Infection of endothelial cells with Anaplasma marginale and A. phagocytophilum

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Abstract

Anaplasma marginale and A. phagocytophilum are obligate intracellular, tick-borne pathogens that target erythrocytes and neutrophil granulocytes, respectively. Because ticks do not directly tap blood vessels, an intermediate tissue may mediate infection of blood cells. We considered that vascular endothelium interacts with circulating blood cells in vivo, and could be involved in pathogenesis and dissemination of the organisms. We used light and electron microscopy and immune labeling to show that A. phagocytophilum invaded rhesus (RF/6A), human (HMEC-1, MVEC), as well as bovine (BCE C/D-1b) endothelial cell lines, whereas A. marginale infected rhesus and bovine endothelial cells. A. marginale formed large intracellular inclusions that appeared smooth and solid at first, and subsequently coalesced into discrete granules. A. phagocytophilum formed numerous smaller inclusions in each cell. Within 1–3 weeks, the monolayers were destroyed, and lysed cultures were diluted onto fresh monolayers. Electron microscopy demonstrated uneven distribution of A. marginale inside large inclusions, with reticulated forms grouped more tightly than denser cells, whereas in A. phagocytophilum individual organisms appeared more evenly spaced. Specific polyclonal and monoclonal antibodies both labeled A. marginale and A. phagocytophilum in endothelial cells, and oligonucleotide primers complimentary to either A. marginale or A. phagocytophilum amplified their expected target from these cultures. In conclusion, we demonstrate that relevant microvascular endothelium is susceptible to anaplasmas in vitro and may present a link that could explain development of the immune response and persistent infection.

Introduction

Anaplasma marginale (Theiler, 1910) and Anaplasma phagocytophilum (Dumler et al., 2001) are closely related, obligate intracellular, tick-borne rickettsial animal pathogens that parasitize two very different host cell types. Only ruminants are known to be susceptible to A. marginale (Kuttler, 1984) whereas the spectrum of species that can be infected with the zoonotic A. phagocytophilum includes small rodents, ruminants, dogs, horses and humans (Dumler et al., 2001). A. marginale is thought to exclusively infect bovine erythrocytes. Acute bovine erythrocytic anaplasmosis is characterized by severe anemia, icterus and hemoglobinuria due to removal of infected cells (de la Fuente et al., 2002). Animals that recover remain chronically infected for life, and experience regular, but low level waves of parasitemia (Barbet et al., 2000). Each rickettsemic peak is composed of new antigenic variants that are selected following a specific antibody response by the host. By contrast, A. phagocytophilum infections produce an acute, febrile illness accompanied by appearance of characteristic colonies of the microbes (referred to as morulae) in peripheral blood neutrophil granulocytes as well as their precursors in the bone marrow (Chen et al., 1994, Klein et al., 1997) with concomitant impairment of resistance to secondary infection (Gokce and Woldehiwet, 1999).

A. phagocytophilum can be cultured in vitro in a human promyelocytic cell line, HL-60 (Goodman et al., 1996), as well as in cell lines ISE6 and IDE8 from the North American tick vector, Ixodes scapularis (Munderloh et al., 1996b, Munderloh et al., 1999). These cultures have facilitated the identification of the cell surface receptor complex for A. phagocytophilum on granulocytes and promyelocytic cell lines (Goodman et al., 1999, Herron et al., 2000), and allowed an in vitro analysis of the cycling of the pathogen between human patient and arthropod vector. Thus, host cell dependent differential expression of specific antigens (DESA) was demonstrated (Jauron et al., 2001). A. marginale can likewise be propagated in Ixodes tick cell lines (Munderloh et al., 1996a), and the subunits of the heterodimeric major surface protein, MSP1, of A. marginale have been identified as ligands to both erythrocytes and tick cells (MSP1a), or erythrocytes alone (MSP1b; de la Fuente et al., 2001). However, a continuous mammalian culture system has been lacking for A. marginale, and the corresponding cell surface receptors are unknown. Moreover, no nucleated host cells of A. marginale have been identified, and none of the cell lines that support A. phagocytophilum could be infected with A. marginale (our unpublished results).

Because ticks do not directly pierce blood vessels, but instead feed on a pool of tissue fluid or blood that seeps from damaged capillaries, we considered that transfer of Anaplasma microorganisms from tick saliva to their ultimate destination in blood cells was likely to involve a type of antigen-presenting cell closely associated with the tick bite site. Transient infection of endothelial cells with A. marginale has been reported before, but in these cultures, the organisms did not persist and disappeared (Blouin et al., 1993, Waghela et al., 1997). Here, we describe, for the first time, productive infection of bovine and primate microvascular endothelial cell lines with A. marginale and A. phagocytophilum, and continuous cultivation of these microbes in rhesus and human endothelial lines.

Section snippets

Host cell lines

Tick cell line ISE6, from embryos of the black-legged tick, I. scapularis, was used for propagation of both A. marginale and A. phagocytophilum (Munderloh et al., 1996a, Munderloh et al., 1996b, Munderloh et al., 1999). Uninfected cells were grown in L15B300 with 5% tryptose phosphate broth (Difco Laboratories, Detroit, MI, USA), 5% heat-inactivated fetal bovine serum (FBS, Harlan, Indianapolis, IN, USA), and 0.1% bovine lipoprotein concentrate (ICN, Irvine, CA, USA), pH 7.2. Medium for

Growth of Anaplasma in endothelial and turbinate cell lines

Both A. marginale and A. phagoytophilum that had been continuously propagated in I. scapularis cell line ISE6, when inoculated onto RF/6A rhesus and BCE C/D-1b bovine endothelial cell layers, invaded these cells and replicated inside intracellular inclusions, whereas only A. phagocytophilum infected HMEC-1 human cells. After the initial inoculation with infected tick cells, inclusions of either Anaplasma sp. could be detected by phase contrast microscopy in RF/6A cells within several days. The

Discussion

This report demonstrates that both A. marginale and A. phagocytophilum readily infect bovine and primate microvascular endothelial cell lines. Invasion occurred rapidly, and replication was prolific. Other cell lines tested, i.e., bovine turbinate cells, line BT, and the cow pulmonary aorta endothelium cell line CPAE, did not support continuous development of either Anaplasma, in agreement with results reported previously (Blouin et al., 1993). Microvascular endothelial cells could play a role

Acknowledgements

The research presented in this publication was supported by Public Health Service grants from NIH (AI42792 and AI40952) to J.L. Goodman and U.G. Munderloh.

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    Present address: Department of Pedriatics, University of Minnesota, 420 Delaware Street SE, Minneapolis, MN 55455, USA.

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