Monocytes regulate T cell migration through the glia limitans during acute viral encephalitis

Abstract

Leukocyte access into the central nervous system (CNS) parenchyma is tightly regulated by the blood-brain barrier (BBB). Leukocyte migration through the endothelial cell wall into the perivascular space is well characterized; however, mechanisms regulating their penetration through the glia limitans into the parenchyma are less well studied, and the role of monocytes relative to neutrophils is poorly defined. Acute viral encephalitis was thus induced in CCL2-deficient (CCL2(-/-)) mice to specifically abrogate monocyte recruitment. Impaired monocyte recruitment prolonged T cell retention in the perivascular space, although no difference in overall CNS accumulation of CD4 or CD8 T cells was detected by flow cytometry. Delayed penetration to the CNS parenchyma was not associated with reduced or altered expression of either matrix metalloproteinases (MMP) or the T cell chemoattractants CXCL10 and CCL5. Nevertheless, decreased parenchymal leukocyte infiltration delayed T cell-mediated control of virus replication as well as clinical disease. These data are the first to demonstrate that the rapid monocyte recruitment into the CNS during viral encephalitis is dispensable for T cell migration across the blood vessel endothelium. However, monocytes facilitate penetration through the glia limitans. Thus, the rapid monocyte response to viral encephalitis constitutes an indirect antiviral pathway by aiding access of effector T cells to the site of viral infection.

FIG. 1.

Monocytes represent the major population of CNS-infiltrating leukocytes early after infection. (A) Representative density plot of CNS-derived cells at 3 day p.i. stained with anti-CD45 and Ly-6C MAbs. CNS-infiltrating leukocytes (CD45^hi) contain a prominent population of Ly6C^hi monocytes (R8; 68.13% of CD45^hi) and a minor proportion of Ly6C^int neutrophils (R7; 5.25% of CD45^hi). (B) F4/80 and MHC class II expression within the CD45^hi Ly6C^int and the CD45^hi Ly6C^hi myeloid populations at days 3 and 5 p.i. (C) Kinetics of MHC class II expression by CD45^hi Ly6C^hi F4/80⁺ monocytes during the course of infection. Data represent means for three mice at each time point.

FIG. 2.

CCL2 is required for CNS monocyte recruitment. CNS inflammation in infected CCL2^−/− and WT was mice analyzed by flow cytometry at the indicated times p.i. Numbers of total inflammatory leukocytes (CD45^hi), macrophages (F4/80⁺), neutrophils (Ly-6G⁺), CD4⁺ T cells, CD8⁺ T cells, and Db/S510 tetramer-positive virus-specific CD8 T cells per brain are shown. Data represent means (± standard errors of the means) from three separate experiments with three pooled mice per time point per experiment (n = 9 per group). *, P < 0.05.

FIG. 3.

Impaired monocyte recruitment delays disease onset and virus clearance. (A) Clinical symptoms were monitored daily according to the following grades: 0, healthy; 1, hunched back; 2, partial hind limb paralysis or inability to maintain the upright position; 3, complete hind limb paralysis; 4, moribund or dead. Data represent means (± standard errors of the means) for 60 WT mice and 54 CCL2^−/− mice from three separate experiments. ***, P < 0.001. (B) Virus replication in brains of WT and CCL2^−/− mice analyzed by plaque assay. Data represent the averages (± standard deviations) for three mice per time point per experiment from two separate experiments. (n = 6 per group) **, P < 0.01.

FIG. 4.

Leukocyte retention in the perivascular space of CCL2^−/− mice. (A) Brain sections of WT and CCL2^−/− mice at day 5 p.i. were stained with hematoxylin and eosin (H&E). Perivascular inflammation was prominent in CCL2^−/− compared to WT mice. Pictures are representative of three mice from each group. Scale bar, 300 μm. (B) Leukocyte localization was analyzed using anti-CD45 (green) and anti-laminin (red) antibodies. Scale bar, 25 μm. (C to E) CD45⁺ cells (C) and CD8 (D) and CD4 (E) T cells in perivascular space versus parenchyma quantified in CCL2^−/− and WT mice at days 5, 7, and 10 p.i. For quantification, 10 pictures per animal were analyzed in areas of inflammation at each time point. Data are representative of three mice from each group (means ± standard errors of the means). *, P < 0.05.

FIG. 5.

Monocytes do not affect mRNA CNS chemokine expression. Expression of CXCL10, CCL5, CCL3, and CXCL12 chemokines relative to ubiquitin mRNA in brains of naive (n = 4) and WT and CCL2^−/− mice at days 3, 5, 7, and 10 p.i. (n = 3 per time point) was measured by real-time PCR. No differences in CXCL10, CCL5, or CCL3 upregulation, or in CXCL12 downregulation, were observed between WT and CCL2^−/− mice during the course of infection.

FIG. 6.

MMP-independent disruption of glia limitans. (A) MMP9 activity was analyzed by zymography from purified populations of monocytes and neutrophils isolated from WT mice at 3 days p.i. (B) Relative mRNA expression of MMP12, MMP3, MMP2, and TIMP1 was analyzed by quantitative real-time PCR. Total RNA was extracted from brains of naive mice (n = 4) and WT and CCL2^−/− mice at days 3, 5, 7, and 10 p.i. (n = 3 for each time point).

FIG. 7.

Neutrophils do not compensate for the absence of monocytes. (A) CNS inflammation of neutrophil-depleted CCL2^−/− mice and controls (WT mice treated with isotype control antibody) analyzed by flow cytometry. Graphs represent the number of neutrophils (Ly-6G⁺) and macrophages (F4/80⁺) in the infiltrating CD45^high population. Data represent means for three mice per group at each time point. (B) The percentage of maximal inflammation relative to that in WT mice was compared between CCL2^−/− and CCL2^−/− or WT neutrophil-depleted mice. Data represent the averages for three mice at each time point.