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1.
Leukemia ; 25(8): 1268-77, 2011 Aug.
Article in English | MEDLINE | ID: mdl-21546901

ABSTRACT

Several chemokines/chemokine receptors such as CCR7, CXCR4 and CXCR5 attract chronic lymphocytic leukemia (CLL) cells to specific microenvironments. Here we have investigated whether the CX(3)CR1/CX(3)CL1 axis is involved in the interaction of CLL with their microenvironment. CLL cells from 52 patients expressed surface CX(3)CR1 and CX(3)CL1 and released constitutively soluble CX(3)CL1. One third of these were attracted in vitro by soluble CX(3)CL1. CX(3)CL1-induced phosphorylation of PI3K, Erk1/2, p38, Akt and Src was involved in induction of CLL chemotaxis. Leukemic B cells upregulated CXCR4 upon incubation with CX(3)CL1 and this was paralleled by increased chemotaxis to CXCL12. Akt phosphorylation was involved in CX(3)CL1-induced upregulation of CXCR4 on CLL. In proliferation centers from CLL lymph node and bone marrow, CX(3)CL1 was expressed by CLL cells whereas CX(3)CR1 was detected in CLL and stromal cells. Nurselike cells (NLCs) generated from CLL patient blood co-expressed surface CX(3)CR1 and CX(3)CL1, but did not secrete soluble CX(3)CL1. Only half of NLC cell fractions were attracted in vitro by CX(3)CL1. In conclusion, the CX(3)CR1/CX(3)CL1 system may contribute to interactions between CLL cells and tumor microenvironment by increasing CXCL12-mediated attraction of leukemic cells to NLC and promoting directly adhesion of CLL cells to NLC.


Subject(s)
Cell Communication , Chemokine CX3CL1/physiology , Leukemia, Lymphocytic, Chronic, B-Cell/pathology , Receptors, Chemokine/physiology , Tumor Microenvironment , Adult , Aged , Aged, 80 and over , Antigens, CD/analysis , Antigens, Differentiation, Myelomonocytic/analysis , CX3C Chemokine Receptor 1 , Chemotaxis , Female , Humans , Leukemia, Lymphocytic, Chronic, B-Cell/immunology , Lymph Nodes/metabolism , Male , Middle Aged , Phosphorylation , Proto-Oncogene Proteins c-akt/metabolism , Signal Transduction
2.
Clin Exp Pharmacol Physiol ; 36(11): e72-7, 2009 Nov.
Article in English | MEDLINE | ID: mdl-19671069

ABSTRACT

1. Chlorhexidine digluconate has been used as a topical antiseptic in the treatment of acne vulgaris and periodontitis. The acute phase of these diseases involves neutrophilic infiltration. Neutrophil activation and recruitment to inflammatory sites are crucial in both protection against bacterial infection and the induction of hystotoxic damage. Activated neutrophils release several enzymes, including elastase and myeloperoxidase (MPO), which contribute to tissue injury via direct toxic actions, the generation of oxidants and inactivation of protective factors, such as alpha1-antitrypsin (alpha1-AT). In the present study, we investigated whether chlorhexidine can modulate neutrophil-mediated histotoxicity. 2. Human primary neutrophils were isolated from healthy donors. Inactivation of alpha1-AT by neutrophils or hypochlorous acid (HOCl) was evaluated by spectrophotometry and sodium dodecyl sulphate-polyacrylamide gel electrophoresis analysis of its capacity to complex with porcine pancreatic elastase (PPE). Neutrophil generation of HOCl, superoxide anion and MPO release were assessed spectrophometrically. 3. Chlorhexidine (0, 0.5, 1, 5 and 10 micromol/L) dose-dependently prevented HOCl-induced inactivation of alpha1-AT and reduced HOCl recovery from phorbol myristate acetate (PMA)-treated human neutrophils, but did not inhibit superoxide anion and MPO release. Chlorhexidine directly inhibited HOCl recovery from neutrophils and HOCl-induced inactivation of alpha1-AT in a cell-free assay. Accordingly, chlorhexidine reversed HOCl-mediated inhibition of alpha1-AT capacity to complex with PPE. 4. These data suggest that chlorhexidine prevents neutrophil-induced alpha1-AT inactivation via a direct inhibitory action on HOCl. Although highly speculative, the present study indicates that chlorhexidine may protect inflamed tissues not only through its antimicrobial properties, but also via a direct anti-inflammatory effect on neutrophil toxic products.


