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Integrins are transmembrane proteins regulating cellular shape, mobility and the cell cycle. A highly conserved signature motif in the cytoplasmic tail of the integrin alpha-subunit, KXGFFKR, plays a critical role in regulating integrin function. To date, six proteins have been identified that target this motif of the platelet-specific integrin alpha(IIb)beta(3). We employ peptide-affinity chromatography followed-up with LC-MS/MS analysis as well as protein chips to identify new potential regulators of integrin function in platelets and put them into their biological context using information from protein:protein interaction (PPI) databases. Totally, 44 platelet proteins bind with high affinity to an immobilized LAMWKVGFFKR-peptide. Of these, seven have been reported in the PPI literature as interactors with integrin alpha-subunits. 68 recombinant human proteins expressed on the protein chip specifically bind with high affinity to biotin-tagged alpha-integrin cytoplasmic peptides. Two of these proteins are also identified in the peptide-affinity experiments, one is also found in the PPI databases and a further one is present in the data to all three approaches. Finally, novel short linear interaction motifs are common to a number of proteins identified.

A synthetic cell-permeable peptide corresponding to the highly conserved alpha-integrin signature motif, Palmityl-K(989)VGFFKR(995) (Pal-FF), induces integrin activation and aggregation in human platelets. Systematic replacement of the F(992)-F(993) with amino acids of greater or lesser hydrophobicity to create Pal-KVGxxKR peptides demonstrate that hydrophobic amino acids (isoleucine, phenylalanine, tyrosine, tryptophan) are essential for agonist potency. In marked contrast, substitution with small and/or hydrophilic amino acids (glycine, alanine, serine) causes a switch in the biological activity resulting in inhibition of platelet aggregation, adhesion, ADP secretion, and thromboxane synthesis. These substituted, hydrophilic peptides are not true pharmacological antagonists, as they actively induce a phosphotyrosine signaling cascade in platelets. Singly substituted peptides (Pal-AF and Pal-FA) cause preferential retention of pro- or anti-thrombotic properties, respectively. Because the alpha-integrin signature motif is an established docking site for a number of diverse cytoplasmic proteins, we conclude that eliminating critical protein-protein interactions mediated through the hydrophobic amino acids, especially F(993), favors an anti-thrombotic pathway in platelets. Agents derived from the inhibitory peptides described in this study may represent a new therapeutic strategy for anti-platelet or anti-integrin drug development.

The paper describes a method for the separation of 18 nucleotides, nucleosides and nucleobases by isocratic solvent-generated ion-pair chromatography in 55 min. It has been developed for pharmacodynamic monitoring of mycophenolic acid, the active metabolite of the immunosuppressant mycophenolate mofetil. The method was applied for the detection of mycophenolic acid-induced changes in inosine 5′-monophosphate dehydrogenase (IMPDH) activity in the lysate of human peripheral blood mononuclear cells.

Background : Mycophenolic acid selectively inhibits inosine 5′-monophosphate dehydrogenase leading to a shortage of guanosine nucleotides. Since GTP is required for the synthesis of glycoproteins, this immunosuppressive drug also influences the production of several cell adhesion molecules. Method : Soluble endothelial cell adhesion molecules released into cell culture supernatants after an incubation period of 16 h are assessed via a standard ELISA procedure applying test kits for E-selectin, VCAM-1 and ICAM-1. Results : Treatment with TNF-α leads to the induction of E-selectin and causes a significant increase in VCAM-1 and ICAM-1 content in the supernatant in relation to the level of unstimulated cells. Due to the inhibitory effects of MPA-applied either alone or in combination with cyclosporin A and prednisolone-sE-selectin is significantly reduced and sVCAM-1 is slightly but not significantly decreased, whereas sICAM-1 levels remain unchanged. Conclusions : We demonstrate that the influence of MPA on endothelial cell adhesion molecules can readily be determined via ELISA. The results indicate that the immunosuppression by MPA is also achieved by slightly reducing the expression and consequent release of E-selectin, a pivotal molecule in the first step of leucocyte–endothelial interactions.

