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Xenograft Rejection: A Study of Cytokine mRNA Expression in Experimental Models
Uppsala University, Medicinska vetenskapsområdet, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology.
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 [en]
Oncology, Type 1 diabetes, Parkinson’s disease, xenotransplantation, islets, neural tissue, heart, mRNA, cytokines, rejection.
Keyword [sv]
Onkologi
National Category
Cancer and Oncology
Research subject
Clinical Immunology
Identifiers
URN: urn:nbn:se:uu:diva-2686ISBN: 91-554-5433-X (print)OAI: oai:DiVA.org:uu-2686DiVA: diva2:162038
Public defence
2002-11-01, Rudbeck salen, Uppsala, 13:15
Opponent
Available from: 2002-10-11 Created: 2002-10-11Bibliographically approved
List of papers
1. A distinct Th1 immune response precedes the described Th2 response in Islet xenograft rejection
Open this publication in new window or tab >>A distinct Th1 immune response precedes the described Th2 response in Islet xenograft rejection
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2002 (English)In: Diabetes, ISSN 0012-1797, E-ISSN 1939-327X, Vol. 51, no 1, 79-86 p.Article in journal (Refereed) Published
Abstract [en]

Previous studies using semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) have demonstrated that islet xenograft rejection in mice is dominated by Th2-associated cytokines, i.e., interleukin (IL)-4 and IL-10. However, immunohistochemical stainings show that the morphological pattern in this model is more reminiscent of a delayed-type hypersensitivity (DTH) reaction, which is associated with a Th1 response. This study was designed to resolve the mechanisms of acute cellular xenograft rejection in rats transplanted with fetal porcine islet-like cell clusters (ICCs). Real-time quantitative RT-PCR was used to quantify the mRNA expression of cytokines in the grafts and lymph nodes, and the findings were related to the immunopathology of the rejecting grafts. By day 1, mRNA expression levels of IL-1 beta, IL-2, IL-12p40, interferon-gamma, and tumor necrosis factor-alpha were already induced in the lymph nodes. From days 3 to 12, an increasing amount of activated macrophages was seen in the grafts, whereas T- and NK-cells were fewer and mainly accumulated in the periphery of the grafts. Most of the ICCs were rejected by day 5. Transcripts of Th1-associated cytokines were dominant in both regional lymph nodes and in the grafts, with peak levels on days 3 and 5, respectively. The mRNA expression of IL-4 was increased on day 12, and it correlated with the infiltration of eosinophils and an increased level of xenoreactive IgG. The data presented indicate that an islet xenograft triggers a sequential activation of 1) a Th1-associated response characterized by graft destruction in a DTH-like reaction and then 2) a subsequent Th2-associated response characterized by increased levels of xenoreactive antibodies.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-90015 (URN)10.2337/diabetes.51.1.79 (DOI)11756326 (PubMedID)
Available from: 2002-10-11 Created: 2002-10-11 Last updated: 2017-12-14Bibliographically approved
2. Immunosuppressive drugs in islet xenotransplantation: A tool for gaining further insights in the mechanisms of the rejection process
Open this publication in new window or tab >>Immunosuppressive drugs in islet xenotransplantation: A tool for gaining further insights in the mechanisms of the rejection process
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2002 (English)In: Transplantation, ISSN 0041-1337, E-ISSN 1534-6080, Vol. 74, no 8, 1084-1089 p.Article in journal (Refereed) Published
Abstract [en]

BACKGROUND:

The aim of the present study was to examine the effect of tacrolimus (TAC) and prednisolone (PRE) in islet xenotransplantation and to use the immunosuppressive effects of these drugs and others to further characterize the mechanisms behind islet xenograft rejection.

METHODS:

Fetal porcine islet-like cell clusters (ICCs) were transplanted under the kidney capsule in Lewis rats. The animals were treated with TAC, cyclosporine A (CsA) plus 15-deoxyspergualin (DSG), CsA plus sirolimus (SIR) or CsA plus leflunomide (LEF), with or without the addition of PRE. Rejection was assessed by immunohistological evaluation 12 days after transplantation. In selected groups, the intragraft cytokine mRNA expression was analyzed with real-time quantitative reverse-transcriptase polymerase chain reaction (RT-PCR).

RESULTS:

In untreated rats, the ICC xenografts were completely rejected. Treatment with PRE alone had no, or only a marginal, protective effect. TAC alone at a dose of 1 or 0.5 mg/kg of body weight (BW) prevented xenograft rejection. The addition of PRE to TAC treatment had a paradoxical unfavorable effect. In contrast, when PRE was added to CsA-based protocols (CsA+DSG, CsA+SIR, or CsA+LEF), the immunosuppressive effect was slightly enhanced. In comparison with untreated rats, the messengers for interleukin (IL)-1beta, IL-2, IL-4, IL-10, interferon (IFN)-gamma, and tumor necrosis factor (TNF)-alpha were reduced in both CsA and TAC treated rats. Notably, the amount of IL-12p40 transcripts was only inhibited in rats given TAC alone, whereas this messenger was increased to approximately the same levels in untreated, CsA treated, and TAC plus PRE treated rats.

CONCLUSIONS:

TAC exerted a pronounced immunosuppressive effect in the pig-to-rat islet xenotransplantation model. So far, no other single drug protocol has shown a comparable efficacy. Notably, the graft protective effect of TAC was markedly abrogated when PRE was added to the treatment protocol, suggesting that TAC exerts its effect by a unique mechanism of action. In contrast with the other studied immunosuppressive regimens, treatment with TAC alone inhibited intragraft mRNA expression of all the Th1 associated cytokines, indicating that Th1 response is one important rejection mechanism that needs to be inhibited to achieve islet xenograft survival.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-90016 (URN)12438951 (PubMedID)
Available from: 2002-10-11 Created: 2002-10-11 Last updated: 2017-12-14Bibliographically approved
3. Intragraft cytokine mRNA expression in rejecting and non-rejecting Vascularised Xenografts
Open this publication in new window or tab >>Intragraft cytokine mRNA expression in rejecting and non-rejecting Vascularised Xenografts
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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.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-90017 (URN)10.1034/j.1399-3089.2003.02032.x (DOI)12795680 (PubMedID)
Note

De två första författarna delar förstaförfattarskapet.

Available from: 2002-10-11 Created: 2002-10-11 Last updated: 2017-12-14Bibliographically approved
4. Intracerebral cytokine profiles in adult rats grafted with neural tissue of different immunological disparity
Open this publication in new window or tab >>Intracerebral cytokine profiles in adult rats grafted with neural tissue of different immunological disparity
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Manuscript (Other academic)
Identifiers
urn:nbn:se:uu:diva-90018 (URN)
Available from: 2002-10-11 Created: 2002-10-11 Last updated: 2010-01-13Bibliographically approved

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