Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 10 de 10
Filter
Add more filters










Publication year range
1.
Ann Rheum Dis ; 68(10): 1644-50, 2009 Oct.
Article in English | MEDLINE | ID: mdl-18957484

ABSTRACT

OBJECTIVE: To study the effects of different disease-modifying antirheumatic drugs (DMARD) on different events mediated by IL-15-activated lymphocytes. METHODS: Peripheral blood lymphocytes (PBL) were isolated from healthy donors and activated with IL-15 after exposure to different DMARD: leflunomide, cyclosporin A, methotrexate, mycophenolic acid, FK-506, sulphasalazine and sodium aurothiomalate. The expression of different surface molecules on the PBL was then determined by flow cytometry. Cells were also co-cultured with the monocytic cell line THP-1 and the tumour necrosis factor (TNF) concentration in the supernatant was measured after 24 h using an immunoenzyme assay. The effect of the aforementioned drugs on IL-17 production by IL-15-activated PBL was also studied. RESULTS: Treatment of PBL with leflunomide, cyclosporin A and FK-506 inhibited the IL-15-induced expression of both CD54 and CD69 by PBL, as well as TNF production in co-cultures of activated PBL and THP-1 cells. The downregulation of CD54 and CD69 in PBL was correlated with the inhibition of TNF production. Likewise, leflunomide, cyclosporin A and FK-506 all inhibited IL-17 production in IL-15-activated PBL. Interestingly, the effect of leflunomide was not reverted by the presence of uridine in the medium. In addition, leflunomide inhibited the phosphorylation of STAT6 in vitro. CONCLUSION: Inhibition of the JAK/STAT pathway may represent an additional effect of leflunomide in chronic polyarthritis because it impairs certain events that control proinflammatory TNF and IL-17 cytokine production.


Subject(s)
Antirheumatic Agents/pharmacology , Interleukin-17/biosynthesis , Isoxazoles/pharmacology , Lymphocytes/drug effects , Tumor Necrosis Factor-alpha/biosynthesis , Antigens, CD/biosynthesis , Antigens, Differentiation, T-Lymphocyte/biosynthesis , Cells, Cultured , Coculture Techniques , Humans , Intercellular Adhesion Molecule-1/biosynthesis , Interleukin-15/immunology , Lectins, C-Type , Leflunomide , Lymphocyte Activation/drug effects , Lymphocytes/immunology , Lymphocytes/metabolism , Phosphorylation/drug effects , STAT6 Transcription Factor/metabolism , Signal Transduction/drug effects , Up-Regulation/drug effects
3.
Blood ; 96(10): 3592-600, 2000 Nov 15.
Article in English | MEDLINE | ID: mdl-11071659

ABSTRACT

L-selectin is an adhesion molecule that plays an essential role in the early events of the inflammatory response. Our group has recently described that several nonsteroidal anti-inflammatory drugs (NSAIDs) are able to induce both in vivo and in vitro the shedding of L-selectin in neutrophils through an unknown mechanism. In this work, we have studied potential mechanisms involved in the shedding of L-selectin induced by NSAIDs. This effect of NSAIDs did not involve any detectable intracellular calcium flux. Pretreatment of neutrophils either with Ro 31-8220 and H7, 2 specific inhibitors of protein kinase C (PKC), or with inhibitors of protein tyrosine kinases such as tyrphostin A25 or herbimycin A did not prevent the NSAID-mediated L-selectin shedding. However, the KD-IX-73-4, an inhibitor of L-selectin proteolysis was able to block the effect of NSAIDs on L-selectin expression. Remarkably, NSAIDs caused a variable reduction in the neutrophil intracellular ATP concentration that highly correlated with the differential ability of NSAIDs to trigger L-selectin shedding (r = 0.8, P <.01). In agreement with this finding, azide plus 2-deoxy-D-glucose, 2 metabolic blockers, also induced a rapid L-selectin shedding (65% +/- 8%) without affecting the neutrophil viability, activation, or expression level of other surface molecules with soluble isoforms such as CD16 and CD59. These data indicate that the maintenance of L-selectin on the neutrophil surface requires energy consumption, which suggests that L-selectin is shed in neutrophils by default. Interestingly, NSAIDs seem to cause the shedding of L-selectin, at least in part, through the reduction of the intracellular ATP concentration.


