Lewis rat pancreas, but not cardiac xenografts, are resistant to anti-gal antibody mediated hyperacute rejection.

BACKGROUND: The objective of this study was to evaluate the role of anti-Gal Abs and non-anti-Gal Abs in hyperacute rejection (HAR) of concordant pancreas xenografts compared with heart xenografts. In addition, we tested whether rejection of Lewis rat pancreas grafts was T-cell dependent and could be prevented by anti-T-cell treatment. METHODS: To determine the role of anti-Gal Abs in the induction of HAR, Lewis rat pancreas and heart xenografts were transplanted into alpha1,3Galactosyltransferase knockout (GT-Ko) mice treated with normal human serum (NHS) or hyperimmune serum, or into presensitized GT-Ko mice. To investigate whether rejection of pancreas xenograft was mediated by a T-cell dependent response, Lewis rat pancreas grafts were transplanted into streptozotocin (STZ)-induced diabetic GT-Ko mice treated with FK506, anti-CD4 mAbs (GK1.5), and thymectomy. Antidonor-specific IgM and IgG and anti-Gal Abs were analyzed by flow cytometry. Rejected and long-term surviving pancreas xenografts were assessed by functional (blood glucose) and histopathological examination. RESULTS: HAR of Lewis rat pancreas xenografts could not be induced by NHS (0.4 ml), whereas NHS (0.2 ml) resulted in HAR of Lewis heart xenografts. Infusion of Lewis rat-specific hyperimmune serum (0.2 ml) resulted in HAR of Lewis rat pancreas xenografts. In addition, second Lewis rat pancreas grafts were hyperacutely rejected by presensitized GT-Ko mice. Immunohistochemical staining showed a low expression of Galalpha1,3Gal antigen in the endocrine tissue compared with that in the cardiac grafts. The levels of anti-Gal Abs in pancreas xenograft transplantation did not increase in GT-Ko mice after pancreas xenograft transplantation that was significantly increased after heart transplantation. FK506 treatment induced long-term survival of Lewis pancreas xenografts (mean survival time (MST) >90 days). Anti-CD4 treatment delayed rejection of Lewis rat pancreas xenografts with MST of 34.3 days, whereas anti-CD4, in combination with thymectomy, synergistically prolonged survival of pancreas xenograft (MST=70.4 days). CONCLUSION: Pancreas xenograft is resistant to anti-Gal Abs-induced HAR but is susceptible to anti-donor specific Abs. Rejection of Lewis pancreas xenograft in STZ-induced, diabetic, GT-Ko mice is T-cell dependent.

Non-depleting anti-CD4, but not anti-CD8, antibody induces long-term survival of xenogeneic and allogeneic hearts in alpha1,3-galactosyltransferase knockout (GT-Ko) mice.

The anti-galactose-alpha1,3-galactose (Gal) antibody (Ab) response following pig-to-human transplantation is vigorous and largely resistant to currently available immunosuppression. The recent generation of GT-Ko mice provides a unique opportunity to study the immunological basis of xenograft-elicited anti-Gal Ab response in vivo, and to test the efficacy of various strategies at controlling this Ab response [1]. In this study, we compared the ability of non-depleting anti-CD4 and anti-CD8 to control rejection and antibody production in GT-Ko mice following xenograft and allograft transplantation. Hearts from baby Lewis rat or C3H mice were transplanted heterotopically into GT-Ko. Non-depleting anti-CD4 (YTS177) and anti-CD8 (YTS105) Abs were used at 1 mg/mouse, and given as four doses daily from day -2 to 1 then q.o.d. till day 21. Xenograft rejection occurred at 3 to 5 days post-transplantation in untreated GT-Ko recipients, and was histologically characterized as vascular rejection. Anti-CD4, but not anti-CD8, Ab treatment prolonged xenograft survival to 68 to 74 days and inhibited anti-Gal Ab as well as xeno-Ab production. In four of the five hearts from anti-CD4 mAbs-treated GT-Ko mice, we observed classic signs of chronic rejection, namely, thickened intima in the lumen of vessels, significant IgM deposition, fibrosis and modest mononuclear cell infiltrate of Mac-1+ macrophages and scattered T cells (CD8>CD4). Xenograft rejection in untreated, as well as anti-CD4- and anti-CD8-treated, recipients was associated with increased intragraft IL-6, IFN-gamma and IL-10 mRNA. C3H allografts were rejected in 7 to 9 days by untreated GT-Ko mice and were histologically characterized as cellular rejection. Treatment with anti-CD4 and anti-CD8 mAb resulted in graft survivals of >94.8 and 11.8 days, respectively. Anti-CD4 mAb treatment resulted in a transient inhibition of alloreactive and anti-Gal Ab production. The presence of circulating alloreactive and anti-Gal Abs at >50 days post-transplant was associated with significant IgM and IgG deposition in the graft. Yet, in the anti-CD4 mAb-treated group, the allografts showed no signs of rejection at the time of sacrifice (>100 days post-transplantation). All rejected allografts had elevated levels of intragraft IL-6, IFN-gamma and IL-10 mRNA, while the long-surviving anti-CD4-treated allografts had reduced mRNA levels of these cytokines. Collectively, our studies suggest that the elicited xeno-antibody production and anti-Gal Ab production in GT-Ko mice are CD4+ T-cell dependent. The majority of xenografts succumbed to chronic rejection, while allografts survived with minimal histological change, despite elevated levels of circulating alloAbs. Thus, immunosuppression with anti-CD4 mAb therapy induces long-term survival of allografts more effectively than to xenografts.

