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The brain is an immunoprivileged organ isolated from the peripheral immune system. However, it has been shown that resident cells, notably astrocytes and microglia, can express numerous innate immune molecules, providing the capacity to generate a local antipathogen system. Perforin is a cytolytic protein present in the granules of cytotoxic T lymphocytes and natural killer cells. Expression in cells other than those of the hemopoetic lineage has not been described. We report here that fetal astrocytes in culture (passages 2 to 15), astrocytoma, and adult astrocytes expressed perforin. Reverse transcriptase polymerase chain reaction followed by Southern blot was carried out using multiple specific primers and all cDNAs were cloned and sequenced. Human fetal astrocyte perforin cDNA sequence was
100% identical to the reported perforin cDNA cloned from T cells. Western blot analysis using monoclonal and polyclonal antiperforin peptide antibodies revealed a protein of 65 kD in both human fetal astrocyte and rat natural killer cell lysates (n = 4). Immunostaining followed by FACS® and confocal and electron microscopy analysis revealed that perforin was expressed by 40–50% of glial fibrillary acidic protein positive cells present in the fetal brain culture (n = 11). Perforin was not localized to granules in astrocytes but was present throughout the cytoplasm, probably in association with the endoplasmic reticulum. Perforin was not detected in normal adult brain tissue but was present in and around areas of inflammation (white and grey matter) in multiple sclerosis and neurodegenerative brains. Perforin-positive cells were identified as reactive astrocytes. These findings demonstrate that perforin expression is not unique to lymphoid cells and suggest that perforin produced by a subpopulation of astrocytes plays a role in inflammation in the brain.
This work was supported by grants from the Medical Research Council (P. Gasque), the Wellcome Trust (B.P. Morgan), the Arthritis and Rheumatism Council (J. Jones), and the Welsh Scheme for the Development of Health and Social Research (S.K. Singhrao).
Address correspondence to Dr. Philippe Gasque, University of Wales College of Medicine, Department of Medical Biochemistry, Brain Inflammation and Immunity Group (BIIG), Tenovus Building, Heath Park, Cardiff, CF4 4XX, UK. Phone: 44-1-222-744236; Fax: 44-1-222-744305; E-mail: wmbpg{at}cardiff.ac.uk
1 Abbreviations used in this paper: AD, Alzheimer's disease; BCIP, 5-bromo-4-chloro-3indolyl phosphate; C4bp, C4B binding protein; CRP, C-reactive protein; DAB, diaminobenzidine; FH, complement factor H; GC, galactocerebroside; GFAP, glial fibrillary acidic protein; HD, Huntington's disease; MS, multiple sclerosis; NBT, nitroblue tetrazolium; NSE, neuron-specific enolase; PD, Pick's disease; RT, reverse transcriptase.
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