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Crry and CD59 regulate complement in rat glomerular epithelial cells and are inhibited by the nephritogenic antibody
R J Quigg1, V M Holers, B P Morgan
1Department of Medicine, University of Chicago, IL 60637.
Insights
Rat glomerular epithelial cells (GEC) possess complement regulators Crry and CD59. Crry controls complement activation, while CD59 limits cell damage, especially when Crry is inhibited or the classical pathway is activated.
Area of Science:
- Immunology
- Nephrology
- Cell Biology
Background:
- Human glomerular epithelial cells (GEC) express complement regulatory proteins.
- Crry is the rodent homolog of human decay-accelerating factor and membrane cofactor protein.
- The nephritogenic antibody anti-Fx1A impairs complement regulation in rat GEC.
Purpose of the Study:
- To investigate the role of complement regulators Crry and CD59 in rat GEC.
- To examine the functional activity of Crry and CD59 in both alternative and classical complement pathways.
- To understand how anti-Fx1A interacts with these complement regulators.
Main Methods:
- Immunoprecipitation of 125I-labeled GEC membrane proteins using specific antibodies (anti-Crry, anti-CD59, anti-Fx1A).
- Assessment of alternative complement pathway activation by incubating GEC in rat serum.
- Functional inhibition of Crry and CD59 using antibodies to evaluate cytotoxicity.
Main Results:
- Crry and CD59 were confirmed to be present and functional in rat GEC.
- Inhibition of Crry led to significant cytotoxicity via alternative pathway activation.
- CD59 inhibition enhanced cytotoxicity when Crry was inactive or the classical pathway was activated.
- Anti-Fx1A was found to bind to both Crry and CD59.
Conclusions:
- Crry and CD59 are active complement regulators in GEC.
- Crry plays a crucial role in restricting complement activation through both pathways.
- CD59 acts as a critical limiter of C5b-9-mediated cytotoxicity, particularly under specific conditions.
- The interaction of anti-Fx1A with Crry and CD59 may explain its in vitro alternative pathway activation and in vivo nephritogenicity.
Abstract:
Human glomerular epithelial cells (GEC) contain CD59, decay-accelerating factor, and membrane cofactor protein. Crry is the rodent analogue to the latter two proteins. We have previously shown that the nephritogenic Ab of passive Heymann nephritis, anti-Fx1A, impairs C regulation in rat GEC. Here we examined rat GEC C regulation. 125I-labeled GEC membrane proteins were immunoprecipitated with anti-Crry, anti-CD59, or anti-Fx1A. Crry and CD59 were present in GEC. Anti-Fx1A reacted with both Crry and CD59 from GEC, as well as with purified rCrry and CD59. The alternative C pathway was studied by incubating GEC in rat serum in Mg(++)-EGTA buffer. To inhibit the function of the C regulators, anti-Crry or anti-CD59 Ab were added to GEC. Inhibition of CD59 function alone had no effect on C regulation, whereas inhibition of Crry led to significant cytotoxicity from alternative pathway activation. Under conditions in which Crry was inactive, inhibition of CD59 further enhanced cytotoxicity. When the classical pathway of C was activated by GEC-bound IgG Ab, inhibition of either Crry or CD59 enhanced cytotoxicity, whereas inhibition of both Crry and CD59 together was additive. Therefore, Crry and CD59 are present and functionally active in GEC. Crry restricts C activation via both alternative and classical pathways. When the classical pathway of C is activated, or when Crry function is inhibited, CD59 limits C5b-9-mediated cytotoxicity. Anti-Fx1A binds to both Crry and CD59, which may account for its ability to activate the alternative pathway in vitro, and for its superior nephritogenicity in vivo.