclear leukocytes normally activate the NADPH oxidase enzyme complex and generate superoxide anion (O2−). However, HL-60 cells infected with HGE bacteria did not produce O2− upon activation with PMA. RT-PCR demonstrated that HGE organisms inhibited mRNA expression of a single component of NADPH oxidase, gp91phox, and FACS analysis showed that plasma membrane-associated gp91phox protein was reduced on the infected cells. Infection with HGE organisms also decreased gp91phox mRNA levels in splenic neutrophils in a murine model of HGE, demonstrating this phenomenon in vivo. Therefore, HGE bacteria repress the respiratory burst by down-regulating gp91phox, the first direct inhibition of NADPH oxidase by a pathogen.

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Primary Bone Marrow Progenitors of Both Granulocytic and Monocytic Lineages Are Susceptible to Infection with the Agent of Human Granulocytic Ehrlichiosis

Cited by 71 publications

References 14 publications

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

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

A simian model of human granulocytic ehrlichiosis.

Cutting Edge: Infection by the Agent of Human Granulocytic Ehrlichiosis Prevents the Respiratory Burst by Down-Regulatinggp91phox

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