Celli, S

Fung, JJ

Kelly, RH

Nalesnik, M

Pan, F

Rao, AS

Starzl, TE

Sun, H

Valdivia, LA

Wakizaka, Y

English

PubMed

D-Scholarship@Pitt

The Antigenicity of Serum Proteins and Their Role in Xenograft Rejection S. Celli, R.H. Kelly, A.S. Rao, F. Pan, H. Sun, M. Nalesnik, Y. Wakizaka, J.J. Fung, T.E. Starzl , and L.A. Valdivia I T HAS been reported previously that following xeno-transplantation. liver secretes proteins of donor origin into the serum of xenograft recipients. thus allowing them to retain their species-specificity. I The persistence and the function of hamster xenoproteins in long-surviving hamster --> rat liver transplant recipients has already been well establishedK ~ In the present study, we have extended this concept further by evaluating the possible immunogenicity o f donor (hamster) serum proteins . To this end. we have attempted to ( I) characterize the hamster serum proteins that elicit an antigenic response in the rat and (2) identify do nor proteins in th e recipient serum that share common epitopes with those on the cell surface of the vascular endothelium. which are the primary targets of antibody-mediated rejection of vascularized xenografts. MATERIALS AND METHODS Animals and Operative Procedures Syrian hamsters and LEW rats were used as donors and hetero-topic hean transplants recipients. respectively . Based on the treat-ment regimen. the transplant recipients (rats) were grouped as follows : Group I (n = 6): sensitized with hamster serum (HS) alone: Group II (n = 10) : sensitized with HS emulsified in Freund's incomplete adjuvant (F!A): Group III (n ~ 5): sensitized with rat serum + FIA: Group IV (n = 5): FIA alone. For sensitization. three injections of cell-free HS were administered to rats at 14-day intervals. and the challenge hearts were transplanted 5 days after thc last immunization . HAMSTER SERUM v ~ 2 globulins area Immunofluorescence (IF) Using indirect IF technique. the deposition of rat o·hamster IgG and IgM antibodies was determined by incubating (ex vivo) naive hamster heans with hypenmmune serum (HIS) obtaincd from an imals in Group II. Similar analyses were also performed in hamster hearts transplanted into rat recipients treated postrevas· cularization with 2 mL (IV ) of HIS. Immunoelectrophoresis (IEP) Normal HS was electrophoresed against rat-o·hamster IgG anti-bodies. the bands of precipitation being indicative o f the recogni-tion by the latter of hamster protems. Western Blot (WB) The HS used for this assay was depleted of IgG (by absorption with Protein G; Sigma. SI. Louis. MO) and albumin (by absorption with Cibacron Blue 3 GA: Sigma). The treated HS was electrophoresed in SDS-PAGE. transferred onto a membrane. and then stained with Ponce au red or incubated with HIS. or with a similar amount From the Pittsburgh Transplantation Institute and the Depart-ments of Surgery and Pathology. University of Pittsburgh Med-ical Center, Pittsburgh. Pennsylvania. This work was aided by Prolect Grant DK29961 from the National Institutes of Health, Bethesda, Maryland. Address reprint requests to Luis A. Valdivia, MD, Pittsburgh Transplantation Institute, E1546 Biomedical Science Tower, 200 Lothrop St.. Pittsburgh, PA 15213. © 1996 by Appleton & Lange 0041-1345/96/$3.00/ +0 RAT IgG a HAM PROTEINS Fig 1. Immunoelectrophoresis of normal hamster serum with that obtained from a rat immu-nized with hamster sera. There are four distinct bands of pre-cipitation, three of which are in the area of the 132 -globulins. Transplantation Proceedings. Vol 28, No 2 (April), 1996: pp 669-670 669 , I I; i , I 1, 1, II I'i " KI~t , [ Il q 670 of IgG eluted from rejected (hamster -> rat) liver or heart xenografts. RESULTS AND DISCUSSION Accelerated (within 48 hours) rejection (AR) of the trans-planted hamster heart was witnessed in 50% of Group I and 30% of Group II animals. On the contrary, hamster hearts transplanted into rats in Groups III and IV maintained a strong palpable beat until the day of rejection (day 3 or 4). Interestingly, hearts transplanted into Group I and Group II animals that had escaped AR showed a significantly weaker pulsation with fibrillation by the third postoperative day, suggesting that these organs were undergoing "slow motion" rejection. When tested in IEP assays, the serum obtained from normal hamsters showed four distinct arcs of precipitation, three of which were in the area of /32-globulin (Fig 1). This result indicates that within the serum of normal hamsters there are at least four proteins (and perhaps more), which by virtue of being immunog~nic eli.cit an antibody response after infusion of this serum mto naive rats. Western blot analysis using HIS confirmed the pres-ence of four predominant bands with apparent molecular CELLI, KELLY, RAO ET AL weights of '180, 100, 90, and 48 kDa and four additional bands of relatively lower intensity. Of greater interest, however, was the recognition by IgG antibodies eluted from rejected hamster liver or heart xenografts of three of the four primary bands revealed by HIS. Immunohis-, tochemistry revealed the deposition (primarily) of rat IgG in normal hamster hearts incubated ex vivo with HIS and also in the hearts transplanted into rats passively treated with HIS. Taken together, the results of this study indicate that even in closely related species such as hamster and rats, there are some serum proteins that may be immunogenic. However, our ability to prolong graft survival (with appropriate immunosuppression) in the hamster -> rat strain combination suggests that. despite their obvious immunogenicity, the serum proteins do not playa major role in the initiation of xenograft rejection. Further characteriza-tion of these serum proteins is currently underway. REFERENCES 1. Valdivia LA. Fung 11, Demelris N. et al: Transplantation 57(6):918. 1994 2. Celli S. Valdivia LA. Kelly RH. cl al: Xenotransplantation 2:46, 1995 I ·i 1 , 

The antigenicity of serum proteins and their role in xenograft rejection

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The antigenicity of serum proteins and their role in xenograft rejection

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