Markus Raab

Variation of adhesion molecule expression on human umbilical vein endothelial cells upon multiple cytokine application.

BACKGROUND A variety of cytokines, mediators, activators, growth factors and other products are simultaneously released into circulation with the activation of the cellular immune system during rejection or infection. The secretion of these biochemical markers potentiates the immunological events associated with these processes. Among other things some cytokines demonstrate regulatory effects on the expression of endothelial cell adhesion molecules. METHOD Endothelial cells are detached by trypsinisation and adhesion molecule expression is assessed by means of flow cytometry. Fluorescence-conjugated mouse monoclonal antibodies directed against VCAM-1, ICAM-1, PECAM-1, CD34, E- and P-selectin are used. RESULTS The combined application of different cytokines synergistically evokes P-selectin expression after a chosen incubation period of 16 h, while under single cytokine treatment P-selectin induction is not observed. Co-stimulation with TNF-alpha and a second cytokine reduces its influence on E-selectin. IL-1 beta/IFN-gamma lead to E-selectin levels higher than those under treatment with one of the both alone. Concomitant incubation with all cytokines synergistically down-regulates PECAM-1 referred to each cytokine alone. CONCLUSION Our investigations in some cases clearly demonstrate that the combination of a second cytokine with TNF-alpha, IL-1 beta or IFN-gamma can either synergistically or antagonistically modulate the expression of adhesion molecules on HUVECs.

Endothelial adhesion molecule expression in an in vitro model of inflammation.

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-alpha, IL-1beta and IFN-gamma-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.

RN181, a novel ubiquitin E3 ligase that interacts with the KVGFFKR motif of platelet integrin αIIbβ3

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.

In vitro effects of cyclosporin A on the expression of adhesion molecules on human umbilical vein endothelial cells.

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-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) have been reported to demonstrate profound effects on this cell type. It has been shown that the increased release of IFN-alpha/gamma and TNF-alpha 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-alpha, IL-1beta or with a cytokine mixture consisting of those stimulants present at an elevated level in sera of patients during allograft rejection (i.e. IL-1beta, IL-2, IL-4, IL-6, IL-10, TNF-alpha and IFN-gamma). 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 microg/ml revealed a significant down-regulating influence on the surface expression of E-selectin and VCAM-1.

Protein interactions with the platelet integrin αIIb regulatory motif

Integrins are transmembrane proteins regulating cellular shape, mobility and the cell cycle. A highly conserved signature motif in the cytoplasmic tail of the integrin α‐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 αIIbβ3. 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 α‐subunits. 68 recombinant human proteins expressed on the protein chip specifically bind with high affinity to biotin‐tagged α‐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.

Ligand Switching in Cell-Permeable Peptides: Manipulation of the α-Integrin Signature Motif

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.

Differential binding of ICln in platelets to integrin-derived activating and inhibitory peptides

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.