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

Transplant Immunology 1998; 6: 251-259

Inhibition of endothelial receptor expression and of T-cell ligand activity by mycophenolate mofetil Roman A Blahetaa, Kerstin Leckel’, Bianca Wittigb, Dietmar Zenkerc, Elsie Oppermann”, Sebastian Harderd, Martin Scholze, Stephan Webera, Horst Schuldes”, Albrecht Enckea and Bernd H Markusa aDepartment of General Surgety, Johann Wolfgang Goethe- University,Frankfurt am Main, bGerman Cancer Institute, Heidelberg, ‘Georg-Speyer-Haus, Frankfurt am Main, dDepartment of Clinical Pharmacology and eDepan?nent of Medical Virology, Johann Wol&ang Goethe- University,Frankfurt am Main Received 6 October 1998; accepted 15 October 1998

Abstract: The novel immunosuppressive drug mycophenolate mofetil (CelICept”, 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 celhtlar infiltrates in the course of transplant rejection. lb evaluate the therapeutic value of MMF, we investigated to what extent MMF blocks T-lymphocyte infiltration in vitm 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 CD@ T-cell adhesion and penetration by MMF. The &value for CD4+ T-cell adhesion was calculated to be 0.03 pM and the IDss value for CD4+ Tell penetration 1.21 @vI.MMF did not significantly intluence 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 @Al), very late antigen 4 (VLA-4) and PSGL-1 (P-selectin glycoprotein ligand 1) to immobilized intercellular adhesion molecule 1 (ICAM-l), vascular cell adhesion molecule 1 (VCAM-1) and P-selectin was impaired. Moreover, MMF prevented u-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.

Address for correspondence: R Blaheta, J.W. Goethe-UniversityHospital, Department of General Surgery, Transplant-Immunology Laboratory, Building 23 A, EG 7, Theodor-Stem-I&i 7, D-60590 Frankfurt am Main, Germany. E-mail: [email protected] 0 Arnold 1998

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Introduction

Materials and methods

Mycophenolate mofetil (CellCepte, MMF) is a rationally designed immunosuppressive drug which potentially blocks the proliferative activity of allosensitized responder lymphocytes.’ The mode of action of MMF is based on the inhibition of the enzyme inosine monophospbate dehydrogenase (IMDH). IMDH catalyses the conversion of inosine to guanosine monophosphate, which is required for purine synthesis during cell division. However, it is also known that IMDH is additionally involved in the synthesis of membrane glycoproteins, some of which are adhesion receptors.’ Several reports demonstrate the important role of adhesion molecules in the process of recruitment and transendothelial infiltration of activated leucocytes into the transplanted organ?-4 Therefore, MMF could modulate the immune response, not only at the level of cell mitosis, but also at the level of cell infiltration. This bivalent mode of action would be in strong contrast to the one-sided action ‘of the immunosuppressive drugs cyclosporine A or tacrolimus (FK506), which both interfere with the immune system by blocking cell proliferation, but fail to block cell emigration.5 It is important that optimal immunosuppressive therapy should incorporate different aspects of the cellular immune reaction. Therefore, MMF could, at least in part, fuhil the desired multipotent characteristics. However, in order to clearly evaluate the therapeutic value of MMF, its influence on the process of lymphocyte infiltration into donor organs needs exact analysis. The relevant adhesion receptors involved in the regulation of the cellular infiltration cascade are endothelial intercellular adhesion molecule 1 (ICAM-l), vascular cell adhesion molecule 1 (VCAM-1) and/or E-selectin, which interact with their respective lymphocytic ligands leucocyte function associated antigen 1 (LFA-l), very late antigen 4 (VLA-4) or sLeX antigen. Recent publications speculate that endothelial P-selectin might also trigger cell transmigration via its counterpart PSGL-1 (P-selectin glycoprotein ligand 1): Beside receptor-ligand interactions, changes in the intracellular F-actin (filamentous actin) content are necessary to allow lymphocyte movement along the endothelial cell borders (horizontal locomotion).“’