Subject(s)
Anti-Inflammatory Agents/pharmacology , Chlorhexidine/analogs & derivatives , Neutrophil Activation/drug effects , Neutrophils/drug effects , alpha 1-Antitrypsin/metabolism , Cells, Cultured , Chlorhexidine/pharmacology , Dose-Response Relationship, Drug , Enzyme Inhibitors/pharmacology , Humans , Hypochlorous Acid/antagonists & inhibitors , Hypochlorous Acid/metabolism , Hypochlorous Acid/pharmacology , Models, Immunological , Neutrophils/enzymology , Neutrophils/metabolism , Peroxidase/metabolism , Superoxides/metabolism , Tetradecanoylphorbol Acetate/pharmacology
3.
Clin Exp Pharmacol Physiol ; 36(11): 1132-5, 2009 Nov.
Article in English | MEDLINE | ID: mdl-19473188

ABSTRACT

1. Neutrophils release several histotoxic molecules that cause tissue injury. Neutrophil apoptosis is a crucial process that governs the persistence of inflammatory disorders and tissue damage. Thus, in the present study, we investigated whether the anti-inflammatory drug sulphasalazine (SSZ) affects neutrophil apoptosis in the presence of insoluble immune complex (IC). 2. Neutrophils were obtained from healthy donors. Neutrophils were resuspended in incubation medium and incubated for 2-12 h with or without 10, 30 or 100 micromol/L SSZ and 25 microg/mL IC. In some experiments, cells were co-incubated with 20 micromol/L Z-IETD-fmk (a caspase 8 inhibitor) or 20 micromol/L Z-LEHD-fmk (a caspase 9 inhibitor). Apoptosis was evaluated morphologically on cytological preparations stained with May-Grünwald-Giemsa as well as by flow cytometry analysis of annexin V and propidium iodide staining. Caspase 3 activity was determined spectrophotometrically. 3. At 100 micromol/L, SSZ significantly accelerated IC-induced neutrophil apoptosis. Treatment of neutrophils with 20 micromol/L of the caspase 8 or 9 inhibitors Z-IETD-fmk or Z-LEHD-fmk, respectively, demonstrated that the SSZ-induced pro-apoptotic effect was mediated by a caspase 8- but not caspase 9-dependent pathway. The caspase 3 activity assay showed that treatment with 100 micromol/L SSZ increased caspase 3 activation. 4. In conclusion, the results of the present study indicate that it is possible that the molecular mechanism underlying SSZ protection against neutrophil-mediated tissue injury inflammatory disorders, such as rheumatoid arthritis and inflammatory bowel diseases, involves a caspase 8-dependent pathway.


Subject(s)
Antigen-Antibody Complex/pharmacology , Apoptosis/drug effects , Caspase Inhibitors , Enzyme Inhibitors/pharmacology , Neutrophils/drug effects , Signal Transduction/drug effects , Sulfasalazine/pharmacology , Apoptosis/physiology , Caspases/metabolism , Cell Survival/drug effects , Cells, Cultured , Humans , Neutrophils/metabolism , Neutrophils/pathology , Oligopeptides/pharmacology
4.
Curr Drug Targets ; 7(1): 81-90, 2006 Jan.
Article in English | MEDLINE | ID: mdl-16454701

ABSTRACT

Chemokines are low molecular weight cytokines specialized in leukocyte recruitment. Recent studies have shown that tumor cells of hematopoietic and non hematopoietic origin express different chemokine receptors that may be involved in neoplastic cell growth, metastasis and angiogenesis. Human lymphoproliferative disorders arise from the malignant transformation of normal lymphoid cells frozen at discrete maturational stages. Studies performed with acute or chronic lymphoproliferative disorders have shown that CXCR4, the unique receptor for CXCL12, is up-regulated in many B and T cells malignancies and may be involved in metastatic localization of the neoplastic elements. Additional chemokine receptors are expressed in the individual lymphoproliferative disorders, but some of these are often non functional. Here we shall review the state of the art on chemokine receptor expression and function in human lymphoproliferative disorders, stressing the potential value of chemokines receptors as novel therapeutic targets. In this respect, small antagonistic peptides are being produced by pharmaceutical companies and hold great promise for clinical application.


