PECAM-KO mice exhibit early onset of EAE and early inflammatory cell migration into CNS.
To examine the role of PECAM-1 in MOG-induced EAE, C57BL/6 WT and PECAM-KO mice were immunized with MOG peptide antigen and monitored daily for clinical symptoms of EAE (Figure a). The PECAM-KO mice were susceptible to MOG-induced EAE and developed clinical disease symptoms significantly earlier than did WT mice. The differences in the mean values among the groups (WT and KO) are greater than would be expected by chance (F
= 24.17, P
< 2.23 × 10–30
). An all-pairwise multiple comparison procedure resulted in significant differences between the two groups from days 12 through 22. By day 24 the average severity of disease scores was comparable between WT and PECAM-KO mice. Both groups of mice exhibited chronic disease, which is typical of MOG-induced EAE in the C57BL/6 model (40
To determine the extent of inflammatory cell trafficking into the CNS parenchyma, brain tissues of WT and PECAM-KO mice were examined (Figure , b–j). In comparison to WT mice, perivascular mononuclear cell infiltrates were noted earlier in PECAM-KO tissues. Specifically, at 4 days after immunization neither WT nor PECAM-KO mice exhibited appreciable perivascular or parenchymal invasion of inflammatory cells into the CNS (Figure , b and c). At day 8, the brains of WT mice were mostly devoid of inflammatory infiltrates, while substantial perivascular cuffing of mononuclear cells was observed in the brains of PECAM-KO mice (Figure , d and e). By day 20, brains of both WT and PECAM-KO mice exhibited modest meningeal and submeningeal perivascular infiltrates (Figure , f and g). However, brains of PECAM-KO mice exhibited more robust invasion around larger, deeper vessels compared with WT mice, corresponding to significantly higher disease severity scores (Figure , h and i). Lytic foci were also apparent in brains of PECAM-KO mice (Figure j). At later time points, when the average clinical disease scores were similar for both groups of mice (days 24–30), the inflammatory infiltrates were similar to those illustrated in Figure , i and j, and were indistinguishable from each other (data not shown).
Leukocytes were isolated from the CNS of WT and PECAM-KO mice in the early stages of EAE to determine the composition of invading cells. In comparison to WT mice, increased numbers of total leukocytes, CD4+ and CD8+ T cells, B cells, total macrophages, TNF-α–producing (activated) macrophages, and neutrophils were present in the CNS of PECAM-KO mice (Table ). Increases in MOG-specific CD4+ T cells were also seen earlier and in greater numbers in the CNS of PECAM-KO mice (data not shown).
PECAM-KO mice have more leukocytes in the CNS early in EAE
The increased presence of inflammatory cells in the brain tissue of PECAM-KO mice may be due to either an enhanced initial immune response to antigen or an increased ability of inflammatory cells to transmigrate across the blood-brain barrier. Because PECAM-1 possesses the ability to modulate signal transduction, the initial response of MOG-specific T cells to antigen may be increased. Following immunization with MOG peptide, cells were isolated from draining lymph nodes. There is no significant difference between WT and PECAM-KO lymph node cells in their ability to proliferate in response to a range of concentrations of MOG peptide antigen (Figure a). In addition, we have demonstrated, using adoptive transfer techniques (47
), an earlier onset of EAE in KO mice than in WT mice, regardless of whether the mice were injected intravenously with antigen-specific (MOG) WT or PECAM-KO T lymphocytes (Figure b). The differences in the mean values among the animal groups (WT and KO mice) are greater than would be expected by chance (F
= 3.85, P
< 7.1 × 10-9
). An all-pairwise multiple comparison procedure resulted in significant differences between the two groups from days 13 through 16. No differences were noted in KO mice given WT or KO MOG-specific T lymphocytes. Further, using WT mice lethally irradiated and engrafted with PECAM-KO marrow and KO mice lethally irradiated and engrafted with WT marrow (47
), we demonstrated an earlier disease onset of EAE in the KO mice following direct immunization with MOG peptides (Figure c). The differences in the mean values among the marrow recipient groups (WT mice engrafted with KO marrow and KO mice engrafted with WT marrow) are greater than would be expected by chance (F
= 7.9, P
< 3.1 × 10–22
). An all-pairwise multiple comparison procedure resulted in significant differences between the two groups from days 13 through 17. These studies support the concept that while EAE is a T cell–mediated disease process, it is dependent, in part, upon the presence or absence of endothelial PECAM-1. Therefore, we investigated a second possibility that transendothelial migration of immune system cells may play a role in the early onset of EAE in the PECAM-KO mice.
