Biomedical subjects
Publications and source records attributed to T B Strom.
Although Th1- and Th2-type cytokine profiles have been associated with rejection and tolerance respectively, this paradigm may not be completely accurate. Instead, recent studies suggest that there could be a hierarchy of T-cell growth factors with regard to their ability to block tolerance and induce rejection (rather than a polarized Th1/Th2 demarcation).
Murine CTLA4/Fc therapy leads to permanent engraftment of islet allografts in interleukin 4 (IL-4) knockout (IL-4-/- mice. Interestingly, IL-4+/- hosts were more resistant to tolerance induction than IL-4-/- mice. An IL-2/Fc fusion protein abrogates the effect of CTLA4/Fc therapy while an IL-4/Fc fusion protein tends to inhibit rather than enhance the effect of CTLA/Fc treatment in IL-4-/- recipients. We conclude that allograft acceptance requires principally a blockade of T cell activation rather than 'immune deviation' of the T cell activation program to Th2 cytokines (i.e. IL-4).
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T-cell activation is the key event in the development of acute allograft rejection and precedes clinically apparent organ damage. We have performed competitive RT-PCR to quantify the intragraft gene expression for T-cell associated cytokines (IL-2, IL-4, IL-7, IL-15), CTLA4 and cytotoxic lymphocyte specific molecules to test their potential as rejection markers and to further elucidate mechanisms involved in graft rejection. RNA was isolated from snap-frozen portions of core biopsies obtained for the evaluation of graft dysfunction in 34 adults and 8 children. Reverse transcription derived cDNA was coamplified with a known amount of a competitor (a mutated target gene fragment) and normalized for the house keeping gene GAPDH. IL-2, the principal T-cell growth factor and IL-4 were not detectable in any biopsy at the time of histologically apparent rejection. Transcripts of the novel cytokine IL-15 were found in all dysfunctioning grafts and in two donor kidneys prior to reperfusion. CTLA-4, expressed in activated T-cells after costimulation by CD28 was uniformly present post transplantation, but not in the two donor kidneys. Transcripts for IL-7 (p < 0.001), IL-15 (p < 0.0005), CTLA4 (p = 0.04), granzyme B (p < 0.00015) and perforin (p < 0.0003) showed a significant correlation to acute rejection episodes. Heightened gene expression declined rapidly after initiation of rejection treatment. Fas-ligand mRNA gene expression was upregulated in both acute and chronic rejections. While this study shows that competitive RT-PCR is a reliable diagnostic tool to detect acute rejection in renal core biopsies, a future challenge will be to identify molecular markers of evolving rejections utilizing RT-PCR in sequential samples of fine needle aspirations, urine and blood.
To test the hypothesis that blockade of B7-triggered costimulation by donor cells could preclude allograft rejection, we coated crude islet allograft preparations in vitro for 1 h with a murine CTLA4/Fc fusion protein. Murine CTLA4/Fc blocks the proliferative response in primary mixed lymphocyte cultures (MLC) and Con A-stimulated murine spleen cell cultures by 85 to 95%. Responder cells from a primary MLC containing mCTLA4/Fc were hyporesponsive upon restimulation to the same stimulator cells in a secondary MLC lacking mCTLA4/Fc. Because of mutations in the Fc gamma RI and C'1q binding sites of the Fc portion of the murine CTLA4/Fc fusion protein, the molecule binds to, but does not target, cells for Ab-dependent cellular cytotoxicity or complement-directed cytolysis. Although systemic immunosuppression was not applied, 42% (10 of 24) of B6AF1 recipients of islet allografts pretreated with CTLA4/Fc were permanently engrafted. Further, 50% of hosts bearing functioning islet allografts more than 150 days post-transplant were formally proved to be tolerant to donor tissues. A persistent CD4+ and CD8+ T cell infiltrate surrounding, but not invading, islet grafts in tolerant hosts was discerned. In control experiments, 89% (8 of 9) of islet allografts coated with mIgG3, and 100% (n = 10) pretreated with media alone were rejected. Thus, we conclude that 1) B7-triggered costimulation by donor APCs is an important element of rejection, and 2) blockade of the B7 pathway by in vitro allograft manipulation is able to induce tolerance.
