Endothelial damage from cytomegalovirus-specific host immune response can be prevented by targeted disruption of fractalkine-CX3CR1 interaction

Abstract

Human cytomegalovirus (CMV) infection has been linked to inflammatory diseases, including vascular disease and chronic transplant rejection, that involve vascular endothelial damage. We have previously shown that the host CD4(+) T-cell response to CMV antigen can produce IFNgamma and TNFalpha at levels sufficient to drive induction of fractalkine, a key marker of inflammation in endothelial cells. We have also observed a major pathogenic effect in which endothelial cell damage and loss follow the induction of fractalkine and up-regulation of cell adhesion markers in the presence of peripheral blood mononuclear cells (PBMCs) from donors with a high CMV-specific T-cell frequency. In this report, we show that the fractalkine-CX(3)CR1 interaction resulting in recruitment of natural killer (NK) cells and monocyte-macrophages plays an important role in mediating this endothelial damage. Supportive evidence for frac-talkine's key role is shown by the ability of specific antibody to CX(3)CR1 to reduce significantly CX(3)CR1(+)-bearing cell chemoattraction and to protect against endothelial damage. These findings support CMV as a member of a class of persistent pathogens in which a high T-cell response and chemokine-mediated effects are a risk factor for development of chronic inflammation and endothelial cell injury.

Figure 1

CMV-seropositive donor PBMCs exposed to CMV antigen results in endothelial cell damage in cocultures. (A) PBMCs isolated from a CMV-seropositive donor and cocultured with endothelial cells after 5 days are shown. (B) The same donor PBMCs cocultured on endothelial monolayers for 5 days are shown, in the presence of CMV antigen. (C) Control in which endothelial monolayers alone were maintained for 5 days. (D) CMV-seronegative donor PBMC exposed to the same conditions had no effect on endothelial cell monolayers and are indistinguishable from panel C. Results are representative of 6 different CMV-seropositive and -seronegative donor pairs.

Figure 2

Factors present in supernatants from antigen-stimulated CMV-seropositive donor PBMCs support chemoattraction of PBMCs. Results are shown for transmigrated populations from cell migration assays with coculture supernatants representative of a CMV-seropositive donor and -seronegative donor. PBMCs were loaded into the upper chambers of transwells to test migration into coculture supernatants as described in “Transwell migration assays and specific antibody neutralization assays.” Sample 1 was CMV-seropositive donor PBMCs + AECs + CMV antigen, 2 was CMV-seropositive donor PBMCs + AECs, 3 was CMV-seronegative donor PBMCs + AECs + CMV antigen, 4 was CMV-seronegative donor PBMCs + AECs, 5 was AEC cultures, resting, and sample 6 was RPMI/1% FCS. For each sample, 3 transmigration wells were set up, and the transmigrated populations were counted from each well independently. Results are shown as the mean (± SD) in cells per milliliter and are representative of 3 independent assays with different CMV-seropositive and -seronegative donor pairs compared in each.

Figure 3

Fractalkine has the greatest effect on chemoattraction. Transwell migration assay results after 3 hours, showing total cell numbers of PBMC populations migrating in response to chemokines present in coculture supernatants from the CMV antigen-stimulated CD4⁺ T cells with endothelial monolayer samples. Sample sets on x-axis are as follows. Sample set 1 was total cell numbers of PBMC populations transmigrating in response to untreated coculture supernatants; sample set 2 was PBMCs treated with anti–human CX₃CR1 antibody transmigrating in response to coculture supernatants; sample set 3 was PBMCs transmigrating in response to coculture supernatants treated with anti–human RANTES antibody; sample set 4 was PBMCs treated with anti–human CX₃CR1 antibody and anti–human RANTES antibody; and sample set 5 was PBMCs treated with isotype antibodies rabbit IgG and mouse IgG. Each sample represents the results from 3 replicate transwell migrations in which the transmigrated populations were counted from each well independently. Results are shown as the mean (± SD) and are representative of 5 assays with different seropositive donors.

Figure 4

Migration of different PBMC cell types in response to chemokine gradients in coculture supernatants. Results are shown as mean (± SD) of transmigrated populations from cell migration assays of 3 CMV-seropositive donors. PBMCs from each donor were prepared for transwell migration assays, as described in “Transwell migration assays and specific antibody neutralization assays.” PBMCs were loaded into the upper chambers of transwells to test migration into coculture supernatants prepared previously from the same donor, or negative control media. The “+” and “−” symbols refer to migration results using coculture supernatants or negative control media, respectively. Culture supernatants from fractalkine-induced cultures were used for the endothelial damage assays. Percentage values with each cell type represent the mean number of cells migrating of each cell type (ie, mean cells migrated in lower chamber/mean cell initially in upper chamber × 100).

Figure 5

CX₃CR1 block has greatest inhibition effect on migration of CD14⁺ cells compared with other PBMC subsets. Untreated PBMCs, CX₃CR1 blocked, or RANTES blocked cells or supernatants were compared for migration into antigen-stimulated coculture supernatants. Migrated cells were then collected, stained, and analyzed by flow cytometry to identify migrated subsets of CD14⁺, NK (CD16⁺/CD56⁺), CD4⁺ T cells, and CD8⁺ T cells. Treated samples for each analysis are grouped together for PBMCs or each subset as no antibody treatment, anti–CX3CR1-neutralizing antibody treatment, and anti–RANTES-neutralizing antibody. Results are shown as mean (± SD) percentage of migrated cell numbers relative to untreated PBMC migrations for PBMCs, CD14⁺ cells, NK cells, CD4⁺ T cells, and CD8⁺ T cells.

Figure 6

Endothelial damage is associated with CD14⁺ or NK populations. (A) Endothelial damage by CMV antigen–stimulated PBMC subset populations shows NK and CD14⁺ monocyte-macrophage populations associated with damage after 5 days of coculture. Negative controls without CMV antigen stimulation are indicated. (B) Endothelial damage assays per subset populations in which results were quantitated by counting remaining endothelial cells per field of view (FOV) under the microscope (20× objective). Results are representative of 3 independent assays, each with different CMV-seropositive donors. Error bars represent SD.

Figure 7

CMV-seropositive donor PBMCs exposed to CMV antigen results in endothelial damage in cocultures that is mediated predominantly by the fractalkine-CX₃CR1 interaction. Each micrograph panel shows the resulting effects of specific neutralizing antibody blocks and controls on CMV-induced endothelial damage in PBMC-AEC cocultures at day 6. Negative control was transmigrated PBMC populations using negative control media lacking chemokines; positive control was transmigrated PBMCs using untreated coculture supernatants; isotype control was PBMCs treated with isotype control antibodies for CX₃CR1-specific and RANTES-specific neutralizing antibodies, followed by transmigration into coculture supernatants; anti-CX₃CR1 block was PBMCs treated with neutralizing antibody specific for CX₃CR1, followed by transmigration into coculture supernatants; anti-RANTES block was PBMCs treated with neutralizing antibody specific for RANTES, followed by transmigration into coculture supernatants; dual block was PBMCs treated with both neutralizing antibody specific for CX₃CR1 and neutralizing antibody specific for RANTES, followed by transmigration into coculture supernatants. Extensive endothelial damage and loss are seen in positive control, isotype control, and anti-RANTES block. In contrast, protection against endothelial damage is observed in the presence of the anti–CX₃CR1-blocked samples.