Identification and characterization of undifferentiated mast cells in mouse bone marrow.

Sequential immunomagnetic isolation with 2 monoclonal antibodies was used to purify and characterize an undifferentiated mast cell in adult mouse bone marrow that had not been previously recognized. This cell represents 0.02% of the cells in the bone marrow, is CD34(+), CD13(+), and c-kit(+), and does not express FcepsilonRI. However, by polymerase chain reaction (PCR) the cell contains message for the alpha and beta subunits of FcepsilonRI, mast cell-specific proteases, and carboxypeptidase A. Morphologically, this cell has a large nucleus, little cytoplasm, few cytoplasmic organelles, and no cytoplasmic granules. In vitro, in the presence of interleukin-3 (IL-3) and stem cell factor (SCF) these cells differentiate only into a granulated mast cell that now expresses CD13, c-kit, mast cell-specific gangliosides, FcepsilonRI, and binds immunoglobulin E (IgE). When injected into lethally irradiated mice, these cells are able to reconstitute the mast cell population in the spleen.

Selenium-binding protein-1 in smooth muscle cells is downregulated in a rhesus monkey model of chronic allograft nephropathy.

Treating patients with kidney failure by organ transplantation has been extraordinarily successful. Although, current immunosuppressants have improved short-term allograft survival, most transplants are eventually lost due to chronic allograft nephropathy (CAN). The molecular mechanisms underlying CAN are poorly understood. Smooth muscle cells (SMC) play a major role in the pathogenesis of CAN by contributing to the thickening of the intima and narrowing of the lumen of blood vessels. We show that selenium-binding protein-1 (SBP-1), a protein implicated in protein trafficking and secretion, is localized primarily to SMC in vivo. SBP-1 was heavily tyrosine-phosphorylated in vivo. Remarkably, SBP-1 was absent or strongly downregulated in vascular SMC in monkey kidney allografts with CAN. In contrast, the SMC alpha-actin was strongly expressed in the vascular SMC of the same allografts, indicating that the decrease in SBP-1 was not due to a global decrease in SMC proteins. Out of four growth factors implicated in the pathogenesis of CAN, only TGF-beta blocked the expression of SBP-1; thus, TGF-beta could regulate the expression of SBP-1 in CAN. These results show that SBP-1 localizes primarily to SMC in vivo and implicate this phosphoprotein in the effects of TGF-beta on SMC and in the process of CAN.

Surveillance of acute rejection in baboon renal transplantation by elevation of interferon-gamma inducible protein-10 and monokine induced by interferon-gamma in urine.

BACKGROUND: CXCR3 binding chemokines play a key role in recruitment of inflammatory cells into an organ transplant. This study addresses the question of whether urinary excretion of these chemokines correlates with acute rejection in a baboon kidney transplantation model. METHODS: Seven outbred baboons underwent renal allotransplantation from major histocompatibility complex (MHC)-mismatched donors. The treatment of baboons consisted of anti-CD4 monoclonal antibody (mAb), anti-CD8 mAb, rapamycin, and mycophenolate mofetil (MMF). Urinary levels of interferon-gamma inducible protein-10 (IP-10) and monokine induced by interferon-gamma (Mig) were determined by ELISA. Renal biopsies were examined by immunohistochemical staining for CXCR3 and Mig. RESULTS: Urinary levels of IP-10 and Mig increased significantly in all of the five baboons at the time of acute rejection of renal transplant. The IP-10 and Mig levels did not rise in two nonrejecting baboons. In two baboons, urinary levels of IP-10 and Mig rose before the elevation of the serum creatinine. In renal biopsies, expression of Mig was detected in glomeruli, tubules, and infiltrating cells, and the expression was significantly elevated in biopsies with acute rejection (P<0.01). CXCR3 was constitutively expressed in tubular cells in biopsies derived from both normal grafts and grafts with acute rejection. Whereas the infiltrating cells were increased in the biopsies with acute rejection, the expression of CXCR3 was also significantly higher (P<0.01) in these infiltrating cells compared with those in the normal controls. CONCLUSIONS: This study shows an important correlation between urinary excretion of IP-10 and Mig and acute rejection in baboon kidney transplantation.

Immune status assay (ISA): a noninvasive procedure for studying allograft rejection.