Anti-galactose-alpha(1,3) galactose antibody production in alpha1,3-galactosyltransferase gene knockout mice after xeno and allo transplantation.

Antibodies (Abs) that mediate the hyperacute rejection and acute vascular rejection/delayed xenograft rejection of pig organs in humans and Old World primates are predominantly directed at a single carbohydrate epitope, galactose-alpha1,3-galactose (alpha1,3Gal). The T-cell dependence of elicited anti-alpha1,3Gal Ab responses in humans and Old World primates is controversial. In this study we have characterized anti-alpha1,3Gal Ab production in mice with disrupted alpha1,3-galactosyltransferase genes (GT-Ko mice) and determined the T-cell dependence of anti-alpha1,3Gal Ab responses, following xenograft and allograft transplantation. GT-Ko mice produce natural anti-alpha1,3Gal IgM and IgG in an age-dependent manner, however, these Abs could not elicit hyperacute rejection nor affect the rate of cardiac xenograft (3-5 days) or allograft rejection (7-9 days). Transplantation of xenogeneic Lewis rats hearts elicited modest anti-alpha1,3Gal Ab, but vigorous xenoAb responses. The anti-alpha1,3Gal Ab response was restricted to the IgM and IgG3 subclass while the xenoAb response comprised IgM and all four IgG subclasses. Transplantation of allogeneic C3H hearts elicited weak anti-alpha1,3Gal Ab responses that were primarily IgM, but vigorous alloAb responses. Despite the restriction of elicited anti-alpha 1,3Gal Ab responses to the IgM and IgG3 isotypes, these responses are T-cell dependent. The ability of allografts to elicit weak anti-alpha1,3Gal but strong allo-Ab responses, can be explained by the dependence of alpha1,3Gal-specific B cells on cognate help from T cells.

Tolerance of T-independent xeno-antibody responses in the hamster-to-rat xenotransplantation model is species-restricted but not tissue-specific.

