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. 2001 Mar 19;193(6):713-26.
doi: 10.1084/jem.193.6.713.

Absence of monocyte chemoattractant protein 1 in mice leads to decreased local macrophage recruitment and antigen-specific T helper cell type 1 immune response in experimental autoimmune encephalomyelitis

D R Huang  1 , J Wang, P Kivisakk, B J Rollins, R M Ransohoff
Affiliations

Absence of monocyte chemoattractant protein 1 in mice leads to decreased local macrophage recruitment and antigen-specific T helper cell type 1 immune response in experimental autoimmune encephalomyelitis

D R Huang et al. J Exp Med. .

Abstract

Monocyte chemoattractant protein (MCP)-1 plays a critical role in innate immunity by directing the migration of monocytes into inflammatory sites. Recent data indicated a function for this chemokine in adaptive immunity as a regulator of T cell commitment to T helper cell type 2 (Th2) effector function. Studies in a Th1-dependent animal model, experimental autoimmune encephalomyelitis (EAE), showed that MCP-1 was highly expressed in the central nervous system (CNS) of affected rodents, and MCP-1 antibodies could block relapses of the disease. Mice deficient for the major MCP-1 receptor, CC chemokine receptor (CCR)2, did not develop EAE after active immunization but generated effector cells that could transfer the disease to naive wild-type recipients. We analyzed EAE in mice deficient for MCP-1 to define the relevant ligand for CCR2, which responds to murine MCP-1, MCP-2, MCP-3, and MCP-5. We found that C57BL/6 MCP-1-null mice were markedly resistant to EAE after active immunization, with drastically impaired recruitment of macrophages to the CNS, yet able to generate effector T cells that transferred severe disease to naive wild-type recipients. By contrast, adoptive transfer of primed T cells from wild-type mice into naive MCP-1-null recipients did not mediate clinical EAE. On the SJL background, disruption of the MCP-1 gene produced a milder EAE phenotype with diminished relapses that mimicked previous findings using anti-MCP-1 antibodies. There was no compensatory upregulation of MCP-2, MCP-3, or MCP-5 in MCP-1-null mice with EAE. These results indicated that MCP-1 is the major CCR2 ligand in mice with EAE, and provided an opportunity to define the role of MCP-1 in EAE. Compared with wild-type littermates, MCP-1-/- mice exhibited reduced expression of interferon gamma in draining lymph node and CNS and increased antigen-specific immunoglobulin G1 antibody production. Taken together, these data demonstrate that MCP-1 is crucial for Th1 immune responses in EAE induction and that macrophage recruitment to the inflamed CNS target organ is required for primed T cells to execute a Th1 effector program in EAE.

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Figures

Figure 1
Figure 1
Effect of MCP-1 gene disruption on MOG35-55–induced EAE. F9 MCP-1+/+, MCP-1−/+, and MCP-1−/− mice were immunized with MOG35–55 emulsified in CFA and intravenously injected with pertussis toxin (PT) on the day of immunization and 48 h later (500 ng/injection). Three wild-type mice died of EAE and another three had to be killed due to severe EAE attack. None of the MCP-1−/+ mice died of EAE, but three were killed. None of the MCP-1−/− mice died of EAE or had to be killed. Shown are EAE score (mean ± SD) in each group of mice that had been followed throughout the experiment (n = 3, 6, and 8 for MCP-1+/+, MCP-1−/+, and MCP-1−/− group, respectively). This graph is representative of three experiments with similar results.
Figure 2
Figure 2
Spinal cord histology of MCP-1+/+ and MCP-1−/− mice with EAE score 4.0. Hematoxylin and eosin staining of longitudinal cryosections. Note the numerous perivascular cuffs and subpial infiltrates (arrows) as well as leukocytes disseminated in the white matter of MCP-1+/+ mice (A), whereas merely fewer inflammatory infiltrates were found in MCP-1–deficient mice (B).
Figure 4
Figure 4
Milder disease and reduced number of relapses in PLP-induced EAE MCP-1−/− SJL mice. MCP-1−/− SJL mice and their littermate wild-type controls were immunized with PLP139–151 in CFA plus intravenous injection of pertussis toxin as described in Materials and Methods. Mice were monitored for 90 d after immunization. MCP-1−/− SJL mice showed significantly decreased EAE index, reduced number of relapses, and nonsignificantly delayed disease onset.
Figure 3
Figure 3
Attenuated MOG35–55–induced EAE in MCP-1–null mice. F10 MCP-1−/− mice (n = 6) and their littermate wild-type controls (n = 6) were immunized with MOG35–55 in CFA and intravenously injected with pertussis toxin (PT; 200 ng/injection). Shown are EAE score (mean ± SD) of individuals in each group.
Figure 5
Figure 5
Clinical course of EAE in MCP-1−/− and littermate control recipient mice that received MOG35–55–reactive T cells generated from MCP-1+/+ or MCP-1−/− littermates. Data are presented as the EAE score (mean ± SD) in each group. Each group consists of four recipient mice.
Figure 6
Figure 6
Altered pattern of CNS cell infiltrates in MCP-1–null mice. MCP-1−/− and MCP-1+/+ littermate controls were immunized with MOG35–55 and pertussis toxin (500 ng/injection) and killed at the peak of EAE (score 4). Cells were isolated from the CNS and stained with anti-CD4–FITC, anti-CD11b–PE, and anti-CD45–Cy mAbs. Compared with wild-type controls (A), the percentages of CD11b+CD4− cells were significantly decreased whereas the percentages of CD4+ T cells increased in MCP-1−/− mice (B). In contrast to MCP-1+/+ mice that developed full-blown EAE with numerous CNS CD11b+CD45high macrophages/activated microglia and T cells (C), the majority of cells isolated from CNS tissues of EAE symptom-free MCP-1−/− mice on day 14 pi were CD11b+CD45low microglia (D).
Figure 8
Figure 8
Cytokine levels in cell culture supernatants of lymphocytes from MOG35–55–immunized MCP-1–deficient mice and their littermate controls. Cells from draining lymph nodes were restimulated with MOG35–55 peptide in vitro. Supernatants were collected at different times after immunization as indicated and measured for IFN-γ, IL-10, and IL-4 (undetectable, data not shown). Data represent means ± SD and are representative of two experiments with similar results. WT, wild-type; KO, knockout.
Figure 7
Figure 7
Quantitation of circulating cytokine levels in MCP-1−/− and MCP-1+/+ mice immunized with MOG35–55 in CFA and pertussis toxin (500 ng/injection). Sera were collected at different times after immunization and measured for levels of IFN-γ (A), IL-10 (B), and IL-4 (undetectable, data not shown). Data are mean ± SD of five individual mice in each group. This histogram is representative of two experiments with similar results. WT, wild-type; KO, knockout.
Figure 9
Figure 9
Chemokine expression in spinal cords from MCP-1−/− and MCP-1+/+ mice immunized with MOG35–55 and pertussis toxin (500 ng/injection). Spinal cords were collected from mice that were equally affected by EAE (score 4) and levels of chemokine mRNA were measured using RPA. Data are presented as mean chemokine products ± SD. WT, wild-type; KO, knockout.
Figure 10
Figure 10
Serum concentrations of anti-MOG35–55 IgG, IgG1, and IgG2a Abs in MCP-1−/− and MCP-1+/+ littermate controls after immunization with MOG35–55 plus pertussis toxin (500 ng/injection). Indicated are days pi when sera were collected. Data are expressed as mean ± SD, n = 6, in each group. *P = 0.08, # P < 0.01. WT, wild-type; KO, knockout.
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