Chlorhexidine prevents hypochlorous acid-induced inactivation of alpha1-antitrypsin.

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.

Sulphasalazine accelerates apoptosis in neutrophils exposed to immune complex: Role of caspase pathway.

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.

Dexamethasone -induced apoptosis of human monocytes exposed to immune complexes. Intervention of CD95- and XIAP-dependent pathways.

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.

Chimaeric Lym-1 monoclonal antibody-mediated cytolysis by neutrophils from G-CSF-treated patients: stimulation by GM-CSF and role of Fc gamma -receptors.

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.

Immune complex stimulation of neutrophil apoptosis: investigating the involvement of oxidative and nonoxidative pathways.

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.

Monoclonal Lym-1 antibody-targeted lysis of B lymphoma cells by neutrophils. Evidence for two mechanisms of FcgammaRII-dependent cytolysis.

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.

Cefoperazone prevents the inactivation of alpha(1)-antitrypsin by activated neutrophils.

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.

FMLP- and TNF-stimulated monoclonal Lym-1 antibody-dependent lysis of B lymphoblastoid tumour targets by neutrophils.

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.

Monoclonal Lym-1 antibody-dependent cytolysis by neutrophils exposed to granulocyte-macrophage colony-stimulating factor: intervention of FcgammaRII (CD32), CD11b-CD18 integrins, and CD66b glycoproteins.

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.

Chemoattractant-induced release of elastase by tumor necrosis factor-primed human neutrophils: auto-regulation by endogenous adenosine.

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.

Activation of neutrophil respiratory burst by cytokines and chemoattractants. Regulatory role of extracellular matrix glycoproteins.

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.

Prostaglandin E2 inhibits apoptosis in human neutrophilic polymorphonuclear leukocytes: role of intracellular cyclic AMP levels.

Human neutrophilic polymorphonuclear leukocytes (neutrophils) are terminally differentiated cells that die by undergoing apoptosis. At present, the intracellular pathways governing this process are only partially known. In particular, although the adenylate cyclase-dependent generation of cyclic AMP (cAMP) has been implicated in the triggering of apoptosis in lymphoid cells, the role of the intracellular cAMP pathway in neutrophil apoptosis remains controversial. In the present study, we found that two cAMP-elevating agents, prostaglandin E2 (PGE2) and the phosphodiesterase type IV inhibitor RO 20-1724, inhibit neutrophil apoptosis without inducing cell necrosis. When administered in combination, PGE2 and RO 20-1724 displayed additive effects. Moreover, neutrophil apoptosis was inhibited by a membrane-permeable analog of cAMP, dibutyryl-cAMP, in a dose-dependent manner. Finally, treatment of neutrophils with the protein kinase A inhibitor H-89 prevented PGE2- and RO 20-1724-induced inhibition of cell apoptosis. In conclusion, taking into account that PGE2 and other cAMP-elevating agents are well known downregulators of neutrophil functions, our results suggest that conditions favoring a state of functional rest, such as intracellular cAMP elevation, prolong the life span of neutrophils by delaying apoptosis.

Chemoattractant-induced release of elastase by lipopolysaccharide (LPS)-primed neutrophils; inhibitory effect of the anti-inflammatory drug nimesulide.

Human neutrophils, pre-exposed to low concentrations (1-10 ng/ml) of bacterial LPS in the presence of 1% autologous serum, released elastase activity in response to N-formyl-met-leu-phe (fMLP). Both cell incubation with LPS without subsequent fMLP stimulus and fMLP stimulation without prior exposure to LPS failed to promote significant elastase release. Therefore, LPS primes neutrophils for the subsequent release of elastase in response to fMLP. Compared with fMLP, human recombinant C5a had a slight although not significant activity, whereas other chemoattractants such as IL-8, platelet-activating factor and leukotriene B4 were ineffective. The fMLP-induced response of LPS-primed neutrophils was susceptible to suppression by the methane-sulphonanilide anti-inflammatory drug nimesulide and RO 20-1724, which selectively inhibit cAMP-catabolizing phosphodiesterase type IV. This suggests that the elastase release by LPS-primed neutrophils is likely to be controlled by intracellular cAMP, and raises the possibility of limiting pharmacologically the elastase-mediated tissue injury during neutrophilic inflammation.

Inhibitory effect of salmeterol on the respiratory burst of adherent human neutrophils.

Human neutrophils, plated in fibronectin-coated wells and stimulated with N-formyl-methionylleucyl-phenylalanine (fMLP), were found to undergo a massive and prolonged respiratory burst, as measured by monitoring superoxide production. The beta 2-agonist salmeterol inhibited the respiratory burst in a dose-dependent manner. In contrast, salbutamol was ineffective. Moreover, the neutrophil respiratory burst was partially suppressed by prostaglandin E2 (PGE2) and the phosphodiesterase type IV (PDE-IV) inhibitor RO 20-1724. When salmeterol was used in combination with PGE2 or RO 20-1724, additive inhibitory effects were observed. The inhibitory activity of salmeterol was not reversed in the presence of the beta-blocker propranolol, and did not correlate with its ability of increasing cyclic AMP (cAMP) levels. Finally, the compounds used did not affect neutrophil adherence to fibronectin-coated wells. The results suggest that salmeterol is capable of down-regulating the neutrophil oxidative response to fMLP, also of co-operating with PGE2 and PDE-IV inhibitor RO 20-1724 in a manner not related to its beta 2-receptor binding activity. In other words, salmeterol displays neutrophil-directed effects, susceptible to be amplified by natural mediators such as PGE2 or PDE-IV inhibitors, consistent with possible anti-inflammatory properties of the drug.