Control of early acute xenograft rejection xenoreactions in the hamster-to-Lewis rat xenotransplantation model with cyclosporine (CsA) and leflunomide subdues early T-independent xenoreactivity and uncovers a late immune response that can be controlled by CsA alone. We had attributed this acquired responsiveness to CsA to the induction of tolerance of T-independent xeno-antibody responses in the recipient and recently reported that this tolerance is species-specific. Here we have further characterized the specificity and nature of this tolerant state. Lewis rats transplanted with either hearts, skin, kidney or spleen/pancreas from Golden Syrian hamsters were treated with leflunomide (5 mg/kg/day by gavage) for 14-21 days and CsA (20 mg/kg/day by gavage) continuously from the day of transplant. Some Lewis rats received a second graft of hearts or skin from Golden Syrian hamsters (day 21-30 after first transplant), and a third heart graft from Balb/c mice (day 60 after the first transplant). Serum was harvested and the titers of xenoreactive antibodies were quantified by flow cytometry. All grafts were harvested at the end of each experiment and examined by histological and immunohistochemical methods. The combination of CsA and leflunomide was able to completely inhibit the rejection of kidney, spleen and pancreas xenografts in this hamster-to-rat xenotransplantation model. In addition, only a transient treatment with leflunomide was necessary, and long-term graft survival could subsequently be maintained by CsA alone. Histological examination of these grafts at > 80 days post-transplantation indicated minimal signs of rejection. These immediately vascularized organs induced T-independent B-cell tolerance, so that second grafts of hamster hearts and skin could be maintained with CsA alone. Under the same immunosuppressive regimen, only four out of nine Lewis rats exhibited long-term hamster skin survival, probably reflecting the increased immunogenicity of the skin compared with other vascularized grafts. Nonetheless, all rats that did not reject the hamster skin graft also did not reject the hamster heart while on CsA alone. Finally, we demonstrate that the tolerant state could be maintained for up to 30 days in the absence of xenograft. The vigorous T-independent antibody response that mediates acute xenograft rejection in the hamster-to-rat model can be tolerized by the immunosuppressive regimen of CsA and leflunomide. The lack of organ specificity for the induction of this tolerance suggests that the xenoantigens inducing tolerance may be common endothelial cell antigens. Finally, the presence of the xenograft has been previously shown to be critical for the induction of T-independent B-cell tolerance, however, the tolerant state is relatively stable and persists after the removal of the xenograft.

The portosystemic shunt protects liver against ischemic reperfusion injury.

BACKGROUND: The goal of this study was to characterize the importance of splanchnic viscera in liver ischemic reperfusion injury and to enhance the tolerance of liver to warm ischemia injury with portosystemic shunt. METHODS: The hepatic blood flow of male Sprague Dawley rats was subjected to 45, 60, 120, and 150 min liver warm ischemia with or without portosystemic shunt (splenic-caval shunt). The production of tumor necrosis factor a (TNFa), nuclear factor-kappaB activation, inducible NO synthase (iNOS) expression, and apoptosis were examined. RESULTS: A total of 67% of rats with 45 min liver warm ischemia (n=6) and 100% of rats with 60 min liver warm ischemia (n=6) died within 1 day. However, all rats with 120 min (n=8) liver warm ischemia in splenic-caval shunt group survived for over 1 day, 6/8 for over 3 days, and 5/8 for over 5 days without significant histological changes of the liver. Serum tumor necrosis factor levels in liver warm ischemic rats were increased, This increase was significantly reversed after portosystemic shunt. After challenge with lipopolysaccharide (1 mg/kg, p.v.), naive rats survived for over 5 days (n=4) with the peak value of rat tumor necrosis factor (240 pg/ml) at 90 min. In contrast, all rats died within one day (n=5) with the peak value of rat tumor necrosis factor a (465 pg/ml) at 45 min after administration of lipopolysaccharide in the rats with liver warm ischemia plus splenic-caval shunt. iNOS expression and nuclear factor-kappaB activation were very strongly increased in the hepatocytes after liver warm ischemia with portosystemic shunt, compared with liver ischemia without portosytemic shunt. CONCLUSIONS: We conclude that the splanchnic viscera can contribute to liver ischemic reperfusion injury. Portosystemic shunt enhances the tolerance of liver to warm ischemia through the protective role of iNOS and nuclear factor-kappaB (NF-kappaB).

In vitro and in vivo antitumor activity of a novel immunomodulatory drug, leflunomide: mechanisms of action.