Cell culturea Human umbilical vein endothelial cells (HUVEC) were harvested by enzymatic treatment14 and grown in Medium 199 (Biozol, Munich, Germany), 10% fetal calf serum (Gibco, Karlsruhe, Germany), 10% pooled human serum (Blood Bank of the Red Cross, Frankfurt, Germany), 28 pg/ml endothelial cell growth factor (Boehringer, Mannheim, Germany), 0.1% heparin (Roche, Basel, Switzerland), 100 n&l gentamycin (Gibco) and 2% 1 M HEPES-buffer (Seromed, Berlin, Germany). Peripheral blood lymphocytes were isolated by FicollHypaque centrifugation and resuspended at 1 x lo6 cells/ml in complete HUVEC-medium. The lymphocytes were further separated into CD4+ and CD8+ T-cells on high gradient magnetic columns (MACS CD4/CD8-microbeads; Miltenyi Biotec GmbH, Bergisch Gladbach, Germany). The purity of CD4” or CD8+ T-cell populations was controlled by FACS analysis and was always >95%.

Objective The aim of this study was to investigate to what extent MMF blocks CD4+ and CD8+ Tlymphocyte intiltration with regard to (a) adhesion to endothelial cells, (b) horizontal migration along these cells and (c) penetration through endothelial cells. lb emulate the transplaut situation, an in vitro coculture model was used throughout the study, based upon peripheral blood Tlymphocytes and allogeneic human vascular endotbelial cells (HUVEC). In order to evaluate the cellular mechanisms underlying those putative infiltration-blocking effects of MMR we also investigated (a) the expression of endothelial adhesion receptors ICAM-1, VCAM-1, E-selectin, P-selectin, (b) the binding activity of the respective lymphocytic counter receptors LFA-1, vLA-4, sLeX, PSGL-1 to immobilized IgG fusion proteins, and (c) the expression of cytoskeletal F-actin-filaments.

Monolayer invaeion 8nay Round cover slips were treated with 3-aminopropyl-triethoxysilan (2%; Sigma, Munich, Germany)-acetone solution for 60 min (20°C) to allow firm adhesion of HUVEC and then placed into sir-well multiplates (Falcon Primaria, Be&on Dickinson). HUVEC subcultures were transferred to the prepared multiplates in complete HUVEC-medium. When confluency was reached, 1 x lo6 CD4+ or CD8+ T&Wwell were carefully added to the allogeneic HUVEC monolayer for 60-120 min. In order to investigate effects of MMF on cellular infiltration dynamics the following experiments were carried out: (a) T-cells were pretreated for 12 h with MMF, (b) HUVEC were pretreated for 12 h with MMF, or (c) both T-cells and HUVEC were pretreated for 12 h with MMF (O-100 PM). In some experiments cell cultures were incubated simultaneously with 500 U/ml gamma-interferon (r_IFN; Sigma) or 100 U/ml interleukin 1 (IL-l; Biozol). After 60-120 min incubation (37“C) nonadherent lymphocytes were washed off using warmed (37°C) Medium 199. The remaining cells were fixed with 1% glutaraldehyde (Merck, Darmstadt, Germany). Bound CD4+ or CD8+ T-cells (= adherent and penetrated cells) were counted in five different fields (5 x 0.25 mm2) using a phase contrast microscope (20 x objective) and mean cellular adhesion rate was calculated as follows: total adhesion [%] = n x a x 100/z where n = counted cells/o.25 mm2; u = conversion factor to calculate cell number/total area, and z = T-cells added. A reflection interference contrast microscope with a Ploem apparatus enable separate assessment of the CD4+ or CD8+ Tcells which penetrated the HUVEC monolayer. The relevant theoretical evaluation of reflection contrast microscopy has been described by Bereiter-Hahn et al. and Gingell and Todd?,” The number of T-cells which penetrated the monolayer were counted in five different fields (5 x 0.25 mm’, uhc objective), and the mean penetration rate was calculated as follows: total penetration where a = penetrated cells/area.