Background: Inosine 5′-monophosphate dehydrogenase (IMPDH) catalyses the oxidation of inosine 5′-monophosphate (IMP) to xanthosine 5′-monophosphate (XMP). Thus, this enzyme plays an important role in the rate-limiting step of the de novo guanine nucleotide biosynthesis, making it a potent target for immunosuppressive drugs. Mycophenolic acid (MPA) is the most potent and specific inhibitor of IMPDH. Method: IMPDH activity is determined via evaluation of XMP formation and the inhibitory influence of MPA in human peripheral blood mononuclear cells (PBMCs) is assessed by means of high-performance liquid chromatography (HPLC). For this objective, we have optimised a method based on solvent-generated ion exchange chromatography by cautiously varying mobile phase parameters. Results: The optimised method renders it possible to separate 18 analytes in 54 min in a single isocratic experiment and to measure the IMPDH activity in the lysate of human PBMCs in dependence on incubation time, substrate, co-substrate and inhibitor concentrations. In this way, we have determined the Michaelis–Menten constants KM and Vmax for IMP and β-NAD+ and the inhibitor constant Ki for MPA. Conclusions: The chromatographic method presented in this report allows a rapid, reliable and reproducible quantification of IMPDH activity in PBMCs and therefore represents an attractive tool for the pharmacodynamic monitoring of the effects of MPA in patients under immunosuppressive therapy.

Background: Among the different factors playing crucial roles in endothelial cell activation, cytokines such as interleukin-1 (IL-1), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ) have been reported to demonstrate profound effects on this cell type. It has been shown that the increased release of IFN-α/γ and TNF-α causes structural and functional modulations of the endothelial cell. These cytokines participate in the recruitment and activation of the immune system. CsA is an immunosuppressive drug that is necessary at high levels in human recipients of vascularised xenografts. This drug could contribute to a prolonged graft survival by modulation of endothelial cell activation. Methods: The present study deals with the effects of cyclosporin A on adhesion molecule expression (i.e. ICAM-1, VCAM-1, E-selectin, P-selectin, PECAM-1 and the L-selectin ligand CD 34) on the surface of cytokine stimulated HUVECs. The in vitro model described herein mimics the stimulation of endothelial cells by cytokines as seen during inflammatory processes after transplantation. Therefore, HUVECs were activated either with TNF-α, IL-1β or with a cytokine mixture consisting of those stimulants present at an elevated level in sera of patients during allograft rejection (i.e. IL-1β, IL-2, IL-4, IL-6, IL-10, TNF-α and IFN-γ). Results: The results obtained show that the immunosuppression of CsA is not only achieved by inhibiting lymphocyte proliferation, but also by decreasing the expression of adhesion molecules on endothelial cells, which are the first target of the cellular rejection process. Conclusion: Co-incubation of stimulated endothelial cells with a final CsA concentration of 5 μg/ml revealed a significant down-regulating influence on the surface expression of E-selectin and VCAM-1.

The immunosuppressant mycophenolic acid (MPA) selectively inhibits proliferation of T- and B-lymphocytes by blocking inosine 5'-monophosphate-dehydrogenase (IMPDH), the key enzyme for de-novo-synthesis of guanine nucleotides. In an in vitro study the effects of MPA on human peripheral blood lymphocyte activation markers and on cell cycle characteristics were investigated. Mononuclear cells from healthy volunteers were incubated with phytohaemagglutinin (PHA) and increasing doses of MPA. After 72 h incubation an aliquot of the cells was stained with propidium iodide and measured by FACS analyses to assess the DNA shape. In addition, the expression of the activation markers HLA-DR and CD25 on T- and B-lymphocytes was determined by flow cytometry analysis.PHA stimulation led to a significant increase of the S-phase of cell cycle. PHA stimulation clearly increased mean fluorescence intensity (MFI) of HLA-DR expression on B-lymphocytes. PHA stimulation also elevated the number of CD25 positive B-lymphocytes. Expression of HLA-DR on T-lymphocytes was not influenced by PHA, whereas CD25 expression and MFI significantly increased. All the observed PHA induced effects were reduced by co-incubation with increasing doses of MPA. The data presented show that in vitro the immunosuppressive effect of MPA can be demonstrated using FACS technology on a cellular level. MPA leads to an inhibition of cell cycle proliferation in peripheral blood lymphocytes.

