Increased calpain expression in activated glial and inflammatory cells in experimental allergic encephalomyelitis

Abstract

In demyelinating diseases such as multiple sclerosis (MS), myelin membrane structure is destabilized as myelin proteins are lost. Calcium-activated neutral proteinase (calpain) is believed to participate in myelin protein degradation because known calpain substrates [myelin basic protein (MBP); myelin-associated glycoprotein] are degraded in this disease. In exploring the role of calpain in demyelinating diseases, we examined calpain expression in Lewis rats with acute experimental allergic encephalomyelitis (EAE), an animal model for MS. Using double-immunofluorescence labeling to identify cells expressing calpain, we labeled rat spinal cord sections for calpain with a polyclonal millicalpain antibody and with mAbs for glial (GFAP, OX42, GalC) and inflammatory (CD2, ED2, interferon gamma) cell-specific markers. Calpain expression was increased in activated microglia (OX42) and infiltrating macrophages (ED2) compared with controls. Oligodendrocytes (galactocerebroside) and astrocytes (GFAP) had constitutive calpain expression in normal spinal cords whereas reactive astrocytes in spinal cords from animals with EAE exhibited markedly increased calpain levels compared with astrocytes in adjuvant controls. Oligodendrocytes in spinal cords from rats with EAE expressed increased calpain levels in some areas, but overall the increases in calpain expression were small. Most T cells in grade 4 EAE expressed low levels of calpain, but interferon gamma-positive cells demonstrated markedly increased calpain expression. These findings suggest that increased levels of calpain in activated glial and inflammatory cells in EAE may contribute to myelin destruction in demyelinating diseases such as MS.

Figure 1

H&E and calpain immunoperoxidase staining of Lewis rat spinal cord (×200). (A) Normal rat spinal cord white matter with H&E stain. (B) Spinal cords of Lewis rats with acute EAE demonstrated perivascular cuffing and increased numbers of glial and inflammatory cells with H&E staining. (C) Immunoperoxidase staining of control spinal cords showed some calpain expression in glial cells of white matter. (D) In white matter of spinal cords from animals with EAE, a greater number of cells demonstrated markedly increased calpain expression with immunoperoxidase staining.

Figure 2

Double-immunofluorescent staining of Lewis rat spinal cord for calpain (green) and cell-specific markers (red) (×200). (A) Calpain and GFAP (astrocytes) were colocalized (yellow) in normal spinal cord white matter. (B) Markedly increased calpain expression in reactive astrocytes of EAE (arrow). (C) Microglia (red) in normal spinal cord white matter. (D) Spinal cords from animals with EAE showed markedly increased calpain expression in reactive microglia (arrow). (E) Normal spinal cords stained for calpain and galactocerebroside (oligodendrocytes). (F) Oligodendrocytes in EAE showed a small increase in calpain immunofluorescence. (G) Calpain and CD2 (T cells) antibodies in controls often detected no T cells. (H) Spinal cords from EAE animals contained many T cells with limited calpain expression. (I) Normal spinal cords labeled with calpain and IFN-γ antibodies. (J) In EAE, calpain and IFN-γ expression were colocalized as both proteins appear inside and outside the activated T cells (arrow). (K) Macrophage (red) in normal spinal cord white matter. (L) Spinal cords of rats with EAE showed calpain expression in an activated macrophage (arrow).