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1998; 6: 251-259

[%]= a/b x 100-’

cells/area, b = adherent

+ penetrated

Inhibition

of endothelial

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

Lymphocytemotility away HUVEC subcultures were transferred to six-well multiplates and cultured to co&rent monolayers. Subsequently, 1 x lo6 allogeneic C!D4+ or C!D8+ T-cells were added to each well, together with 2 ml fresh HUVEC medium, according to three different protocols: (a) HUVEC + T-cells in conventional HUVEC medium (control), (b) HUVEC + T-cells, simultaneously incubated with MMF, (c) HUVEC + T-cells, preincubated for 12 h with MMF. After 2 h under standard incubation conditions the multiplates were transferred to a phase contrast microscope (Axiovert 35, Zeiss, Oberkochen, Germany; 20 x objective) and positioned in a heated chamber (37°C). A video camera and monitor were connected to the microscope and the image registered at 24x real time. Five fields of observation were chosen at random in each well, and each field was registered for 10 min. Motility, i.e. horizontal locomotion of lymphocytes along HUVEC were tracked on the monitor and the distance of each track was measured. Migration distance divided by the total time of observation was calculated for each lymphocyte. Factin expreeeion To quantify the F-actin content of lymphocytes, isolated CD4+ or CD@ T-lymphocytes (1 x lO$nl) or isolated HUVEC were transferred to polypropylene tubes (500 p.l/tube). MMF (CnOO PM) was added to the unstimulated cells, or to cells simultaneously stimulated with IL-l (100 U/ml). Lymphocytes were washed and fixed after 12 h and subsequently stained with FITClabelled phalloidin (Sigma; 500 r&ml; 60 min at 4°C). Fluorescence intensity of the cells was then detected using a FACscan (Becton Dickinson; 1 x 104cells/scan). spot-adheeion aeeay ‘Ib investigate the binding capacity of lymphocytes to isolated adhesion proteins in the presence or absence of MMF, chimeric receptor obulins were constructed as has previously been described.“- $3 Bound culture dishes (Becton Dickinson) were then incubated with a spot of goat-anti-human IgG (Sigma; 10 mg/ml in 50 mM ‘B-is,pH 9.5; 90 min at 20’C), and subsequently washed with PBS (Biochrom) and 1% bovine serum albumin (Merck). The dishes were then incubated with 5 pg/ml E-selectin, ICAM-1, VCAM-1 or P-Selectin IgG fusion protein, for 30 minutes at 20°C and subsequently washed with a buffer containing 150 mM NaCl and 2 mM CaClr. CD4+ or CD@ T cells were resuspended at a density of 3 x lo6 cells/ml in complete HUVEC-medium without heparin and were incubated with various concentrations of MMF (O-100 ulb4). The cell suspension was then washed in binding buffer containing 50 mM HEPES (pH 7.4), 100 mM NaCl, 1 mg/ml bovine serum albumin, 2 mM MgQ, 1 mM CaCl*, 3 mM l&C&, 0.02% Na-Azid, and 0.2 mM PMSF and transferred to the culture dishes for 30 min. After 20 min, i.e. 10 min before the end of the incubation time, cells were stimulated with la6 M PMA. Nonadherent cells were then washed off with buffer and the remaining cells were counted using a phase contrast microscope (x20 objective). Five fields of observation were chosen at random in each dish, and the mean value was calculated. Adheeion and penetrationblockade Several reports describe the importance of ICAM-1, E-selectin and VCAM-1 for the lymphocytic infiltration cascade. lb show that T-cell adhesion and penetration in our coculture model is also P-selectin dependent, 0.5 x 106 CD4+ or CD8+ Tcells were Transplant Immunology 1998; 6: 251-259