The capacity of platelets to form a thrombus is mediated by integrin alpha(IIb)beta(3). The cytoplasmic tail of alpha(IIb) contains a highly conserved motif, (989)KVGFFKR(995), which plays a critical role in regulating integrin activation and acts as a recognition site for various intracellular proteins, e.g. CIB1, PP1, ICln and RN181. Previously, we demonstrated that a cell-permeable integrin-derived activating (IDA) peptide, KVGFFKR, induces platelet activation, whereas an integrin-derived inhibitory (IDI) peptide, KVGAAKR, is antithrombotic. To elucidate the molecular mechanism underlying these opposite effects we investigate the affinity of known integrin alpha(IIb) binding proteins for the two immobilized peptides in dependence on the activation state of platelets by means of peptide-affinity chromatography, blotting techniques and protein:peptide docking studies. Our results provide a model for the inhibition of ICln interaction with the integrin in activated platelets by the IDI-peptide. Thus, ICln:IDI-peptide interaction profiles can have a pivotal purpose in the search for consensus pharmacophores specifically inhibiting ICln function in platelets potentially leading to the development of integrin-derived antithrombotic drugs.

We previously identified proteins that bind with high affinity to a peptide corresponding to the cytoplasmic regulatory domain (KVGFFKR) of the platelet-specific integrin subunit alpha(IIb). These included a hypothetical protein termed HSPC238, recently renamed as RING finger protein, RN181. Here, we establish the presence of RN181 in human platelets by RT-PCR, Western blotting and mass spectrometry and confirm its affinity for the platelet integrin. We demonstrate that RN181 has ubiquitin E3 ligase activity and that all other components of the ubiquitination pathway are abundant in platelets, suggesting a novel link of integrin signal transduction pathways with ubiquitin-conjugation events.

To understand the regulation of integrin alpha(IIb)beta(3), a critical platelet adhesion molecule, we have developed a peptide affinity chromatography method using the known integrin regulatory motif, LAMWKVGFFKR. Using standard Fmoc chemistry, this peptide was synthesized onto a Toyopearl AF-Amino-650 M resin on a 6-aminohexanoic acid (Ahx) linker. Peptide density was controlled by acetylation of 83% of the Ahx amino groups. Four recombinant human proteins (CIB1, PP1, ICln and RN181), previously identified as binding to this integrin regulatory motif, were specifically retained by the column containing the integrin peptide but not by a column presenting an irrelevant peptide. Hemoglobin, creatine kinase, bovine serum albumin, fibrinogen and alpha-tubulin failed to bind under the chosen conditions. Immunodetection methods confirmed the binding of endogenous platelet proteins, including CIB1, PP1, ICln RN181, AUP-1 and beta3-integrin, from a detergent-free platelet lysate. Thus, we describe a reproducible method that facilitates the reliable extraction of specific integrin-binding proteins from complex biological matrices. This methodology may enable the sensitive and specific identification of proteins that interact with linear, membrane-proximal peptide motifs such as the integrin regulatory motif LAMWKVGFFKR.