Subject(s)
Chemokines/physiology , Lymphoproliferative Disorders/physiopathology , Receptors, Chemokine/physiology , Animals , Antineoplastic Agents/therapeutic use , B-Lymphocytes/physiology , Hodgkin Disease/physiopathology , Humans , Lymphoproliferative Disorders/drug therapy , Lymphoproliferative Disorders/pathology , Neoplasms/pathology , Neoplasms/physiopathology , Receptors, Chemokine/drug effects
5.
Int J Immunopathol Pharmacol ; 18(3): 403-15, 2005.
Article in English | MEDLINE | ID: mdl-16164824

ABSTRACT

Monocytes and macrophages play a key role in the initiation and persistence of inflammatory reactions. The possibility to interfere with the survival of these cells, once recruited and activated at sites of inflammation, is an attractive therapeutic option. Although resting monocytes are susceptible to pharmacologically induced apoptosis, no data are available about the possibility to modulate the survival of activated monocytes. The present work was planned to investigate if dexamethasone is able to promote apoptosis of human monocytes activated by immune complexes. When monocytes were cultured with immune complexes, a dose-dependent inhibition of apoptosis was observed. Dexamethasone stimulated apoptosis of resting and activated monocytes in a dose-dependent manner. Both the immune complex inhibitory activity and dexamethasone stimulatory properties depend on NF-kappaB/XIAP and Ras/MEK/ERK/CD95 pathways. In fact, the exposure of monocytes to immune complexes increased NF-kB activation and XIAP expression, which in turn were inhibited by dexamethasone. On the other hand, immune complex-stimulated monocytes displayed a reduced expression of CD95, which is prevented by dexamethasone, as well as by MEK inhibitor U0126. Furthermore, anti-CD95 ZB4 mAb prevented dexamethasone-induced apoptosis in immune complex stimulated monocytes. Similarly, ZB4 inhibited dexamethasone-mediated augmentation of caspase 3 activity. The present findings suggest that Fc triggering by insoluble immune complexes result in the activation of two intracellular pathways crucial for the survival of monocytes: 1. Ras/MEK/ERK pathway responsible for the down-regulation of CD95 expression; 2. NF-kappaB pathway governing the expression of XIAP. Both the pathways are susceptible to inhibition by monocyte treatment with pharmacologic concentrations of dexamethasone.


Subject(s)
Anti-Inflammatory Agents/pharmacology , Apoptosis/immunology , Dexamethasone/pharmacology , Monocytes/immunology , X-Linked Inhibitor of Apoptosis Protein/immunology , fas Receptor/immunology , Acridine Orange/metabolism , Anti-Inflammatory Agents/metabolism , Antigen-Antibody Complex/immunology , Apoptosis/drug effects , Blotting, Western , Cells, Cultured , Densitometry , Dexamethasone/metabolism , Dose-Response Relationship, Drug , Electrophoretic Mobility Shift Assay , Flow Cytometry , Fluorescent Antibody Technique, Direct , Humans , Monocytes/drug effects
6.
Br J Cancer ; 92(10): 1948-52, 2005 May 23.
Article in English | MEDLINE | ID: mdl-15841077

ABSTRACT

In cancer patients, the ability to detect disseminated tumour cells in peripheral blood or bone marrow could improve prognosis and consent both early detection of metastatic disease and monitoring of the efficacy of systemic therapy. These objectives remain elusive mainly due to the lack of specific genetic markers for solid tumours. The use of surrogate tissue-specific markers can reduce the specificity of the assays and give rise to a clinically unacceptable false-positive rate. Mammaglobin (MAM) and maspin are two putative breast tissue-specific markers frequently used for detection of occult tumour cells in the peripheral blood, bone marrow and lymph nodes of breast cancer patients. In this study, it was evaluated whether MAM and maspin gene expression may be induced in the normal blood and bone marrow cells exposed to a panel of cytokines, including chemotactic factors (C5a, interleukin (IL)-8), LPS, proinflammatory cytokines (TNF-alpha, IL-1beta) and growth factors (IL-3, granulocyte-macrophage colony-stimulating factor, granulocyte colony-stimulating factor). The experimental data show that all cytokines included in the panel, except for IL-8, were able to induce maspin expression; on the contrary, MAM gene was never induced. These results suggest that MAM is more specific than maspin and that the possible interference of cytokines should be taken into account in interpreting molecular assays for detection of isolated tumour cells.