253

added to allogeneic HUVEC monolayers, pretreated with antiP-selectin monoclonal antibody (clone CLBThromb& 200 pg, 40 CLgor 20 ug protein/ml; control = no anti-P-se&tin pretreatment). In order to induce P-selectin expression on HUVEC, HUVEC were incubated with lo* M prostaglandin &for 10 min (control = no prostaglandin &incubation). T-cell adhesion and penetration rate were calculated after 60 ruin, as described above. Eadothelial membrane receptor moleculee MMF was added to HUVEC, simultaneously stimulated with either IL-l (10 U/ml, to induce E-select& VCAM-1 and ICAM1 expression) or prostaglandin & ( 10d M, to induce P-selectin expression). After various periods of incubation (12 h for Eselectin, VCAM-1, ICAM-1, 10 min for P-selectin), HUVEC were washed in buffer consisting of PBS, 1% bovine serum albumin, 0.1% NaN3 and 0.2% ‘Bveen 20, and were then fixed. For quantitative imm~ostainmg, HUVEC were incubated for 1 h at 4°C with the following primary monoclonal antibodies: antiICAM- (clone BBIg-II), anti-E-selectin (clone BBIg-E6), antiVCAM-1 (clone BBIg-Vt; all Biermann, Bad Nauheim, Germany), or anti-P-selectin (clone CLBThromb/@ Immunotech, Hamburg, Germany). HUVEC were then washed and incubated with FITC-conjugated goat-anti-mouse IgG (Zymed, Munich, Germany) for 30 min at 4°C. Fluorescence analysis was performed using a FACscan. lymphocytic adheeion receptors PSGL-1, VLA-4,

sLeX,andLFA-1 Isolated CD4+ or CDS+ T-cells (1 x 106/ml) were stimulated with IL-l, y-IFN, PMA (lo+ M) or prostaglandin Er. Additionally, the cells were treated with MMF for 10 min, 12, 24, or 48 h. The cells were then washed, fixed and subsequently stained with: l

l

l l

anti CDlla (anti LFA-l)-FITC (clone: 25.3.1, Immunotech, Hamburg); anti CD 15s (anti sLeX)-FITC (clone CSLEXl, Becton Dickinson, Heidelberg); anti CD 49d (anti VLA-4)FI’lC (clone HP2/1, Immunotech); anti CD 162 (anti PSGL-l)-FITC (clone PL2, Immunotech).

Fluorescence intensity (MFU) was detected using a FACscan (1 x 104 cells/scan). lb analyse the receptor distribution on the T-cell membrane in the presence of MMF, HUVEC were transferred to round cover glasses as described above. When confluency was reached, 1 x lo6 CD4+ or CD@ T-cells/ml, treated for 12 h with 1 or 100 uM MMF (control = untreated cells), were added to each well. After 60-120 min, the cell cocultures were washed and subsequently incubated for 60 mm with monoclonal antibodies anti sLeX, anti LFA-1, anti VLA-4, or anti PSGL-1. Indocxbocyanine (Cys’“; Dianova; working dilution: 1:50) conjugated goat-anti-mouse IgG was used as the secondary antibody. To prevent photobleaching of the fluorescent dyes, cover glasses with stained cells were taken out of the wells, the residual liquid was removed, and these were then embedded in an antifade reagent/mounting medium mixture (ProLong” Antifade Kit, MoBiTec, Giittingen, Germany) and mounted on slides. The slides were viewed using a confocal laser scanning microscope (LSM 10; Zeiss, Jena, Germany) with a plan-neofluar x100/1.3 oil immersion objective.

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Evaluationof MMF toxicity MMF toxicity was investigated by morphological analysis following trypan blue dye exclusion, or by quantitative fluorescence analysis of enzyme-catalysed fluorescein-diacetate-metabolism (FDA, Sigma). FDA was dissolved in methanol and diluted in Medium 199. HUVEC or Tcells were incubated under standard conditions with MMF (O-100 uM) for different time periods. The medium was then removed and the cells were washed three times with Medium 199. FDA (1 ug/ml) was then pipetted on to the monolayer. After incubating for 1 h, fluorescence was analysed by a cytofluor scanner (Cytofluor 2300 system, Millipore, Eschbom, Germany; h, = 485 nm, &.,, = 530 nm). StatiStiClB All experiments were carried out from three to six times. Mean values f SD were calculated. A value of 50% inhibition of lymphocyte adhesion, penetration or binding to immobilized adhesion receptors (IDso) by MMF was calculated using the modified Hill equation: effect = (1 - C?j/(ID/

40 -

30 -

+

Adheston

--8-

Penetration

+ c”) x 100

where N describes a slope factor indicating the steepness of the concentration/response curve. Statistical significance was investigated by the Wilcoxon signed rank test showing two-sided probabilities and using normal approximation. Differences were considered statistically significant at a p value less than 0.05. Intra-assay variation of all tests was usually around 10%.