Background: Cytokines influence the expression of adhesion molecules and hence, regulate the passage of leucocytes from the blood to the site of inflammation causing leucocyte accumulation and the modulation of the nature and progression of inflammatory responses. They form a complex communication network causing results which are not determined by the effects of a single cytokine but especially by the interaction of several cytokines. Method: For the determination of adhesion molecule expression on the surface of enzymatically detached endothelial cells, flow cytometry is applied. Fluorescence-conjugated mouse monoclonal antibodies directed against VCAM-1, ICAM-1, PECAM-1, CD34, E- and P-selectin are used. Results: We clearly demonstrate that ICAM-1, PECAM-1, P-selectin and CD34 are-in relation to an incubation cocktail containing solely TNF-α, IL-1β and IFN-γ-altered antagonistically by the supplementary addition of the inflammatory cytokines IL-2 and IL-6 as well as the anti-inflammatory cytokines IL-4 and IL-10, whereas VCAM-1 is synergistically enhanced under the same test conditions. Conclusion: The results of our in vitro investigations show that the effects of a single cytokine within a multi-component cytokine combination on endothelial adhesion molecule expression are strongly influenced by the nature of the other cytokines present in the combination tested.

Immunosuppressive drugs are needed to prevent the rejection of transplanted organs by the immune system. Immunosuppressive antimetabolites act by interrupting cell metabolism. Their mechanism of action can be studied in vitro by measuring the inhibition of biochemical activities which is reflected by changes in the nucleotide content. In our experiments, human peripheral blood mononuclear cells (PBMC) isolated from healthy volunteers were used. After PBMC stimulation with phytohaemagglutinin (PHA) to mimic activation occurring at a rejection crisis, cells were exposed to varying concentrations of different immunosuppressants (i.e., mycophenolic acid, cyclosporin A and prednisolone) for 68 h at 37 degrees C. Changes in nucleotide content were observed by determining the concentrations of 15 nucleotides using a newly developed HPLC method. The results obtained for mycophenolic acid (MPA; final concentrations in a range between 0.1 and 5 micromol/l), cyclosporin A (CsA; final concentrations between 100 ng/ml and 1 microg/ml) and prednisolone (final concentrations between 0.5 and 10 micromol/l) are given as percentage changes in nucleotide content versus controls and are expressed as mean +/- confidence interval. The possibility of synergistic effects was investigated by incubating the cells with mixtures of all three immunosuppressive drugs varying the amount of mycophenolic acid. In addition, we have shown the effects of MPA/guanosine co-incubation on the intracellular nucleotide levels. Stimulation of peripheral blood mononuclear cells with phytohaemagglutinin led to a significant increase of pyrimidine and purine nucleotides versus control values (100%). Pyrimidine (CTP, UDP, UTP) and purine nucleotides (GDP, GTP, ADP, ATP) were elevated up to 153+/-14% and 142+/-17%, respectively. Under co-incubation of cells with MPA, the GTP level decreased in a dose-related manner to 56+/-3% of control at a MPA final concentration of 5 micromol/l. Concomitantly, an increase of UTP values to 203+/-18% versus control was observed under co-incubation with 1 micromol/l MPA. Co-incubation of mononuclear cells with guanosine (50 micromol/l) compensated for the effects of MPA on intracellular GTP levels. Combination of MPA, CsA and prednisolone did not alter intracellular nucleotide profiles of PBMC compared to those under MPA incubation alone. The depletion of the guanine nucleotide pool and concomitant increase of uridine nucleotides under the influence of the immunosuppressive drug mycophenolic acid is caused by its inhibitory effects on the key enzyme of de novo purine biosynthesis, inosine 5'-monophosphate dehydrogenase (IMPDH).

