C4d Deposition without Recruitment of Inflammatory Cells Is Insufficient To Trigger Microcirculation Injury in Mouse Kidney Allografts
Aaron Chow, Paula Blanco, Lin-Fu Zhu, Banu Sis. University of Alberta, Edmonton, AB, Canada
Background: C4d staining is a diagnostic marker for antibody-mediated rejection in human allografts, but has limitations i.e. not highly sensitive or specific (such as in ABO incompatible allografts).
We hypothesized that C4d deposition in capillaries without recruitment of intracapillary inflammatory cells is not sufficient to trigger allograft injury.
Design: We developed an MHC class I mismatched allograft model in the mouse by transplanting CBA (H-2k) kidneys into B6.RAG1KO (H-2b) recipients who lack mature B and T cells. To induce the effects of antibody-mediated rejection, three different doses of anti-mouse H-2Kk monoclonal antibodies were injected: a single injection of 100ug on day 6 post-transplant and harvesting of kidneys 24 hours later (n=3), three injections of 300ug on days 3,5,7 and harvesting 45 minutes after the last injection (n=8), or a single injection of 750ug on day 6 and harvesting 24 hours later (n=3). C4d deposition was visualized by immunohistochemistry. We also examined soluble serum complement component C5a levels by ELISA, and related C4d staining to histopathology, serum C5a levels, and intragraft transcriptome changes, measured by Affymetrix microarrays.
Results: Normal kidneys, CBA or RAG1KO isografts, and RAG1KO allografts without alloantibody treatment were negative for C4d staining. However, all RAG1KO allografts treated with alloantibody showed diffuse C4d staining in glomerular and peritubular capillaries. By histopathology, RAG1KO allografts with alloantibody treatment were negative for increased microcirculation inflammation, thrombi, congestion or other histological features of rejection, thus did not significantly differ from isografts or untreated RAG1KO allografts. Serum C5a levels did not increase in mice with RAG1KO allografts after 100ug and 300ug alloantibody injections (p>0.05) and mildy increased following injection of 750ug of alloantibody, but the difference did not reach a statistical significance when compared to serum from untreated RAG1KO allograft recipients. By microarrays, expression of Ifng inducible transcripts, endothelial cell-, and macrophage-associated transcripts did not differ between RAG1KO allografts with alloantibody injection and untreated RAG1KO allografts (p>0.05).
Conclusions: We showed that alloantibody-induced capillary C4d deposition is not associated with microcirculation inflammation and intragraft gene expression changes when there is incomplete activation of the complement cascade. Thus C4d protein is an inert molecule and incapable of triggering allograft injury.
Category: Kidney (does not include tumors)
Tuesday, March 20, 2012 8:45 AM
Platform Session: Section H, Tuesday Morning