Results hfluameof MMF on Tcell infbation The upper right-hand corner of Figure 1 shows adhesion and penetration kinetics of allogeneic PBL acting on unstimulated HUVEC (control). Analysis of the lymphocyte subpopulations revealed: CD4+ cells: 70%, CD8+ cells: 2-S%, CD16+ cells: cl%, CD22+ cells: S-10%.14 The number of adhering PBL increased after 1 h and reached a plateau after 4 h. At this time 32.1 f 4.1% of all lymphocytes added were attached. Less than 10% of these (mean: 6.5 & 1.9%) penetrated the HUVEC monolayer. No difference could be found between the adhesion and penetration capacity of isolated CD4+ and CD8+ T-cells. After 4 h the adhesion rates were 36.8 + 6.8% (CD4+ cells) and 35.1 + 4.2% (CD8+ cells), penetration rates were 5.7 -C 2.3% (CD4+ cells) and 7.1 + 2.8% (CD8+ cells). The addition of MMF to the cell cultures resulted in a dosedependent reduction of the amount of adherent and penetrated CD4+ or CD8+ T-cells. The infiltration blocking effect of MMF was most pronounced when both HUVEC and T-cells were incubated with this compound (Figure 1, representative for CD4+ T-cells). The IDHl value for CD4+ T-cell adhesion was calculated to be 0.03 PM, IDsovalue for CD4+ T-cell penetration 1.21 uM. MMF did not significantly influence the horizontal migration of T-lymphocytes along the HUVEC cell borders (cell motility, Figure 2a). Additionally, intracellular F-actin filaments were not significantly impaired by MMF (Figure 2b). lnffuence of MMF on endothelial adhesion receptors

P-selectin is strongly involved in the process of T-cell infiltration. Blocking of the endothelial P-selectin receptor by monoclonal antibodies potently prevented T-cell binding to HUVEC (Figure 3, upper right-hand corner, representative for CD4+ T Transplant Immunology 1998; 6: 251-259

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absence (control) or presence of MMF.Nonadherent cells were washed off after 60-120 min according to the protocol described in ‘Materials and methods’. Adherent cells were counted using phase contrast optics and penetrating cells were visualized and axmted using rekction interference contrast optics. Control values were set to be 100%. The upper right-hand comer of the figure shows the kinetica of lymphocyte adhesion to and penetration through allogeneic endothelial cells. Lymphocytes were incubated with allogeneic HUEC for up to 12 h and nonadherent PBL were washed off accorclmgto the standard protocol. Each point represents the mean 2 SD of four experiments.

cells). The incubation of prostaglandin & activated HUVEC with MMF led to a distinct and dose-dependent suppression of this receptor on the cellular membrane (Figure 3). The optimum concentration was 10 pM (ID50 = 0.197 uM). The same concentration also blocked membrane E-selectin expression (IDsc = 0.008 PM), but did not influence ICAM- and VCAM-1 expression (Figure 4). Influence of MMF on lymphocytic ligmuds

Pilot experiments demonstrated that the lymphocytic adhesion receptors LFA-1, sLeX, VLA-4 and PSGL-1 cannot be up- or down-regulated by the cytokines IL-l or g-IFN. However, a 48 h incubation of T-cells with prostaglandin lZc significantly reduced LFA-1 and enhanced VLA-4 and sLeX. In contrast, a 48 h incubation of TcelIs with PMA reduced m-4, sLeX and PSGL-1 and enhanced LFA-I on the cell membrane (Figure 5a-d). The additional incubation of the cell cultures with MMF prevented the receptor regulation only when applied in high concentrations (> 10 l&I). However, MMF dose-dependently diminished the binding activity of CD4+ or CD8+ T-cells to immobilixed endothelial adhesion receptors in the following order: P-selectin (via PSGL-1) > VCAM-1 (via VLA-4) > ICAM- (via LFA-1) > E-se&tin (via sLeX). ID~avalue was calculated to be 0.928 pM for the PSGL-1-P-selectin interaction and 0.308 uM for the VCAM-1-m-l interaction (Figure 4).

