C4d deposition without recruitment of inflammatory cells is insufficient to trigger microcirculation
injury in mouse kidney allografts
Aaron Chow, Luis Hidalgo, Lin-Fu Zhu, Phil Halloran, Banu Sis
Department of Laboratory Medicine and Pathology, University of Alberta, Edmonton, Alberta, Canada
C4d staining is a diagnostic marker for antibody-mediated rejection in human allografts, but has
limitations i.e. not sensitive nor 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.
We developed an MHC class I mismatched allograft model in the mouse. Male CBA (H-2k)
kidneys were transplanted into to male B6.RAG1KO (H-2b) recipients. The B6.RAG1KO mice do not have
mature B or T cells. A single dose of 100 g of anti-mouse H-2Kk mAb was injected to B6.RAG1KO
recipients at posttransplant day 6, and kidneys were harvested 24 hours later. To increase alloantibody
binding onto the MHC mismatched allograft endothelium, we repeated the experiment with the addition of
interferon-gamma (Ifng) pretreatment in recipient mice prior to alloantibody injection. C4d
immunohistochemistry was performed using rat anti-mouse monoclonal C4 antibody, which cross-reacts
with C4d. We related C4d staining to histopathology and transcriptome changes, measured by Affymetrix
microarrays.
Normal kidneys, isografts, and RAG1KO allografts without alloantibody treatment were negative
for C4d staining. However, RAG1KO allografts treated with alloantibody or Ifng plus alloantibody showed
diffuse C4d staining in glomerular and peritubular capillaries. By histopathology, RAG1KO allografts with
alloantibody treatment were negative for microcirculation inflammation, thrombi, congestion or other
histological features of rejection, thus did not differ from RAG1KO allografts without alloantibody or
isografts. By microarrays, expression of Ifng inducible transcripts, endothelial cell-, and macrophageassociated transcripts did not differ between RAG1KO allografts with alloantibody injection and untreated
RAG1KO allografts (p>0.05).
We showed that alloantibody induced capillary C4d deposition is not associated with
microcirculation inflammation and intragraft gene expression changes. Thus C4d protein is an inert
molecule and incapable of triggering allograft injury in the absence of microcirculation inflammation.