Endothelial cells derived from PECAM-KO mice support enhanced in vitro transendothelial migration of T cells.
We performed in vitro transmigration assays using low-passage endothelial cells and MOG-specific T cells derived from WT and PECAM-KO mice (Figure , a–c). Interestingly, endothelial cells derived from PECAM-KO mice supported a significantly higher level of T cell transmigration (~42%) compared with WT endothelial cells (~31%), regardless of expression of PECAM-1 on the T cells. Two-way ANOVA, with two between-group factors, revealed a significant main effect for the endothelial cell type (WT versus KO) [F1,28 = 113.02, P < 0.001]. The main effect for T cell type (WT versus KO) proved to be insignificant, [F1,28 = 0.04, P = 0.85], as did the interaction between endothelial cell type (WT versus KO) and T cell type (WT versus KO) [F1,28 = 0.10, P = 0.76].
In addition, we performed adhesion and transmigration assays using immortalized endothelioma cell lines derived from brain microvasculature of WT and PECAM-KO mice (Figure a) and lung microvasculature of PECAM-KO mice, and the same cells reconstituted with PECAM-1 (PECAM-RC) (Figure b). We found the level of adhesion of MOG-specific T cells derived from WT (Figure c) and PECAM-KO (Figure e) animals to these four endothelioma lines indistinguishable (Figure , c and e). Analyses using two-way ANOVA revealed no significant differences between groups. In contrast, migration of T cells across PECAM-KO endotheliomas derived from brain (bEnd.PECAM-1.2) and lung (luEnd.PECAM-1.1) was significantly increased in comparison with transmigration across WT endothelioma monolayers derived from brain (bEnd.WT) and KO endothelioma monolayers derived from lung (luEnd.PECAM-1.1) that were transfected and stably expressing PECAM-1 (PECAM-RC) (Figure , d and f). These endothelioma lines express equivalent levels of VE-cadherin, ICAM-1, ICAM-2 (not shown), and VCAM-1 (not shown), but only WT and PECAM-RC cells express PECAM-1 (Figure , a and b). In these transmigration assays the percent transmigration was also noted to be dependent upon PECAM-1 expression on the MOG-specific T lymphocyte clones, with the WT T lymphocytes exhibiting a greater percent transmigration than the KO T lymphocytes (Figure , compare d with f). Two-way ANOVA, with two between-group factors, revealed a significant main effect for endothelial cell type (brain WT versus KO) [F1,13 = 23.56, P = 0.0007] and a significant main effect for T cell type (WT versus KO) [F1,13 = 11.77, P = 0.0064]. The interaction between endothelial cell type and T cell type proved to be insignificant [F1,13 = 0.67, P = 0.43]. In transmigration assays using lung-derived endothelial cells, two-way ANOVA, with two between-group factors, also revealed a significant main effect for endothelial cell type (lung RC versus KO) [F1,15 = 27.39, P = 0.0002] and a significant main effect for T cell type (WT versus KO) [F1,15 = 16.71, P = 0.0015]. The interaction between EC type and T cell type proved to be insignificant [F1,15 = 1.68, P = 0.22].
Similar assays were performed in a second laboratory using PLP-specific T lymphoblasts isolated from PLP-immunized SJL mice transmigrating across these immortalized endothelioma cell lines. These endothelioma lines were also noted to express equivalent levels of ICAM-1, ICAM-2, and VCAM-1, but only WT cells express PECAM-1 (data not shown). Migration of these T cells across PECAM-KO endotheliomas derived from brain (bEnd.PECAM-1.2), mesenteric lymph nodes (mlEnd.PECAM-1.1), and lung (luEnd.PECAM-1.1) was significantly increased in comparison with transmigration across WT endothelioma monolayers (bEnd.WT) (data not shown). Interestingly, while treatment of the endotheliomas with TNF-α increased surface expression of ICAM-1 and VCAM-1, it did not affect transmigration levels (data not shown) (44
The differences in transmigration rates noted in Figures and likely reflect several differences in the endothelial cells and the T lymphocytes used. The endothelial cells used in Figure were immortalized, cloned cells, whereas low-passage endothelial cells were used in Figure . Further, in other experiments T lymphoblasts isolated from PLP-immunized SJL mice were used, while the T lymphocytes used in Figures and were C57BL/6-derived MOG-specific T lymphocytes activated with MOG peptide and IL-2. These experiments, utilizing different endothelial cells and different T lymphocyte populations, would be expected to yield disparate levels of transmigration. However, in all experiments performed, transmigration rate is dependent upon the presence or absence of endothelial PECAM-1. ICAM-1 levels were found to vary modestly on the cells. Increasing ICAM-1 and VCAM-1 expression by TNF-α treatment did not alter the transmigration rates, further supporting a role for endothelial PECAM-1 in this process.