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

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80

In the presence of MMF confocal laser-scan analysis revealed a large loss of lymphocytic PSGL-1 and VIA-4 capping in the coculture invasion model (Figure 6a-d).

70 MlUF toxicity The MMF concentrations used in the study were not toxic to either HUVEC or T-lymphocytes.

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F&me 3 Ekpression of P-selectin on HUVEC membranes. The expression of P-selectin on HUVEC under the h&tenor of MMF was investigated by quantitative imrnunofluorescence analysis according to the protocol given in (‘Materials and methods’). For maximal P-selectin expression, HUVEC were stimulated with prostaglandin Q ( lo4 M) for 10 min. Control value (stimulation without addition of MMF) was defined in the x-axis to be 1, e-3. The upper right-hand comer of the field shows blocking of T-cell adhesion to HUVEC by anti-P-selectin monoclonal antibodies. T-cells were added to allogeneic HUVEC monolayers, pretreated with anti-P-&e&r monoclonal antibody (200 pg, [200], 40 ug, [40], or 20 pg protein/ml [20]; control = no anti-P-selectin pretreatment). In order to induce P-selectin expression, HUVFKJwere incubated with lo4 M prostaglandin & for 10 min (control [ctr.] = no prostaglandin & incubation). T-cell adhesion rate is given in cells/mm* (mean + SD of three experiments).

Discussion

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MMF[WI (a) Horizontal locomotion (motility) of Wyrnphocytes in the presence of MMP. The motility of Tcells was measured videomicroscopicahy by registration of the track lengths of migrating CD4+ or CD8+ T-cells on allogeneic HUWC and division by the time of registration (velocity; pm/min). Experiments were performed six times. In Flgmx 2

each experiment tIve fields of observation were chosen and each field was registered for 10 min. (b) Quantitication of F-actin in T-lymphocytes and I-IUVEC. The amount of F-actin inside CD4+ or CDS+ Tkxlls or HUVEC under the influence of MMF was measured by quantitative immunofluorescence following incubation of cells with FITC-phalloidin (RFu= relative fluorescence unit). Each point represents the mean f SD of three experiments.

Tmnspht

Immunology 1998; 6: 251-259

MMF is a potent reversible inhibitor of the enzyme IMDH which catalyses the wnversion of inosine to guanosine monophosphate, required for purine synthesis during cell division. Since T- and B-lymphocytes preferentiahy use the de nova pathway of purine synthesis, these cells are particularIy sensitive to the inhibitory action of MMF.‘5 Our results demonstrate that MMF also suppresses the adhesion and penetration of CD4+ and CD8+ Tcells through allogeneic endothelium in an in vitro invasion model. In good accordance, MMF has been shown to decrease T-cell attachment to IL-l stimulated endothelial ce1ls.r Furthermore, MMF reduces the leucocytic intihrate hi acute rejection of rat kidney allografts. l6 In contrast to the anti-mitotic effects of this compound, MMF acts on both T-cells and HUVEC. MMF inthrences the endothelial adhesion receptors E-sclectin and P-selectin, as well as the lymphocytic ligands LFA-1, VLA-4 and PSGL-1. Several reports reveal that E-selectiu acts as an addressin for T+mphocytes.‘7*‘8 Our experiments show strong binding of CD4+ and CD8+ T-cells, not only to immobilized E-selectin, but also to immobilized Pselectin.‘9’~ Blocking of these receptors by monoclonal antibodies strongly reduced the cellular interaction with the respective ligands (Figure 3).20 Therefore, MMF-evoked

