ABSTRACT
We have recently reported that complement factor H, a negative regulator of complement-mediated cytotoxicity, is produced and secreted by most bladder cancers. This observation was exploited in the development of the BTA stat and BTA TRAK diagnostic assays, both of which make use of two factor H-specific monoclonal antibodies in sandwich format. Here we show that both antibodies exert interesting effects on the biochemistry of complement activation in in vitro systems. Antibody X13.2 competes with C3b for association with factor H and strongly inhibits factor H/factor I-mediated cleavage of C3b, thereby evidently inactivating a negative regulator of complement; yet, the antibody strongly inhibits complement-mediated lysis as well. Conversely, antibody X52. 1, which does not compete with C3b and has no effect on solution-phase cleavage of C3b, is capable of enhancing complement-mediated lysis of various cell types, including cancer cells, by over 10-fold. Our observations indicate that it is possible to deconvolute the biochemical roles of factor H in complement by means of appropriate inhibitors, a finding with potentially valuable implications for both basic research and cancer therapy.
Subject(s)
Antibodies, Monoclonal/immunology , Complement Activation/drug effects , Complement Factor H/immunology , Animals , Antibody Specificity , Blotting, Western , Complement C3b/metabolism , Dose-Response Relationship, Drug , Erythrocytes/immunology , HL-60 Cells , Hemolysis/immunology , Humans , Hybridomas/immunology , Kinetics , Models, Biological , Reverse Transcriptase Polymerase Chain Reaction , Sheep , Time Factors , Tumor Cells, Cultured , Up-Regulation , Zymosan/metabolismABSTRACT
The Salmonellae PhoP-PhoQ virulence regulators induce resistance to host cationic antimicrobial peptides (CAMP) after infection of vertebrate tissues, and Mg2+ or Ca2+ limitation. The PhoP-PhoQ activated gene, pagP, was identified as important to inducible CAMP resistance and increased acylation of lipid A, the major component of the outer leaflet of the outer membrane. pagP mutants demonstrated increased outer membrane permeability in response to CAMP, supporting the hypothesis that increased lipid A acylation is a CAMP resistance mechanism. Similarly, in response to Mg2+ limited growth, other enteric Gram-negative bacteria demonstrated increased lipid A acylation. Compounds that inhibit the ability to increase lipid A acylation may have utility as new antimicrobial agents.