Clusterin and the terminal complement pathway synthesized by human umbilical vein endothelial cells are closely linked when detected on co-cultured agarose beads.
Clusterin and the terminal complement pathway synthesized by human umbilical vein endothelial cells are closely linked when detected on co-cultured agarose beads. Clusterin is a multifunctional regulatory protein rendering the terminal complement complex (TCC) soluble and unable to insert into cell membranes. The aim of the present study was to examine whether clusterin was an integral part of serum-derived TCC bound to agarose beads which activate the alternative pathway of complement. Further, we searched for evidence of clusterin synthesis in human umbilical vein endothelial cells (EC) and whether this synthesis was regulated by various proinflammatory cytokines (IL-1, IL-6, and TNF) and IFN-gamma. The clusterin and TCC on co-incubated beads were measured by radioimmunoassay based on primary anti-complement antibodies (anti-C3c, anti-TCC, anti-clusterin). We found that clusterin in serum experiments is bound to C9 in agarose bound TCC and not directly to the agarose. Addition of the protein synthesis inhibitor cycloheximide to cultured human umbilical vein cells resulted in a strong reduction (about 70%) of anti-clusterin binding to co-cultured beads, which strongly supports de novo synthesis of clusterin in EC. The results indicate that clusterin derived from the EC is linked with the TCC on the co-incubated beads for the following reasons: First, in serum experiments clusterin like vitronectin, was co-deposited with C9 in agarose-bound TCC. Second, cytokine stimulation of the EC with proinflammatory cytokines such as IL-1, IL-6 and TNF, known to increase the detection of bound TCC, also increased the amount of clusterin detected on the beads. Third, IFN-gamma, which reduces the concentration of bound TCC, exhibited the same effect on the amount of clusterin detected on such beads. There was a strong and dose-dependent reduction of anti-TCC binding from about 45% to about 95% when clusterin (5-40 micrograms/ml) was added to EC cultures. This effect was also evident (about 40-50% inhibition of bound TCC) using human serum as complement source. These results are probably mainly caused by clusterin binding to C5b-7, making this complex soluble without the capacity to bind to the agarose surface. This study supports the view that clusterin is a potent regulator of TCC at the levels of C5b-7 and C9.
Berge
V
V
Department of Surgery, Ullevål Hospital, Norway.
Johnson
E
E
Høgåsen
K
K
D002478:Cells, Cultured; D051152:Clusterin; D003176:Complement C3 / Q000378:metabolism; D015938:Complement Membrane Attack Complex / Q000737:chemistry*; D003170:Complement Pathway, Alternative; D016207:Cytokines / Q000494:pharmacology; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism*; D005312:Fetal Blood; D006023:Glycoproteins / Q000096:biosynthesis* / Q000276:immunology / Q000378:metabolism; D006801:Humans; D008863:Microspheres; D018832:Molecular Chaperones; D012685:Sepharose
C495242:CLU protein, human; D051152:Clusterin; D003176:Complement C3; D015938:Complement Membrane Attack Complex; D016207:Cytokines; D006023:Glycoproteins; D018832:Molecular Chaperones; D012685:Sepharose