Chemokine expression by glial cells directs leukocytes to sites of axonal injury in the CNS - PubMed (original) (raw)

Chemokine expression by glial cells directs leukocytes to sites of axonal injury in the CNS

Alicia A Babcock et al. J Neurosci. 2003.

Abstract

Innate responses in the CNS are critical to first line defense against infection and injury. Leukocytes migrate to inflammatory sites in response to chemokines. We studied leukocyte migration and glial chemokine expression within the denervated hippocampus in response to axonal injury caused by entorhinodentate lesions. A population of Mac1/CD11b+ CD45high macrophages (distinct from CD45low microglia) was specifically detected within the lesion-reactive hippocampus by 12 hr after injury. Significant infiltration by CD3+ T cells did not occur in the denervated hippocampus until 24 hr after axotomy. A broad spectrum of chemokines [RANTES/CCL5, monocyte chemoattractant protein (MCP)-1/CCL2, interferon gamma inducible protein (IP)-10/CXCL10, macrophage inflammatory protein (MIP)-1alpha/CCL3, MIP-1beta/CCL4, and MIP-2/CXCL2] was induced at this time. RANTES/CCL5 was not significantly elevated until 24 hr after axotomy, whereas MCP-1/CCL2 was significantly induced before leukocyte infiltration occurred. Neither T cells nor macrophages infiltrated the denervated hippocampus of CCR2-deficient mice, arguing for a critical role for the CCR2 ligand MCP-1/CCL2 in leukocyte migration. Both T cells and macrophages infiltrated CCR5-deficient hippocampi, showing that CCR5 ligands (including RANTES/CCL5) are not critical to this response. In situ hybridization combined with immunohistochemistry for ionized binding calcium adapter molecule (iba)1 or glial fibrillary acidic protein (GFAP) identified iba1+ microglia and GFAP+ astrocytes as major sources of MCP-1/CCL2 within the lesion-reactive hippocampus. We conclude that leukocyte responses to CNS axonal injury are directed via innate glial production of chemokines.

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Figures

Figure 1.

Figure 1.

Macrophages infiltrate the lesion-reactive hippocampus. Flow cytometry profiles show live cells on the basis of forward and side scatter gating. Shown are representative profiles from individual unmanipulated (U, left column), contralateral (C, middle column), or lesion-reactive (L, right column) hippocampi from C57BL/6 (A), CCR2-deficient (B), or CCR5-deficient (C) mice at 24 hr after axotomy. Macrophages were identified on the basis of expression of Mac1/CD11b and high levels of CD45 (top right quadrant). D shows proportions of macrophages within groups of hippocampi between 3 hr and 14 d after axotomy (mean ± SE). Quadrants were set on the basis of fluorescence levels using isotype-matched control antibodies. Data are representative of 3-11 animals per group. *p < 0.05-0.001 versus U and C_; **p_ < 0.05-0.001 versus U, C, and L of C57BL/6 at 24 hr_; ***p_ < 0.05-0.001 versus U, C, and L.

Figure 2.

Figure 2.

T cells infiltrate the lesion-reactive hippocampus. Flow cytometry profiles show live cells on the basis of forward and side scatter gating. Shown are representative profiles from individual unmanipulated (U, left column), contralateral (C, middle column), or lesion-reactive (L, right column) hippocampi from C57BL/6 (A), CCR2-deficient (B), or CCR5-deficient (C) mice at 24 hr after axotomy. T cells were identified on the basis of expression of CD3 and CD45 (box). D shows T cell proportions within groups of hippocampi between 3 hr and 14 d after axotomy (mean ± SE). Boxes were drawn on the basis of fluorescence levels using isotype-matched control antibodies. Data are representative of three to seven animals per group. *p < 0.05 versus U and C_; **p_ < 0.05-0.001 versus U, C, and L of C57BL/6 at 3, 12, and 24 hr.

Figure 3.

Figure 3.

Axonal injury induces chemokine expression. RPA of chemokine expression in perfused hippocampi of C57BL/6 mice at 24 hr after axotomy shows that MCP-1/CCL2, RANTES/CCL5, IP-10/CXCL10, MIP-1α/CCL3, MIP-1β/CCL4, and MIP-2/CXCL2 were specifically induced in the lesion-reactive hippocampus and at the site of axonal transection. Results are representative of four separate experiments. U, Unmanipulated hippocampus; C, contralateral unlesioned hippocampus; L, lesion-reactive hippocampus; T, site of axonal transection. *p < 0.05-0.01 versus U and C.

Figure 4.

Figure 4.

Message for MCP-1/CCL2 and RANTES/CCL5 is induced by axotomy. A, RT-PCR analysis of MCP-1/CCL2, RANTES/CCL5, and β-actin mRNA from perfused hippocampus of C57BL/6 mice at 3, 12, 24, or 48 hr after axotomy. Levels of MCP-1/CCL2 (B) and RANTES/CCL5 (C) amplimers were determined by fluroimager analysis and then normalized to β-actin. Data are representative of 2-14 animals per group and plotted as arbitrary fluroimager units, with bars representing SE. U, Unmanipulated hippocampus; S, sham-operated hippocampus; C, contralateral unlesioned hippocampus; L, lesion-reactive hippocampus; T, site of axonal transection. *p < 0.01-0.001 versus U; **p < 0.01 versus U, S, C; ***p < 0.01-0.001 versus U, S, C, and L.

Figure 5.

Figure 5.

Axotomy induces MCP-1/CCL2 expression within the denervated hippocampus. MCP-1/CCL2+ cells were not detected by in situ hybridization in coronal sections from unmanipulated hippocampus (A) or contralateral unlesioned hippocampus after axotomy (C). Numerous robustly positive cells were found in the lesion-reactive hippocampus (D), located distally to the site of axonal transection (B). Original magnification, 10×.

Figure 6.

Figure 6.

Microglia and astrocytes express MCP-1/CCL2 transcripts. Immunohistochemistry and in situ hybridization were combined to determine the cellular source of chemokine expression. Brown immunoreactivity for GFAP or iba1 identifies astrocytes (A-C) and microglia (D, E), respectively. Clusters of silver grains identify MCP-1/CCL2-positive cells. Gray arrowheads identify GFAP+ astrocytes (A, C), and black arrowheads identify iba1+ microglia (D, E) that express MCP-1/CCL2. Black arrows show GFAP- (B, C) and gray arrows show iba1- (D, E) MCP-1/CCL2+ cells. In D, a microglial cell and an astrocyte are juxtaposed. Original magnification, 60×; except C (40×).

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