Celli, S

Demetris, AJ

Fung, JJ

Reilly, MJ

Simmons, RL

Starzl, TE

Thai, NL

Valdivia, LA

Wang, SC

English

PubMed

D-Scholarship@Pitt

A-' :; I ~ : .. Cytokine Messenger RNA Profiles in Hamster-to-Rat Liver Xenografts N.L. Thai, S.C. Wang, L.A. Valdivia, S. Celli, M.J. Reilly, A.J. Demetris, R.L. Simmons, T.E. Starzl, and J.J. Fung XENOGENEIC transplant rejection is thought to be mediated primarily by a humoral mechanism. Dis-cordant xenografts are rejected hyperacutely by preformed natural antibodies recognizing genetically disparate donor cells. Concordant xenografts are also destroyed by anti-bodies; however, this rejection process takes place over a longer span of time, during which T-helper cells playa crucial role in mounting the antibody response. I The concordant xenograft antibody response contains two components: one results in an immunoglobulin M (lgM) response and is primarily T cell independent. whereas the other results in an IgG response and is T cell dependent. 2 This helper cell-evoked antibody response is presumably due to helper cell production of B-cell growth factors such as interleukin-4 (lL-4), IL-6, and IL-lO. To investigate the immune mechanism underlying the xenogeneic response, we used the hamster-to-rat liver xenograft model. After transplantation, serum antibody titers of both IgM and IgG were markedly elevated as early as day 3 and peaked around day 6. These grafts invariably reject by day 7 posttransplant with histologic findings suggestive of anti-body-mediated and cellular-mediated injury. The presence of high levels of IgM and IgG in our hamster-to-rat animals suggested involvement of T cells in the xenograft immu-nity. To determine the pattern of T-helper activity, we examined cytokine messenger RNA (mRNA) profiles in liver xenografts. METHODS Liver grafts from Syrian Golden hamsters were transplanted into male Lewis rats using the cuff technique described by Kamada. 3 No immunosuppressants were administered. and animals were killed on day 5 postgrafting. Total RNA was extracted from liver grafts using RNAzol (CinnaiBiotecx) and quantitated spectropho-tometrically. Five micrograms of RNA was reverse transcribed into cDNA and f-~ aliquots subjected to polymerase chain reaction (PCR) amplification for 25 cycles using 32P-labeled prim-ers specific for rat cytokines and glyceraldehyde-3-phosphate dehydrogenase (G3PD), a housekeeping gene. Quantitation of amplification product was performed on a radioanalytic scanner after polyacrylamide gel electrophoresis. Specificity of amplifica-tion was confirmed by sizing and restriction analysis. RESULTS AND DISCUSSION Examination of cytokine mRNA levels in hamster-to-rat liver xenografts using rat-specific radiolabeled reverse-transcription PCR (RT-PCR) demonstrates the presence of detectable recipient rat T-helper cell cytokine mRNAs (Table I) at day 5 posttransplant. We were more easily able to detect IL-4 and IL-lO mRNA than either IL-2 or interferon-y (IFN-y) mRNAs. Helper T cells are divided Table 1. Cytoklne mRNA Levels Detected by Radlolabaled Reverse-Transcription peR In Hamster-to-Rat Liver Xenogratts Grafts Mean Cytokine A B C Counts IFN--y 259 80 280 206 IL-2 87 272 89 149 IL-4 3923 381 1,661 1.988 IL-l0 13,976 3,351 11,132 8,765 G3PD 108,840 60,480 87,720 85,680 L VG hamster livers were transplanted into leWis rat recipients and the xenografts harvested on day 5 posttransplant. One-microgram aliquots of RNA were subjected to reverse·transcription peR amplification for 25 cycles using 32P·labeled primers targeted to rat cytokines and G3PD. Ouantrtanon of amplifi-cation product was per10rmed on a radioanalytic scanner after polyacrylamide get electrophoresis. Data from a representative experiment are expressed above in total counts of radioactivity over background. into two major subsets based on their pattern of cytokine production.4 The T-helper I (THl) subset of cells is characterized by the production of IL-2 and IFN--yand are responsible for the induction of the delayed-type hyper-sensitivity. In contrast, the T-helper 2 (TH2) subset of cells is characterized by the production of IL-4. IL-5, IL-6. and IL-lO. Because IL-4, IL-6, and IL-lO are potent B-cell growth factors, the TH2 subset is thought to mediate humoral immunity. The vigorous humoral response ob-served after xenotransplantation and our ability to easily detect the TH2 cytokines IL-4 and IL-lO suggest that TH2 cells may play an important role in mediating xenograft rejection. Whether TH2 activity predominates over THI activity in the xenograft remains to be determined; direct comparison between cytokines measured by peR cannot be made unless the amplification efficiency for each cyto-kine is determined and taken into account. We have previously shown in vitro with xenogeneic mixed lympho-cyte response (MLR) (mouse-anti-rat) that there is a high expression ofTH2 cytokine mRNAs. 5 In contrast. mouse-anti-mouse allogeneic MLR showed a relatively higher expression of THI cytokine mRNAs using the same as-says. These previous findings indicated that TH2 cells may From the Departments of Surgery and Pathology, and the Pittsburgh Transplantation Institute, University of Pittsburgh, Pitts-burgh, Pennsylvania. Supported by an American College of Surgeons Scholarship and National Institutes of Health Grants AI-01030 and AI-16869. Address reprint requests to Ngoc L. Thai, Department of Sur-gery, W1504 Biomedical Science Tower, University of Pittsburgh, Pittsburgh. PA 15260. © 1993 by Appleton & Lange 0041-1345/931$3.001 +0 444 Transplantation Proceedings, Vol 25, No 1 (February), 1993: pp 444-445 .. • CYTOKINE MRNA PROFILES differentially respond more during the xenogeneic than allogeneic response. Although it is possible that our MLR findings were due to the necessarily artificial conditions in vitro, our current findings in vivo with hamster-to-rat xenografts are also consistent with increased TH2 activity relative to TH I activity in xenogeneic responses. The reason for increased TH2 reactivity after xenografting is unknown. but ligand binding by responding T cells may playa role. Bottomly has shown that high antigen ligand density binding of T-cell receptors leads to a predomi-nantly THI reaction, whereas low ligand density TCR binding leads to a predominantly TH2 reaction.6 After xenografting, poor interaction between accessory mole-cules due to species differences may interfere with the 445 normally high-affinity T-cell receptor binding and thus effectively decreases perceived antigen ligand density. REFERENCES I. Cramer DV. Wu GD. Chapman FA. et al: Transplantation 24:533. 1991 2. Langer A. Valdivia LA. Murase N. et al: (submitted for publication) 3. Kamada N. Caine RY: Transplantation 28:47, 1979 4. Mosmann TR. Coffman RL: Ann Rev Immunol 7:145, 1988 5. Wren SM. Wang SC. Thai NL. et al: American Society of Transplant Surgeons. 1992 (Abstr) 6. Pfeiffer C. Murray J. Madri J. Botlomiy K: Immunol Rev 123:65. 1991 

Cytokine messenger RNA profiles in hamster-to-rat liver xenografts.

Name not available

Cytokine messenger RNA profiles in hamster-to-rat liver xenografts

http://d-scholarship.pitt.edu/4852/1/31735062112531.pdf

Cytokine messenger RNA profiles in hamster-to-rat liver xenografts.

Abstract

Similar works

Full text

Available Versions

D-Scholarship@Pitt

Name not available

Name not available