BACKGROUND: There is a need for a simple, sensitive, noninvasive technique for monitoring graft function. We report here on a simple assay called immune status assay (ISA) that determines the status of the graft by simply examining the activation status of blood T cells. METHODS: Graft-derived fibroblasts were used as a source of alloantigens and the recipient blood as a source of allograft-specific peripheral blood lymphocytes (PBL). PBL were added to wells containing donor or third-party graft-derived fibroblasts in the presence or absence of interleukin-2 (IL-2). On day 4 [(3)H]thymidine incorporation was quantified after the cells were incubated for 3 days at 37 degrees C, in a 5% CO(2) water-jacketed incubator. The results were analyzed using the following equation: %IL2 - /IL2+ = ((mean[(3)H]thymidine uptake in the absence of IL - 2) / (mean [(3)H]thymidine uptake in the presence of IL - 2)) x 100. RESULTS: The ISA score (%IL-2 - /IL-2+) correlated strongly with the outcome of the graft, as it had a sensitivity of 82% for detecting rejections (14/17), and a specificity of 81% (30/37) for detecting non-rejections. Notably, the ISA detected immune T cell activation in the blood of graft rejecting subjects, which were not detected by currently used techniques such as mixed lymphocytes reaction. CONCLUSION: The ISA is a straightforward procedure that detects allograft rejection with high specificity and sensitivity.

Monotherapy with the novel human anti-CD154 monoclonal antibody ABI793 in rhesus monkey renal transplantation model.

BACKGROUND: This study assesses the safety and efficacy of the novel human anti-human CD154 monoclonal antibody ABI793 in rhesus monkeys. METHODS: Outbred rhesus monkeys were used for renal transplantation from major histocompatibility complex-mismatched donors. Seven recipients were treated with ABI793, and six untreated recipients were used as controls. Graft function was monitored by urine output, serum creatinine, and renal biopsy. Phenotypic analysis of peripheral blood lymphocytes and mixed lymphocyte reaction were performed before transplantation and periodically after transplantation. Anti-donor major histocompatibility complex class I antibody levels were measured at the time of sacrifice. RESULTS: Monkeys in the treated group demonstrated prolonged graft survival compared with controls. One monkey was sacrificed because of a urine leak on postoperative day 13. Three monkeys were sacrificed because of acute rejection (days 44, 149, and 158). Two monkeys were sacrificed because of chronic active rejection (days 154 and 221). One monkey was sacrificed on day 139 without rejection to observe the effects of ABI793 in the absence of rejection. There were no obvious clinical side effects of ABI793, but microscopic thromboembolic changes were observed in two monkeys. Lymphocyte subsets remained unaltered in all monkeys. Mixed lymphocyte reaction showed nonspecific suppression 6 weeks after transplantation. The monkeys with chronic active rejection showed relatively strong alloantibody responses. CONCLUSIONS: ABI793 induces prolonged renal allograft survival in rhesus monkeys. Nevertheless, thromboembolic complications may occur and chronic allograft nephropathy may develop after anti-CD154 treatment is discontinued.

Metastable tolerance to rhesus monkey renal transplants is correlated with allograft TGF-beta 1+CD4+ T regulatory cell infiltrates.

Approaches that prevent acute rejection of renal transplants in a rhesus monkey model were studied to determine a common mechanism of acceptance. After withdrawal of immunosuppression, all 14 monkeys retained normal allograft function for >6 mo. Of these, nine rejected their renal allograft during the study, and five maintained normal function throughout the study period. The appearance of TGF-beta 1(+) interstitial mononuclear cells in the graft coincided with a nonrejection histology, whereas the absence/disappearance of these cells was observed with the onset of rejection. Analysis with a variety of TGF-beta 1-reactive Abs indicated that the tolerance-associated infiltrates expressed the large latent complex form of TGF-beta 1. Peripheral leukocytes from rejecting monkeys lacking TGF-beta 1(+) allograft infiltrates responded strongly to donor Ags in delayed-type hypersensitivity trans-vivo assays. In contrast, allograft acceptors with TGF-beta 1(+) infiltrates demonstrated a much weaker peripheral delayed-type hypersensitivity response to donor alloantigens (p < 0.01 vs rejectors), which could be restored by Abs that either neutralized active TGF-beta 1 or blocked its conversion from latent to active form. Anti-IL-10 Abs had no restorative effect. Accepted allografts had CD8(+) and CD4(+) interstitial T cell infiltrates, but only the CD4(+) subset included cells costaining for TGF-beta 1. Our data support the hypothesis that the recruitment of CD4(+) T regulatory cells to the allograft interstitium is a final common pathway for metastable renal transplant tolerance in a non-human primate model.

Elevation of CXCR3-binding chemokines in urine indicates acute renal-allograft dysfunction.