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down-regulation of specific endothelial membrane molecules might be one mode of action responsible for diminished T-cell adhesion and penetration. Concerning the lymphocytic receptors, it has been speculated that MMF-mediated guanosine monophosphate and triphosphate depletion decreases glycosylation of VL4-4, in decreased lymphocyte binding to endothelial resultin celIs.lY’sgOur studies showed that MMF, given in concentrations below 10 nM, did not interfere with the PMA or prostaglandin Q-evoked quantitative changes of VIA-4 expression. This implies that either the process of glycoprotein synthesis is not impaired by MMF or guanosine triphosphate is not depleted sufficiently by MMF to induce a significant down-regulation of the VLA-4 receptor. It should also be mentioned in this context that T-cell binding to immobilixed E-selectin via the sLeX ligand is not influenced by MMF, although sLeX also requires glycosylation to become activated. The data shown in Figure 4 indicate that the main mechanism of MMF action on lymphocyte infiltration is a reduced binding capacity of VLA-4, LFA-1 and PSGL-1 to their counterparts VCAM-1, ICAM- and P-selectin. Details about activation and turnover of the lymphocytic membrane proteins are not known. However, it is hypothesized that rapid changes in the chemical configuration or assembly of these membrane proteins have to occur to initiate a high binding activity and firm contact of T-cells to the endothelium.21*22 Lymphocytes typically each

extend one pseudopodium after binding to HUVEC.‘4 Our confocal images revealed an accumulation of VIA-4, LFA-1 and PSGL-1 molecules on the membrane of the pseudopodium. This receptor localization seems to be a necessary prerequisite to direct cell movement.” Interestingly, in presence of MMF, the accumulation of VLA-4 and PSGL-1 disappeared. Therefore, one possible explanation for the distinct effects of MMF on the lymphocytic binding activity might be the loss of protein localization in the protrusions of these cells. Because LFA-1 capping was not inlIuenced by MMF, effects of this drug on the chemical configuration of T-cell receptors should also be taken into account. It can be concluded that MMF possesses potent infiltration inhibiting properties. Concerning the desired dualistic modulation of the immune system, this compound seems a good support for the current immunosuppressive protocol. Further studies are now underway to investigate possible effects of MMF on other infiltration-dependent processes. Acknowledgements

We thank R Schrijder and K Wilhelm for their excellent technical assistance. We would also like to thank Mrs K Nelson for critically reading the manuscript. This work was supported by the Messer Stiftung, the Paul und Ursula Klein Stiftung, the foundation Hilfe ftir krebskranke Kinder e.V and Verein Hilfe fir krebskranke Kinder e.V

Flgmrc4 Inhence of MMP (10 PM) on endothelial and lymphocytic adhesion receptors. The figure indicates %inhibition of endothelial ICAM-1, VCAM-1, E-selectin and P-selectin expression and of the binding activity of iymphocytic LPA-1, U-4, sL.eX and PSGL-1 ligands. Control values were set to be 100%. IDx values are partially included above the columns. Each column represents the mean 2 SD of three experiments.

Transplant Immunology 1998; 6: 251-259

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Figure 5 Quantification of the lymphocytic adhesion receptors VLA-4, sLeX, PSGL-1 and LFA-1 (A-D, representative for CD4+ T-cells). Isolated T-cells were stimulated with IL-l (100 U/ml), g-IF’N (g-IFN; 500 U/ml), PMA (lo+ M) or prostaglandin I$ (l@ M) with or without (control) MMF (concentration > 10 pM)_ Receptor expression (%) were measured by quantitative immunofluorescence. The unstimulated controls were set to be 100%. Each point represents the mean f SD of three experiments. Transplant Immunology 1998; 6: 251-259

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Figure 6 Analysis of PSGL-1 and VLA-4 distribution in lymphocytes before and after MMF treatment. Control experiments revealed an accumulation of PSGL-1 (a) and VLA-4 (c) in the region of the anterior pseudopodium of a lymphocyte (representative: CD4+ T-cells). Treatment with MMF reduced the PSGL-1 capping (MMF concentration: 1 pM, b) and VLA-4 capping (MMF concentration: 100 uhf, d). Scale bar: 10 urn.

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