Kerstin Leckel

Inhibition of endothelial receptor expression and of T-cell ligand activity by mycophenolate mofetil

The novel immunosuppressive drug mycophenolate mofetil (CellCept, MMF) blocks DNA-synthesis by the inhibition of the enzyme inosine monophosphate dehydrogenase (IMDH). IMDH is also involved in the synthesis of adhesion receptors which are known to play an important role in the regulation of cell-cell contacts. Therefore, application of MMF might lead to a reduction of cellular infiltrates in the course of transplant rejection. To evaluate the therapeutic value of MMF, we investigated to what extent MMF blocks T-lymphocyte infiltration in vitro with regard to (a) adhesion to endothelial cells, (b) horizontal migration along these cells and (c) penetration through the endothelial cells. The results demonstrated a strong inhibition of both CD4+ and CD8+ T-cell adhesion and penetration by MMF. The ID50 value for CD4+ T-cell adhesion was calculated to be 0.03 microM and the ID50 value for CD4+ T-cell penetration 1.21 microM. MMF did not significantly influence the horizontal migration of T-lymphocytes along the human vascular endothelial cell (HUVEC) borders. FACS-analysis revealed a diminished E-selectin and P-selectin expression on endothelial cell membranes in the presence of MMF. Although MMF did not interfere with the synthesis of T-cell adhesion ligands, the binding activity of lymphocytic leucocyte function associated antigen 1 (LFA-1), very late antigen 4 (VLA-4) and PSGL-1 (P-selectin glycoprotein ligand 1) to immobilized intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1) and P-selectin was impaired. Moreover, MMF prevented VLA-4 and PSGL-1 receptor accumulation on the membranes of T-cell pseudopodia. It can be concluded that MMF possesses potent infiltration blocking properties. MMF evoked down-regulation of specific endothelial membrane molecules and the loss of protein localization in the lymphocyte protrusions might be predominantly responsible for the observed blockade of cell adhesion and penetration.

In vitro analysis of verapamil-induced immunosuppression: Potent inhibition of T cell motility and lymphocytic transmigration through allogeneic endothelial cells

BACKGROUND Cyclosporine A (CsA) and tacrolimus prevent proliferation but not transendothelial migration of alloreactive lymphocytes into donor organs. As a result, serious adverse effects, such as nephrotoxicity and neurotoxicity, have been observed under CsA/tacrolimus therapy. The incorporation of new drugs with infiltration blocking properties might enhance the efficacy of the current immunosuppressive protocol, allowing lower CsA/tacrolimus dosage. Because Ca2+ plays a critical role in cell-cell interaction, the Ca2+-channel blocker verapamil might be a good cany. didate for supporting CsA/tacrolimus-based therapy. METHODS A T-cell endothelial cell coculture model or immobilized immunoglobulin G globulin chimeras were employed to investigate how S- and R- verapamil interfere with the lymphocytic infiltration process. The expression and arrangement of membranous adhesion receptors and cytoskeletal F-actin filaments were analyzed by fluorometric method in the presence of. verapamil. RESULTS Both verapamil enantiomers strongly inhibited lymphocyte infiltration. CD4+ and CD8+ T-cells were influenced to a similar extent with regard to horizontal locomotion (CD4+=CD8+), but to a different extent with regard to adhesion and penetration (CD4+ > CD8+). Moreover, penetration was blocked to a higher extent than was adhesion. ID50-values were 31 microM (CD4+-adhesion) and 11 microM (CD4+-penetration). Verapamil reduced P-selectin expression on endothelial cells and effectively down-regulated binding of T-cells to immobilized P-selectin immunoglobulin G globulins (ID50=4.4 microM; CD4+). A verapamil-induced reduction of intracellular F-actin in T-lymphocytes was proven to be mainly responsible for diminished cell locomotion. CONCLUSIONS The prevention of CD4+ T-cell penetration by verapamil might argue for its use as an adjunct to CsA/tacrolimus-based immunosuppressive therapy.

Mycophenolate mofetil increases adhesion capacity of tumor cells in vitro.