Subject(s)
Biomarkers, Tumor/biosynthesis , Breast Neoplasms/genetics , Breast Neoplasms/pathology , Cytokines/pharmacology , Gene Expression Regulation/drug effects , Neoplasm Metastasis/diagnosis , Neoplasm Proteins/biosynthesis , Serpins/biosynthesis , Uteroglobin/biosynthesis , Biomarkers, Tumor/analysis , False Positive Reactions , Genes, Tumor Suppressor , Humans , Mammaglobin A , Neoplasm Metastasis/genetics , Neoplastic Cells, Circulating , Reverse Transcriptase Polymerase Chain Reaction , Sensitivity and Specificity , Tumor Cells, Cultured
7.
Rheumatology (Oxford) ; 41(11): 1249-60, 2002 Nov.
Article in English | MEDLINE | ID: mdl-12421997

ABSTRACT

OBJECTIVE: In synovial fluid (SF) from patients with rheumatoid arthritis (RA), neutrophils are exposed to proinflammatory mediators endowed with either anti-apoptotic or pro-apoptotic properties. We investigated neutrophil apoptosis in the presence of SF from 11 RA patients. METHODS: SF was obtained from affected knees of 11 patients with RA. Human neutrophil apoptosis was evaluated by light microscopic examination and flow-cytometric analysis of annexin V binding. Immune complex-induced neutrophil activation was evaluated as superoxide anion production. Adenosine levels in SF were detected by chromatographic analysis and cytokine levels were studied by enzyme-linked immunosorbent assay. RESULTS: Spontaneous and immune complex-triggered neutrophil apoptosis was reduced by SF from eight out of 11 patients. Immune complex-induced neutrophil activation was unaffected by SF. The cytokines tested had no role in promoting the anti-apoptotic activity of SF. On the contrary, the anti-apoptotic activity of SF was found to depend on the presence of adenosine. Adenosine levels detected in the various samples of SF correlated significantly with the anti-apoptotic activity of the fluids and with the number of apoptotic neutrophils detected in the articular exudate. CONCLUSION: The microenvironment of rheumatoid SF is a proinflammatory milieu responsible for the in loco persistence of activated and long-surviving neutrophils. Adenosine plays a crucial role in this phenomenon, which is related to anti-apoptotic activity.


Subject(s)
Adenosine/metabolism , Apoptosis/drug effects , Arthritis, Rheumatoid/physiopathology , Cytokines/metabolism , Neutrophils/drug effects , Neutrophils/physiology , Synovial Fluid/chemistry , Synovial Fluid/cytology , Adult , Cells, Cultured , Culture Media , Female , Flow Cytometry , Fluorescent Antibody Technique , Granulocyte-Macrophage Colony-Stimulating Factor/pharmacology , Humans , Interleukin-15/pharmacology , Interleukin-2/pharmacology , Interleukin-8/pharmacology , Knee Joint , Male , Middle Aged , Sensitivity and Specificity , Tumor Necrosis Factor-alpha/pharmacology
8.
Br J Cancer ; 85(3): 463-9, 2001 Aug 03.
Article in English | MEDLINE | ID: mdl-11487281