The IL-2 pathway is portrayed often as central to allograft rejection. To test this hypothesis, we studied IL-2-deficient mice as allograft recipients. IL-2 gene knockout (KO) mice reject islet allografts and demonstrate a classical mononuclear leukocytic infiltrate, containing CD4+ and CD8+ T cells, surrounding and invading the islet allografts. Moreover, allograft rejection in the IL-2 KO mouse is associated with intragraft expression of certain cytokine and CTL attack molecule genes (e.g. IFN-gamma, IL-4, IL-7, IL-10, and granzyme B). In separate experiments, IL-2 KO mice generated CTLs in response to in vivo challenge with allogeneic tumor cells. Although IL-2 KO mice reject allografts in vivo, spleen cells from immunologically naive IL-2 KO mice exhibit a diminished proliferative response to mitogens in vitro that could be restored largely by exogenous IL-2, IL-4, or IL-7. The paradoxical ability to execute graft rejection in vivo despite near absent T cell proliferative responses in vitro may result from the expression of IL-7 in vivo, but not in vitro. Con A-stimulated bulk spleen cell cultures derived from IL-2 KO mice were essentially devoid not only of IL-2 but also IL-7 gene transcripts. These data indicate that 1) IL-2 is not the sole T cell growth factor capable of supporting allograft rejection and 2) expression of IL-4, but not IL-2, during the allograft response does not lead inevitably to tolerance.
Numerous studies have suggested the potential application of IL-10 as an anti-inflammatory and as an antirejection agent. Unfortunately, cytokines have short circulating t1/2 We developed a murine IL-10/Fc gamma 2a immunoligand that possesses the biologic functions of IL-10 and the long circulating t1/2 in vivo, characteristic of Igs. We mutated the Fc gamma 2a fragment to render the immunoligand ineffective in directing Ab-dependent cell-mediated cytotoxicity and complement-directed cytolysis (noncytolytic IL-10/Fc (IL-10/Fc2-)). In terms of IL-10 activity, IL-10/Fc2- was as effective as rIL-10 mole per mole in preventing lethal septic shock, but the immunoligand had a prolonged period of efficacy in accord with its extended circulating half-life. Contrary to expectations, IL-10/Fc2- treatment tended to accelerate the destruction of islet cell allografts and increase the levels of granzyme B gene expression in local draining lymph nodes. These data suggest that the enhanced cytotoxic activity of allograft-destroying CTLs may contribute to the accelerated allograft rejection. Finally, our studies suggest that a noncytolytic IL-10/Fc fusion protein provides a useful tool to study the biologic effects of IL-10 in vivo and may provide a useful agent for the prevention and treatment of septic shock.
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An earlier reported trial suggests that omega-3 fatty acids in fish oil supplements at 6 g/day with administration commencing at the time of engraftment may reduce acute CsA renal dysfunction. When started at the time of renal transplant, there are improvements in renal hemodynamics and blood pressure, and a decrease in rejection episodes. To examine the effect of later introduction of omega-3 fatty acids, 133 cadaver renal transplant recipients received CsA, prednisone, and AZA for 16 weeks (period 1). If patients were stable without rejection or infection activity, they were randomized to 9 g of eicosapentanoic acid (EPA), 18 g of EPA, 9 g of corn oil, or 18 g of corn oil in 1-g capsules as supplements. Glomerular filtration rate, renal blood flow, number of rejection episodes, blood pressure, and episodes of CsA nephrotoxicity were followed for 26 weeks in a double-blind manner (period 2). Ninety patients were evaluable and completed the protocol. There were 50 corn oil placebo patients, 22 low dose EPA patients, and 18 high dose EPA patients. In period 1, there were 27 rejection episodes in 21 patients without differences among subsequent treatment groups. In period 2, there were 13 rejection episodes in 4 patients. No patient with an EPA level in plasma statistically higher than placebo had a rejection episode. All allografts functioned for the entire 6 months with none lost to rejection. All 5 episodes of acute CsA nephrotoxicity occurred in placebo-treated patients without differences in whole blood CsA among toxic patients, other placebo patients, and EPA-treated recipients. At the end of the study, there were no differences in glomerular filtration rate, renal blood flow, or creatinine clearance among groups. Diastolic blood pressure fell by 9 mmHg during period 2 in high dose fish oil recipients and by 10 mmHg in low dose fish oil recipients (P < 0.05), while it rose by 2 mmHg in placebo patients. No serious adverse effects of EPA supplements were noted, although compliance based on plasma EPA was erratic. Based on our experience and that in the literature, administration of omega-3 fatty acids for purposes of kidney protection would seem to be most useful when started early after surgery. Late administration in our study was associated with minor clinical benefits.
