Expression and function of membrane regulators of complement on rat astrocytes in culture.
Human astrocytes express CD59, decay accelerating factor and membrane cofactor protein to restrict the damaging effect of complement (C) activation on their cell surface. 5I2 antigen (5I2 Ag) is the functional analogue of the latter two proteins in rats. We here demonstrate the surface expression on rat astrocytes of CD59 and 5I2 Ag and use sodium dodecyl sulphate-polyacrylamide gel electrophoresis and Western blotting to confirm their identity and to quantify expression. Rat CD59 (MW 20,000) was expressed at 720 x 10(3) molecules per cell and 5I2 Ag (MW 58,000 and 64,000) at 625 x 10(3) molecules per cell. Reverse transcription-polymerase chain reaction using specific oligonucleotide primers demonstrated expression of mRNA for each protein. Twenty-four-hour stimulation with inflammatory cytokines (interferon-gamma, tumour necrosis factor-alpha, interleukins-1 beta, -2 and -6) or phorbol myristate acetate had no significant effect on the level of expression of either protein as determined by Western blotting. Lysis caused by classical pathway activation of C in human or rat serum was enhanced by blocking the function of CD59 and 5I2 Ag on rat astrocytes with monoclonal antibodies.
Rogers
C A
CA
Department of Medical Biochemistry, University of Wales College of Medicine, Cardiff, UK.
Gasque
P
P
Piddlesden
S J
SJ
Okada
N
N
Holers
V M
VM
Morgan
B P
BP
D000818:Animals; D000911:Antibodies, Monoclonal / Q000494:pharmacology; D000951:Antigens, Neoplasm / Q000032:analysis* / Q000235:genetics / Q000276:immunology; D001253:Astrocytes / Q000737:chemistry*; D001483:Base Sequence; D015153:Blotting, Western; D018957:CD59 Antigens / Q000032:analysis* / Q000235:genetics / Q000276:immunology; D002462:Cell Membrane / Q000737:chemistry; D002478:Cells, Cultured; D003167:Complement Activation; D003169:Complement Inactivator Proteins / Q000032:analysis* / Q000235:genetics / Q000276:immunology; D017931:DNA Primers / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D017208:Rats, Wistar
D000911:Antibodies, Monoclonal; D000951:Antigens, Neoplasm; D018957:CD59 Antigens; D003169:Complement Inactivator Proteins; D017931:DNA Primers; D012333:RNA, Messenger