A noninvasive urinary test that diagnoses acute renal allograft dysfunction would benefit renal transplant patients. We aimed to develop a rapid urinary diagnostic test by detecting chemokines. Seventy-three patients with renal allograft dysfunction prompting biopsy and 26 patients with stable graft function were recruited. Urinary levels of CXCR3-binding chemokines, monokine induced by IFN-gamma (Mig/CXCL9), IFN-gamma-induced protein of 10 kDa (IP-10/CXCL10), and IFN-inducible T-cell chemoattractant (I-TAC/CXCL11), were determined by a particle-based triplex assay. IP-10, Mig and I-TAC were significantly elevated in renal graft recipients with acute rejection, acute tubular injury and BK virus nephritis. Using 100 pg/mL as the threshold level, both IP-10 and Mig had diagnostic value (sensitivity 86.4%; specificity 91.3%) in differentiating acute graft dysfunction from other clinical conditions. In terms of monitoring the response to antirejection therapy, this urinary test is more sensitive and predictive than serum creatinine. These results indicate that this rapid test is clinically useful.

Rapamycin antagonizes cyclosporin A- and tacrolimus (FK506)-mediated augmentation of linker for activation of T cell expression in T cells.

The discovery of new immunosuppressive drugs such as rapamycin, cyclosporin A (CsA) and tacrolimus (FK506) has been very useful for preventing graft rejection and autoimmune disease. However, these drugs are not specific, and are associated with side-effects and toxicities. Therefore, understanding the molecular mechanisms of these drugs is important for designing specific immunosuppressants. Here, we show that in contrast to CsA and FK506, rapamycin blocks activation-induced expression of the linker for activation of T cells (LAT), a signaling molecule critical for initiating TCR signaling. Thus, whereas CsA and FK506 strongly enhanced TCR- and phorbol myristate acetate-induced LAT expression in T cells, rapamycin effectively inhibited activation-induced LAT expression. Importantly, these opposite effects were mutually antagonistic, as rapamycin acted as a potent antagonist for both CsA and FK506. Because CsA, unlike FK506 and rapamycin, does not bind to the intracellular immunophilin FK-binding protein (FKBP), the antagonism between these drugs is not simply due to competition for intracellular FKBP. Accordingly, RNA and protein stability analyses suggest inhibition by rapamycin at the translational level. Given the important role of LAT in initiating T cell activation, our data suggests that the effects of rapamycin, CsA and FK506 on T cell activation involve regulating early T cell signaling. These findings refine our understanding of the manifold effects of these immunosuppressants, thus providing insight into the drastic physiological contrasts observed between these drugs.

Molecular actions of calcineurin inhibitors.

The immunosuppressive drugs cyclosporin A and FK-506, also called calcineurin inhibitors, have been useful for treating immune system-mediated diseases and have truly revolutionized allograft transplantation. Both drugs block T-cell proliferation by mechanisms that involve the inhibition of the key signaling phosphatase calcineurin, hence, the name calcineurin inhibitors. The inhibition of calcineurin activation by cyclosporin A and FK-506 blocks T-cell receptor-mediated production of interleukin-2 (IL-2), a growth factor critical for T-cell proliferation. Recent studies, however, suggest that the effects of the drugs are not limited to blocking calcineurin activation and IL-2 production. This review discusses the molecular actions of cyclosporin A and FK-506.

Immunotoxin-treated rhesus monkeys: a model for renal allograft chronic rejection.

BACKGROUND: Unlike acute and hyperacute rejection, chronic rejection (CR) still constitutes a poorly understood process. The onset is insidious, occurs in a period of months to years and, because the pathophysiology is not well understood, is untreatable. A reliable large-animal model for renal allograft CR is needed and has not been reported in the literature yet. METHODS: CR biopsy changes were studied in major histocompatibility complex-mismatched renal allografts performed in nine rhesus monkeys that received CD3 T-lymphocyte depletion therapy with immunotoxin on the day of the transplantation (n=7) or 7 days before transplant (n=2). RESULTS: Mean graft survival time was 613.77 days. Biopsy changes of CR were identified as soon as 84 days after transplant (mean, 336 days; range, 84-896 days). Most of the experimental animals had severe interstitial fibrosis, tubular atrophy, chronic transplant glomerulopathy, and chronic vascular rejection changes at the time of necropsy. A significant positive correlation between the severity of CR and the degree of CD68+ macrophage infiltrate of renal parenchyma and the degree of anemia and serum creatinine level elevations were also observed. CONCLUSIONS: Our findings are similar to those seen in human renal chronic allograft nephropathy, but in contrast, our model excludes all the nonimmune factors associated with chronic allograft nephropathy, including donor disease, injury from prolonged preservation, drug toxicity, and underlying recipient disease. Immunotoxin-treated rhesus monkeys emerge as an outstanding animal model for assisting us in understanding the pathophysiology of CR.