Subject(s)
Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Down-Regulation/drug effects , L-Selectin/drug effects , L-Selectin/physiology , Neutrophils/drug effects , Adenosine Triphosphate/metabolism , Adenosine Triphosphate/physiology , Diglycerides/pharmacology , Energy Metabolism , Enzyme Inhibitors , Flow Cytometry , Humans , Inhibitory Concentration 50 , Intracellular Fluid/chemistry , L-Selectin/metabolism , Metalloendopeptidases/antagonists & inhibitors , Metalloendopeptidases/pharmacology , Neutrophils/chemistry , Neutrophils/metabolism , Protein Kinase C/pharmacology , Protein-Tyrosine Kinases/pharmacology , Sodium Azide/pharmacology
4.
Blood ; 95(7): 2413-9, 2000 Apr 01.
Article in English | MEDLINE | ID: mdl-10733515

ABSTRACT

In response to the chemoattractants interleukin 8, C5a, N-formyl-methionyl-leucyl-phenylalanine, and interleukin 15, adhesion molecules P-selectin glycoprotein ligand 1 (PSGL-1), intercellular adhesion molecule 3 (ICAM-3), CD43, and CD44 are redistributed to a newly formed uropod in human neutrophils. The adhesion molecules PSGL-1 and ICAM-3 were found to colocalize with the cytoskeletal protein moesin in the uropod of stimulated neutrophils. Interaction of PSGL-1 with moesin was shown in HL-60 cell lysates by isolating a complex with glutathione S-transferase fusions of the cytoplasmic domain of PSGL-1. Bands of 78- and 81-kd were identified as moesin and ezrin by Western blot analysis. ICAM-3 and moesin also coeluted from neutrophil lysates with an anti-ICAM-3 immunoaffinity assay. Direct interaction of the cytoplasmic domains of ICAM-3 and PSGL-1 with the amino-terminal domain of recombinant moesin was demonstrated by protein-protein binding assays. These results suggest that the redistribution of PSGL-1 and its association with intracellular molecules, including the ezrin-radixin-moesin actin-binding proteins, regulate functions mediated by PSGL-1 in leukocytes stimulated by chemoattractants.


Subject(s)
Antigens, CD , Antigens, Differentiation , Cell Adhesion Molecules/blood , Membrane Glycoproteins/blood , Microfilament Proteins/blood , Neutrophils/chemistry , Phosphoproteins/blood , Antibodies, Monoclonal , Blotting, Western , Cell Membrane/chemistry , Chromatography, Affinity , Complement C5a/pharmacology , Cytoplasm/chemistry , Cytoskeletal Proteins , Humans , Immunoassay , Interleukin-8/pharmacology , N-Formylmethionine Leucyl-Phenylalanine/pharmacology , Neutrophils/ultrastructure , Recombinant Proteins/blood
5.
FEBS Lett ; 446(2-3): 318-22, 1999 Mar 12.
Article in English | MEDLINE | ID: mdl-10100866

ABSTRACT

We have studied the effect of non-steroidal antiinflammatory drugs (NSAIDs) on alphaII(b)beta3 integrin activation and platelet aggregation. NSAIDs such as meloxicam, piroxicam, indomethacin and aspirin, but not aceclofenac or diclofenac interfered with the activation state of alphaII(b)beta3. NSAIDs that inhibited alphaII(b)beta3 activation were also able both to partially inhibit platelet primary aggregation and to accelerate platelet deaggregation. These effects of NSAIDs were not dependent on cyclooxygenase inhibition. The results obtained indicate that some NSAIDs exert a specific action on alphaII(b)beta3 activation, and provide an additional mechanism that accounts for their beneficial effects in diseases in which platelet activation is involved.


