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1.
J Immunol ; 166(3): 1457-61, 2001 Feb 01.
Article in English | MEDLINE | ID: mdl-11160184

ABSTRACT

Chemokine-induced eosinophil chemotaxis is mediated primarily through the C-C chemokine receptor, CCR3. We have now detected CCR3 immunoreactivity on epithelial cells in biopsies of patients with asthma and other respiratory diseases. CCR3 mRNA was detected by Northern blot analysis after TNF-alpha stimulation of the human primary bronchial epithelial cells as well as the epithelial cell line, BEAS-2B; IFN-gamma potentiated the TNF-alpha-induced expression. Western blots and flow cytometry confirmed the expression of CCR3 protein. This receptor is functional based on studies demonstrating eotaxin-induced intracellular Ca(2+) flux and tyrosine phosphorylation of cellular proteins. The specificity of this functional response was confirmed by blocking these signaling events with anti-CCR3 mAb (7B11) or pertussis toxin. Furthermore, (125)I-eotaxin binding assay confirmed that CCR3 expressed on epithelial cells have the expected ligand specificity. These studies indicate that airway epithelial cells express CCR3 and suggest that CCR3 ligands may influence epithelial cell functions.


Subject(s)
Bronchi/immunology , Bronchi/metabolism , Chemokines, CC/metabolism , Epithelial Cells/immunology , Epithelial Cells/metabolism , Receptors, Chemokine/biosynthesis , Binding, Competitive/immunology , Bronchi/cytology , Bronchi/pathology , Calcium/metabolism , Calcium Signaling/immunology , Cell Line , Chemokine CCL11 , Chemokines, CC/pharmacology , Cytokines/agonists , Cytokines/antagonists & inhibitors , Cytokines/metabolism , Cytokines/pharmacology , Epithelial Cells/pathology , Humans , Immunohistochemistry , Inflammation/immunology , Inflammation/metabolism , Intracellular Fluid/metabolism , RNA, Messenger/biosynthesis , Receptors, CCR3 , Receptors, Chemokine/agonists , Receptors, Chemokine/genetics
2.
J Allergy Clin Immunol ; 106(5): 918-24, 2000 Nov.
Article in English | MEDLINE | ID: mdl-11080715

ABSTRACT

BACKGROUND: The endothelial cell adhesion molecule P-selectin may contribute to selective leukocyte migration in allergic diseases by binding to its ligand, P-selectin glycoprotein ligand 1 (PSGL-1), on eosinophils and other leukocytes. Although expression of PSGL-1 on basophils has been detected in leukocyte typing workshops, its function on basophils has not been explored. OBJECTIVE: We sought to characterize the expression and function of PSGL-1 on human basophils and a basophil-like cell line (KU812) and to compare these characteristics with those for PSGL-1 on eosinophils and neutrophils. METHODS: Basophils, eosinophils, and neutrophils were enriched from peripheral blood by using density gradient centrifugation and immunomagnetic negative selection. KU812 cells were cultured by using standard techniques. Indirect immunofluorescence and flow cytometry were used to determine surface PSGL-1 expression under various conditions, and Western blotting was used to analyze the molecular forms of PSGL-1 on each cell type. Static adhesion assays were performed by using immobilized recombinant P-selectin and relevant blocking antibodies. Histamine release assays were done by using adherent and nonadherent basophils to determine whether adhesion by means of PSGL-1 altered basophil releasability. RESULTS: The expression of PSGL-1 on basophils was similar to that on neutrophils but was approximately 30% less bright than levels on eosinophils. Levels on basophils were 10-fold higher than on KU812 cells. Basophil activation by means of IgE cross-linking resulted in reductions in surface expression of PSGL-1 and L-selectin, as well as increased CD11b expression. Western blot analysis of PSGL-1 revealed that the molecular weights of the bands for neutrophils and basophils were similar, whereas those for eosinophils were of greater molecular weights. Static adhesion assays demonstrated that basophils bound well to P-selectin, whereas KU812 cells bound poorly. Adhesion of basophils to P-selectin was completely blocked by antibodies to either P-selectin or PSGL-1. Finally, adhesion to P-selectin did not alter the magnitude or kinetics of anti-IgE-induced histamine release. CONCLUSION: Expression of PSGL-1 on basophils is more similar to that on neutrophils than that on eosinophils. KU812 cells express much lower levels of this molecule but, like basophils and other cells, bind to P-selectin by means of PSGL-1. P-selectin expression at sites of allergic inflammation is likely to play an important role in human basophil recruitment, but adhesion by means of PSGL-1 does not alter IgE-dependent basophil histamine release.