Background. The immunosuppressive drug mycophenolate mofetil (MMF) reduces expression of the heterophilic binding elements intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 and thereby prevents attachment of alloactivated leukocytes to donor endothelium. The authors speculated that MMF might further diminish receptors of the immunoglobulin superfamily which, however, act as homophilic binding elements. Because decrease of homophilic adhesion receptors correlates with tumor dissemination and metastasis, MMF could trigger development or recurrence of neoplastic tumors. Methods. The authors analyzed the influence of MMF on homotypic adhesion receptors and its consequence for tumor cell attachment to an endothelial cell monolayer. Neuroblastoma (NB) cells, which self-aggregate by means of the homophilic-binding element neural cell adhesion molecule (NCAM), were used. Effects of MMF on the 140- and 180-kDa NCAM isoforms were investigated quantitatively by flow cytometry, Western blot, and reverse-transcriptase (RT) polymerase chain reaction (PCR). The relevance of NCAM for tumor cell binding was proven by treating NB with NCAM antisense oligonucleotides. Results. MMF profoundly increased the number of adherent NB cells, with a maximum effect at 0.1 &mgr;M, compared with controls. Decrease of NCAM on the cell surface was detected by flow cytometry. Western blot and RT-PCR demonstrated reduced protein and RNA levels of the 140- and 180-kDa isoforms. Treatment of NB cells with NCAM antisense oligonucleotides showed that reduced NCAM expression leads to enhanced tumor cell adhesion. Conclusions. MMF decreases NCAM receptors, which is associated with enhanced tumor cell invasiveness. The authors conclude that an MMF-based immunosuppressive regimen might increase the risk of tumor metastasis if this process is predominantly conveyed by means of homophilic adhesion proteins.

Mycophenolate mofetil decreases endothelial prostaglandin E2 in response to allogeneic T cells or cytokines

BACKGROUND Prostaglandin E2 (PGE2) is a powerful endogenous immune suppressant and interferes with various T-cell functions. However, it is not known in detail whether immunosuppressive drugs influence the PGE2-driven immune response in transplant patients. Therefore, we investigated the effect of several immunosuppressive compounds, in particular the novel drug mycophenolate mofetil (MMF), on endothelial PGE2 release. METHODS Endothelial cells (HUVEC) were activated by either allogeneic CD4+ or CD8+ T cells, or by the cytokines interleukin-1 or gamma-interferon. Using an enzyme-linked immunosorbent assay, we analyzed PGE2 release of the activated HWEC in the presence of MMF, cyclosporine, or tacrolimus. As verapamil and mibefradil also possess immunosuppressive properties, they were included in the study as well. RESULTS Activation of HUVEC with interleukin-1 or T cells resulted in a drastic accumulation of PGE2 in the supernatant. Cyclosporine or tacrolimus had no effect on PGE2 release. However, Ca2+ channel blockers, when applied at higher dosages, caused a significant increase in PGE2. Interestingly, MMF strongly diminished the PGE2 level in the cell culture supernatant in a concentration-dependent manner. CONCLUSION The results demonstrate an inhibitory effect of MMF on PGE2 production, which may lower the benefits of the PGE2-triggered immune response after organ transplantation.

Tacrolimus, daclizumab, sirolimus, and budesonide after small bowel transplantation in order to reduce nephrotoxicity

An isolated intestinal allograft transplant was performed in September 2000 in a patient with a history of ischemic necrosis of the whole small bowel including the ascending and transverse colon plus left kidney after an aortic dissection (Stanford B). The donor was a cadaveric source who was ABO identical with a random HLA match. The patient was not preimmunized. No attempts were made to alter the graft immunologenicity. Whole jejunum and ileum were transplanted anastomosing the superior mesenteric artery to the aorta and the portal vein to the vena cava. Lymphovenous anastomosis of a lymph node to the hypogastric vein was performed. Immunosuppression consisted of tacrolimus, given intravenously for 3 days and then orally. Blood levels 12 hours after the last dose were maintained at 15 to 20 ng/mL for the first 2 months and between 10 and 15 for the next 6 months. Intraoperatively, daclizumab, a monoclonal antibody against the interleukin-2 receptor, was administered at a dose of 1 mg/kg in addition to prednisolone at a dose of 500 mg intravenously (IV). Four further doses of IV daclizumab were delivered every weeks. Prednisolone doses were tapered to 20 mg/day on day 5 post-SBTx and stopped at 6 months. In addition, topical budesonide 9 mg/d was administered initially through the enterostoma and from the third week as an oral medication. The initial creatinine clearance value was 30 mL/min and blood creatinine concentration 1.8 mg%, leading to consideration of addition of add immunosuppression with MMF in order to reduce the dose of tacrolimus. However, in spite of 3 g/d MMF, the blood concentrations of MMF remained low. Therefore, MMF was discontinued and 2 mg/d sirolimus was started. The ileostomy was closed 10 months after the transplant operation. At that time, renal function had gradually deteriorated, particularly when amphotericin B treatment had to be given due to candidae colonization of the bowel. A further effort to spare calcineurin inhibitory agents was made by reducing the tacrolimus dose and reintroducing MMF. Protocol biopsies were performed, weekly for the first month after transplantation and thereafter only when clinically indicated. Immunoprophylaxis with IgG against CMV and ganciclovir was given for the first 3 months and CMV and EBV surveillance performed thereafter.

