Background/Purpose

Complement plays a central role in the pathogenesis of systemic lupus erythematosus (SLE) wherein inappropriate activation of complement leads to substantial tissue damage, especially in the kidney. Factor H is a complement regulatory protein that controls activation of the alternative pathway in the fluid phase and on cell surfaces. Recent human linkage data implicate a role for a group of genes, encoding a family designated Factor H-Related (FHR) proteins, in the pathogenesis of SLE. Specifically, a FHR3-1Δ deletion is associated with a higher risk for the development of SLE, while also demonstrating a protective association in age-related macular degeneration. To date, few functional studies have been carried out on the mouse FHR proteins, and these molecules have not been studied in any in vivo models of inflammatory or autoimmune disease. Our central hypothesis is that FHR proteins act as antagonists of FH function and increase complement deposition which exacerbates inflammation and injury.

Methods

To test our hypothesis that FHR proteins compete with FH-mediated complement regulation we: 1) Generated recombinant forms of the murine FHR proteins (mFHR-A and mFHR-B) using transiently transfected 293-F cells grown in serum free media. 2) Evaluated the capacity of each of these molecules to inhibit FH function using a hemolysis protection assay. 3) Performed ELISA assays to detect cross-species reactivity between murine FHRs and human complement components. 4) Used flow cytometry to evaluate C3b deposition on the surface of nucleated cells (Retinal Pigment Epithelial (ARPE-19) and/or murine tubular epithelial cells (TEC)) upon addition of mFHRs under both oxidatively stressed and non-stressed conditions.  We are also producing antibodies directed to the murine FHRs in order to better understand how the FHRs may be involved in SLE and other autoimmune diseases and whether FHR levels are altered during different disease activity states (i.e. during SLE disease flares).

Results

Addition of 1uM of mFHR-A or mFHR-B results in 100% and 50% hemolysis, respectively, of sheep red blood cells which are normally resistant to complement mediated lysis due to their lack of membrane associated complement regulators. We also observe a significant increase in C3b deposition under both stressed and non-stressed conditions on the surface of at least one type of nucleated cell (ARPE-19) upon addition of mFHR-A (mean fluorescence intensity is two-fold greater than serum only control). These results suggest that the murine FHRs are excellent surrogates by which to interrogate the underlying mechanisms linking variations within the human CFH gene family to complement deregulation in tissues such as the kidney or eye.

Conclusion

Our preliminary work supports recent studies which have shown that the FHR proteins modulate complement by competing with FH for binding to its major ligand, complement component C3b, likely disrupting FH-driven complement regulation on specific biological surfaces. The long-term objective of this work is to determine whether the FHR proteins are suitable therapeutic targets for the treatment of complement-driven inflammatory diseases such as SLE.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/investigating-the-roles-of-factor-h-related-proteins-in-systemic-lupus-erythematosus-sle-and-other-autoimmune-diseases/