Subject(s)
Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Blood Platelets/metabolism , Platelet Aggregation Inhibitors/pharmacology , Platelet Glycoprotein GPIIb-IIIa Complex/metabolism , Blood Platelets/drug effects , Humans , Platelet Aggregation/drug effects
6.
J Biol Chem ; 272(40): 24832-6, 1997 Oct 03.
Article in English | MEDLINE | ID: mdl-9312081

ABSTRACT

The region of fibronectin encompassing type III repeats 4-6 contains a low affinity heparin binding domain, but its physiological significance is not clear. We have studied whether this domain is able to interact with cells as already shown for other heparin binding domains of fibronectin. A computer search based on homologies with known active sites in fibronectin revealed the sequence KLDAPT located in FN-III5. A synthetic peptide containing this sequence induced lymphoid cell adhesion upon treatment with the activating anti-beta1 monoclonal antibody (mAb) TS2/16 or with Mn2+, indicating that KLDAPT was binding to an integrin. A recombinant fragment containing repeat III5 (FN-III5) also mediated adhesion of TS2/16/Mn2+-treated cells while the FN-III6 fragment did not. Soluble KLDAPT peptide inhibited cell adhesion to FN-III5 as well as to a 38-kDa fibronectin fragment and VCAM-1, two previously known ligands for alpha4beta1 integrin. KLDAPT also competed with the binding of soluble alkaline phosphatase-coupled VCAM-Ig to Mn2+-treated alpha4beta1. Furthermore, mAbs anti-alpha4 and anti-alpha4beta7, but not mAbs to other integrins, inhibited cell adhesion to FN-III5 and KLDAPT. These results therefore establish a cell adhesive function for the FN-III5 repeat and show that KLDAPT is a novel fibronectin ligand for activated alpha4 integrins.


Subject(s)
Cell Adhesion , Fibronectins/chemistry , Fibronectins/metabolism , Integrins/metabolism , Receptors, Lymphocyte Homing/metabolism , Amino Acid Sequence , Antibodies, Monoclonal , B-Lymphocytes , Binding Sites , Cell Adhesion/drug effects , Cell Line , Humans , Integrin alpha4beta1 , Jurkat Cells , Ligands , Manganese/pharmacology , Molecular Sequence Data , Peptide Fragments/chemistry , Peptide Fragments/metabolism , Peptide Fragments/pharmacology , Peptides/chemistry , Peptides/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Sequence Alignment , T-Lymphocytes , Tumor Cells, Cultured , Vascular Cell Adhesion Molecule-1/metabolism
7.
Cell Adhes Commun ; 4(4-5): 251-67, 1996 Nov.
Article in English | MEDLINE | ID: mdl-9117345

ABSTRACT

The lymphocyte integrin alpha 4 beta 1 is the receptor for the Hep II domain and CS-1 site in fibronectin (Fn) as well as for VCAM-1. We recently showed that upon activation with anti-beta 1 mAb TS2/16, alpha 4 beta 1 also recognizes the RGD Fn sequence. To determine the physiological role of these multiple interactions, we have now studied some intracellular events induced by "resting" and activated alpha 4 beta 1 binding to its different ligands. Analyses of actin and tubulin reorganization upon adhesion of B lymphoid cells to Fn fragments or VCAM-1 showed that VCAM-1, a 38 kDa fragment (Hep II+CS-1), and the CS-1 synthetic peptide induced formation of transient cytoplasmic projections; however, cells attached to a 58 kDa (Hep II) or 80 kDa (RGD) fragments remained rounded. Using transfilter assays, we showed that VCAM-1, 38 kDa and CS-1 also induced dose-dependent B cell migration mediated by alpha 4 beta 1. Furthermore, these three ligands, but not the 80 kDa fragment or a synthetic peptide (H1) containing a sequence from Hep II shown to bind alpha 4 beta 1, induced tyrosine phosphorylation of a 110 kDa protein. Activation of alpha 4 beta 1 with TS2/16 inhibited the cytoplasmic protrusions and cell migration but did not affect the pattern of phosphorylation. Our results indicate that the various alpha 4 beta 1 ligands induce different cellular responses. Most importantly they show that alpha 4 beta 1 interaction with CS-1 is sufficient to trigger intracellular events in B cells. Furthermore, they suggest a regulation by the activation form of the receptor as well as by the ligand in events involving lymphocyte adhesion and migration.


