Divergent interactions of Ehrlichia chaffeensis- and Anaplasma phagocytophilum-infected leukocytes with endothelial cell barriers

Abstract

Human anaplasmosis (formerly human granulocytic ehrlichiosis) and human monocytic ehrlichiosis (HME) are emerging tick-borne infections caused by obligate intracellular bacteria in the family Anaplasmataceae. Clinical findings include fever, headache, myalgia, leukopenia, thrombocytopenia, and hepatic inflammatory injury. Whereas Ehrlichia chaffeensis (HME) often causes meningoencephalitis, this is rare with Anaplasma phagocytophilum infection. The abilities of infected primary host monocytes and neutrophils and of infected HL-60 cells to cross human umbilical vein endothelial cell-derived EA.hy926 cell barriers and human brain microvascular cells (BMEC), a human blood-brain barrier model, were studied. Uninfected monocyte/macrophages crossed endothelial cell barriers six times more efficiently than neutrophils. More E. chaffeensis-infected monocytes transmigrated than uninfected monocytes, whereas A. phagocytophilum suppressed neutrophil transmigration. Differences were not due to barrier dysfunction, as transendothelial cell resistivities were the same for uninfected cell controls. Similar results were obtained for HL-60 cells used as hosts for E. chaffeensis and A. phagocytophilum. Differential transmigration of E. chaffeensis- and A. phagocytophilum-infected leukocytes and HL-60 cells confirmed a role for the pathogen in modifying cell migratory capacity. These results support the hypothesis that Anaplasmataceae intracellular infections lead to unique pathogen-specific host cell functional alterations that are likely important for pathogen survival, pathogenesis, and disease induction.

FIG. 1.

Total transmigration of infected and uninfected primary monocyte/macrophage cells and neutrophils through TNF-α-stimulated in vitro models of systemic endothelial cells (EA.hy926) (A) and endothelium of the blood-brain barrier (BMEC) (B) after 18 h. The number of transmigrating cells was counted after 5 to 8 h and again after 18 h. E. chaffeensis-infected (Echaff-monocytes) and uninfected monocytes transmigrated through both endothelial cell lines to a greater degree than either uninfected or A. phagocytophilum-infected neutrophils (Aphag-neutrophils) (P < 0.001). Although E. chaffeensis-infected monocytes migrated through both endothelial cell lines more rapidly than uninfected monocytes, by 18 h the difference was significant only for BMEC (P = 0.014).

FIG. 2.

Compared to uninfected neutrophils, A. phagocytophilum infection of neutrophils (Aphag-neutrophils) suppresses transmigration through both EA.hy926 systemic endothelial cell barrier (A and B) and BMEC blood-brain barrier (B) models (P < 0.012) at 18 h. Panel B shows the neutrophil data from panel A expanded to a different scale. Note the significant difference in migration through EA.hy926 cells compared with that through BMEC cells regardless of infection status of neutrophils.

FIG. 3.

Transmigration of uninfected, A. phagocytophilum-infected (Ap-HL60), and E. chaffeensis-infected HL-60 (Ech-HL60) cells through both EA.hy926 systemic endothelial cell barrier and BMEC blood-brain barrier models mimics the findings with infected primary leukocytes. Note the significantly increased transmigration of E. chaffeensis-infected HL-60 cells through EA.hy926 endothelial cells after 5 h, compared with that of either uninfected or A. phagocytophilum-infected cells (P < 0.006) (A). Similarly, E. chaffeensis-infected HL-60 cells, but not A. phagocytophilum-infected HL-60 cells, transmigrated by 18 h through the BMEC blood-brain barrier model more than uninfected HL-60 cells (P < 0.04), regardless of pretreatment of endothelial cells with TNF-α. A. phagocytophilum-infected HL-60 cells migrated through both EA.hy926 and BMEC endothelial cells to the same degree or less than uninfected cells (B). The results are averages of triplicate cultures and are representative of at least four replicated experiments.