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. 2014 Aug:40:61-73.
doi: 10.1016/j.bbi.2014.02.003. Epub 2014 Feb 24.

Leukocyte infiltration into spinal cord of EAE mice is attenuated by removal of endothelial leptin signaling

Suidong Ouyang  1 , Hung Hsuchou  1 , Abba J Kastin  1 , Pramod K Mishra  1 , Yuping Wang  1 , Weihong Pan  2
Affiliations

Leukocyte infiltration into spinal cord of EAE mice is attenuated by removal of endothelial leptin signaling

Suidong Ouyang et al. Brain Behav Immun. 2014 Aug.

Abstract

Leptin, a pleiotropic adipokine, crosses the blood-brain barrier (BBB) and blood-spinal cord barrier (BSCB) from the periphery and facilitates experimental autoimmune encephalomyelitis (EAE). EAE induces dynamic changes of leptin receptors in enriched brain and spinal cord microvessels, leading to further questions about the potential roles of endothelial leptin signaling in EAE progression. In endothelial leptin receptor specific knockout (ELKO) mice, there were lower EAE behavioral scores in the early phase of the disorder, better preserved BSCB function shown by reduced uptake of sodium fluorescein and leukocyte infiltration into the spinal cord. Flow cytometry showed that the ELKO mutation decreased the number of CD3 and CD45 cells in the spinal cord, although immune cell profiles in peripheral organs were unchanged. Not only were CD4(+) and CD8(+) T lymphocytes reduced, there were also lower numbers of CD11b(+)Gr1(+) granulocytes in the spinal cord of ELKO mice. In enriched microvessels from the spinal cord of the ELKO mice, the decreased expression of mRNAs for a few tight junction proteins was less pronounced in ELKO than WT mice, as was the elevation of mRNA for CCL5, CXCL9, IFN-γ, and TNF-α. Altogether, ELKO mice show reduced inflammation at the level of the BSCB, less leukocyte infiltration, and better preserved tight junction protein expression and BBB function than WT mice after EAE. Although leptin concentrations were high in ELKO mice and microvascular leptin receptors show an initial elevation before inhibition during the course of EAE, removal of leptin signaling helped to reduce disease burden. We conclude that endothelial leptin signaling exacerbates BBB dysfunction to worsen EAE.

Keywords: Autoimmunity; Blood–spinal cord barrier; EAE; Endothelia; Leptin receptor.

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Conflict of interest statement

Conflict of Interest

There is no conflict of interest.

