Inhibition of complement alternative pathway function with anti-properdin monoclonal antibodies.

Complement activation products appear to contribute to the pathology of several acute and chronic inflammatory conditions. The relative contributions of the classical and alternative complement pathways to these pathologies have, in large part, been undefined. Considerable progress has been made recently in identifying inhibitors of complement activation and demonstrating that such molecules can attenuate inflammation in various models of disease. However, most of these complement inhibitors affect aspects of both the classical and alternative pathways. In an effort to better define the role of the alternative complement pathway in complement-mediated inflammatory conditions, we have developed monoclonal antibodies that specifically inhibit alternative pathway function. These blocking antibodies bind human properdin with high avidity and prevent its interaction with the alternative pathway C3 convertase. This results in a cessation of alternative pathway function in several in vitro assay systems. When tested in a model of cardiopulmonary bypass, in which human blood passes through tubing, a selected antiproperdin antibody caused nearly complete inhibition of the C3a and C5b-9 formation that was seen in untreated blood. Moreover, the anti-properdin agent resulted in a dramatic reduction of neutrophil and platelet activation in the bypass model. Surprisingly, the monoclonal antibody also caused a significant inhibition of C5b-9 generation when classical pathway activators, such as heparin-protamine or immune complexes, were added to human blood. These latter data suggest that the alternative pathway contributes significantly to the formation of complement activation products in blood when the classical pathway is initially triggered.

Congo red inhibits proteoglycan and serum amyloid P binding to amyloid beta fibrils.

Various data suggest that Alzheimer's disease results from the accumulation of amyloid beta (A beta) peptide fibrils and the consequent formation of senile plaques in the cognitive regions of the brain. One approach to lowering senile plaque burden in Alzheimer's disease brain is to identify compounds that will increase the degradation of existing amyloid fibrils. Previous studies have shown that proteoglycans and serum amyloid P (SAP), molecules that localize to senile plaques, bind to A beta fibrils and protect the amyloid peptide from proteolytic breakdown. Therefore, molecules that prevent the binding of SAP and/or proteoglycans to fibrillar A beta might increase plaque degradation and prove useful in the treatment of Alzheimer's disease. The nature of SAP and proteoglycan binding to A beta is defined further in the present study. SAP binds to both fibrillar and nonfibrillar forms of A beta. However, only the former is rendered resistant to proteolysis after SAP association. It is interesting that both SAP and proteoglycan binding to A beta fibrils can be inhibited by glycosaminoglycans and Congo red. Unexpectedly, Congo red protects fibrillar A beta from breakdown, suggesting that this compound and other structurally related molecules are unlikely to be suitable for use in the treatment of Alzheimer's disease.

Amyloid beta protein (A beta) removal by neuroglial cells in culture.

Because the mechanisms of A beta degradation in normal and Alzheimer's disease brain are poorly understood, we have examined whether various cortical cells are capable of processing this peptide. Rat microglia and astrocytes, as well as the human THP-1 monocyte cell line, degraded A beta 1-42 added to culture medium. In contrast, neither rat cortical neurons or meningeal fibroblasts effectively catabolized this peptide. When A beta fibrils were immobilized as plaque-like deposits on culture dishes, both microglia and THP-1 cells removed the peptide. Astrocytes were incapable of processing the A beta deposits, but these cells released glycosaminoglycase-sensitive molecules that inhibited the subsequent removal of A beta by microglia. This implied that astrocyte-derived proteoglycans associated with the amyloid peptide and slowed its degradation. The addition of purified proteoglycan to A beta that was in medium or focally deposited also resulted in significant inhibition of peptide removal by microglia. These data suggest that A beta can be catabolized by microglia and proteoglycans which co-localize with senile plaques may slow the degradation of A beta within these pathologic bodies.

Proteoglycan-mediated inhibition of A beta proteolysis. A potential cause of senile plaque accumulation.

Senile plaques of Alzheimer's disease brain contain, in addition to beta amyloid peptide (A beta), multiple proteoglycans. Systemic amyloidotic deposits also routinely contain proteoglycan, suggesting that these glycoconjugates are generally involved in amyloid plaque formation and/or persistence. We demonstrate that heparan sulfate proteoglycan (HSPG) and chondroitin sulfate proteoglycan (CSPG) inhibit the proteolytic degradation of fibrillar, but not non-fibrillar, A beta at physiological pH. In accordance with the proteolysis studies, high affinity binding of proteoglycans to fibrillar A beta(1-40) and A beta(1-42) is observed from pH 4 to 9, whereas appreciable binding of HSPG or CSPG to non-fibrillar peptide is only seen at pH < 6. This differing pH dependence of binding suggests that a lysine residue is involved in proteoglycan association with fibrillar A beta, whereas a protonated histidine appears to be needed for binding of the glycoconjugates to non-fibrillar peptide. Scatchard analysis of fibrillar A beta association with proteoglycans indicates a single affinity interaction, and the binding of both HSPG and CSPG to fibrillar A beta is completely inhibited by free glycosaminoglycan chains. This implies that these sulfated carbohydrate moieties are primarily responsible for proteoglycan.A beta interaction. The ability of proteoglycans to bind fibrillar A beta and inhibit its proteolytic degradation suggests a possible mechanism of senile plaque accumulation and persistence in Alzheimer's disease.