Molecular control of physiological and pathological T-cell recruitment after mouse spinal cord injury

Abstract

The intraspinal cues that orchestrate T-cell migration and activation after spinal contusion injury were characterized using B10.PL (wild-type) and transgenic (Tg) mice with a T-cell repertoire biased toward recognition of myelin basic protein (MBP). Previously, we showed that these strains exhibit distinct anatomical and behavioral phenotypes. In Tg mice, MBP-reactive T-cells are activated by spinal cord injury (SCI), causing more severe axonal injury, demyelination, and functional impairment than is found in non-Tg wild-type mice (B10.PL). Conversely, despite a robust SCI-induced T-cell response in B10.PL mice, no overt T-cell-mediated pathology was evident. Here, we show that chronic intraspinal T-cell accumulation in B10.PL and Tg mice is associated with a dramatic and sustained increase in CXCL10/IP-10 and CCL5/RANTES mRNA expression. However, in Tg mice, chemokine mRNA were enhanced 2- to 17-fold higher than in B10.PL mice and were associated with accelerated intraspinal T-cell influx and enhanced CNS macrophage activation throughout the spinal cord. These data suggest common molecular pathways for initiating T-cell responses after SCI in mice; however, if T-cell reactions are biased against MBP, molecular and cellular determinants of neuroinflammation are magnified in parallel with exacerbation of neuropathology and functional impairment.

Figure 1.

Migration of T-cells to the injured spinal cord is accelerated in Tg mice and is associated with earlier expression of T-cell chemoattractant mRNAs. At 7 dpi, CD4⁺ T-cells are absent from the injury site of nTg mice (A). In contrast, small T-cell clusters form within the injury site of Tg mice (B). Accelerated T-cell recruitment in Tg mice is associated with increased expression of IP-10/CXCL10 mRNA at 7 dpi in Tg but not nTg mice (C; ^*p < 0.05 vs uninjured and nTg; n = 4 per group). By 21 dpi, IP-10/CXCL10 mRNA is elevated in Tg and nTg mice; however, the magnitude of change is greater in Tg mice (C; ^*p < 0.05 vs uninjured and nTg; n = 4 per group). Expression of RANTES/CCL5 mRNA increases as a function of time after injury in both groups but to a greater extent in Tg mice (D; ^*p < 0.05 vs uninjured and nTg; n = 4 per group). Scale bar, 25 μm.

Figure 2.

SCI-induced priming of MBP-reactive T-cells increases T-cell infiltration throughout the injured spinal cord. CD4⁺ T-cells are increased throughout the rostrocaudal spinal cord in Tg and nTg mice at 70 dpi (A). However, more T-cells penetrate both intact (inset in A; p < 0.05) and lesioned spinal parenchyma in Tg mice (A; p < 0.05 vs nTg, two-way ANOVA; n = 4 per group). An example of how lesioned tissue can be distinguished from nonlesioned tissue using luxol fast blue histochemistry is shown in B (∼3 mm rostral to the injury site; dotted line delineates boundary between lesioned and nonlesioned tissue). An adjacent section stained with anti-CD4 antibodies show that CD4⁺ T-cells are prominent within lesioned tissue (within boundaries of dotted line; nTg mouse; C). In Tg mice, T-cells also penetrate into uninjured parenchyma (boxed region in D shown in high power in E). Arrows highlight a meningeal lymphocyte cluster that appears to migrate into nearby gray matter (arrowheads in D and E point to neuron profile in adjacent ventrolateral gray matter). Scale bars: B, C, 100 μm; D, 50 μm; E, 25 μm.

Figure 3.

Cellular and molecular indices of CNS macrophage activation are increased in the injured spinal cord of Tg mice. At 7 dpi, MCP-1/CCL2 mRNA is reduced relative to uninjured controls in both groups (not significant); however, by 21 dpi, CCL2 mRNA is increased in Tg mice (A). Expression of CD80/B7.1 and CD86/B7.2 mRNA (B, C) is increased in nTg and Tg mice at 7 and 21 dpi but with 10- to 40-fold greater changes noted in Tg mice at 21 dpi. Confocal analysis of immunofluorescent double-labeled sections reveal MHC class II expressing microglia/macrophages (CD11b⁺) in and nearby lesioned tissue in Tg and nTg mice (only nTg sample shown; D-F). MHC class II/CD86 double-positive cells were limited in nTg tissues and, when present, were restricted to zones of fibrosis (area above dotted line; G-I). Conversely, MHC class II/CD86 double labeling was seen throughout lesioned and intact spinal cord (boundary demarcated by arrows/lesioned tissue above arrows) of Tg mice (J-L). For D-L, the first two micrographs in a row are single channel (red or green) images with the merged image shown in F, I, or L.(^*p < 0.05 vs uninjured or nTg in A-C; two-way ANOVA) Scale bar: D-F, 25 μm; G-L, 50 μm.

Figure 4.

mRNAs encoding mediators of tissue repair and immunoregulation are upregulated chronically in the injured mouse spinal cord. Induction of TGF-β1 (A) and IGF-1 (B) mRNA are increased at 7 and 21 dpi in nTg and Tg mice (p < 0.05 vs uninjured controls) but continue to rise at 21 dpi only in Tg mice (p < 0.05 vs nTg). Expression of endogenous IL-1 receptor antagonist (C) is upregulated after SCI in nTg and Tg mice (p < 0.05) but is declining by 21 dpi in Tg mice (p < 0.05 vs nTg). n = 3 per group.