Vasoactive intestinal peptide loss leads to impaired CNS parenchymal T-cell infiltration and resistance to experimental autoimmune encephalomyelitis

Abstract

The neuropeptide vasoactive intestinal peptide (VIP) has been shown to inhibit macrophage proinflammatory actions, promote a positive Th2/Th1 balance, and stimulate regulatory T-cell production. The fact that this peptide is highly efficacious in animal models of inflammatory diseases such as collagen-induced arthritis and experimental autoimmune encephalomyelitis (EAE) suggests that the endogenous peptide might normally provide protection against such pathologies. We thus studied the response of VIP-deficient (i.e., VIP KO) mice to myelin oligodendrocyte protein-induced EAE. Surprisingly, VIP KO mice were almost completely resistant to EAE, with delayed onset and mild or absent clinical profile. Despite this, flow cytometric analyses and antigen-rechallenge experiments indicated that myelin oligodendrocyte protein-treated VIP KO mice exhibited robust Th1/Th17 cell inductions and antigen-specific proliferation and cytokine responses. Moreover, adoptive transfer of lymphocytes from immunized VIP KO mice to WT recipients resulted in full-blown EAE, supporting their encephalitogenic potential. In contrast, transfer of encephalitogenic WT cells to VIP KO hosts did not produce EAE, suggesting that loss of VIP specifically affected the effector phase of the disease. Histological analyses indicated that CD4 T cells entered the meningeal and perivascular areas of VIP-deficient mice, but that parenchymal infiltration was strongly impaired. Finally, VIP pretreatment of VIP KO mice before immunization was able to restore their sensitivity to EAE. These results indicate that VIP plays an unanticipated permissive and/or proinflammatory role in the propagation of the inflammatory response in the CNS, a finding with potential therapeutic relevance in autoimmune neuroinflammatory diseases such as multiple sclerosis.

Fig. 1.

Comparison of EAE in WT and VIP KO mice. Mice (n = 9–10 per group) were immunized s.c. with 100 μg of MOG_35–55 in CFA and injected with pertussis toxin on days 0 and 2. (A) Representative experiment shows the mean clinical scores ± SEM in a range from 0 to 4 as described in Materials and Methods (*P < 0.05, Student t test). (B) Overall percent incidence of disease over time (summary of three experiments; *P < 0.05 by log-rank test). Histological changes in transverse sections of spinal cords from MOG-immunized WT and VIP KO mice. (C and D) Low-magnification photomicrographs (4×) of thoracolumbar spinal cord from WT (C) and VIP KO (D) mice. WT mice show multifocal inflammation in the meninges and white matter parenchyma. (E and F) Higher-magnification photomicrographs (20×) of spinal cords of WT and VIP KO mice, respectively. Whereas evidence of intense inflammation is present in the leptomeninges and white matter of the spinal cord of the WT mice (E), inflammation, when present, was much less severe in VIP KO mice, and was primarily in the leptomeninges, sometimes invading the anterior fissure (F). (G) Mean histological score of sections from WT and VIP KO mice ± SEM (**P < 0.01, Student t test). ND, no inflammation detected.

Fig. 2.

Th profiles and MOG-induced proliferative and cytokine responses in lymph node cultures in WT versus VIP KO mice. (A) Photograph of WT and VIP KO spleen and draining lymph nodes (DLN) 14 d after EAE induction. Cell suspensions from DLN from EAE-induced WT and VIP KO mice (day 14) were prepared. For flow cytometry Th profile analyses (B), cells were stimulated for 5 h with PMA/ionomycin/brefeldin, and then stained for CD4 and IFNγ, IL-17, or IL-4. For ex vivo studies (C), cells were stimulated with MOG_35–55 peptide or ovalbumin (10 μg/mL). Proliferation was measured 72 h later by [³H]thymidine incorporation. Cytokines released to the culture supernatant were measured by ELISA 48 h after stimulation with MOG_35–55. IL-4 expression was detected in cell extracts by real-time PCR. Data shown are representative of three independent experiments (*P < 0.05 and **P < 0.01, Student t test). In WT mice, all parameters were significantly induced by MOG (*P < 0.05). NS, not significant.

Fig. 3.

Adoptive transfer of immune cells from WT or VIP KO MOG-injected mice triggered EAE in WT but not in VIP KO recipients. Encephalitogenic cells were prepared by immunizing WT or VIP KO mice and culturing their spleen and lymph node cells harvested on day 14 in the presence of 30 μg/mL of MOG for 3 d. Cells were injected i.v. into WT and/or VIP KO C57BL/6 mice, and EAE clinical scores were assessed as described in Fig. 1A. Data are shown as a mean clinical score ± SEM (n = 4 per group). One of three similar experiments is shown.

Fig. 4.

Immune cell infiltration is impaired in VIP KO mice. EAE was induced to WT and VIP KO mice as in Fig. 1 legend, and CNS tissues were collected on days 7, 10, and 14. (A–E) Total numbers of mononuclear cells, as well as CD11b, CD3, CD4, and CD8 cells. (F) Total numbers of Th1 and Th17 cells in the CNS on day 14. The number of infiltrating immune cells was remarkably lower in VIP KO mice (*P < 0.05, **P < 0.01, and ***P < 0.001; Student t test). (G) Photomicrographs at magnifications of 4× (Upper) and 20× (Lower) of spinal cord sections from WT (Left) and VIP KO mice (Right) collected 14 d after immunization and stained by immunofluorescence for CD4 (Alexa 594), laminin (FITC), and DAPI. Thoracic level is shown. Top: CD4 staining only. Lower: Triple staining of laminin, CD4, and DAPI. Note in particular the high abundance of CD4⁺ cells in the parenchyma of WT but not VIP KO mice (lower two panels), and their apparent nonassociation with laminin-positive blood vessels. Similar results were found at all levels of spinal cords of four sets of WT and VIP KO mice.

Fig. 5.

Pretreatment of VIP KO mice with VIP restores their sensitivity to EAE. Mice were injected i.p. with 10 nmoles of PBS solution or VIP every day for 2 wk, and treatment was stopped 1 d before EAE immunization. One of three representative experiments is shown. Scores in VIP-treated VIP KO mice were higher than in PBS-treated VIP KO mice on days 10 to 30 (*P < 0.05, Student t test) and higher than in WT mice on days 16 to 20.