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1.
J Leukoc Biol ; 68(5): 662-8, 2000 Nov.
Article in English | MEDLINE | ID: mdl-11073105

ABSTRACT

Human neutrophils incubated with the anti-HLA-DR mAb Lym-1, plus PMA, induced significant cytolysis of B lymphoma cells compared with Lym-1 and PMA alone. The effect of PMA was independent of the ability of the compound to stimulate neutrophil-respiratory burst. In fact, first, neutrophils from a patient with chronic granulomatous disease were cytolytically effective in spite of their inability to produce oxidants. Second, various kinase inhibitors exerted different effects on the PMA-stimulated cytolytic system and neutrophil-oxidative burst. Previous studies have shown the involvement of the FcgammaRII, CD11b-CD18 integrins, and CD66b glycoproteins in the Lym-1 mAb-dependent cytolysis by GM-CSF-stimulated neutrophils. The present PMA-stimulated system was inhibited by the anti-FcgammaRII mAb IV.3, the anti-CD18 mAb MEM 48, and the anti-CD11b mAb 2LPM19c but not by the anti-CD66b mAb 80H3 and N-acetyl-D-glucosamine. Furthermore, the PMA- and GM-CSF-stimulated cytolysis was insensitive and sensitive to inhibition by pertussis toxin, respectively. Thus, the use of PMA and GMCSF as neutrophil stimulants uncovers the existence of distinct mechanisms of Lym-1 mAb-mediated cytolysis.


Subject(s)
Antibodies, Monoclonal/immunology , Antibody-Dependent Cell Cytotoxicity/immunology , Antigens, Neoplasm , B-Lymphocytes/immunology , Burkitt Lymphoma/immunology , Cell Adhesion Molecules , Neutrophils/immunology , Receptors, IgG/immunology , Adjuvants, Immunologic/pharmacology , Antibodies, Monoclonal/pharmacology , Antibodies, Monoclonal, Murine-Derived , Antibody-Dependent Cell Cytotoxicity/drug effects , Antigens, CD , B-Lymphocytes/cytology , Burkitt Lymphoma/pathology , CD18 Antigens/immunology , GPI-Linked Proteins , Granulocyte-Macrophage Colony-Stimulating Factor/pharmacology , Humans , Macrophage-1 Antigen/immunology , Membrane Glycoproteins/immunology , Neutrophil Activation/drug effects , Neutrophil Activation/immunology , Neutrophils/drug effects , Pertussis Toxin , Signal Transduction/drug effects , Signal Transduction/immunology , Tetradecanoylphorbol Acetate/pharmacology , Virulence Factors, Bordetella/pharmacology
2.
Br J Cancer ; 80(3-4): 331-7, 1999 May.
Article in English | MEDLINE | ID: mdl-10408834

ABSTRACT

Human neutrophils, incubated with Cr51-labelled B lymphoblastoid Raji cells in the presence of the anti-target monoclonal antibody (mAb) Lym-1 plus formyl-methionyl-leucyl-phenylalanine (FMLP) or tumour necrosis factor alpha (TNF-alpha), were found to induce significant C51 release, i.e. significant cytolysis. The lytic process was inhibited by mAb IV.3, specific for the Fcgamma receptor (FcgammaR) type II. The mAb 3G8, which reacts with FcgammaR type III, was ineffective. Moreover, the lysis was inhibited by the anti-CD18 mAb MEM-48. These data suggest that FMLP/Lym-1 as well as TNF-alpha/Lym-1 cytolytic systems strictly require FcgammaRII and CD18 integrins. As the lysis induced by TNF-alpha/Lym-1 was prevented by pertussis toxin (PT), PT-sensitive G-proteins are likely to intervene in post-FcgammaRII signal transduction. Both the FMLP- and the TNF-alpha-dependent systems were also found to be equally susceptible to inhibition by various inhibitors of kinases (genistein, staurosporin, 1-(5-isoquinolinnylsulphonyl)-2-methylpiperazine and wortmannin). On the contrary, an inhibitor of protein kinase C (bis-indolyl-maleimide, BIM) was effective only in the FMLP/Lym-1 cytolytic system. Therefore, it appears that signals delivered by FMLP or TNF-alpha, BIM-sensitive and insensitive respectively, converge and synergize with those from G-protein-coupled FcgammaRII and, probably, CD18-integrins to promote the expression of the neutrophil cytolytic potential.


