title: Passive transfer of alloantibodies restores acute cardiac rejection in IgKO mice.
abstract: BACKGROUND
  Alloantibody is an intrinsic component of the immune response to organ transplants. Although alloantibodies have been correlated with decreased graft survival, the mechanisms of alloantibody-mediated injury remain largely undefined in vivo. In the present study, we have established a model of alloantibody-mediated graft injury using B10.A (H-2a) hearts transplanted to wild type (WT) or immunoglobulin knock out (IgKO) C57BL-Igh-6 (H-2b) mice.
  
  
  METHODS
  Alloantibodies were measured in the circulation and graft by flow cytometry and in immunofluorescence staining, respectively. Intragraft cytokine mRNA expression was evaluated using a competitive template reverse transcriptase polymerase chain reaction (RT-PCR) technique. P-selectin and von Willebrand factor expression were localized by immunoperoxidase staining. The capacity of alloantibodies to restore acute cardiac allograft rejection was tested by passive transfer of monoclonal antibodies (mAbs) against donor major histocompatibility complex (MHC) class I antigens to IgKO recipients.
  
  
  RESULTS
  B10.A cardiac allografts are rejected acutely by WT C57BL/6 recipients, but over 50% of the cardiac allografts survived more than 50 days after transplantation in IgKO mice. Competitive template RT-PCR on the cardiac transplants demonstrated similar levels of IL-1-alpha, IL-12 (p40), TNF-alpha, IL-2, IFN-gamma, IL-4, and IL-10 mRNA in WT and IgKO recipients 8-10 days after transplantation, indicating that macrophage- and T-cell-dependent immune responses were intact in IgKO recipients. The rejection of B10.A hearts in WT recipients was characterized by interstitial and perivascular cellular infiltration; IgG, IgM, and complement (C3) deposition; vascular cell injury and intravascular platelet aggregation; and release of von Willebrand factor and P-selectin. In IgKO recipients the lower degree of vascular injury in the absence of alloantibody responses was reflected by the lack of release of von Willebrand factor and P-selectin, which remained confined to cytoplasmic storage granules of endothelial cells and platelets. Acute rejection of cardiac allografts was restored to IgKO recipients by passive transfer of proinflammatory IgG2b mAbs against donor MHC; recipients injected with isotype-matched control mAbs did not reject. In contrast, passive transfer of IgG1 mAbs against donor MHC failed to restore acute rejection of cardiac allografts to IgKO recipients. Passive transfer of IgG2b, but not IgG1 mAbs was associated with endothelial cell activation and plate. let aggregation together with the release of preformed von Willebrand factor and P-selectin from storage granules.
  
  
  CONCLUSIONS
  Acute rejection of cardiac allografts can be reconstituted in IgKO recipients by passive transfer of IgG2b, but not IgG1 antibody. This model allows the mechanism of alloantibody-mediate graft injury to be dissected in vivo.
authors.affiliation: Department of Pathology, The Johns Hopkins University School of Medicine, Baltimore, MD 21205-2196, USA.
authors.forename: B A
authors.initials: BA
authors.lastname: Wasowska
authors.forename: Z
authors.initials: Z
authors.lastname: Qian
authors.forename: D L
authors.initials: DL
authors.lastname: Cangello
authors.forename: E
authors.initials: E
authors.lastname: Behrens
authors.forename: K
authors.initials: K
authors.lastname: Van Tran
authors.forename: J
authors.initials: J
authors.lastname: Layton
authors.forename: F
authors.initials: F
authors.lastname: Sanfilippo
authors.forename: W M
authors.initials: WM
authors.lastname: Baldwin
mesh_terms: D000208:Acute Disease; D000818:Animals; D000917:Antibody Formation; D004730:Endothelium, Vascular / Q000166:cytology / Q000293:injuries; D006084:Graft Rejection / Q000276:immunology; D006085:Graft Survival / Q000276:immunology; D016027:Heart Transplantation / Q000276:immunology*; D007116:Immunization, Passive; D007136:Immunoglobulins / Q000172:deficiency; D007518:Isoantibodies / Q000276:immunology*; D008264:Macrophages / Q000276:immunology; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout / Q000276:immunology*; D019007:P-Selectin / Q000378:metabolism; D013601:T-Lymphocytes / Q000276:immunology; D014841:von Willebrand Factor / Q000378:metabolism
chemical_list: D007136:Immunoglobulins; D007518:Isoantibodies; D019007:P-Selectin; D014841:von Willebrand Factor