ABSTRACT

Chimaeric Lym-1 (chLym-1) is a monoclonal antibody generated by fusing the variable region genes of murine Lym-1 to human gamma1 and kappa constant regions. Owing to its selectivity and avidity for human malignant B cells, it is an attractive candidate for developing immune-interventions in B-lymphomas. In the attempt to identify rational bases for optimizing potential chLym-1 related therapeutic approaches, we studied the ability of this ch-mAb to trigger neutrophil-mediated Raji cell cytolysis in cooperation with two neutrophil-related cytokines, G-CSF and GM-CSF. ChLym-1 triggered low levels of cytolysis by normal neutrophils but induced consistent cytolysis in neutrophils from individuals treated with G-CSF. When exposed to GM-CSF, neutrophils from subjects treated with G-CSF became potent effectors, also leading to 75% lysis. By using mAbs specific for distinct FcgammaRs, normal neutrophils were inhibited by mAb IV.3, suggesting the intervention of FcgammaRII, constitutively expressed on the cells. On the other hand, neutrophils from patients treated with G-CSF were inhibited by mAb IV.3 plus mAb 197, a finding consistent with a cooperative intervention of FCgammaRII and G-CSF-induced FcgammaRI. The anti-FcgammaRIII mAb 3G8 promoted significant enhancement of the neutrophil cytolytic efficiency. Therefore, neutrophil FcgammaRIII behaves as a down-regulator of the cytolytic potential. The present findings suggest new attempts to develop mAb-based and G-CSF/GM-CSF combined immune-interventions in B lymphomas.


Subject(s)
Antibodies, Monoclonal/immunology , Antibody-Dependent Cell Cytotoxicity/immunology , Burkitt Lymphoma/therapy , Granulocyte Colony-Stimulating Factor/therapeutic use , Granulocyte-Macrophage Colony-Stimulating Factor/pharmacology , Neutrophils/immunology , Receptors, IgG/physiology , Antibodies, Monoclonal, Murine-Derived , Antibody Specificity , Antigens, CD/physiology , B-Lymphocytes/cytology , B-Lymphocytes/immunology , Burkitt Lymphoma/immunology , Burkitt Lymphoma/pathology , Cytotoxicity, Immunologic/immunology , Humans , Neutrophil Activation/drug effects , Neutrophil Activation/immunology , Phagocytes/immunology , Phagocytes/metabolism , Tumor Cells, Cultured
9.
J Pharm Biomed Anal ; 24(5-6): 1143-6, 2001 Mar.
Article in English | MEDLINE | ID: mdl-11248511

ABSTRACT

A high-performance liquid chromatographic method has been developed for the quantitative determination of adenosine in human synovial fluid. The method is simple, rapid and, overall, selective. No interference with the components of the biological matrix was observed in these chromatographic conditions. An ODS (250 x 4.6 mm) 5 microm column was used with an isocratic elution of a phosphate buffer-acetonitrile mobile phase. Detection was carried out on a UV detector at 260 nm. Calibration curve was found to be linear in the 0.7--70 microg ml(-1) range. Linear regression analysis of the data demonstrates the efficacy of the method in terms of precision and accuracy. The precision of this method, calculated as the relative standard deviation (RSD) of the recoveries (1.57--2.21%), was excellent. The limits of quantitation (LOQ) and detection (LOD) were respectively 0.7 and 0.2 microg ml(-1). The method was applied to some samples of synovial effusion from patients affected by rheumatoid arthritis. The concentrations of adenosine which were found were included in the range of the calibration curve.


Subject(s)
Adenosine/analysis , Chromatography, High Pressure Liquid/methods , Synovial Fluid/metabolism , Arthritis, Rheumatoid/metabolism , Calibration , Humans , Reproducibility of Results , Sensitivity and Specificity , Spectrophotometry, Ultraviolet
10.
Free Radic Biol Med ; 30(2): 161-9, 2001 Jan 15.
Article in English | MEDLINE | ID: mdl-11163533

ABSTRACT

Neutrophils are involved in the pathogenesis of various inflammatory diseases. One of the mechanisms by which neutrophilic inflammation is generated is immune complex (IC) deposition in tissue. As the clearance of apoptotic neutrophils from inflamed sites is considered a crucial determinant for the resolution of inflammation, we investigated the effects of IC-induced neutrophil activation on apoptosis and the mechanisms regulating neutrophil survival. Our results show that IC stimulated apoptosis efficiently. The percentage of apoptotic neutrophils was reduced by the anti-FcgammaRII mAb IV.3, but not by anti-FcgammaRIII mAb 3G8. The spontaneous apoptosis was completely inhibited by the antioxidant compound catalase, which in turn prevented only partially the apoptosis in presence of IC. The oxidative metabolism triggered by IC was inhibited only blocking both FcgammaRII and FcgammaRIII. Neutrophils from patients with chronic granulomatous disease, congenitally incapable of producing oxidants, showed low level of spontaneous apoptosis, but underwent a nearly 3-fold increment in the apoptosis rate when incubated with IC. In conclusion, neutrophil apoptosis appears to be a process governed by multiple pathways, some of which are strictly ROS-dependent, others acting in a nonoxidative manner. In particular, the herein shown FcgammaRII-dependent, ROS-independent, signal-inducing neutrophil apoptosis may uncover new pharmacological targets for the promotion of cell removal from sites of inflammation, thereby favoring the resolution of the inflammatory process.


