Diminished adhesion of Anaplasma phagocytophilum-infected neutrophils to endothelial cells is associated with reduced expression of leukocyte surface selectin

Abstract

Anaplasma phagocytophilum propagates within neutrophils and causes a disease marked by inflammatory tissue injury or complicated by opportunistic infections. We hypothesized that infection with A. phagocytophilum modifies the binding of neutrophils to endothelial cells and the expression of neutrophil adhesion molecules and studied these changes in vitro. Infected dimethyl sulfoxide-differentiated HL-60 cells and neutrophils showed reduced binding to cultured brain and systemic endothelial cells and lost expression of P-selectin glycoprotein ligand 1 (PSGL-1, CD162) and L-selectin (CD62L) (to 33 and 5% of control values, respectively), at a time when the levels of beta(2) integrin and immunoglobulin superfamily adhesion molecules and activation markers Mac-1 and intercellular adhesion molecule 1 increased (5 to 10 times that of the control). The loss of CD162 and CD62L expression was inhibited by EDTA, which suggests that neutrophil activation and sheddase cleavage occurred. The loss of selectin expression and the retained viability of the neutrophils persisted for at least 18 h with A. phagocytophilum infection, whereas Escherichia coli and Staphylococcus aureus rapidly killed neutrophils. The adhesion defect might increase the numbers of infected cells and their persistence in the blood prior to tick bites. However, decreased CD162 expression and poor endothelial cell binding may partly explain impaired host defenses, while simultaneous neutrophil activation may aggravate inflammation. These observations may help us to understand the modified biological responses, host inflammation, and immune response that occur with A. phagocytophilum infections.

FIG. 1.

A. phagocytophilum infection of neutrophils and DMSO-differentiated HL-60 cells inhibits adhesion to rTNF-α-activated endothelial cell monolayers under static and low-shear-force conditions. Uninfected and infected cells were labeled with PKH67 green fluorescent dye and incubated on confluent endothelial cell monolayers for 1 h at 37°C (static assay) or for 45 min at 4°C under static conditions followed by 15 min on a rotating platform at 4°C (low-shear-force assay), after which nonadherent cells were removed. The fluorescence retained was measured and expressed as a percentage of that of the cells adherent to monolayers treated with uninfected neutrophils or DMSO-differentiated HL-60 cells. Cells were described as having a low infection rate when 10 to 20% of the HL-60 cells contained morulae and a high infection rate when >70 to 80% were infected. Error bars indicate standard errors of the means; static, static assay; shear force, low-shear-force assay. (A) Adhesion of A. phagocytophilum-infected DMSO-differentiated HL-60 cells and infected neutrophils to rTNF-α-activated human BMEC (Neutrophils-1 and -2 indicate the results of two representative experiments). (B) Adhesion of A. phagocytophilum-infected neutrophils and DMSO-differentiated HL-60 cells to rTNF-α-activated EA.hy926 cells (HL-60 static-1 and static-2 indicate the results of two representative experiments). (C) Adhesion of uninfected DMSO-differentiated HL-60 cells to rTNF-α-activated human BMEC and EA.hy926 cells under low shear force is inhibited (P < 0.03) by the function-blocking CD162 MAbs KPL-1 and PL-1. Separate experiments are designated by the endothelial cell line used and the suffix -1 or -2. KPL-1+PL-1, mixture of both blocking antibodies.

FIG. 2.

Changes in expression of CD162 and CD62L on A. phagocytophilum-infected cells. Surface expression of CD162 and CD62L was analyzed by flow cytometry. (A and B) Neutrophils were either not stimulated or stimulated with LPS (10 μg/ml) or live bacteria (E. coli, S. aureus) and infected with cell-free A. phagocytophilum or stimulated with PFA-fixed A. phagocytophilum for 3 h at 37°C. Surface expression of both CD162 (A) and CD62L (B) was decreased on A. phagocytophilum-infected neutrophils. (C) HL-60 cells were cultivated with 1.25% DMSO for 3 days to induce granulocytic differentiation and to achieve various degrees of infection. Expression of CD162 was reduced in A. phagocytophilum-infected DMSO-differentiated HL-60 cells after 3 days. SSC-H, side scatter.

FIG. 3.

A. phagocytophilum-infected and LPS-stimulated neutrophil cultures that lose selectin expression remain viable for at least 18 h after infection, whereas infection by E. coli and S. aureus rapidly kills neutrophils. Viability is expressed as the ratio of viable cells (as determined by trypan blue staining) at each interval to the total quantity of viable cells added at the beginning of the experiments. The data represent the means ± standard errors of the means (error bars) of determinations performed in at least two and up to five separate experiments.

FIG. 4.

CD162-expressing and non-CD162-expressing A. phagocytophilum-infected differentiated HL-60 cells have different levels of infection. More non-CD162-expressing cells (A) were infected and contained larger numbers of morulae than did CD162-expressing cells (B), as determined by flow cytometry and cell sorting (Romanowsky stain; magnification, ×25). However, approximately 20 to 25% of uninfected cells also lacked CD162 expression (A, arrows).

FIG. 5.

Surface expression of Mac-1 (CD-18) and ICAM-1 (CD54) in A. phagocytophilum-infected cells detected by flow cytometry; the results of CD18 staining are shown, but similar results were obtained for CD11b. The panels show the results of representative flow cytometric analyses of experiments conducted at least three times. Expression levels of Mac-1 (A) and ICAM-1 (B) were significantly increased on A. phagocytophilum-infected DMSO-differentiated HL-60 cells. The intensity of constitutively expressed Mac-1 (C) on A. phagocytophilum-infected neutrophils was increased, and new ICAM-1 expression (D) was markedly increased. Similarly, stimulation with live E. coli or S. aureus resulted in moderate increases in ICAM-1 expression, but stimulation with PFA-inactivated A. phagocytophilum did not. FITC, fluorescein isothiocynate; PE, phycoerythrin; SSC-H, side scatter.

FIG. 6.

CD162 (PSGL-1) is shed from surfaces of A. phagocytophilum-infected neutrophils and DMSO-differentiated HL-60 cells in the culture supernatant. (Top) CD162 is lost from cells with A. phagocytophilum infection. Lane 1, uninfected neutrophil lysates; lane 2, LPS-stimulated neutrophil lysates; lane 3, A. phagocytophilum-infected neutrophil lysates; lane 4, uninfected DMSO-differentiated HL-60 cell lysates; lane 5, A. phagocytophilum-infected DMSO-differentiated HL-60 cell lysates. (Bottom) CD162 appears in cell culture supernatants after A. phagocytophilum infection. Lane 1, recombinant CD162; lane 2, uninfected neutrophils; lane 3, A. phagocytophilum-infected neutrophils; lane 4, uninfected DMSO-differentiated HL-60 cells; lane 5, A. phagocytophilum-infected DMSO-differentiated HL-60 cells. MW, molecular weight (in thousands).

FIG. 7.

The divalent cation chelator EDTA inhibits the reduction in surface expression of CD162 in A. phagocytophilum-infected neutrophils. Neutrophils were preincubated with cell-free A. phagocytophilum for 3 h and then treated with either 14.3 or 1.43 mM EDTA. Incubation in the presence of 14.3 mM EDTA inhibits the loss of CD162 expression after A. phagocytophilum infection. Error bars, standard errors of the means.