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Intragraft cytokine mRNA expression in rejecting and non-rejecting Vascularised Xenografts
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Transplantation Surgery.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Clinical Immunology.
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2003 (English)In: Xenotransplantation, ISSN 0908-665X, E-ISSN 1399-3089, Vol. 10, no 4, 311-324 p.Article in journal (Refereed) Published
Abstract [en]

BACKGROUND:

The aim of the present study was to further investigate the characteristics of both graft-infiltrating cells and splenocytes during acute vascular rejection (AVR), cell-mediated rejection and non-rejection of vascularized concordant xenografts, by analysing both proinflammatory [interleukin-1beta (IL-1beta) and tumour necrosis factor (TNF-alpha)] and more specific [(IL-2, IL-4, IL-10, IL-12p40 and interferon-gamma (IFN-gamma)] cytokines. A parallel investigation was made of the antibody response of IgM and IgG to the xenografts.

METHODS:

Mouse hearts were heterotopically transplanted to the neck vessels of recipient rats. Grafts, spleens and sera were collected from untreated (AVR) and cyclosporin A (CyA) treated animals on day 2 after transplantation. Organs from rats treated with 15-deoxyspergualin (DSG) or CyA and DSG in combination were harvested on both day 2 and day 8. Grafts from DSG-treated rats undergo cell-mediated rejection and stop beating on day 9 and forth, while CyA + DSG treatment results in long-term graft survival. Real-time quantitative reverse transcriptase polymerase chain reaction (RT-PCR) was applied for analysis of intragraft and splenic cytokine messenger RNA (mRNA) expression. The phenotypes of the graft infiltrating cells were characterized by immunohistochemistry. The antibody response was investigated by means of immunofluorescence, haemagglutination and flow cytometry.

RESULTS:

All the studied cytokines (IL-1beta, IL-2, IL-4, IL-10, IL-12p40, IFN-gamma and TNF-alpha) were up-regulated in the grafts from rejecting untreated (day 2) and DSG-treated animals (day 8) in comparison with grafts from CyA + DSG treated animals (day 8). On day 2 under immunosuppression with CyA, DSG or CyA + DSG no or low cytokine mRNA levels were found. The mRNA levels of IL-2, IL-4 and IFN-gamma in the spleens were suppressed under both DSG- and CyA + DSG treatment on day 8. Immunofluorescence showed deposits of both IgM and IgG in grafts from untreated, CyA-treated (day 2) and DSG-treated (day 8) animals, while CyA + DSG treatment diminished these deposits on both day 2 and day 8. No circulating antibodies were identified in either group.

CONCLUSION:

We hereby conclude that both AVR on day 2 and cell-mediated rejection on day 8 (under DSG treatment) in a concordant cardiac mouse-to-rat xenotransplantation model are associated with an increase of proinflammatory cytokines, T helper 1 (Th1)-associated cytokines as well as IL-10, while immunosuppression with CyA + DSG diminishes the levels of all examined cytokines. Grafts undergoing AVR or cellular rejection are subjected to deposits of both IgM and IgG, although circulating donor specific antibodies are undetectable in serum.

Place, publisher, year, edition, pages
2003. Vol. 10, no 4, 311-324 p.
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:uu:diva-90017DOI: 10.1034/j.1399-3089.2003.02032.xPubMedID: 12795680OAI: oai:DiVA.org:uu-90017DiVA: diva2:162036
Note

De två första författarna delar förstaförfattarskapet.

Available from: 2002-10-11 Created: 2002-10-11 Last updated: 2017-12-14Bibliographically approved
In thesis
1. Xenograft Rejection: A Study of Cytokine mRNA Expression in Experimental Models
Open this publication in new window or tab >>Xenograft Rejection: A Study of Cytokine mRNA Expression in Experimental Models
2002 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Acute cellular xenograft rejection of ICCs triggered mRNA expression of proinflammatory cytokines (IL-1b and TNF-a), Th1 associated cytokines (IL-12p40, IL-2 and IFN-g) and Th2 associated cytokines (IL-4 and IL-10). The peak values and the kinetics of the mRNA expression indicated that acute cellular xenograft rejection is enhanced by the proinflammatory response and mediated by the Th1 associated response, whereas the function of the Th2 associated response most likely is to counteract the Th1 associated cytokines and thereby inhibit bystander damage by the delayed type hypersensitivity like rejection process. Tacrolimus was shown to have a protective effect on the ICC xenografts, which was linked with inhibition of Th1 associated cytokines. This beneficial effect was paradoxically abrogated by prednisolone. The cytokines were interpreted to mediate the same response during cellular rejection of vascularized xenografts in the mouse-to-rat heart transplant model as in the non vascularized ICC model, even though the the response includes intragraft antibody deposits. Also the xenogeneic neural grafts implanted into the CNS displayed a similar cytokine response, but this process was slower and had less distinguished peak values compared to the ICC xenografts.

