Sensitization to brain antigens after stroke is augmented by lipopolysaccharide

Abstract

After stroke, the blood-brain barrier is transiently disrupted, allowing leukocytes to enter the brain and brain antigens to enter the peripheral circulation. This encounter of normally sequestered brain antigens by the systemic immune system could therefore present an opportunity for an autoimmune response to brain to occur after stroke. In this study, we assessed the immune response to myelin basic protein (MBP) in animals subjected to middle cerebral artery occlusion (MCAO). Some animals received an intraperitoneal injection of lipopolysaccharide (LPS; 1 mg/kg) at reperfusion to stimulate a systemic inflammatory response. At 1 month after MCAO, animals exposed to LPS were more likely to be sensitized to MBP (66.7% versus 22.2%; P=0.007) and had more profound and persistent neurologic deficits than non-LPS-treated animals. Exposure to LPS was associated with increased expression of the costimulatory molecule B7.1 early after stroke onset (P=0.009) and increased brain atrophy 1 month after MCAO (P=0.03). These data suggest that animals subjected to a systemic inflammatory insult at the time of stroke are predisposed to develop an autoimmune response to brain, and that this response is associated with worse outcome. These data may partially explain why patients who become infected after stroke experience increased morbidity.

Figures

Figure 1

Regions of the brain evaluated for quantitative ICC; the ischemic hemisphere areas 1 to 4 are in the ischemic or right hemisphere; areas 5 and 6 in the nonischemic or left hemisphere.

Figure 2

MNCs extracted from the brains of LPS(+) animals 30 days after MCAO evidence a Th1 response to MBP, which is more robust than sham-operated animals and LPS(–) animals at the same time point (A). The Y-axis represents the ratio of the number of MBP-specific cells secreting IFN-γ to that of the number of MBP-specific cells secreting TGF-β1. Lipopolysaccharide(–) animals, however, develop a Th2/Th3 response to MBP, as indicated by an increase in the ratio of TGF-β1- to IFN-γ-secreting MBP-reactive MNCs in brain (B). The Y-axis represents the ratio of the number of MBP-specific cells secreting TGF-β1 to that of the number of MBP-specific cells secreting IFN-γ. There is an absolute increase in the number of MBP-specific Th1 cells in the brains of LPS(+) animals 1 month after MCAO (C). There was also a trend for increased numbers of MBP-specific cells and TGF-β1-secreting cells in the brains of LPS(–) animals at the same time point (P=0.08) (D). The Y-axis represents the number of MBP-reactive cells per 1 × 105 total cells secreting either IFN-γ (C) or TGF-β1 (D). *P<0.05 in comparison to control/sham-operated LPS(–)animals; †P<0.05 in comparison to each other.

Figure 3

Neurologic scores were higher in LPS(+) animals after MCAO (A). Similarly, Th1(+) animals, irrespective of LPS treatment status, had higher neurologic scores as long as 1 week after MCAO (B). The degree of sensitization to MBP (the ratio of MBP-specific IFN-γ-secreting to TGF-β1-secreting cells) 1 month after MCAO was strongly correlated with neurologic scores 72h (P=0.002) and 7 days (P=0.02) after MCAO (C, D). Lipopolysaccharide treatment had no effect on body weight 1 month after MCAO (E), but animals that were sensitized to MBP were less likely to gain weight (F). *P<0.05.

Figure 4

More CD4+ cells are present in both the ischemic (A) and nonischemic (B) hemispheres of LPS(+) animals early after MCAO. CD4+ cells can be seen in approximation to neurons 24h after MCAO; CD4+ cells labeled with DAB, neurons labeled with CV (C), or CD4+ cells labeled FITC and neurons labeled with cy-3-tagged antibodies to NSE (D) (100×). The number of CD8+ cells peaks late after MCAO; more CD8+ cells are present in the brains of LPS(+) animals 1 month after MCAO (E, F). CD8+ cells are seen in the periinfarct region of LPS(+) animals 1 month after MCAO (100×)(G, H). The Y-axis represents the total number of cells counted (as described in Materials and methods). *P<0.05; **P<0.001.

Figure 5

Lipopolysaccharide significantly increases the expression of B7.1 early after MCAO (A, B). There is increased expression of B7.2 1 month (720h) after MCAO in LPS(–) animals (C, D). B7.1 staining 24h after MCAO in LPS(+) animals (40×)(E). B7.1 is expressed on microglia (100×)(F to H). B7.2 is expressed on microglia-like cells 1 month after stroke in LPS(–) animals; ×40(I) and × 100 (J). Major histocompatibility class I(K, L) and MHC II (M, N) expression is enhanced shortly after LPS administration. Major histocompatibility class II expression 24h after MCAO in LPS(+) animals; ×40 (O) and × 100 (P). The Y-axis represents the total number of cells counted using the protocol described in Materials and methods. *P<0.05; **P<0.001.

Figure 6

Animals treated with LPS have more brain atrophy after stroke. Infarct volume did not differ between LPS(+) and LPS(–) animals at any time point after MCAO (A). At 1 month after MCAO, the ischemic hemisphere of LPS(+) animals evidences more atrophy than that of LPS(–) animals (B). There are more pyknotic/apoptotic neurons per total number of neurons in both the ischemic and nonischemic hemispheres of LPS(+) animals (C). There is little evidence of apoptosis in LPS(–) animals (D), while apoptotic cells are numerous in LPS(+) animals (E).