The immunosuppressive drug mycophenolate mofetil (MMF) and its active metabolite mycophenolic acid (MPA) selectively inhibit inosine 5'-monophosphate dehydrogenase (IMPDH), and therefore interfere with cellular guanine nucleotide biosynthesis. IMPDH is additionally involved in the synthesis of membrane glycoproteins, some of which are adhesion receptors known to play an active part in the regulation of cell-cell contacts, which are crucial in the process of recruitment and transendothelial infiltration of activated leucocytes in the transplanted organ. As a consequence, MPA leads to a reduction of cellular infiltrates in the course of transplant rejection. In the present study, the effects of MPA on human umbilical vein endothelial cells (HUVEC) are investigated at both molecular and cellular levels. In our experiments, HUVECs are treated with tumor necrosis factor-alpha (TNF-alpha; 10 ng/ml) in order to mimic activation occurring at a rejection crisis. The dose-dependent influence of concomitant incubation with MPA (5-20 micromol/l; 48 h, 37 degrees C, 5% CO2) on their intracellular nucleotide profile is observed by determining the concentrations of purine and pyrimidine nucleotides, using a HPLC method based on solvent generated ion-exchange. The possibility of synergistic effects is investigated by incubating endothelial cells with mixtures of three different immunosuppressants (mycophenolic acid; cyclosporin A, 100 ng/ml; prednisolone, 1 micromol/l)--a combination commonly used after transplantation--varying the amount of MPA (5-20 micromol/l). Stimulation with TNFalpha does not significantly modulate the intracellular levels of nucleotides quantitated. In the presence of MPA concentrations of at least 5 micromol/l, GTP levels (68+/-12%) are significantly decreased compared to controls (100%). At a concentration of 20 micromol/l MPA, the GTP amount is reduced to 58+/-7%. In contrast to these observations, the levels of UDP and UTP are increasing significantly under coincubation with MPA concentrations greater than 5 micromol/l. At 20 micromol/l MPA, UDP and UTP are increased to 147+/-19% and 114+/-11%, respectively. All other nucleotides (CTP, ADP, ATP) reveal no significant alterations in their intracellular concentrations under the conditions applied. Incubation of TNFalpha-treated HUVEC monolayers, with a mixture of three immunosuppressive drugs varying the amount of MPA, show no significant differences compared with the data observed after incubation with MPA alone. In addition, the influence of MPA (10 micromol/l) on a cellular level is observed by measuring the cell surface expression of adhesion molecules on cytokine-stimulated HUVECs, using TNFalpha (10 ng/ml), interferon-gamma (100 ng/ml), interleukin-1beta (10 ng/ml) and interleukin-8 (20 ng/ml). Expression of the intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), endothelial leucocyte adhesion molecule-1 (ELAM-1) and platelet endothelial cell adhesion molecule-1 (PECAM-1) was assessed by flow cytometry. Activation of endothelial cell monolayers with TNFalpha significantly increases the mean fluorescence intensity of VCAM-1 (361+/-14%) and ICAM-1 (429+/-47%) surface expression, compared to controls, and additionally induces E-selectin expression (2919+/-134%). The same tendencies, but in a lesser degree, are observed under stimulation of cells with either IFNgamma or IL-1beta. Incubation with a combination of TNFalpha and MPA leads to a significant reduction in VCAM-1 (329+/-13%) and E-selectin (2613+/-167%) expression, compared to the values obtained for HUVEC incubated with the cytokine alone. Treatment of the cells with IL-1beta/MPA also reduces the expression of VCAM-1 to a level significantly lower than the level observed after stimulation with IL-1beta. Incubation with MPA alone reveals no significant modulation in the expression of all surface molecules tested compared to the values of unstimulated HUVECs. The experiments show that the immunosuppressive action of MPA not only inhibits lymphocyte proliferation but also decreases the expression of adhesion molecules on endothelial cells, which are the first target of the cellular rejection process.