The shortage of organs is the major limitation for clinical allotransplantation. For making clinical xenotransplantation possible and thereby eliminate the organ shortage, issues regarding ethical dilemmas, microbiological hazards, physiological incompatibilities and immunological problems need to be addressed. The aim of the studies in this thesis was to investigate the mechanisms behind acute cellular xenograft rejection. The immunological response, with a focus on the cytokine mRNA expression, was analysed at different time points in rats transplanted with fetal porcine islet like cell clusters (ICCs), mouse hearts, or syngeneic, allogeneic, concordant or discordant xenogeneic neural tissue grafts.

In conclusion, both the cytokine mRNA expression and the morphology of acute cellular xenograft rejection bear a close resemblance to a delayed type hypersensitivity reaction, primarily mediated by indirect antigen presentation. This might explain why clinically used immunosuppressive protocols normally are inefficient for use in xenotransplantation, since these are presumed to primarily suppress T cell response triggered direct antigen presentation, which dominates during allogeneic acute rejection.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2002. 61 p.
Series
Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 0282-7476 ; 1193
Keyword
Oncology, Type 1 diabetes, Parkinson’s disease, xenotransplantation, islets, neural tissue, heart, mRNA, cytokines, rejection., Onkologi
National Category
Cancer and Oncology
Research subject
Clinical Immunology
Identifiers
urn:nbn:se:uu:diva-2686 (URN)91-554-5433-X (ISBN)
Public defence
2002-11-01, Rudbeck salen, Uppsala, 13:15
Opponent
Available from: 2002-10-11 Created: 2002-10-11Bibliographically approved
2. Studies of Rejection in Experimental Xenotransplantation
Open this publication in new window or tab >>Studies of Rejection in Experimental Xenotransplantation
2002 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

One main hurdle to xenotransplantation, i.e. transplantation between different species, is the immunological barrier that the organ meets in the recipient. The aim of this thesis was to characterise xenogeneic rejection mechanisms by using the concordant mouse-to-rat heart transplantation model.

Graft-infiltrating immune cells could be isolated from both rejecting and non-rejecting grafts using ex vivo propagation, a technique based on incubation of graft biopsies in culture medium for 48 hours. The numbers of recovered T lymphocytes were considerably higher in grafts undergoing cell-mediated rejection than in grafts undergoing acute vascular rejection (AVR) or in non-rejecting transplants. Thus, ex vivo propagation should be a valuable tool for further studies of cell-mediated rejection.

Cytokine patterns in the grafts, as measured by a quantitative real-time RT-PCR method, showed that AVR and cell-mediated rejection are associated with an increase of both pro-inflammatory cytokines (IL-1β and TNF-α) and more specific cytokines (IL-2, IL-10, IL-12p40 and IFN-γ). These data differed considerably from the patterns seen in the spleens of the recipients. Cell-mediated xenograft rejection was also found to be associated with a local accumulation of hyaluronan.

Oral administration of xenogeneic cells stimulated a production of antibodies that could induce hyperacute rejection of cardiac xenografts when passively transferred to graft recipients. This is in contrast to several models for autoimmune diseases and allogeneic transplantation where oral administration of antigens is an effective way to induce unresponsiveness. Hence, future attempts to induce oral tolerance in xenotransplantation should be done with caution.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2002. 60 p.
Series
Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 0282-7476 ; 1211
Keyword
Surgery, Antibodies, cell culturing, cytokines, hyaluronan, immunosuppression, mouse, rat, rejection, T lymphocytes, xenotransplantation., Kirurgi
National Category
Surgery
Research subject
Surgery
Identifiers
urn:nbn:se:uu:diva-2891 (URN)91-554-5476-3 (ISBN)
Public defence
2002-12-13, Rosénsalen, Akademiska sjukhuset, ingång 95/96, Uppsala, 09:15
Opponent
Available from: 2002-11-21 Created: 2002-11-21 Last updated: 2013-06-19Bibliographically approved

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Lorant, TomasTufveson, GunnarKorsgren, Olle

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