Leflunomide, a novel immunomodulatory drug, has two biochemical activities: inhibition of tyrosine phosphorylation and inhibition of pyrimidine nucleotide synthesis. In the present study, we first showed that A77 1726 [N-(4-trifluoromethylphenyl-2-cyano-3-hydroxycrotoamide)], the active metabolite of leflunomide, was more effective at inhibiting the tyrosine kinase activity of platelet-derived growth factor (PDGF) receptor than that of epidermal growth factor (EGF) receptor, and had no effect on the tyrosine kinase activity of the fibroblast growth factor receptor. In the presence of exogenous uridine, A77 1726 was more effective at inhibiting the PDGF-stimulated proliferation of PDGF receptor-overexpressing C6 glioma than the EGF-stimulated proliferation of EGF receptor-overexpressing A431 cells. In vivo studies demonstrated that leflunomide treatment strongly inhibited the growth of the C6 glioma but had only a modest effect on the growth of the A431 tumor. Uridine co-administered with leflunomide did not reverse the antitumor activity of leflunomide on C6 and A431 tumors significantly. Quantitation of nucleotide levels in the tumor tissue revealed that leflunomide treatment significantly reduced pyrimidine nucleotide levels in the fast-growing C6 glioma but had no effect on the relatively slow-growing A431 tumor. Whereas uridine co-administration normalized pyrimidine nucleotide levels, it had minimal effects on the antitumor activity of leflunomide in both tumor models. Immunohistochemical analysis revealed that leflunomide treatment significantly reduced the number of proliferating cell nuclear antigen-positive cells in C6 glioma, and that uridine only partially reversed this inhibition. These results collectively suggest that the in vivo antitumor effect of leflunomide is largely independent of its inhibitory effect on pyrimidine nucleotide synthesis. The possibility that leflunomide exerts its antitumor activity by inhibition of tyrosine phosphorylation or by a yet unidentified mode of action is discussed.

In vivo activity of leflunomide: pharmacokinetic analyses and mechanism of immunosuppression.

BACKGROUND: Leflunomide is an experimental drug with demonstrated ability to prevent and reverse acute allograft and xenograft rejection. The two biochemical activities reported for the active metabolite of leflunomide, A77 1726, are inhibition of tyrosine phosphorylation and inhibition of dihydroorotate dehydrogenase, an enzyme necessary for de novo pyrimidine synthesis. These activities can be distinctly separated in vitro by the use of uridine, which reverses the anti-proliferative effects of A77 1726 caused by inhibition of de novo pyrimidine synthesis. We report the effect of uridine on the in vivo immunosuppressive activities of leflunomide. METHODS: We first quantified the serum levels of A77 1726, the active metabolite of leflunomide, after a single treatment of leflunomide (5, 15, and 35 mg/kg). Additionally, we quantified the levels of serum uridine and of nucleotide triphosphates in the liver, spleen, and lymph nodes of Lewis rats after the administration of a single dose of uridine (500 mg/kg; i.p.). Lewis rats heterotopically transplanted with brown Norway or Golden Syrian hamster hearts were treated for 50 or 75 days with leflunomide (5, 15, and 35 mg/kg/day; gavage) alone or in combination with uridine (500 mg/ kg/day; i.p.). Hematocrits were determined and the levels of alloreactive or xenoreactive immunoglobulin (Ig)M and IgG were determined by flow cytometric analysis. The allograft and xenografts, small bowel, liver, kidney, and spleen were subjected to pathological examination. RESULTS: A linear relationship was observed between the serum A77 1726 concentrations in Lewis rats and the dose of leflunomide administered. Peak A77 1726 concentrations were 20.9, 71.8 and 129.3 mg/l (77.5, 266.1 and 478.8 microM) for the 5, 15, and 35 mg/kg doses of leflunomide, respectively. The concentration of uridine in the serum of normal Lewis rats is 6.5 microM; after i.p. administration of 500 mg/kg uridine, the serum uridine concentrations peaked at 384.1 microM in 15-30 min. The rapid elimination of uridine was not reflected in the lymphoid compartments, and the pharmacokinetics of pyrimidine nucleotides in the spleen resembled that of A77 1726. This dose of uridine, when administered daily (500 mg/kg/day, i.p.), weakly antagonized the immunosuppressive activities of leflunomide (5, 15, and 35 mg/kg/day) in the allotransplantation model. In contrast, in the xenotransplantation model, the same concentration of uridine completely antagonized the immunosuppressive activities of low-dose leflunomide (15 mg/kg/day) and partially antagonized the immunosuppressive activities of high-dose leflunomide (35 mg/kg/day). Toxicities associated with high-dose leflunomide (35 mg/kg/day) were anemia, diarrhea, and pathological changes in the small bowel and liver. These toxicities were significantly reduced by uridine co-administration. CONCLUSION: These studies reveal that the blood levels of A77 1726 in Lewis rats satisfy in vitro requirements for both inhibition of de novo pyrimidine synthesis and protein tyrosine kinase activity. Our data also illustrate that the in vivo mechanism of immunosuppression by leflunomide is complex and is affected by at least the following four factors: type and vigor of the immune response, availability of uridine for salvage by proliferating lymphocytes, species being investigated, and concentration of serum A77 1726.

