Abstract
Isolevuglandins (isoLG) are products of lipid oxidation that covalently ligate self-proteins and contribute to immune activation in hypertension. We hypothesized that these are recognized as “non-self” elicit an antibody response. To determine the presence of anti-isoLG adduct antibodies in humans, immunoblots were performed using endogenous antibodies present in plasma of healthy and hypertensive subjects against human proteins artificially adducted with isoLG. Protein-G HRP was used to visualize the binding of IgG antibodies. We found that humans with hypertension possess IgG antibodies that bind isoLG protein adducts to a greater extent compared to unadducted protein. To identify unique monoclonal antibodies to isoLG adducts C57Bl/6 mice were made hypertensive via angiotensin II infusion for two weeks and then boosted with kidney protein that was adducted with isoLG. Boosted mice were then sacrificed and splenic B-cells were fused with a myeloma cell line. Individual colonies were screened for reactivity with isoLG adducts identifying 11 monoclonal antibody clones with a high specificity for isoLG adducts. Four clones were chosen for further analysis that exhibited 1.5 to 6-fold affinity to adducted vs unadducted mouse proteins. Western blots of normotensive and hypertensive kidney, bone marrow, and heart protein lysate using one of our monoclonal antibodies (clone 2B11) revealed a 90 kDa protein that is increased in bone marrow of hypertensive mice. We conclude that hypertension results in the production of anti-isoLG adduct antibodies in mice and humans. Identification of unique monoclonal antibodies that react with isoLG adducts suggests clonal expansion of anti-isoLG antibody producing cells. Finally, a hypertensive antigen is recognized by the 2B11 clone. Future studies will utilize these antibodies to identify peptide antigens that drive immune activation in hypertension.
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