A novel model of allograft rejection: immune reconstitution of Rag-1 recipients with 2C transgenic T-cell receptor lymphocytes.

The complexity of allorejection (cell activation, homing, and effector function) makes detailed studies difficult. We have developed a model of allograft rejection using purified monoclonal alloreactive effector cells. Immunodeficient C57Bl/6-Rag-1 (H-2(b)) recipients of Balb/c (H-2(d)) islet or skin grafts were reconstituted via adoptive transfer of splenocytes from 2C transgenic mice containing CD8+ cytotoxic effector cells directed against L(d). Recipients were assessed for engraftment, activation and homing of effector cells, and ability to reject grafts. Both unpurified 2C splenocytes and purified 2C/CD8+ cells durably reconstitute immunodeficient mice. Naive 2C effector cells reject skin grafts, but not islet allografts. However, when effector cells are primed prior to reconstitution, islet allografts are rejected. Using this model, blockade of adhesion molecules LFA-1 and alpha4-integrin delayed infiltration of islet allografts and prolonged allograft survival. This model of allorejection may be useful to study the activation and homing of allospecific cells in vivo.

Immunomodulation through inhibition of multiple adhesion molecules generates resistance to autoimmune diabetes in NOD mice.

The effect of simultaneous blockade of adhesion molecules on the development of long-term resistance to type 1 diabetes was investigated in an adoptive transfer model in NOD mice. Splenocytes isolated from acutely diabetic NOD mice injected into NOD-scid mice caused diabetes at 43 +/- 5.0 days. Treatment with anti-alpha4-integrin monoclonal antibody (mAb) delayed the onset of insulitis and significantly delayed hyperglycemia to 66 +/- 5.8 days. Combination treatment with anti-alpha4-integrin and anti-LFA-1 mAbs delayed the onset of diabetes to >100 days (p<0.0001). Combination-treated mice were subjected to a second challenge with diabetogenic splenocytes after 85 days of normoglycemia. Without additional mAb treatment they developed hyperglycemia after significant delay (72 +/- 8.1 days post-reinoculation). Splenocytes from combination-treated mice transferred protection from diabetes to naive NOD-scid mice when co-transferred with diabetogenic splenocytes. The long-surviving mice showed periislet infiltration with CD62L+ cells, which were not seen in the insulitis developing in control animals. These findings suggest that adhesion molecule blockade does not prevent homing and may affect effector cell action through activation of immunoregulatory suppressor cells, leading to protection against development of diabetes.

Acute nephrotoxicity of tacrolimus and sirolimus in renal isografts: differential intragraft expression of transforming growth factor-beta1 and alpha-smooth muscle actin.

BACKGROUND: Renal dysfunction early after kidney transplantation has multiple causes including ischemia-reperfusion (I/R) injury and drug-induced nephrotoxicity. This study assesses the acute nephrotoxicity of tacrolimus (Tac) and sirolimus (Sir) in a rat renal isograft model. METHODS: Lewis renal isografts and uninephrectomized rats that did not undergo transplantation were treated with various doses of Tac (0.5-5.0 mg/kg/d) or Sir (0.5-6.5 mg/kg/d). Kidneys were examined on day 14 by routine histology and immunohistochemistry for transforming growth factor (TGF)-beta1 and alpha-smooth muscle actin (SMA). RESULTS: Both Tac and Sir demonstrated evidence of nephrotoxicity in the early posttransplant period including increased serum creatinine and morphologic changes in the graft including interstitial inflammation, fibrosis, and tubular vacuolization. Nephrotoxicity was most prominent in the high-dose treatment groups for both drugs and was more severe in transplanted kidneys than in uninephrectomized animals that did not undergo transplantation, suggesting an additive effect of I/R injury and drug nephrotoxicity. Both Tac and Sir increased intragraft TGF-beta1 and alpha-SMA, but there were distinct differences in the patterns of TGF-beta1 expression. Both demonstrated TGF-beta1 in tubular epithelial cells, but Sir was associated with proximal tubular TGF-beta1 localization in a bright granular pattern, whereas Tac was associated with diffuse distal tubular staining. CONCLUSIONS: Both Tac and Sir may be nephrotoxic in the early posttransplant period, especially at high doses and when combined with I/R injury. Immunohistochemical localization of TGF-beta1 in the tubular cells was distinctly different with each drug, suggesting possible differences in the mechanism(s) of nephrotoxicity requiring further study.

Accommodation in ABO-incompatible kidney allografts, a novel mechanism of self-protection against antibody-mediated injury.

To elucidate the mechanism of self-protection against anti-donor blood-group antibody known as accommodation, we studied 16 human ABO-incompatible living-donor kidney transplant recipients at 3 and 12 months post transplantation. Both circulating anti-blood-group antibody and the target blood-group antigen in the graft were demonstrable in all patients after transplantation. Thirteen of 16 grafts had normal renal function and histology, while three grafts with prior humoral rejection demonstrated significant glomerulopathy and thus did not meet the criterion for accommodation. Using microarrays, we compared five 1-year protocol ABO-compatible renal graft biopsies to four accommodated ABO-incompatible graft biopsies. Significant alterations in gene expression in 440 probe sets, including SMADs, protein tyrosine kinases, TNF-alpha and Mucin 1 were identified. We verified these changes in gene expression using RT-PCR and immunohistochemistry. Heme oxygenase-1, Bcl-2 and Bcl-xl were not increased in ABO-incompatible grafts at any time-point. We conclude that accommodation is always present in well-functioning, long-surviving ABO-incompatible kidney transplants. This self-protection against antibody-mediated damage may involve several novel mechanisms including the disruption of normal signal transduction, attenuation of cellular adhesion and the prevention of apoptosis.

Forskolin suppresses insulin gene transcription in islet beta-cells through a protein kinase A-independent pathway.

This work was designed to evaluate the effect of cAMP on insulin gene regulation. We studied the effects of forskolin on insulin gene transcription in the INS-1 beta-cell line, confirming key results in primary cultures of human islet cells. Forskolin increased intracellular cAMP and cAMP-responsive element-binding activity. Insulin gene transcription was studied using a reporter construct in which the human insulin promoter was fused to luciferase. When cells were treated with forskolin for 12 h, insulin promoter activity was decreased 2- to 3-fold, whereas islet amyloid polypeptide promoter activity was significantly increased. This effect of forskolin on the insulin gene was time- and concentration-dependent, and was mimicked by 8-bromo-cAMP. Mutagenesis of the CRE-like elements in the insulin promoter had no effect on the forskolin-induced suppression, but dramatically decreased basal insulin promoter activity. Inhibition of PKA with H-89 also did not reverse the forskolin-induced suppression of insulin transcription. However, this effect was completely reversed by inhibition of cellular MAP kinase activity with PD98059 or U0126. These results demonstrate that forskolin suppresses insulin transcription in INS-1 cells through a PKA-independent mechanism that probably involves MAP kinase signalling.