Inhibition of cytomegalovirus in vitro and in vivo by the experimental immunosuppressive agent leflunomide.

Despite progress in antiviral chemotherapy, cytomegalovirus (CMV) remains a major cause of morbidity and mortality among pharmacologically immunosuppressed transplant recipients, frequently engaging the clinician in a struggle to balance graft preservation with control of CMV disease. Leflunomide, an inhibitor of protein kinase activity and pyrimidine synthesis, is an experimental immunosuppressive agent effective against acute and chronic rejection in animal models. Herein we summarize our recent studies demonstrating that leflunomide inhibits the production of multiple clinical CMV isolates (including multi-drug-resistant virus) in both human fibroblasts and endothelial cells. In contrast to all other anti-CMV drugs currently in use, leflunomide does not inhibit viral DNA synthesis, but rather appears to interfere with virion assembly. Finally, preliminary studies in a rat model suggest that this agent reduces viral load in vivo. These findings imply that leflunomide, an effective immunosuppressive agent, shows potential to concurrently attenuate a major complication of immunosuppression, CMV disease, by a novel mechanism of antiviral activity.

FK506 treatment in combination with leflunomide in hamster-to-rat heart and liver xenograft transplantation.

BACKGROUND: In the experiment described here, we investigated the effects of the immunosuppressants FK506 and leflunomide (Lef) on the survival of hamster hearts and liver xenografts in Lewis rats. METHODS: Lewis rats were used as recipients of hamster heart or liver grafts using different regimens of FK506 and Lef. Donor-matched heart grafts were transplanted into long-term surviving Lewis rat recipients of hamster xenografts to test donor-specific prolongation of xenograft survival. Hyperimmune, late xenograft rejection, and naive sera were transferred into long-term surviving Lewis rat recipients of hamster heart xenografts to determine whether these sera could inhibit the efficacy of donor-specific long-term survival. Anti-donor-specific antibodies were analyzed by flow cytometry. RESULTS: After a short induction with FK506 plus Lef, maintenance treatment with FK506 alone was sufficient to prolong survival of hamster xenografts. All hamster heart and four of six hamster liver xenografts survived for more than 3 months. Second hamster hearts were permanently accepted by Lewis rats bearing long-term surviving hamster heart xenografts when rats were treated with FK506 monotherapy (mean survival time >60 days, n=4). Long-term surviving hamster heart grafts were rejected after transfer of hyperimmune serum but not late xenograft rejection serum or naive serum. Lef and FK506 significantly reduced the production of anti-donor-specific antibodies in Lewis rats transplanted with hamster liver and heart xenografts. CONCLUSION: Long-term survival of hamster liver and heart xenografts in Lewis rats could be induced by a regimen of short-term FK506 in combination with Lef followed by FK506 monotherapy. The acquired sensitivity of late xenoreactivity to FK506 reflects primarily a modification in the host immune response to the hamster graft.

Histological characterization and pharmacological control of chronic rejection in xenogeneic and allogeneic heart transplantation.