Subject(s)
B-Lymphocytes/metabolism , Cytoskeletal Proteins/metabolism , Fibronectins/metabolism , Integrins/metabolism , Receptors, Lymphocyte Homing/metabolism , Vascular Cell Adhesion Molecule-1/metabolism , Amino Acid Sequence , Cell Adhesion , Cell Line , Cell Movement , Cell Size , Fibronectins/genetics , Humans , Integrin alpha4beta1 , Ligands , Molecular Sequence Data , Peptide Fragments/genetics , Peptide Fragments/metabolism , Phosphorylation , Protein Serine-Threonine Kinases/metabolism , Recombinant Proteins/metabolism , Signal Transduction , Structure-Activity Relationship , Vascular Cell Adhesion Molecule-1/genetics
8.
J Cell Biol ; 126(1): 271-9, 1994 Jul.
Article in English | MEDLINE | ID: mdl-7517944

ABSTRACT

Lymphocyte attachment to fibronectin is mainly mediated by the interaction of alpha 5 beta 1 and alpha 4 beta 1 integrins with the RGD and CS-1/Hep II sites, respectively. We have recently shown that the anti-beta 1 mAb TS2/16 can convert the partly active alpha 4 beta 1 present on certain hemopoietic cells that recognizes CS-1 but not Hep II, to a high avidity form that binds both ligands. In this report we have studied whether mAb TS2/16 also affects alpha 4 beta 1 ligand specificity. Incubation of the B cell lines Ramos and Daudi (which lack alpha 5 beta 1) with mAb TS2/16 induced specific attachment to an 80-kD fragment which lacks CS-1 and Hep II and contains the RGD sequence. mAbs anti-alpha 4 and the synthetic peptides CS-1 and IDAPS inhibited adhesion to the 80-kD fragment thus implying alpha 4 beta 1 as the receptor for this fragment. Interestingly, the synthetic peptide GRGDSPC and a 15-kD peptic fibronectin fragment containing the RGD sequence also inhibited B cell adhesion to the 80-kD fragment. Because we have previously shown that RGD peptides do not affect the constitutive function of alpha 4 beta 1, we tested whether TS2/16-activated alpha 4 beta 1 acquired the capacity to recognize RGD. Indeed RGD peptides inhibited TS2/16-treated B cell adhesion to a 38-kD fragment containing CS-1 and Hep II but did not affect binding of untreated cells to this fragment. An anti-fibronectin mAb reactive with an epitope on or near the RGD sequence also efficiently inhibited cell adhesion to the 80-kD fragment, indicating that the RGD sequence is a novel adhesive ligand for activated alpha 4 beta 1. These results emphasize the role of alpha 4 beta 1 as a receptor with different ligand specificities according to the activation state, a fact that may be important for lymphocyte migration, localization, and function.


Subject(s)
B-Lymphocytes/metabolism , Cell Adhesion/physiology , Fibronectins/metabolism , Integrins/metabolism , Amino Acid Sequence , Antibodies, Monoclonal , B-Lymphocytes/drug effects , Cell Line , Dose-Response Relationship, Drug , Fibronectins/immunology , Humans , Integrin alpha4 , Integrin alpha4beta1 , Integrin beta1 , Integrins/immunology , Ligands , Molecular Sequence Data , Peptide Fragments/metabolism , Peptide Fragments/pharmacology , Receptors, Fibronectin/metabolism , Tetradecanoylphorbol Acetate/pharmacology
9.
Blood ; 83(4): 994-1005, 1994 Feb 15.
Article in English | MEDLINE | ID: mdl-8111067