Figures

Fig. 1
Fig. 1
The mRNA level of ObR isoforms showed a dynamic change during the course of EAE. (A) In enriched microvessels from cerebral cortex, there was an initial non-significant increase on day 6, followed by a significant reduction on day 17 and day 24 for all three membrane-bound leptin receptors. The soluble receptor ObRe also was reduced on day 24. (B) In enriched spinal cord microvessels, there was an initial increase of membrane-bound ObRs on day 6, and subsequent reduction of all isoforms on day 17. *p < 0.05; **p < 0.01; ***p < 0.005 in comparison with 0 time.
Fig. 2
Fig. 2
Effects of ELKO on EAE scores and blood leptin concentrations. (A) The EAE symptoms in ELKO mice were less severe than those of WT mice, supported by the significantly lower EAE scores in the ELKO group on days 12,13,17, 18 and 20 after EAE induction (n = 7/group). The cumulative 10-day score (day 11–20) was also lower in the ELKO mice (inset). (B) On day 17 of EAE, both serum leptin and sObR concentrations were higher in the ELKO mice than in the WT mice (n = 2–3/group). *p < 0.05; **p < 0.01; ***p < 0.005.
Fig. 3
Fig. 3
Effects of ELKO and EAE on paracellular permeability of the BSCB. (A) The spinal cord uptake of sodium (Na) fluorescein 30 min after iv injection was higher in EAE mice than in CFA mice. The increase was less pronounced in the ELKO with EAE than in the WT group with EAE. (B) The brain uptake was also higher in EAE than CFA groups, and showed a trend of difference (p = 0.07) between the WT and ELKO groups with EAE. *p < 0.05; **p < 0.01.
Fig. 4
Fig. 4
Leukocyte accumulation in the spinal cord was seen by immunofluorescent staining of CD45 (marker for leukocytes) and CD3 (marker for T cells) against a background of DAPI staining to demarcate all nucleated cells (both infiltrated and CNS residential cells). There was increased cellularity in the WT mice, both for CD45 in the ventral cervical spinal cord (A) and CD3 in the dorsal cervical spinal cord (C). There was also perivascular cuffing in the WT with EAE (arrow). The difference in the number of infiltrated cells in the whole spinal cord was quantified by flow cytometry of cells recovered after vascular washout, homogenization, and Percoll gradient isolation. The number of CD45+ cells (B) and CD3+ cells (D) both showed less increase in ELKO mice with EAE than WT mice with EAE (n = 3/group). *p < 0.05.
Fig. 4
Fig. 4
Leukocyte accumulation in the spinal cord was seen by immunofluorescent staining of CD45 (marker for leukocytes) and CD3 (marker for T cells) against a background of DAPI staining to demarcate all nucleated cells (both infiltrated and CNS residential cells). There was increased cellularity in the WT mice, both for CD45 in the ventral cervical spinal cord (A) and CD3 in the dorsal cervical spinal cord (C). There was also perivascular cuffing in the WT with EAE (arrow). The difference in the number of infiltrated cells in the whole spinal cord was quantified by flow cytometry of cells recovered after vascular washout, homogenization, and Percoll gradient isolation. The number of CD45+ cells (B) and CD3+ cells (D) both showed less increase in ELKO mice with EAE than WT mice with EAE (n = 3/group). *p < 0.05.
Fig. 5
Fig. 5
Flow cytometric analysis of leukocytes recovered from the spinal cord of WT or ELKO mice with EAE. (A) Flow cytometry profiles of the cell surface markers and gating strategy. (B) The percent of CD4+ T cells among all cells analyzed in the same sample showed an increase over time, and was less on day 13 in the ELKO mice with EAE than in the WT mice with EAE. (C) The percent of CD8+ T cells increased over time in both WT and ELKO mice, but the elevation was less pronounced in the ELKO group than in the WT group on day 17 of EAE. (D) In WT mice with EAE, the percentage of CD11b+Gr1+ granulocytes increased over time. In ELKO mice with EAE, the increase was not present on day 13 but became apparent on day 17. At this time, the level remained lower than that in WT mice with EAE (n = 3/time point in each group). *p < 0.05 when WT and ELKO were compared on the same day of EAE.
Fig. 5
Fig. 5
Flow cytometric analysis of leukocytes recovered from the spinal cord of WT or ELKO mice with EAE. (A) Flow cytometry profiles of the cell surface markers and gating strategy. (B) The percent of CD4+ T cells among all cells analyzed in the same sample showed an increase over time, and was less on day 13 in the ELKO mice with EAE than in the WT mice with EAE. (C) The percent of CD8+ T cells increased over time in both WT and ELKO mice, but the elevation was less pronounced in the ELKO group than in the WT group on day 17 of EAE. (D) In WT mice with EAE, the percentage of CD11b+Gr1+ granulocytes increased over time. In ELKO mice with EAE, the increase was not present on day 13 but became apparent on day 17. At this time, the level remained lower than that in WT mice with EAE (n = 3/time point in each group). *p < 0.05 when WT and ELKO were compared on the same day of EAE.
Fig. 6
Fig. 6
Effect of ELKO and EAE on the number of three major cell populations. (A) Among cells recovered from spinal cord 17 days after EAE induction, there were fewer CD4+, CD8+, and CD11b+Gr1+ cells in the ELKO group than in the WT group. (B) In the thymus, EAE (day 17 after induction) reduced the number of CD4+ and CD8+ T cells without affecting the CD11b+Gr1+ granulocytes. There was no difference between ELKO and WT, either in naïve or EAE mice. (C) The lymph nodes showed the same pattern of changes as the thymus. (D) Neither ELKO nor EAE affected the number of CD4+ and CD8+ T cells in the spleen. However, the number of CD11b+Gr1+ granulocytes was decreased by EAE, though ELKO had no additional effect (n = 3/group). *p < 0.05; **p < 0.01; ***p < 0.005.
Fig. 7
Fig. 7
Effects of both EAE and the ELKO mutation on chemokine and cytokine mRNA expression in enriched spinal cord microvessels. (A) CXCL9 and (B) CCL5 were increased on day 17 after EAE induction in comparison with naïve controls. The increase in the ELKO group with EAE was less pronounced than that in the WT group with EAE. (C) CXCL10 and (D) CCL12 were increased by EAE but there was no difference between WT and ELKO groups. (E) IFNγ and (F) TNF showed upregulation in EAE microvessels; the increase was more in WT group and less pronounced in the ELKO group (n = 3/group). *p < 0.05; **p < 0.01; ***p < 0.005.
Fig. 8
Fig. 8
Differential effects of EAE and ELKO mutation were seen on mRNA expression of tight junction proteins in spinal cord microvessels. ZO-1 (A) and occludin (B) were decreased by EAE on day 17. The reduction was less severe in the ELKO than WT mice with EAE. Claudin-1 (C), claudin-2 (D), and claudin-5 (F) showed an effect of EAE but not of the ELKO mutation by 2-way ANOVA. Claudin-3 (E) did not show a difference induced by EAE or ELKO. *p < 0.05 for post hoc comparison between ELKO EAE and WT EAE.
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