BACKGROUND: Chronic allograft rejection remains a major barrier to successful long-term allograft transplantation in humans. Chronic allograft rejection is characterized by the appearance of arterial lesions with concentric intimal thickening. This study investigates the development and control of chronic rejection in hamster cardiac xenografts transplanted into Lewis rats. METHODS: Chronic rejection in the xenograft model involves transplantation of hamster hearts into Lewis rats treated with leflunomide (Lef) continuously at 15 mg/kg/day. The allograft model involves transplantation of Lewis hearts into Fisher-334 rats treated with cyclosporine (CsA) at 2.5 mg/kg for 5 days. RESULTS: The average scores of arterial intimal thickening on day 45 after transplantation were 1.89+/-0.43 in the xenograft and 2.50+/-0.72 in the allograft. The basic pathology of both xenografts and allografts undergoing chronic rejection was arterial intimal thickening comprising smooth muscle cell proliferation, mononuclear cell infiltration, and fibrosis. The majority of cells infiltrating the arterial intima and myocardium were T cells and macrophages. Compared with the allograft, intimal edema, matrix deposition and fibrinoid necrosis were specifically presented in the xenografts and generally involved the larger arteries. The predominant isotype of antibody deposited was IgM in xenografts and IgG in allografts. When combined Lef and CsA therapy was initiated on day 45 after transplantation, the changes of chronic rejection were reversed in both xenografts and allografts by day 90. The scores of intimal thickening were significantly reduced to 0.97+/-0.45 and 1.48+/-0.56, respectively. CONCLUSIONS: We conclude that chronic rejection can be induced in xenografts under conditions of inadequate immunosuppression. Chronic rejection in xenografts involves arterial lesions that bear some histological similarities to, as well as differences from, those observed in chronically rejected allografts. Finally, combination therapy with CsA and Lef reduced the incidence and severity of the intimal lesions in both chronically rejecting xenografts and allografts.

Induction of species-specific host accommodation in the hamster-to-rat xenotransplantation model.

The combination of two immunosuppressants, leflunomide and cyclosporin A (CsA), completely inhibits immune xenoreactions in the hamster-to-Lewis rat xenotransplantation model. In addition, the control of acute xenograft rejection with this combination of immunosuppressants subdues early T-independent xenoreactivity and uncovers a late immune response that can be controlled by CsA alone. We attribute this acquired responsiveness to CsA to a modification in the recipient's humoral response to the xenograft, and refer to this change as host accommodation. Host accommodation can be induced in Lewis rats receiving hamster hearts by the combination of leflunomide and CsA. A 7-day treatment with leflunomide and CsA was able to convert xenoreactivity from one that was resistant to CsA treatment into one that was controlled by CsA. The presence of the hamster xenograft was critical for the induction of host accommodation since the immunosuppressive regimen, either alone or in combination with a transfusion with donor-specific spleen cells, was unable to modify the anti-hamster reactivity in Lewis rats. When accommodation was induced in the presence of hamster hearts, these accommodated rats were able to acutely reject third-party mouse hearts while under CsA therapy, thus indicating that the host accommodation is species specific. Finally, we demonstrate that host accommodation is associated with a loss in the ability to produce species-specific, T-independent xenoantibodies. These novel observations suggest that xenoreactive T-independent humoral responses can be deleted selectively without significant loss of other innate, Ag-specific T-independent humoral responses.

In vitro and in vivo mechanisms of action of the antiproliferative and immunosuppressive agent, brequinar sodium.

Intracellular pyrimidine nucleotides (PyN) can be synthesized de novo from glutamine, CO2, and ATP, or they can be salvaged from preformed pyrimidine nucleosides. The antiproliferative and immunosuppressive activities of brequinar sodium (BQR) are thought to be due to the inhibition of the activity of dihydroorotate dehydrogenase, which results in a suppression of de novo pyrimidine synthesis. Here we describe the effects of the pyrimidine nucleoSide, uridine, on the antiproliferative and immunosuppressive activities of BQR. In vitro reduction of PyN levels in Con A-stimulated T cells and inhibition of cell proliferation by low concentrations of BQR (< or =65 microM) are reversed by uridine. However, uridine is unable to reverse the effects of high concentrations of BQR (> or =65 microM). The ability of BQR to induce anemia in BALB/c mice is prevented by the coadministration of uridine. In contrast, the immunosuppressive activity of BQR is unaffected by similar doses of uridine. PyN levels in the bone marrow, but not in the spleen, are depressed in mice treated with BQR. These observations suggest that the induction of anemia by BQR is due to depletion of intracellular PyN in hemopoietic stem cells located in the bone marrow. They also suggest that the mechanism of immunosuppression by BQR may be only marginally dependent on depletion of intracellular PyN in lymphocytes located in the periphery. We report a novel activity of BQR: inhibition of tyrosine phosphorylation, and hypothesize that the immunosuppressive activity may be due, in part, to this unsuspected ability of BQR to inhibit tyrosine phosphorylation in lymphocytes.