Much has been learned regarding immunobiological mechanisms responsible for the rejection of histoincompatible allografts. There has also been considerable progress in our understanding of mechanisms responsible for tolerance. The new knowledge gained regarding graft destructive alloimmunity process and the mechanisms of action of immunosuppressants have resulted in solid organ graft survival rates that are in excess of 80% at one year posttransplantation. The principles of tolerance mechanism are yet to be successfully applied in the clinic. In this review, molecular and cellular mechanisms of action of clinically useful immunosuppressive drugs are reviewed from the perspective of regulation of the anti-allograft repertory.
This review presents our concepts regarding the mechanisms of rejection and the pathway leading to graft tolerance. Allograft rejection is a T-cell-dependent process. Accordingly, we discuss the current understanding of T cell activation and cytokine production and then apply these concepts to the mechanisms that cause rejection and tolerance. We describe the ability of renal epithelial cells to either induce T cell activation or anergy. Finally, we review potential mechanisms responsible for the maintenance of tolerance.
Allograft rejection is a T cell-dependent process. It is not known whether rejection is mediated by a limited number of T cell clones or by a polyclonal population of T cells. Several studies attempting to answer this question using molecular techniques to analyze the T cell receptor (TCR) population have reached conflicting conclusions. Reverse transcription-assisted polymerase chain reaction (PCR) has been used to quantify T cell infiltration and examine TCR heterogeneity in kidney transplant biopsies from patients experiencing graft dysfunction. RNA from snap-frozen biopsies gathered on 23 transplant patients was reverse transcribed to cDNA and used as the template for PCR. The constant region gene of the TCR beta chain (C beta), 22 different variable region genes of the TCR beta chain (V beta) and the constitutively expressed glyceraldehyde phosphate dehydrogenase (GAPD) gene were amplified. T cell infiltration, as estimated by the ratio of reverse-transcribed cDNA C beta/glyceraldehyde phosphate dehydrogenase, was significantly higher in acute cellular rejection (ACR) (2.25) than in nonrejection (NR) (0.40, P < 0.05). The number of intragraft V beta families was higher in chronic rejection and acute cellular rejection (18 and 16.4, respectively) than in nonrejection (8.7). Five serial biopsies from two patients progressing to immunologic graft loss showed an increase in the number of intragraft V beta families. The finding of increased numbers of TCR V beta families amplified from acutely and chronically rejecting grafts as compared with nonrejecting graft supports the hypothesis that, at the time of clinically apparent rejection, there is a polyclonal infilitration of T cells.
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The antiinflammatory agent sulfasalazine (SS) is prescribed to treat Crohn's disease, ulcerative colitis and rheumatoid arthritis. Activated T cells are present within diseased mucosal and synovial sites. We tested whether SS or its metabolites 5-aminosalicylic acid (5-ASA) and sulfapyridine (SP) inhibited the T-cell activation products interleukin 2 (IL-2) and interleukin 2 receptor alpha-chain (IL-2R alpha). Experiments were performed in phytohemaglutinin- and phorbol ester-stimulated peripheral blood mononuclear cells. Radioactive thymidine and leucine incorporation assayed DNA and protein synthesis, respectively. Enzyme-linked immunosorbent assay and Northern blot analysis measured IL-2 and IL-2R alpha. Lactate dehydrogenase release determined cell viability, and intracellular free calcium was measured by an indole fluorescent indicator. SS and 5-ASA, but not SP, inhibited T-cell proliferation and protein synthesis in phytohemaglutinin- and phorbol ester-stimulated peripheral blood monomuclear cells. 5-ASA (625 microM) markedly reduced culture supernatant IL-2 protein levels by 92% and steady-state IL-2 messenger RNA levels 4.4-fold at 24 and 18 hr, respectively. The supplementation of IL-2 restored T-cell proliferation only in 5-ASA-treated cultures. SS, 5-ASA and SP did not alter intracellular calcium accumulation after mitogenic stimulation. SS and 5-ASA (625 microM) caused 71% and 37% cytotoxicity, respectively, in 72-hr cultures. 5-ASA inhibits T-cell proliferation in part by blocking IL-2 messenger RNA accumulation and protein production downstream of the rise in cytosolic calcium. Inhibition of IL-2 production is an additional mechanism of action for 5-ASA.
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