Endothelial IL-1R1 is a critical mediator of EAE pathogenesis

Abstract

Interleukin-1 (IL-1) has been implicated in the disease progression of multiple sclerosis (MS). In the animal model of MS, experimental autoimmune encephalomyelitis (EAE), the induction of disease is significantly attenuated in mice lacking the type I IL-1 receptor (IL-1R1). In this study, we created a transgenic mouse (eIL-1R1 kd) in which IL-1R1 expression is knocked down specifically in endothelial cells. Induction of EAE in eIL-1R1 kd mice results in a decrease in incidence, severity and delayed onset of EAE. In addition, eIL-1R1 kd mice show significant decrease in VCAM-1 expression and diminished CD45(+) and CD3(+) infiltrating leukocytes in the spinal cord in animals challenged with EAE. Further, IL-1 and IL-23 stimulate IL-17 production by splenocytes from both wild type and the eIL-1R1 kd animals. Similarly, IL-1 and IL-23 synergistically stimulate splenocytes proliferation in these two strains of animals. After immunization with MOG(79-96), although eIL-1R1 kd mice displayed greatly reduced clinical scores, their splenocytes produced IL-17 and proliferated in response to a second MOG challenge, similar to wild type animals. These findings indicate a critical role for endothelial IL-1R1 in mediating the pathogenesis of EAE, and describe a new model that can be used to study endothelial IL-1R1.

Figure 1

A. Schematic drawing of the transgene construct for AntiIL-1R1. The antisense cDNA for IL-1R1 (AntiIL-1R1) was placed between the Tie2 promoter (Tie2P) and the Tie2 enhancer (Tie2E). L: linker sequence. B. Top, realtime PCR results show founder animal contains GFP sequence; bottom, detection of AntiIL-1R1 (via the detection of the linker sequence). These transgenes are absent in WT control animals. C. Transgene expression is found in brain (Fig. 1Ca), thymus (Fig.1Cb), and spleen (Fig. 1Cc) in endothelial cells, as indicated by the cell type-specific appearance of GFP in these cells. Fig. 1Cd show spleen CD3 labeling; CD3⁺ T cells do not show the same morphology as the transgene-expressing cells. Inset in Fig. 1Ca shows double-labeling of GFP (green) and PECAM (red) in eIL-1R1 kd brain tissue; all GFP expressing cells are co-localized with the endothelial marker PECAM.

Figure 2

Representative microphotographs show COX-2 induction in the brain 4 h after icv IL-1β injection in WT (A) and eIL-1R1 kd (B) and animals. Arrow points to labeled COX-2 positive cells.

Figure 3

Detection of IL-1R1 protein by in-cell Western in leukocytes from WT and eIL-1R1 kd animals. # indicates no statistical difference between WT and eIL-1R1 kd leukocytes, p<0.05 by student t-test.

Figure 4

Mean clinical scores of EAE symptoms in WT FVB and eIL-1R1 kd animals. Means and standard error of the mean are presented. Higher EAE scores were found in the WT FVB mice (n =17 for both groups, p<0.05 by two-way ANOVA).

Figure 5

Representative microphotographs of H&E stained and immunohistochemically labeled spinal cord sections at the onset of EAE. A–D are from eIL-1R1 kd animals, and a–d WT FVB animals. A and a: H&E; B and b: CD45, C and c: CD3; D and d: VCAM-1. Arrows point to areas of perivascular cuffing and spinal inflammation in Fig. 5a, CD45+ infiltrating leukocytes in Fig. 5b, CD3+ infiltrating T cells in Fig. 5c, and VCAM positive blood vessels in Figs. 5d and 5D.

Figure 6

IL-1 and IL-23 stimulated splenocytes responses. A. IL-17 levels after splenocytes were stimulated with IL-1, IL-23, and IL-1+IL-23. B. Proliferation of splenocytes after they were treated with IL-1, IL-23, and IL-1+IL-23. # indicates no statistical difference between results obtains from WT FVB and eIL-1R1 kd splenocytes (student t-test, p>0.05).

Figure 7

Antigen specific response. A. IL-17 levels produced in response to MOG stimulation by splenocytes from animals that were previously immunized with MOG. B. Proliferation of the same sensitized splenocytes after the second MOG challenge. # indicates no statistical difference between results obtains from WT FVB and eIL-1R1 kd splenocytes (student t-test, p>0.05).