Control of lymphoproliferative and autoimmune disease in MRL-lpr/lpr mice by brequinar sodium: mechanisms of action.

Brequinar sodium (BQR) was originally developed as an antitumor drug and subsequently as an immunosuppressant for controlling transplant rejection. It has been widely accepted that the antitumor and immunosuppressive activities of BQR are dependent on its ability to inhibit the enzymatic activity of dihydroorotate dehydrogenase, the fourth enzyme in the de novo pyrimidine synthesis pathway. Recently, we discovered that BQR has the ability to inhibit protein tyrosine phosphorylation in anti-CD3-stimulated murine T lymphocytes and to inhibit the activity of src-related protein tyrosine kinases, p56lck and p59fyn. We examined the in vivo activities of BQR in MRL-lpr/lpr mice. We report that the dose of BQR (10 mg/kg/day) that induced anemia, controlled lymphadenopathy and inhibited autoantibody production, also selectively reduced the pyrimidine nucleotide levels in the bone marrow and in the lymph nodes. Coadministration of uridine (1000 mg/kg/day) with BQR completely normalized pyrimidine nucleotide levels in the bone marrow and lymph nodes, and prevented BQR-induced anemia. However, coadministration of uridine with BQR only partially reversed the anti-proliferative effects of BQR, and did not antagonize the inhibitory effect of BQR on autoantibody production. Finally, we report that BQR markedly reduced protein tyrosine phosphorylation in lymph nodes of MRL-lpr/lpr mice. These results collectively suggest that the control of lymphadenopathy and autoantibody production in MRL-lpr/lpr mice by BQR is only partially dependent on inhibition of pyrimidine nucleotide synthesis, and suggest a critical role for in vivo inhibition of protein tyrosine phosphorylation.

Quantitation of the changes in splenic architecture during the rejection of cardiac allografts or xenografts.

BACKGROUND: The spleen plays a central role in the generation of both cellular and antibody responses during graft rejection. Although changes in lymphocyte function have been extensively analyzed in vitro, there have been limited attempts at quantitating the structural changes in the lymphoid compartments within the spleen during graft rejection. METHODS: We describe here a means of quantitating the histological changes in the spleen using immunohistochemical techniques and computerized image analysis. RESULTS: Allograft rejection at 6 days after transplant is characterized by a threefold increase in the T cell-rich areas of the periarteriolar lymphoid sheaths (PALs). The follicular areas are enlarged and germinal centers appear in 55% of the white pulp regions. Acute xenograft rejection, 4 days after transplant, is specifically accompanied by a 2.3-fold increase in the marginal zone (MZ) and an increase in the numbers of B cells in the red pulp of the spleen. The expansion of both PALs and follicular/germinal centers during xenograft rejection is comparable to that observed during allograft rejection. We also investigated the effect of two immunosuppressants, leflunomide and cyclosporine, on the spleen of rats with hamster hearts. Leflunomide, which prevents acute xenograft rejection, prevented the increase in PALs and significantly reduced the areas comprising the MZ and follicles. Cyclosporine, which does not alter the tempo of xenograft rejection and only partially inhibited xenospecific antibody production, inhibited the increase in PALs and the appearance of germinal centers, while permitting a modest increase in the area of MZ and follicles. CONCLUSIONS: These observations collectively suggest that both T cell-dependent and T cell-independent responses are stimulated by the transplanted xenograft. However, the T cell-independent responses that initiate xenograft rejection are characterized by very modest increases in the area of MZ and follicles within the white pulp of the spleen.

Effect of anti-CD4 monoclonal antibody combined with human CTLA4Ig on the survival of hamster liver and heart xenografts in Lewis rats.