Subject(s)
Basophils/metabolism , Membrane Glycoproteins/physiology , P-Selectin/metabolism , Animals , Cell Adhesion , Cell Line , Eosinophils/metabolism , Humans , Ligands , Membrane Glycoproteins/biosynthesis , Mice , Neutrophils/metabolism
3.
J Immunol ; 165(5): 2764-72, 2000 Sep 01.
Article in English | MEDLINE | ID: mdl-10946308

ABSTRACT

P-selectin glycoprotein ligand-1 (PSGL-1), the primary ligand for P-selectin, is constitutively expressed on the surface of circulating leukocytes. The objective of this study was to examine the effect of leukocyte activation on PSGL-1 expression and PSGL-1-mediated leukocyte adhesion to P-selectin. PSGL-1 expression was examined via indirect immunofluorescence and flow cytometry before and after leukocyte stimulation with platelet activating factor (PAF) and PMA. Human neutrophils, monocytes, and eosinophils were all demonstrated to have significant surface expression of PSGL-1 at baseline, which decreased within minutes of exposure to PAF or PMA. PSGL-1 was detected in the supernatants of PAF-activated neutrophils by immunoprecipitation. Along with the expression data, this suggests removal of PSGL-1 from the cell surface. Soluble PSGL-1 was also detected in human bronchoalveolar lavage fluids. Down-regulation of PSGL-1 was inhibited by EDTA. However, inhibitors of L-selectin shedding and other sheddase inhibitors did not affect PSGL-1 release, suggesting that PSGL-1 may be shed by an as yet unidentified sheddase or removed by some other mechanism. Functionally, PSGL-1 down-regulation was associated with decreased neutrophil adhesion to immobilized P-selectin under both static and flow conditions, with the most profound effects seen under flow conditions. Together, these data indicate that PSGL-1 can be removed from the surface of activated leukocytes, and that this decrease in PSGL-1 expression has profound effects on leukocyte binding to P-selectin, especially under conditions of flow.


Subject(s)
Leukocytes/immunology , Leukocytes/metabolism , Membrane Glycoproteins/antagonists & inhibitors , P-Selectin/metabolism , Allergens/administration & dosage , Asthma/immunology , Asthma/metabolism , Bronchial Provocation Tests , Bronchoalveolar Lavage Fluid/chemistry , Cell Adhesion/drug effects , Cell Adhesion/immunology , Cell Separation , Down-Regulation/drug effects , Down-Regulation/immunology , Edetic Acid/pharmacology , Enzyme Inhibitors/pharmacology , Eosinophils/enzymology , Eosinophils/immunology , Eosinophils/metabolism , Humans , L-Selectin/metabolism , Leukocytes/enzymology , Ligands , Membrane Glycoproteins/biosynthesis , Membrane Glycoproteins/metabolism , Monocytes/enzymology , Monocytes/immunology , Monocytes/metabolism , Neutrophil Activation/drug effects , Neutrophil Activation/immunology , Neutrophils/enzymology , Neutrophils/immunology , Neutrophils/metabolism , P-Selectin/biosynthesis , Platelet Activating Factor/pharmacology , Pollen/immunology , Rheology , Solubility , Tetradecanoylphorbol Acetate/pharmacology
4.
J Immunol ; 164(6): 3392-401, 2000 Mar 15.
Article in English | MEDLINE | ID: mdl-10706735

ABSTRACT

Macrophage inflammatory protein (MIP-1 alpha), a member of the CC chemokine subfamily, has been shown to attract T cells and monocytes in vitro and to be expressed at sites of inflammation. Although the in vitro activities of MIP-1 alpha have been well documented, the in vivo biological activities of MIP-1 alpha in humans have not been studied. To address this, we challenged human subjects by intradermal injection with up to 1000 pmol of MIP-1 alpha and performed biopsies 2, 10, and 24 h later. Although no acute cutaneous or systemic reactions were noted, endothelial cell activation, as indicated by the expression of E-selectin, was observed. In agreement with its in vitro activity, monocyte, lymphocyte, and, to a lesser degree, eosinophil infiltration was observed, peaking at 10-24 h. Surprisingly, in contrast to its reported lack of in vitro neutrophil-stimulating activity, a rapid infiltration of neutrophils was observed in vivo. This neutrophil infiltration occurred as early as 2 h, preceding the appearance of other cells, and peaked at 10 h. Interestingly, we found that neutrophils in whole blood, but not after isolation, expressed CCR1 on their cell surface. This CCR1 was thought to be functional as assessed by neutrophil CD11b up-regulation following whole-blood MIP-1 alpha stimulation. These studies substantiate the biological effects of MIP-1 alpha on monocytes and lymphocytes and uncover the previously unrecognized activity of MIP-1 alpha to induce neutrophil infiltration and endothelial cell activation, underscoring the need to evaluate chemokines in vivo in humans.