Subject(s)
Antigen-Antibody Complex/immunology , Apoptosis , Neutrophils/cytology , Neutrophils/immunology , Animals , Antibodies, Monoclonal/immunology , Antibodies, Monoclonal/pharmacology , Antioxidants/metabolism , Caspase 3 , Caspases/metabolism , Catalase/metabolism , Cell Survival , Dose-Response Relationship, Immunologic , Flow Cytometry , Fluoresceins , Granulomatous Disease, Chronic/immunology , Granulomatous Disease, Chronic/metabolism , Humans , Inflammation/immunology , Neutrophils/metabolism , Neutrophils/pathology , Oxidation-Reduction , Rabbits , Reactive Oxygen Species/metabolism , Receptors, IgG/antagonists & inhibitors , Receptors, IgG/immunology , Signal Transduction
11.
J Leukoc Biol ; 68(6): 873-80, 2000 Dec.
Article in English | MEDLINE | ID: mdl-11129655

ABSTRACT

In many types of cells, ligation of human leukocyte antigens (HLA) Class I molecules with specific mAbs results in the transduction of signals that trigger different cell functions. We have investigated the effects of Class I ligation in human neutrophils. After several hours in culture, neutrophils split spontaneously into two subpopulations, one with normal and the other with reduced levels of Class I. The latter subpopulation displayed high binding capacity for Annexin V, showed a hypodiploid peak, electrophoretic DNA fragmentation, and morphological features of apoptotic cells. The addition of drugs known to delay apoptosis (GM-CSF or cAMP) resulted in a reduction of Class I modulation. Furthermore, ligation of surface Class I with F(ab')2 fragments of the anti-Class I mAb W6/32 resulted in a delay in the progression of apoptosis. These data indicate that this surface Class I molecule is a marker of age-related apoptosis, and the ligation of these molecules results in the transduction of a signal that inhibits apoptosis. Thus, the downregulation of HLA Class I molecules in aging neutrophils prevents their halting the apoptotic process.


Subject(s)
Apoptosis/physiology , Histocompatibility Antigens Class I/physiology , Neutrophils/cytology , Adult , Annexin A5/metabolism , Antibodies, Monoclonal/immunology , Antigen-Antibody Reactions , Apoptosis/drug effects , Cellular Senescence , Cyclic AMP/pharmacology , DNA Fragmentation , Down-Regulation , Gene Expression Regulation , Granulocyte-Macrophage Colony-Stimulating Factor/pharmacology , Histocompatibility Antigens Class I/immunology , Humans , Immunoglobulin Fab Fragments/immunology , Lewis X Antigen/analysis , Male , Neutrophils/drug effects , Receptors, IgG/analysis , Receptors, IgG/biosynthesis , Receptors, IgG/genetics , Signal Transduction , Tetradecanoylphorbol Acetate/pharmacology
12.
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
13.
J Natl Cancer Inst ; 92(8): 628-35, 2000 Apr 19.
Article in English | MEDLINE | ID: mdl-10772680