Vascular permeability is increased in vitro and in vivo during development of EAE and dermal histamine challenge in PECAM-KO mice.
Migration of leukocytes across endothelial cells has been shown to be enhanced by vasoactive substances that disrupt endothelial junctions and increase vascular permeability (52
). We investigated whether endothelial junctional integrity may be compromised in PECAM-KO monolayers. Endothelioma cells derived from the lungs of PECAM-KO mice or the same cells reconstituted with PECAM-1 (PECAM-RC) were cultured to confluence on porous membranes as in the transmigration assays. In control samples, Evans blue dye was added to the upper chamber to confirm that the endothelial monolayers restricted its flow into the bottom chamber. Monolayers were treated with histamine, and at various time points later, dye was added. The dye that diffused into the lower wells was collected and measured spectrophotometrically (Figure ). At 30 seconds after histamine exposure, dye flowed freely across either the PECAM-KO or PECAM-RC endothelial layers into the bottom chambers. Over the next 10–15 minutes the monolayers became increasingly impermeable to the dye. The rate at which the endothelium became impermeable was faster for the PECAM-RC monolayer, and the barrier of these cells was completely restored by 15 minutes after histamine challenge. The PECAM-KO endothelial layer remained significantly permeable to the dye up to 20 minutes. The differences in the mean values among the groups (RC and KO lung endothelia) are greater than would be expected by chance (F
= 5.67, P
< 5.4 × 10–5
). An all-pairwise multiple comparison procedure resulted in significant differences between the two groups from 2 through 15 minutes.
We next assessed the integrity of CNS vasculature in vivo using an Evans blue vital dye permeability assay. At various days after immunization with MOG peptide to induce EAE, dye was injected retro-orbitally and mice were perfused with PBS to clear it from the vasculature. The dye that had extravasated into the brain tissue was extracted and measured (Figure a). At early time points after immunization, the CNS vasculature of both WT and PECAM-KO mice was impermeable to dye. By day 6, the vasculature became permeable to the dye, indicating a breach of integrity of the endothelial cell barrier. The brains of the PECAM-KO group exhibited a twofold higher average PI compared with the WT group, and there was a delay in the restoration of vascular integrity in PECAM-KO mice. The vasculature of WT mice became impermeable to the dye by day 12, while the permeability barrier in the PECAM-KO mice was not restored until day 24. The differences in the mean values among the groups (WT and KO mice) are greater than would be expected by chance (F = 17.9, P < 2.9 × 10–7). An all-pairwise multiple comparison procedure resulted in significant differences between the two groups at days 6 and 12. Skin vessel permeability was unaffected in both WT and PECAM-KO mice during the course of EAE, illustrating specificity of the response (Figure b) (F = 0.196, P < 0.82).
In a separate experiment, the skin vessels were challenged with histamine to increase dermal vascular permeability (Figure c). Dye was injected at various time points following intradermal injection of histamine or PBS, and extracted from the skin tissue. At early time points after histamine injection (0 minutes, 2 minutes, 5 minutes), dermal vascular permeability of both the WT and the PECAM-KO mice was increased and dynamic. However, by 10 minutes after histamine injection, the PECAM-KO skin vasculature remained markedly permeable to the dye, while the WT vasculature became impermeable. By 15 minutes, dermal vessel integrity had been restored in both groups of mice. The differences in the mean values among the groups (WT and KO mice) are greater than would be expected by chance (F = 2.86, P < 0.044). An all-pairwise multiple comparison procedure resulted in significant differences between the two groups at 10 minutes.
In Figure d, dye was allowed to circulate in the blood for 45 minutes prior to vessel challenge with histamine so that dye accumulated in the tissue from the moment the junctions were compromised until the mice were sacrificed. Five minutes after histamine injection, equal amounts of dye had extravasated in both groups of mice. However, after 15 minutes an increase in the amount of dye extracted from the PECAM-KO mice indicates a greater accumulation of extravasated dye in this group. This result is consistent with the previous experiment, in that the prolonged time required for restoration of vascular integrity in the PECAM-KO mice would result in a longer period of vascular permeability, leading to greater accumulation of dye in the skin tissue. The dermal vessel experiments confirm the hypothesis that the restoration of vascular integrity is impaired in the PECAM-KO mice, which could lead to an exacerbated inflammatory response.