Campath-1H induction plus rapamycin monotherapy for renal transplantation: results of a pilot study.

Campath-1H, an anti-CD52 monoclonal antibody, was used as induction therapy (40 mg i.v. total dose) in 29 primary human renal transplants, and the patients were maintained on rapamycin monotherapy (levels 8-15 ng/mL) post-transplant. Campath-1H profoundly depletes lymphocytes long-term and more transiently depletes B cells and monocytes. All patients are alive and well at 3-29 months of follow up. One graft was lost because of rejection. There have been no systemic infections and no malignancies. Eight of 29 patients have experienced rejection, which was successfully treated in seven of eight patients. Five of these patients had pathological evidence of a humoral component of their rejection. Seven of the 29 patients were converted to standard triple therapy on account of rejection. Rapamycin was generally well tolerated in that there were no significant wound-healing problems; two lymphoceles required surgical drainage; and most patients were treated with a lipid-lowering agent. Flow crossmatch testing post-transplant revealed evidence of alloantibody in two patients tested with previous combined cellular and humoral rejection. Biopsies have shown no chronic allograft nephropathy to date. In view of the relatively high incidence of early humoral rejection, we plan to modify the immunosuppressive regimen in subsequent pilot studies. This clinical trial provides insight into the use of Campath-1H induction in combination with rapamycin maintenance monotherapy.

Targeting proximal T cell receptor signaling in transplantation.

In the past two decades, an immense amount of information has been generated on the mechanism of T cell receptor (TCR) signaling (also called signal 1). This overview describes the major signalling pathways in the TCR signal transduction cascade and focuses on proximal events in TCR signaling. The review also discusses some of the strategies that target proximal TCR signaling, which are used for preventing graft rejection.

Modulation of the electrophoretic mobility of the linker for activation of T cells (LAT) by the calcineurin inhibitors CsA and FK506: LAT is a potential substrate for PKC and calcineurin signaling pathways.

The linker for activation of T cells (LAT) is essential for T cell activation. Cyclosporin A (CsA) and FK506, inhibitors of T cell proliferation, have been very useful for preventing autoimmune and inflammatory disease and graft rejection. However, both compounds are associated with side effects. We show that TCR ligation in the presence of FK506 or CsA induced rapid modifications in LAT that modulate the electrophoretic mobility of the molecule in SDS-PAGE. Calcineurin, a target for CsA and FK506, dephosphorylated LAT in vitro and restored its electrophoretic mobility. Stimulating T cells with the protein kinase C (PKC) activator PMA induced a shift in the mobility of LAT, whereas inhibitors of PKC blocked the effect of PMA. Thus, manipulating calcineurin or PKC activation alters the electrophoretic mobility of LAT. These results shed light on the molecular actions of CsA and FK506 in T cells and implicate LAT in mediating the drugs' actions.

Piceatannol in combination with low doses of cyclosporine A prolongs kidney allograft survival in a stringent rat transplantation model.

BACKGROUND: The discovery of new immunosuppressive agents has enhanced short-term graft survival. However, current immunosuppressants often induce toxicities that limit their clinical use. Thus, there is a need for new immunosuppressants for use in clinical transplantation. Piceatannol blocks Syk and ZAP-70, tyrosine kinases involved in immune cell activation. We examined whether piceatannol prolongs kidney allograft survival in the stringent ACI-to-Lewis rat model. METHODS: Kidney recipients were divided into four groups. Group 1 (n=8) received piceatannol 30 mg/kg per day intravenously and cyclosporine A (CsA) 2 mg/kg per day intramuscularly from day -3 to day 7 after transplantation. At day 8, piceatannol was reduced to 10 mg/kg per day and the combined treatment continued until day 60. Group 2 (n=9) received 2 mg/kg per day CsA alone from day -3 to day 60. Group 3 (n=4) received piceatannol alone as in group 1. Group 4 (n=2) received only the vehicle dimethyl sulfoxide from day -3 to day 60. Graft rejection was defined as either a serum creatinine level more than 2 mg/dL or animal death. RESULTS: Group 1 animals survived for at least 115 days (n=8, P<0.05), with several animals maintaining their grafts for more than 200 days. In contrast, 8 of 9 animals in group 2 rejected their grafts within 10 days of transplantation; one animal survived for 71 days. Excellent graft function was maintained in group 1 animals despite withdrawal of immunosuppression. CONCLUSIONS: These results are the first to show that piceatannol, when combined with subtherapeutic dosages of CsA, prevents graft rejection, suggesting that targeting Syk and Zap could be useful for preventing graft rejection.