Subject(s)
Adjuvants, Immunologic/pharmacology , Antibodies, Monoclonal/pharmacology , Antibody-Dependent Cell Cytotoxicity/immunology , Lymphoma, B-Cell/immunology , N-Formylmethionine Leucyl-Phenylalanine/pharmacology , Neutrophils/immunology , Tumor Necrosis Factor-alpha/pharmacology , Adult , CD18 Antigens/immunology , Humans , Lymphocyte Activation , Middle Aged , Neutrophils/metabolism , Receptors, IgG/biosynthesis , Receptors, IgG/immunology , Signal Transduction/immunology , Tumor Cells, Cultured
3.
J Immunol ; 162(6): 3601-6, 1999 Mar 15.
Article in English | MEDLINE | ID: mdl-10092820

ABSTRACT

It has been recently shown that Fas ligand (FasL) expression on islet beta grafts results in neutrophilic infiltration and graft rejection. In this study, we show that human recombinant soluble FasL is endowed with potent chemotactic properties toward human neutrophilic polymorphonuclear leukocytes (neutrophils) at concentrations incapable of inducing cell apoptosis. Furthermore, neutrophils exposed to soluble FasL did not display detectable change of intracellular Ca2+ and did not undergo superoxide production or exocytosis of primary and secondary granules. Our results show that FasL is a potent chemoattractant for human neutrophils without evoking their secretory responses. This finding suggests a novel proinflammatory function for this ligand and may help to clarify the mechanism governing FasL-mediated graft rejection, thereby offering rational bases for controlling and modulating FasL-based immunotherapies.


Subject(s)
Chemotaxis, Leukocyte/physiology , Membrane Glycoproteins/physiology , Neutrophils/physiology , fas Receptor/metabolism , Adult , Calcium/metabolism , Cell Movement/physiology , Dose-Response Relationship, Immunologic , Fas Ligand Protein , Humans , Ligands , Neutrophil Activation/physiology , Solubility
4.
Inflamm Res ; 48(12): 637-42, 1999 Dec.
Article in English | MEDLINE | ID: mdl-10669115

ABSTRACT

OBJECTIVE AND DESIGN: In the present work, we studied the role of cell-derived adenosine in both the physiologic regulation and pharmacologic control of the exocytosis of azurophilic granules of neutrophils exposed to tumor necrosis factor alpha (TNF) and stimulated with some chemoattractants. MATERIAL AND METHODS: Human neutrophils were pre-incubated in the absence or presence of TNF. Thereafter, the appropriate chemoattractant was added to the cells. After incubation, the cell-free supernatant was collected for testing elastase activity and intracellular cAMP levels. Results, expressed as mean +/- 1 SD, were evaluated by unpaired, two-tailed Student's t-test and by analysis of variance followed by Student-Newman-Keuls multiple comparisons test. RESULTS: Neutrophil incubation with 10 ng/ml TNF or 0.1 micromol/l N-formyl-met-leu-phe (fMLP) failed to release elastase activity (NE) (NE in absence of stimulus: 23.1 +/- 5.7 nmol/h; TNF-induced NE: 26.4 +/- 14.4 nmol/h; fMLP-induced NE: 27.0 +/- 9.9 nmol/h). Neutrophils, pre-exposed to various amounts of TNF, released elastase in response to 0.1 micromol/l fMLP in a dose-dependent manner (NE in presence of 10 ng/ml TNF and 0.1 micromol/l fMLP: 133.7 +/- 24.0 nmoles/h). As compared with fMLP, C5a had lower activity (NE in presence of 10 ng/ml TNF and 0.1 micromol/l C5a: 66.4 +/- 25.1 nmoles/h), whereas interleukin-8, platelet activating factor and leukotriene B4 were ineffective. The secretory response of TNF-primed neutrophils to fMLP was inhibited by adenosine in a dose-dependent manner (IC50 = 5.18 +/- 7.1 micromol/l). The addition of adenosine deaminase (ADA) to TNF-primed neutrophils resulted in increased secretory response to fMLP (NE in absence and presence of 0.25 U/ml ADA: 71.5 +/- 11.0 and 107.3 +/- 18.6 respectively, P = 0.060). Moreover, two inhibitors of phosphodiesterase type IV (RO 20-1724 and nimesulide) reduced the elastase release only in the absence of ADA (RO 20-1724: percent inhibition in absence or presence of ADA = 20.2 +/- 15.0 and 4.4 +/- 5.1 respectively; nimesulide: percent inhibition in absence or presence of ADA = 22.2 +/- 19.6 and 0.8 +/- 3.0 respectively). Similarly, RO 20-1724 and nimesulide increased intracellular cAMP levels only in absence of ADA (RO 20-1724: percent cAMP increment in absence or presence of ADA = 215.4 +/- 97.5 and 47.3 +/- 53.3 respectively; nimesulide: percent cAMP increment in absence or presence of ADA = 177.7 +/- 19.0 and 19.5 +/- 29.3 respectively). CONCLUSIONS: Endogenous adenosine down-regulates the cell secretory response and is instrumental in uncovering the susceptibility of azurophilic granule exocytosis to control by inhibitors of phosphodiesterase type IV.