ABSTRACT

BACKGROUND: Follicular center lymphoma displays widespread lymph node involvement at diagnosis. The chemoattractants that control the locomotion of follicular center lymphoma B cells have not been established. Stromal cell-derived factor-1 (SDF-1) is a CXC-class chemokine that enhances the migration of normal human B cells and is expressed in peripheral lymphoid tissues. Here we have investigated 1) whether SDF-1 stimulates the in vitro locomotion of follicular center lymphoma B cells and of their presumed normal counterparts (i. e., germinal center B cells) and 2) whether the same cells express SDF-1 transcripts. METHODS: B cells were purified by immunomagnetic bead manipulation. Messenger RNA was detected by reverse transcription-polymerase chain reaction. Migration was assessed by the filter and collagen invasion assays. All P values were two sided. RESULTS: Follicular center lymphoma B lymphocytes showed a statistically significant migratory response to 300 ng/mL SDF-1, both in the filter and in the collagen assays (P =.002 for each). Such response was mediated by the SDF-1 receptor, CXCR4. CD40 monoclonal antibody (MAb) and tonsillar germinal center B cells treated with CD40 MAb and recombinant interleukin 4, but not freshly isolated, migrated statistically significantly faster in the presence than in the absence of SDF-1 (P =.002 in both filter and collagen assays). Freshly isolated follicular center lymphoma and germinal center B cells expressed SDF-1 transcripts. CONCLUSIONS: This study shows that SDF-1 substantially enhances the migration of follicular center lymphoma B cells but not the migration of freshly purified germinal center B cells. This difference may be related to the extended survival of follicular center lymphoma versus germinal center B cells. SDF-1 produced in follicular center lymphoma lymph nodes may play a role in the local dissemination of tumor cells.


Subject(s)
B-Lymphocytes/physiology , Chemokines, CXC/pharmacology , Chemotactic Factors/pharmacology , Lymphoma, Follicular/metabolism , Antibodies, Monoclonal/pharmacology , B-Lymphocytes/metabolism , Base Sequence , CD40 Antigens/immunology , CD40 Antigens/physiology , Chemokine CXCL12 , Chemokines, CXC/genetics , Chemotaxis , Gene Expression , Humans , Lymph Nodes/metabolism , Lymphoma, Follicular/genetics , Molecular Sequence Data , Receptors, CXCR4/metabolism , Receptors, Interleukin-4/immunology , Receptors, Interleukin-4/physiology
14.
Antimicrob Agents Chemother ; 43(9): 2307-10, 1999 Sep.
Article in English | MEDLINE | ID: mdl-10471586

ABSTRACT

At sites of neutrophilic inflammation, tissue injury by neutrophil elastase is favored by phagocyte-induced hypochlorous acid-dependent inactivation of the natural elastase inhibitor alpha(1)-antitrypsin. In the present study, cefoperazone prevented alpha(1)-antitrypsin inactivation by neutrophils and reduced the recovery of hypochlorous acid from these cells. Moreover, the antibiotic reduced the free elastase activity in a neutrophil suspension supplemented with alpha(1)-antitrypsin without affecting the cells' ability to release elastase. These data suggest that the drug inactivates hypochlorous acid before its reaction with alpha(1)-antitrypsin, thereby permitting the antiprotease-mediated blockade of released elastase. In conclusion, cefoperazone appears to have the potential for limiting elastase-antielastase imbalances, attenuating the related tissue injury at sites of inflammation.


Subject(s)
Cefoperazone/pharmacology , Cephalosporins/pharmacology , Neutrophils/drug effects , Pancreatic Elastase/metabolism , alpha 1-Antitrypsin/metabolism , Drug Interactions , Humans , Hypochlorous Acid/antagonists & inhibitors , Hypochlorous Acid/pharmacology , Neutrophils/enzymology , alpha 1-Antitrypsin/drug effects
15.
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
16.
J Immunol ; 162(11): 6510-7, 1999 Jun 01.
Article in English | MEDLINE | ID: mdl-10352266

ABSTRACT

Human C5a is a potent chemoattractant for granulocytes, monocytes, and dendritic cells. In mice C5a has been shown to be chemotactic for germinal center (GC) B cells. To date, no information is available on the effects of C5a on human B cell locomotion. Here we demonstrate that rC5a increases polarization and migration of human tonsillar B cells. The locomotory response was due to both chemokinetic and chemotactic activities of rC5a. Moreover, memory and, at a lesser extent, naive B cell fractions from purified tonsillar populations displayed rC5a-enhanced migratory properties, whereas GC cells did not. Flow cytometry revealed C5aR (CD88) on approximately 40% memory and 10% naive cells, respectively, whereas GC cells were negative. Immunohistochemistry showed that a few CD88+ cells were of the B cell lineage and localized in tonsillar subepithelial areas, where the majority of memory B cells settle. Pretreatment of memory B cells with the CD88 mAb abolished their migratory responsiveness to rC5a. Finally, the C5 gene was found to be expressed in naive, GC, and memory B lymphocytes at both the mRNA and the protein level. This study delineates a novel role for C5a as a regulator of the trafficking of human memory and naive B lymphocytes and supports the hypothesis that the B cells themselves may serve as source of C5 in secondary lymphoid tissues.