Subject(s)
Cell Movement/immunology , Macrophage Inflammatory Proteins/administration & dosage , Monocytes/immunology , Neutrophils/immunology , Adolescent , Adult , Cell Line , Chemokine CCL4 , E-Selectin/biosynthesis , Endothelium, Vascular/cytology , Endothelium, Vascular/drug effects , Endothelium, Vascular/immunology , Endothelium, Vascular/metabolism , Female , Humans , Injections, Intradermal , Macrophage Inflammatory Proteins/pharmacology , Macrophage Inflammatory Proteins/physiology , Male , Middle Aged , Neutrophils/metabolism , Receptors, CCR1 , Receptors, CCR5/biosynthesis , Receptors, Chemokine/biosynthesis , Skin/cytology
5.
J Immunol ; 164(2): 825-32, 2000 Jan 15.
Article in English | MEDLINE | ID: mdl-10623828

ABSTRACT

The mechanism by which glucocorticoids (GC) inhibit IL-4 gene expression is currently unknown. In T lymphocytes, IL-4 gene expression is regulated at the level of transcription by increases in intracellular calcium concentration and by the calcium-activated phosphatase calcineurin. In this paper we report that dexamethasone (Dex) inhibits calcium ionophore-induced activation of the human IL-4 promoter in transiently transfected Jurkat T cells. Inhibition of the promoter by Dex is dependent on expression of the GC receptor (GR), because it does not occur in GR-deficient cells. Dex also represses activation of the promoter induced by cotransfecting cells with a constitutively active mutant of calcineurin. Using a series of deletion constructs, we show that the proximal 95 bp of the IL-4 promoter contain a Dex-sensitive regulatory element. This region contains the P1 sequence, a proximal binding site for NF-AT. A calcium-induced but Dex-inhibited nuclear complex containing NF-AT binds to the P1 element in EMSA. Using immunoprecipitation under nondenaturing conditions, we found that the GRalpha isoform coprecipitates with NF-ATc in nuclear extracts of calcium ionophore- and Dex-treated cells. Taken together, our results show that GC inhibit IL-4 gene expression by interfering with NF-AT-dependent transactivation of the proximal human IL-4 promoter.


Subject(s)
Calcineurin/physiology , Calcium/physiology , Dexamethasone/pharmacology , Immunosuppressive Agents/pharmacology , Interleukin-4/genetics , Lymphocyte Activation/drug effects , Nuclear Proteins , Promoter Regions, Genetic/immunology , Base Composition , Calcineurin/metabolism , Calcineurin Inhibitors , Calcium/antagonists & inhibitors , Cell Nucleus/drug effects , Cell Nucleus/metabolism , DNA-Binding Proteins/antagonists & inhibitors , DNA-Binding Proteins/biosynthesis , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Enzyme Activation/genetics , Enzyme Activation/immunology , Humans , Interleukin-4/metabolism , Jurkat Cells , NFATC Transcription Factors , Promoter Regions, Genetic/drug effects , Protein Kinase C/antagonists & inhibitors , Protein Kinase C/physiology , Response Elements/drug effects , Response Elements/immunology , Transcription Factors/antagonists & inhibitors , Transcription Factors/biosynthesis , Transcription Factors/genetics , Transcription Factors/metabolism , Transfection
6.
Mol Immunol ; 35(8): 459-67, 1998 Jun.
Article in English | MEDLINE | ID: mdl-9798650

ABSTRACT

The ragweed allergens Amb t 5 and Amb a 5 are among the smallest inhaled protein allergens known, containing a single, immunodominant T-cell epitope. In this study we analyzed the B-cell epitope structure of Amb t 5. The three-dimensional structures of Amb t 5 and Amb a 5 have been determined by NMR spectroscopy, providing a rare opportunity to analyze three-dimensional antigenic sites. Amb t 5 residues likely to be important for antigenicity were identified by examining the surface area of Amb t 5 accessible to a probe of the size of an antibody molecule. After changing these residues to the corresponding Amb a 5 residues, recombinant proteins were purified and tested for loss of antigenic activity. Inhibition radio-immunoassays, using sera from 8 individuals who had received immunotherapy with giant ragweed extract, allowed the mutations to be divided into three groups: (1) mutations that had little or no effect on antibody binding, (2) mutations that caused a loss of antigenic activity to a different degree in different sera and (3) mutations that drastically reduced antigenic activity in all sera tested. This last set of mutations clustered in the third loop of Amb t 5, suggesting that antibody recognition of Amb t 5, like T-cell recognition, is primarily directed towards a single, immunodominant site.