In vitro Studien zum Wachstumspotential und Differenzierungsgrad kleiner („small“) humaner Hepatozyten

Transplantation of isolated hepatocytes might be an alternative to orthotopic liver transplantation. However, a cell culture system has to be established which requires a sufficient amount of hepatocytes with stable liver-specific function. In the present study, an in vitro cell culture system has been optimized which allows the induction of cell mitosis of highly differentiated hepatocytes. Hepatocytes were derived from human liver tissue with a high pressure 2 step isolation method and grown on a two-dimensional collagen matrix. Expression and activity of growth receptors HGF-r and EGF-r and amount of CK18, CK19, albumin, factor VIII and fibrinogen were investigated by Facs or Western blot. BrdU-incorporation was evaluated fluorometrically. Interestingly, cultivation of human hepatocytes without medium renewal induced growth of cell islands, consisting of small heoatocytes (single nucleus, more granulated than adult human hepatocytes). Furthermore, albumin, factor VIII and fibrinogen were expressed at a high level. Maximal DNA-synthesis was measured on day 5 (15% of cells were Brdu positive). HGF-r and EGFr showed maximal peaks on days 5 and 9. In parallel, CK18-expression and de novo synthesis of CK19 were enhanced. We postulate that a high pressure isolation technique necessary to obtain hepatocytes with high growth potential. In addition, enrichment of soluble mediators in the cell culture supernatant might provide specific triggering factors responsible for stabilizing cell differentiation.

Die Bindung gastrointestinaler Tumorzellen an endotheliales E- oder P-Selektin induziert eine transiente Reduktion der sLeX Liganden in vitro

Sialyl Lewis X (sLeX) expression was analyzed in several gastrointestinal tumor cell lines and related to the ability of the tumor cells to adhere to endothelial cells in vitro. sLeX expression kinetics during the phase of tumor cell-endothelial cell interaction was also evaluated using a dynamic coculture invasion assay. The tumor cells differentially adhered to endothelial cells. However, although anti-sLeX monoclonal antibodies significantly reduced adhesion of the tumor cells to endothelium and prevented tumor cell binding to immobilized E-selectin, quantification of sLeX demonstrated an inverse correlation between sLeX expression level and adhesion capacity of the tumor cells. Most surprisingly, sLeX was downregulated in the course of heterophilic cell-cell contacts. The process occured transiently, with a maximum effect 30–60 min after the experimental onset. Binding of tumor cells to immobilized E- and P-selectin IgG globulin chimeras was shown to be responsible for this phenomenon. We conclude from our studies that the transient loss of sLeX is necessary to allow gastrointestinal tumor cells to procede in the transendothelial invasion cascade.

In-vitro Analysen zum klinischen Einsatz isolierter humaner Hepatozyten

Introduction: As an alternative to orthotopic liver transplantation, hepatocyte transplantation could be an attractive tool in the treatment of severe liver diseases. However, to allow clinical use, a cell culture system has to be established which guarantees a sufficient amount of hepatocytes with stable liver-specific function. The present study investigates to what extent isolated human hepatocytes can be triggered towards dedifferentiation — a prerequisite for the induction of cell mitosis or differentiation — necessary for physiological activity, in a controlled manner. Material and Methods: Hepatocytes were derived from human liver tissue and grown either on a two-dimensional or in a three-dimensional collagen matrix. Cellular differentiation status was influenced by two different media, which were added to the hepatocytes either separately or alternately. The differentiation medium was based on DMEM and enriched with hormones and human serum. The dedifferentiation medium consisted mainly of DMEM/F12, supplemented with growth factors EGF/HGF. Hepatocyte growth factor receptor (HGF-r), epidermal growth factor-receptor (EGF-r), as well as the intermediate filaments CK18 and CK19 served as differentiation markers. Expression was analyzed by Western blot analysis, FACS measurement and laser-scan microscopy. Results: Human hepatocytes cultured in differentiation medium stably expressed EGF-r and HGF-r. CK18 was detected constantly at a low level, whereas CK19 was not visualized before day 10. In contrast, hepatocytes cultured in dedifferentiation medium showed a significant upregulation of HGF-r and CK18 and the novo CK19 synthesis. This phenomenon was more pronounced when the cells were cultured on a two-dimensional matrix compared to the threedimensional matrix. Discussion: The external modulation of human hepatocytes allows the controlled triggering of the cellular differentiation status. Further studies will investigate to what extent the dedifferentiation status is associated with enhanced mitotic activity.