The central nervous system (CNS) is virtually isolated from circulating immunological factors such as complement (C), an important mediator of humoral immunity and inflammation. In circulation, C is constantly inhibited to prevent attack on host cells. Since a host of diseases produce an abnormal blood-brain/cerebrospinal fluid (blood-brain/CSF) permeability allowing C protein extravasation, we investigated if C activation occurs in CSF in vitro and in CNS in vivo during subarachnoid hemorrhage (SAH) or brain infarction. After SAH (n = 15), the terminal complement complex (TCC) concentration on days 0 to 2 was higher in the CSF, 210 +/- 61 ng/ml, than in the plasma, 63 +/- 17 ng/ml, but null in the CSF of controls (n = 8) or patients with an ischemic stroke (n = 7). TCC was eliminated from the CSF after SAH (24 +/- 10 ng/ml on days 7 to 10). Incubation of normal human CSF with serum in vitro also activated the terminal C pathway. In 10 fatal ischemic brain infarctions, immunohistochemical techniques demonstrated neuronal fragment-associated deposition of C9 accompanied by neutrophil infiltration. We conclude that the C system becomes activated intrathecally in SAH and focally in the brain parenchyma in ischemic stroke. By promoting chemotaxis and vascular perturbation, C activation may instigate nonimmune inflammation and aggravate CNS damage in diseases associated with plasma extravasation.

CD59 (protectin) is a glycophosphoinositol (GPI)-anchored inhibitor of the membrane attack complex of complement found on blood cells, endothelia and epithelial cells. In addition to the lipid-tailed CD59, soluble lipid-free forms of CD59 are present in human body fluids. We have investigated the detailed structural composition of the naturally occurring soluble urinary CD59 (CD59u) using peptide mapping, anion-exchange chromatography, sequential exoglycosidase digestion and matrix-assisted laser-desorption mass spectrometry (MALDI-MS). CD59u exhibited an average M(r) of 12444 in MALDI-MS. Mass analysis of the isolated C-terminal peptide (T9) indicated that a GPI-anchor (at Asn-77) without an inositol-associated phospholipid was present in soluble CD59u. By using residue-specific exoglycosidases, chemical modification and MALDI-MS structures of seven different GPI-anchor variants were determined. Variant forms of the anchor had deletions and/or extensions of one or more monosaccharide units. Sialic acid linked to an N-acetylhexosamine-galactose arm was found in two GPI-anchor variants. The N-linked carbohydrate side chain of CD59u (at Asn-18) also displayed considerable heterogeneity. The predominant oligosaccharide chains were fucosylated biantennary and triantennary complexes with variable sialylation. Mono Q anion-exchange chromatography resolved urinary CD59 into nine different fractions that bound equally well to the terminal complement SC5b-8 complexes. Despite binding to C5b-8, soluble CD59u inhibited complement lysis at an approx. 200-fold lower efficiency than erythrocyte CD59. These results document the structural heterogeneity of both the GPI anchor and N-linked oligosaccharide of CD59 and demonstrate that the phospholipid tail is needed for the full functional activity of CD59. The site of cleavage between the diradylglycerol phosphate and inositol suggests that a mammalian phospholipase D could be involved in the solubilization of GPI-anchored proteins.

Protectin (CD59) is a low molecular weight glycophosphoinositol-anchored inhibitor of the membrane attack complex of complement (MAC) that is present, for example, on the membranes of endothelial cells and on epithelial cells of glomeruli and distal tubuli. To examine for the possibility that CD59 becomes detached from cell surfaces following cell injury, this study evaluated renal excretion of CD59 in patients with idiopathic membranous glomerulonephritis (MGN; N = 21), diabetic nephropathy (DNP; N = 15) and in healthy control subjects (N = 13). CD59 in human urine was quantitated by a competitive solid-phase radioimmunoassay having approximately 13 kDa soluble urinary CD59 as a standard. Immunofluorescence microscopy demonstrated a decreased expression of CD59 in the glomeruli of MGN patients. Using a Triton X-114 phase separation method 91 to 97% of urinary CD59 was found to be in a soluble form without anchor-associated phospholipid. The mean (+/- SEM) level of urinary CD59 was 5.6 +/- 0.2 micrograms/ml in MGN patients, 3.7 +/- 0.4 micrograms/ml in healthy controls (P < 0.001) and 2.6 +/- 0.1 in DNP patients (P < 0.001). When related to urinary creatinine (UCr) the corresponding values were 11.9 +/- 5.6, 4.8 +/- 0.3 (P = 0.021) and 4.4 +/- 0.2 (P < 0.002), respectively. The amount of CD59 in urine correlated with the urinary excretion of soluble terminal complement complexes, SC5b-9 (r = 0.594, P < 0.006) in MGN patients. The excretion of CD59 also correlated with the excretion of the inflammatory mediator IL-1 beta (r = 0.671, P = 0.001) but not with TNF-alpha (r = 0.314, P = 0.178). No correlation of CD59 excretion was observed with duration of the disease level of proteinuria, serum albumin concentration or serum creatinine level. Based on these findings we speculate that the increased excretion of CD59 into urine in MGN patients is due to complement activation and inflammation induced shedding of CD59 from glomerular cells.

Activation of the complement system has been documented in both experimental and clinical studies of acute myocardial infarction (AMI). Our earlier immunohistochemical studies have shown that the deposition of the membrane attack complex (MAC) of complement is associated with the loss of protectin (CD59), a glycosylphosphatidylinositol (GPI)-anchored sarcolemmal regulator of MAC, from the human and rat infarcted myocardium. In this study we detected, using an enzyme immunoassay (EIA), CD59 in the plasma of AMI patients at a concentration of 23.0 6 8.4 ng/ml (mean 6 SD; n 17) at 4 h and 27.3 6 11.8 ng/ml (n 24) at 24 h after AMI. Both values were signi®cantly higher than in healthy controls (7.8 6 6.4 ng/ml; n 20; P < 0.001). The amount of CD59 correlated with the level of soluble terminal complement complexes (SC5b-9; r 0.84; P < 0.01) in the plasmas of AMI patients. Our results suggest that myocardial damage leads to release of CD59 from the sarcolemmal cell membranes during AMI.

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