Subject(s)
Allergens/chemistry , Plant Proteins/chemistry , Pollen/immunology , Amino Acids/genetics , Antibody Formation , Antigen-Antibody Reactions/genetics , Antigens, Plant , Binding Sites, Antibody/genetics , Humans , Mutagenesis , Mutation/physiology , Plant Proteins/genetics , Plant Proteins/immunology , Protein Structure, Tertiary , Recombinant Proteins/genetics
7.
Mol Immunol ; 35(4): 249-57, 1998 Mar.
Article in English | MEDLINE | ID: mdl-9736341

ABSTRACT

The protein tyrosine kinase Csk downregulates the activity of the Src family of kinases and has a negative effect on signal transduction through several Src kinase-associated receptors. Because the Src-family kinase Lyn plays a pivotal role in FcepsilonRI-mediated cellular activation, we examined whether Csk is involved in FcepsilonRI signaling events. Using anti-Csk antibodies and recombinant fusion proteins we detected a single tyrosine-phosphorylated protein of 60 kD (herein referred to as 'p60') that associates with the SH2 domain of Csk after stimulation of the FcepsilonRI. p60 phosphorylation reached a maximum within one minute and remained constant while the receptors were aggregated; disaggregation of the receptors resulted in rapid dephosphorylation of p60. The phosphorylation of p60 was only detected after activation by IgE and antigen and not by stimulation with PMA and/or ionomycin. Phosphorylated p60 was associated entirely with the membrane fraction of the cells. A considerable fraction of Csk was associated with the membrane in both unstimulated and stimulated cells, this fraction did not change upon activation. p60 coprecipitated with Csk from both unstimulated and FcepsilonRI stimulated cells and was phosphorylated by the immunocomplex. Total kinase activity of Csk immunoprecipitates increased upon FcepsilonRI stimulation. p60 did not react with antibodies to a number of known signaling molecules, including the recently cloned, GAP-associated protein, p62dok. Our data demonstrate that Csk associates with a membrane-anchored protein complex that is directly involved in FcepsilonRI signal transduction.


Subject(s)
DNA-Binding Proteins , Protein-Tyrosine Kinases/metabolism , RNA-Binding Proteins , Receptors, IgE/physiology , src-Family Kinases/physiology , Animals , Blotting, Western , CSK Tyrosine-Protein Kinase , Oncogene Protein pp60(v-src)/metabolism , Phosphoproteins/analysis , Phosphoproteins/immunology , Phosphorylation , Precipitin Tests , Signal Transduction , Subcellular Fractions/chemistry , Tumor Cells, Cultured , src Homology Domains/physiology , src-Family Kinases/analysis , src-Family Kinases/immunology
8.
J Biol Chem ; 271(16): 9698-703, 1996 Apr 19.
Article in English | MEDLINE | ID: mdl-8621646

ABSTRACT

The protein-tyrosine kinase Csk is one of the main down-regulators of the Src family of kinases. Csk may be involved in the down-regulation of T cell receptor (TCR) signaling by C-terminal tyrosine phosphorylation of Lck and Fyn; however, it is not known how Csk activity is regulated or how it targets these Src family members. We used Jurkat T cells and normal human T cells to examine proteins that bind to the SH2 domain of Csk. In both Jurkat and normal T cells, the Src homology 2 (SH2) domain of Csk bound constitutively to a tyrosine-phosphorylated protein of 60 kDa (p60). The 60-kDa protein was detected in Csk immunoprecipitates from both unstimulated and CD3-stimulated cells. In addition to p60, a protein of 190 kDa coprecipitated with Csk, and both proteins were phosphorylated on tyrosine residues by the immunocomplex. Small amounts of GTPase-activating protein (GAP) were detected in anti-Csk immunoprecipitates, suggesting that p60 may be a GAP-associated protein. Our data demonstrate that the SH2 domain of Csk specifically associates with at least two tyrosine-phosphorylated proteins in normal human T cells, that this association is independent of TCR/CD3 activation, and that Csk may be a part of a multiprotein complex containing GAP.


Subject(s)
Phosphoproteins/metabolism , Protein-Tyrosine Kinases/metabolism , T-Lymphocytes/enzymology , Amino Acid Sequence , CSK Tyrosine-Protein Kinase , Cell Line , Electrophoresis, Polyacrylamide Gel , Glutathione Transferase/biosynthesis , Glutathione Transferase/isolation & purification , Humans , Molecular Sequence Data , Molecular Weight , Phosphoproteins/isolation & purification , Phosphotyrosine , Protein Binding , Protein-Tyrosine Kinases/biosynthesis , Protein-Tyrosine Kinases/isolation & purification , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/isolation & purification , Recombinant Fusion Proteins/metabolism , Transfection , Tumor Cells, Cultured , src-Family Kinases
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