ABSTRACT

Vascular endothelium forms a dynamic interface between blood and underlying tissues. Endothelial monolayer integrity is required for controlled vascular permeability and to preclude exposure of subendothelial cell matrix to circulating cells. Recent studies have established that cultured human umbilical vein endothelial cells (ECs) express receptors for plasminogen (plg) and urokinase-like plasminogen activator (uPA). In the present study, we provide evidence that in EC, uPA receptor is present in focal contacts and at cell-cell contact sites. In these cells, addition of plg and uPA to confluent EC generates a retraction of the monolayer that is evidenced by loss of cell-cell contacts and increase in monolayer permeability. The phenomenon is reversible even after 6 hours of plg-uPA treatment. Inhibition of plg-uPA effect is obtained with plasmin inhibitors, as well as reagents that block binding of uPA or plg to the cell surface. The retractive effect of plg-uPA is concomitant to surface activation of plasminogen and to the loss of cell-cell activation of plg can induce EC retraction, possibly by causing proteolysis at specific cell-cell contacts and cell-matrix sites. This process may be important in mediating the passage of metastatic tumor cells through an intact EC monolayer as well as in regulating contacts between circulating cells and endothelium.


Subject(s)
Cell Membrane/physiology , Endothelium, Vascular/physiology , Receptors, Cell Surface/metabolism , Urokinase-Type Plasminogen Activator/pharmacology , Cell Membrane/drug effects , Cell Membrane Permeability , Cells, Cultured , Endothelium, Vascular/cytology , Endothelium, Vascular/drug effects , Humans , Plasminogen/drug effects , Plasminogen/physiology , Probability , Receptors, Cell Surface/drug effects , Receptors, Urokinase Plasminogen Activator , Saphenous Vein , Serum Albumin/metabolism , Umbilical Veins
10.
Blood ; 80(2): 437-46, 1992 Jul 15.
Article in English | MEDLINE | ID: mdl-1627801

ABSTRACT

Endothelial cells (EC) form a dynamic interface between blood and the rest of the body. EC surface properties promoting adhesion of reactive plasma proteins and/or circulating cells might be of pivotal importance for the homeostasis of blood and tissues. EC express multiple integrin receptors that promote their attachment to the subendothelial matrix proteins. Among these receptors, alpha v beta 3 is of particular relevance on EC, since it is abundantly expressed and can bind many different matrix and plasma proteins. It is still unknown whether integrin receptors are selectively located to the basal side of EC membrane or may also be exposed on the cell surface in contact with blood. This issue was addressed using different experimental approaches. First, selective surface radioiodination using lactoperoxidase (LPO)-latex beads and immunoprecipitation analysis were performed. We found that cultured EC, similarly to human skin fibroblasts (HSF), expose alpha v beta 3 on both their apical (free) and basal (substratum-attached) surfaces. This held also for other integrins such as alpha 2 beta 1, alpha 3 beta 1, alpha 5 beta 1, and alpha 6 beta 1. Immunoprecipitation data were verified by morphological techniques. Immunofluorescence and immunogold-staining of EC with alpha v beta 3, as well as with beta 1 subfamily antibodies, showed a diffuse and granular distribution of these integrins on EC surface. alpha v beta 3 and beta 1 integrins were also detected on the apical membrane of EC at higher magnification by scanning electron microscopy (SEM). Finally, data obtained on cultured EC were confirmed in vivo on immunogold-labeled ultrathin cryosections of human vessels by transmission electron microscopy (TEM). Data indicate, that in addition to their role in promoting EC attachment to extracellular matrix proteins, integrin receptors of EC can be exposed to blood-stream and eventually be available for binding of plasma proteins and circulating cells.


Subject(s)
Endothelium, Vascular/physiology , Integrins/metabolism , Cell Membrane/physiology , Cell Membrane/ultrastructure , Cells, Cultured , Electrophoresis, Polyacrylamide Gel , Endothelium, Vascular/cytology , Fluorescent Antibody Technique , Humans , Immunohistochemistry , Integrins/analysis , Integrins/isolation & purification , Microscopy, Immunoelectron , Molecular Weight , Umbilical Veins
SELECTION OF CITATIONS
SEARCH DETAIL
...