Subject(s)
B-Lymphocyte Subsets/immunology , Cell Movement/immunology , Complement C5a/physiology , Immunologic Memory , Lymphoid Tissue/immunology , Palatine Tonsil/cytology , Recombinant Proteins/pharmacology , Antigens, CD/biosynthesis , Antigens, CD/physiology , B-Lymphocyte Subsets/cytology , B-Lymphocyte Subsets/metabolism , Cell Movement/drug effects , Cells, Cultured , Chemotactic Factors/pharmacology , Complement C5/biosynthesis , Complement C5/genetics , Complement C5a/genetics , Gene Expression Regulation/immunology , Germinal Center/cytology , Germinal Center/immunology , Humans , Immunologic Memory/drug effects , Lymphoid Tissue/cytology , Palatine Tonsil/immunology , Receptor, Anaphylatoxin C5a , Receptors, Complement/biosynthesis , Receptors, Complement/physiology
17.
Blood ; 93(10): 3505-11, 1999 May 15.
Article in English | MEDLINE | ID: mdl-10233903

ABSTRACT

Murine monoclonal antibody (MoAb) Lym-1 is an IgG2a able to bind HLA-DR variants on malignant B cells and suitable for serotherapeutic approaches in B-lymphoma patients. We have previously shown that Lym-1 can synergize with granulocyte-macrophage colony-stimulating factor (GM-CSF) to trigger neutrophil cytolysis towards Raji cells used as a model of B-lymphoma targets. Here we provide evidence for the intervention of certain neutrophil receptors or surface molecules in this model of cell-mediated lysis. The lysis was completely inhibited by the anti-FcgammaRII MoAb IV.3 and unaffected by the anti-FcgammaRIII MoAb 3G8. This suggests that neutrophil cytolysis involves FcgammaRII without cooperation of this receptor with FcgammaRIII. Moreover, the lysis was inhibited by an anti-CD18 MoAb (MEM48) and by a MoAb specific for carcinoembryonic antigen (CEA)-like and glycophosphatidyl inositol (GPI)-linked glycoproteins (CD66b). Using an immunofluorescence staining procedure, cross-linking of CD66b induced the redistribution of CD11b on neutrophils with distinct areas of CD11b clustering via a process susceptible of inhibition by D-mannose. This is consistent with the ability of CD11b-CD18 and CD66b to undergo lectin-like physical interactions on the neutrophil surface. Such a type of interaction is presumably instrumental for neutrophil cytolytic activity in that the lysis was inhibited by D-mannose and enhanced by the MoAb VIM-12, which mimics the cooperation between CD11b and GPI-anchored molecules by specifically interacting with CD11b lectin-like sites. Therefore, the present results prove the absolute requirement for FcgammaRII in neutrophil GM-CSF/Lym-1-mediated cytolysis and, on the other hand, define the crucial role of CD66b and CD11b/CD18 in the expression of the cell lytic potential.


Subject(s)
Antibodies, Monoclonal , Antibody-Dependent Cell Cytotoxicity , B-Lymphocytes/immunology , CD18 Antigens/physiology , Cell Adhesion Molecules , Granulocyte-Macrophage Colony-Stimulating Factor/pharmacology , Lymphoma, B-Cell/immunology , Macrophage-1 Antigen/physiology , Membrane Glycoproteins/physiology , Neutrophils/immunology , Receptors, IgG/physiology , Antibody Specificity , Antigens, CD , Antigens, Neoplasm/physiology , GPI-Linked Proteins , Glycosylphosphatidylinositols/physiology , HLA-DR Antigens/immunology , Humans , Immunoglobulin G , Lymphocytes/immunology , Neutrophils/drug effects , Recombinant Proteins , Tumor Cells, Cultured
18.
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
19.
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
20.
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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