Subject(s)
Adenosine/physiology , Chemotactic Factors/pharmacology , Homeostasis/physiology , Neutrophils/drug effects , Pancreatic Elastase/metabolism , Tumor Necrosis Factor-alpha/pharmacology , 3',5'-Cyclic-AMP Phosphodiesterases/antagonists & inhibitors , 4-(3-Butoxy-4-methoxybenzyl)-2-imidazolidinone/pharmacology , Adenosine Deaminase/pharmacology , Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Complement C5a/pharmacology , Culture Media , Cyclic AMP/metabolism , Cyclic Nucleotide Phosphodiesterases, Type 4 , Cytoplasmic Granules/drug effects , Cytoplasmic Granules/enzymology , Humans , In Vitro Techniques , Indicators and Reagents , N-Formylmethionine Leucyl-Phenylalanine/pharmacology , Neutrophils/metabolism , Neutrophils/ultrastructure , Phosphodiesterase Inhibitors/pharmacology , Sulfonamides/pharmacology
5.
Inflamm Res ; 47(8): 345-50, 1998 Aug.
Article in English | MEDLINE | ID: mdl-9754869

ABSTRACT

OBJECTIVE AND DESIGN: We investigated the in vitro responsiveness of neutrophils adherent to fibronectin (FN) and laminin (LM), toward natural pro-inflammatory and/or phagocyte-activating agents. MATERIALS AND METHODS: Neutrophils from normal volunteers were layered on polystyrene wells precoated or not with FN and/or LM and tested for their ability of responding to eleven pro-inflammatory mediators by evaluation of superoxide anion (O2-) production and adherence. Results, expressed as mean +/-1SEM, were evaluated by non-parametric analyses (Mann-Whitney U-test or Kruskal-Wallis non-parametric ANOVA analysis) RESULTS: Precoating polystyrene wells with LM or FN prevented the plastic-induced neutrophil (O2-) production. Among eleven agents, tumor necrosis factor-alpha (TNF, 3.0+/-0.3 nmoles (O2-)/5 x 10(4) neutrophils/180 min, p < 0.001), granulocyte-macrophage colony stimulating factor (GM-CSF, 2.1+/-0.3 nmoles (O2-)/5 x 10(4) neutrophils/180 min, p < 0.05) and formyl-peptides (fMLP, 2.5+/-0.5 nmoles (O2-)/5 x 10(4) neutrophils/180min, p < 0.01) caused massive (O2-) production by neutrophils adherent to FN. None of the mediators was capable of triggering (O2-) production by neutrophils adherent to LM. LM, mixed with FN to coat wells, caused a dose-dependent inhibition of the oxidative burst triggered by TNF (IC50 LM: 0.84+/-0.03 microg, mean+/-1 SEM), GM-CSF (IC50 LM: 0.36+/-0.16micro/g, mean+/-1SEM) and fMLP (IC50 LM: 0.54+/-0.008 microg, mean+/-1 SEM). To the contrary, fMLP (85.5+/-27.7%), TNF (163.1+/-67.5%), and GM-CSF (121.8+/-66.4%) caused a significant augmentation of neutrophil adherence to LM, suggesting that LM-mediated inhibition of neutrophil oxidative metabolism does not depend on the concomitant LM-induced inhibition of neutrophil adherence. Finally, neither solid-phase FN nor LM affected (O2-) production by neutrophils in response to immune complexes. CONCLUSIONS: Extracellular matrix glycoproteins dictate the response of neutrophils to soluble mediators but not to immune complexes. This appears to be a biologically meaningful mechanism to localise the risk of cellular reactions to mediators that are able to diffuse easily from tissue sites of generation and become widely distributed in body fluids during inflammatory diseases.


Subject(s)
Chemotactic Factors/pharmacology , Cytokines/pharmacology , Extracellular Matrix Proteins/physiology , Neutrophils/metabolism , Respiratory Burst/drug effects , Respiratory Burst/physiology , Cells, Cultured , Fibronectins/pharmacology , Humans , Laminin/pharmacology , Male
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