Human cerebrospinal fluid central memory CD4+ T cells: evidence for trafficking through choroid plexus and meninges via P-selectin

Abstract

Cerebrospinal fluid (CSF) from healthy individuals contains between 1,000 and 3,000 leukocytes per ml. Little is known about trafficking patterns of leukocytes between the systemic circulation and the noninflamed CNS. In the current study, we characterized the surface phenotype of CSF cells and defined the expression of selected adhesion molecules on vasculature in the choroid plexus, the subarachnoid space surrounding the cerebral cortex, and the cerebral parenchyma. Using multicolor flow cytometry, we found that CSF cells predominantly consisted of CD4+/CD45RA-/CD27+/CD69+-activated central memory T cells expressing high levels of CCR7 and L-selectin. CD3+ T cells were present in the choroid plexus stroma in autopsy CNS tissue sections from individuals who died without known neurological disorders. P- and E-selectin immunoreactivity was detected in large venules in the choroid plexus and subarachnoid space, but not in parenchymal microvessels. CD4+ T cells in the CSF expressed high levels of P-selectin glycoprotein ligand 1, and a subpopulation of circulating CD4+ T cells displayed P-selectin binding activity. Intercellular adhesion molecule 1, but not vascular cell adhesion molecule 1 or mucosal addressin cell adhesion molecule 1, was expressed in choroid plexus and subarachnoid space vessels. Based on these findings, we propose that T cells are recruited to the CSF through interactions between P-selectin/P-selectin ligands and intercellular adhesion molecule 1/lymphocyte function-associated antigen 1 in choroid plexus and subarachnoid space venules. These results support the overall hypothesis that activated memory T cells enter CSF directly from the systemic circulation and monitor the subarachnoid space, retaining the capacity to either initiate local immune reactions or return to secondary lymphoid organs.

Figures

Fig. 1.

Six-color flow cytometry was used to characterize the main subpopulations of lymphocytes in paired blood (Upper) and CSF (Lower) samples. The graph shows mean and SD of eight patients with noninflammatory neurological diseases.

Fig. 2.

(A) The majority of CD4+ T cells in both PB (Left) and CSF (Right) were CD45RO+/CD27+ TCM. Small, but distinct, populations of CD45RO+/CD27- TEM were observed in both compartments. Although ≈50% of PB CD4+ T cells were CD45RO-, this phenotype was rare in the CSF. (B) Expression of CCR7, L-selectin, and CD69 on CD4+/CD45RO+ T cells in PB (hatched plot) and CSF (open plot). Isotype-matched irrelevant Abs were used as negative controls in each experiment (filled plot). A representative histogram (n = 5–14) is shown.

Fig. 3.

CD3+ T cells (dark brown) were detected in the choroid plexus stroma, both adjacent to small vessels in choroid plexus villi (not shown) and surrounding large venules in the choroidal stroma (arrows). CD3+ T cells were localized outside of choroid plexus vessels as demonstrated by staining platelet-endothelial cell adhesion molecule 1 positive endothelial cells (red; Inset).

Fig. 4.

P-selectin (A and B) and E-selectin (E andF) immunoreactivity was detected in large venules in choroid plexus (A and E) and SAS (B and F) from individuals who died without neurological or inflammatory disorders. The staining was predominantly detected in large vessels in the choroidal stroma, whereas smaller vessels in choroid plexus villi were negative (C; staining shows P-selectin). In inflammatory parenchymal lesions, punctate P-selectin immunoreactivity was localized to intravascular platelets, whereas endothelial cells were negative (D). The continuous and linear distribution of P-selectin immunoreactivity along the vascular endothelium indicated surface expression of the protein (G; staining shows vessel in choroid plexus). ICAM-1 expression was detected in endothelial cells at multiple localizations in the noninflamed CNS. Shown is characteristic staining of small vessels in the choroid plexus villi (J), large venules in choroidal stroma (H), and SAS (I).

Fig. 5.

(A) All CD4+/CD45RO+ T cells from PB (hatched plot) and CSF (open plot) express PSGL-1. (B) Approximately one-third of CD4+/CD45RA- T cells in PB bound P-selectin in a solution-phase adhesion assay (hatched plot). Filled histogram shows assay performed in the presence of EDTA as negative control.