We investigated the effects of pretransplant anti-CD4 monoclonal antibody (mAb) combined with human (h) CTLA4Ig on the survival of hamster heart and liver xenografts. Pretransplant anti-CD4 mAb (5 mg/kg x 4 days) or hCTLA4Ig (0.5 mg/rat on days 1, 3, and 5 after transplantation) treatment alone prolonged the survival of hamster liver xenografts in Lewis rats (mean survival time [MST]=10.5 days, n=6, and MST=9.0 days, n=5, respectively, compared with untreated Lewis recipients of hamster liver grafts, MST=6.0 days, n=6). The same regimen could not prevent hamster heart xenorejection. Pretransplant anti-CD4 mAb (5 mg/kg x 4 days) combined with hCTLA4Ig (0.5 mg/rat x 4) treatments increased survival of hamster liver xenograft fourfold (MST=24.2 days, n=5). The current results also show that IgG in the sera from Lewis recipients of hamster liver grafts treated with anti-CD4 mAb and hCTLA4Ig was threefold reduced at 6 days after transplantation compared with untreated Lewis rats. These results suggest a synergistic effect of anti-CD4 mAb combined with hCTLA4Ig in a liver xenograft transplantation model.

In vivo mechanism by which leflunomide controls lymphoproliferative and autoimmune disease in MRL/MpJ-lpr/lpr mice.

Two activities have been identified for the immunosuppressive metabolite of leflunomide, A77 1726: inhibition of dihydroorotate dehydrogenase (DHO-DHase), an enzyme involved in the biosynthesis of pyrimidine nucleotides (PyN); and inhibition of protein tyrosine kinases. The in vitro potency of A77 1726 as a DHO-DHase inhibitor is reported to be 10- to 500-fold greater than as a tyrosine kinase inhibitor. These observations suggested that the immunosuppressive efficacy of leflunomide in vivo is related to inhibition of DHO-DHase. However, observations that patients with disorders in the PyN synthetic pathway are not overtly immunodeficient militate against this hypothesis. We investigated the effects of leflunomide in vivo and report that amelioration of lymphoproliferative and autoimmune diseases in MRL/MpJ-lpr/lpr (lpr/lpr) mice by leflunomide is not accompanied by reduced PyN concentrations in lymph node cells. Our hypothesis that lymphocytes could salvage serum uridine to counter the effects of reduced PyN synthesis in vivo was supported by in vitro studies. Finally, we observed that amelioration of disease correlated with a reduction of tyrosine phosphorylated proteins in lymph node cells of lpr/lpr mice. These observations suggest that the primary mechanism by which leflunomide prevents autoimmune and lymphoproliferative diseases in lpr/lpr mice is not depletion of PyN, but correlates with reduced tyrosine phosphorylation concentrations in lymph node cells.

Modification of humoral responses by the combination of leflunomide and cyclosporine in Lewis rats transplanted with hamster hearts.

BACKGROUND: Vigorous antibody-mediated responses prevent the successful engraftment of hamster hearts transplanted into Lewis rats. Early antibody responses mediating acute rejection of the xenograft are T cell-independent and resistant to the T-cell immunosuppressant, cyclosporine (CsA). Immunosuppression with the combination of leflunomide plus CsA completely prevents xenograft rejection, but when such immunosuppression is stopped the hamster heart is rejected by a process that we term late xenograft rejection. We report here on some of the immunological features of late xenograft rejection. METHODS: Lewis rats transplanted with hamster hearts were treated with leflunomide (5 mg/kg/day by gavage) for 14-21 days and CsA (20 mg/kg/day by gavage) continuously from the day of transplant. Serum was harvested and the functional activities of the xenoreactive antibodies were quantitated by in vivo passive transfer of sera, flow cytometry, in vitro C3 deposition assays, and Western blotting. RESULTS: CsA alone prevented late xenograft rejection and the accompanying production of xenoreactive antibodies. The xenoreactive antibodies accompanying acute or late xenograft rejection were predominantly IgM, but only serum from rats undergoing acute xenograft rejection was able to induce hyperacute rejection. The ability of serum to induce hyperacute rejection correlated with its ability to induce C3 deposition on hamster lymphocytes in vitro. The repertoire of hamster antigens recognized by IgM in the serum of rats undergoing late xenograft rejection is more restricted than that of IgM in the serum of rats undergoing acute xenograft rejection. We additionally demonstrate that long-term graft survival is not dependent on graft accommodation. CONCLUSIONS: These studies demonstrate that a brief treatment with the combination of leflunomide and CsA profoundly modifies the humoral xenoreactivity in the recipient, converting it from a T-independent into a T cell-dependent response. Differences in functional activity of sera from acute or late xenograft rejection suggest that antigenic specificity defines the ability of IgM to induce complement activation and hyperacute rejection.