The interaction between leukocytes and endothelial cells plays the essential role in inflammation. Endothelial cells express a variety of adhesive receptors that regulate their adhesion to leukocytes and also to the extracellular matrix. These interactions are complex phenomena that require multiple recognition mechanisms, and include the first rolling and later the stationary adhesion and transmigration of leukocytes. It is known that cytokines have regulatory effects on cell adhesion molecules expression. In the present study we investigated the influence of cytokines (IL-1b, IL-2, IL-4, IL-6, IL-8 IL-10, TNF-a, IFN-g) and the combined use of IL-2, IL-4, IL-6, IL-8 and IL-10 with IL-1b, TNF-b or IFN-g on the expression of adhesion molecules of cultured human umbilical vein endothelial cells (HUVECs) after stimulation for 16 hours. Likewise, in vitro model described herein is designed to mimic the activation of endothelial cells by cytokines as seen during inflammatory processes. This process is mediated by specific cell adhesion molecules being crucial for the generation of immune and inflammatory responses. Therefore, HUVECs are treated with two different cytokine combinations consisting of either IL-2, IL-6, IL-8 IFN-g and TNF-a or IL-1b, IL-2, IL-4, IL-6, IL-10, IFN-g and TNF-a. Endothelial cells were collected from hunam umbilical vain using collagense type II, and cell cultures in complete medium were kept in the incubator (37.4?C, 5% CO2). After stimulation cells were prepared for analysis using tripsinisation procedure. The surface expression of the following adhesion molecules was determined in cultured human umbilical vein endothelial cells (HUVECs) by means of flow-cytometric analysis: CD 62P (P-selectin), CD 62E (E-selectin, ELAM-1) CD 106CD 34 (L-selectin ligand). The highest CD 62E expression on the surface of HUVECs was found when endothelial cells were stimulated with TNF-a alone. Also they were increased after stimulation with IL-1b, while IL-4 led to down-regulation of CD 62E. Incubation of HUVEC monolayers with IL-1b, IL-4 as well as TNF-a and IFN-g, statistically significant, reduced the surface expression of CD 34 while other cytokines did not affect CD 34 expression. Incubation of HUVECs with a single cytokine caused no statistically significant changes in CD 62P expression compared to controls. The most potent effect on CD 54 expression was found under TNF-a stimulation; IL-1b and IFN-g had also amplifying effects, while all other tested cytokines caused no significant changes in surface molecule expression. Surface expression of CD 106 was amplified during incubation with IL-1b, IL-4, TNF-a and IFN-g. Single stimulation of tested cytokines did not significantly alter the cell surface expression of CD 31. Concomitant stimulation with IL-2, IL-4, IL-6, IL-8 or IL-10 with IL-1b, TNF-a or IFN-g led to different effects compared with effects of single cytokine stimulation: CD 62E were up-regulated under co-stimulation with combination of IL-1b and IFN-g, IL-6 and IL-1b, and also in all combinations with TNF-a. Statistically significant differences were found in CD 62P surface expression after concomitant stimulation with IL-1b and IFN-g, and in combinations with TNF-a. Co-stimulation with IL-10 and IL- 1b, TNF-a or IFN-g, or IL-8 with IL-1b or IFNg, IL-6 with IFN-g, IL-4 with TNF-a or IFN-g and IL-2 with IFN-g significantly decreased the level of CD 34 surface expression. (VCAM-1), CD 54 (ICAM-1), CD 31 (PECAM-1) and CD 54 expression was up-regulated after stimulation with IL-1b and IFN-g, and under concomitant stumulation with TNF-a. Surface expression of molecule CD 106 was higher after co-stimulation of cytokines with TNF-a, and IL-4 or IL-10 with IL-1b. These effects indicate modulation of single cytokine effects. Intracellular mechanisms included in those effects need to be investigated. Also there were found modulatory effects of cytokine combinations. Some effects of cytokine combinations were different in comparison to single cytokine effect. This finding indicates that intracellular mechanisms are present and responsible for signal modulation of single cytokine. The application of these two cytokine combinations mimicing inflammation reactions results in effects of comparable dimensions significantly increasing the mean fluorescence intensity of E-selectin, VCAM-1 and ICAM-1 surface expression accompanied by the induction of P-selectin expression. The experiments reveal a strong up-regulation of these cell surface antigens under conditions mimicing inflammation. This is an essential finding stressing the importance of endothelial cells during inflammatory processes.