Monoclonal Lym-1 antibody-dependent lysis of B-lymphoblastoid tumor targets by human complement and cytokinine-exposed mononuclear and neutrophilic polymorphonuclear leukocytes.

Lym-1 is a murine IgG2a monoclonal antibody that recognizes a polymorphic variant of HLA-DR antigens on malignant B cells, with minimal cross-reactivity with normal tissues. Because it can be safely administered in vivo, a detailed knowledge of its ability to recruit and trigger the antitumor immune effector systems is required to optimize potential serotherapeutic approaches in B-lymphoma patients. By using Raji cells as a model of B-lymphoma targets, we found that Lym-1 activates complement-mediated lysis efficiently. Moreover, Lym-1 was capable of triggering the antibody-dependent cellular cytolysis (ADCC) by peripheral blood mononuclear cells (MNCs). On the contrary, it failed to trigger neutrophilic polymorphonuclear leukocyte (PMN)-mediated ADCC activity. In an attempt to enhance Lym-1 ADCC by MNCs and PMNs, nine biologic response modifiers were tested. MNC-mediated Lym-1 ADCC was significantly stimulated by interleukin-2 (IL-2) and unaffected by other mediators, including gamma-interferon (gamma-IFN), tumor necrosis factor a (TNFalpha), and granulocyte-macrophage colony-stimulating factor (GM-CSF). On the other hand, PMN-mediated Lym-1 ADCC was induced or significantly augmented by various cytokines, such as GM-CSF, TNFalpha, and gamma-IFN, and chemotaxins, such as formyl peptides (FMLP), complement fragment C5a, and IL-8. Both MNC- and PMN-mediated ADCC was unaffected by granulocyte colony-stimulating factor (G- CSF) and insulin-like growth factor-1 (IGF-1). Finally, only GM-CSF and TNFalpha augmented the number of PMNs actually engaged in the binding of Raji target cells. The findings presented here, in particular those showing stimulatory activity of biologic response modifiers, may inspire new attempts for developing Lym-1 antibody-based approaches to the therapy of B lymphomas.

Tumour necrosis factor alpha-induced oxidative burst in neutrophils adherent to fibronectin: effects of cyclic AMP-elevating agents.

Human neutrophils, plated on fibronectin-coated polystyrene wells, were found to exhibit a prolonged production of superoxide anion (O2-) in response to tumour necrosis factor-alpha (TNF). The TNF-triggered O2- production was significantly reduced by 10 microM prostaglandin E2 (PGE2), which was ineffective at lower doses. Moreover, the O2- production was slightly reduced by the phosphodiesterase type IV (PDE IV) inhibitor RO 20-1724. When PGE2 and RO 20-1724 were added together to TNF-triggered neutrophils they caused a marked synergistic inhibition of O2- production. The action of PGE2 could be mimicked by forskolin (FK), a well-known direct activator of adenylate cyclase. These results suggest that cyclic AMP (cAMP)-elevating agents (PGE2, FK, RO 20-1724) down-regulate the capacity of adherent neutrophils to mount the respiratory burst in response to TNF. Consistent with this interpretation, PGE2 and RO 20-1724 increased the intracellular levels of cAMP displaying synergistic activity. Moreover, the membrane-permeable analogue of cAMP, dibutyryl cAMP, was found to inhibit the TNF-induced O2- production in a dose-dependent manner. As all the aforementioned cAMP-elevating agents did not affect the O2- production in response to phorbol myristate acetate, they appear to act by interfering with the assembly of the O2(-)-generating NADPH oxidase complex rather than by directly inhibiting the activity of already working oxidase complex. In conclusion, taking into account the TNF capacity to promote PGE2 formation at sites of inflammation, our observations suggest the existence of a negative PGE2-dependent feed-back, potentially capable of controlling the neutrophil response to TNF and susceptible to amplification by PDE IV-inhibiting compounds.

Cyclic AMP-elevating agents down-regulate the oxidative burst induced by granulocyte-macrophage colony-stimulating factor (GM-CSF) in adherent neutrophils.

Human neutrophils, plated on fibronectin-precoated wells, were found to release large quantities of superoxide anion (O2-) in response to GM-CSF. O2- production was reduced by prostaglandin E2 (PGE2) and the phosphodiesterase type IV (PDE IV) inhibitor RO 20-1724. Both agents are known to increase intracellular cyclic AMP (cAMP) levels by inducing its production (PGE2) or blocking its catabolism (RO 20-1724). When added in combination, PGE2 and RO 20-1724 had a marked synergistic inhibitory effect, which was reproduced by replacing PGE2 with a direct activator of adenylate cyclase, i.e. forskolin (FK). Moreover, the neutrophil response to GM-CSF was inhibited by a membrane-permeable analogue of cAMP in a dose-dependent manner. As GM-CSF and PGE2 are known to be generated at tissue sites of inflammation, the results suggest the existence of a PGE2-dependent regulatory pathway potentially capable of controlling the neutrophil response to GM-CSF, in turn limiting the risk of local oxidative tissue injury. Moreover, owing to its susceptibility to amplification by RO 20-1724, the PGE2-dependent pathway and in particular PDE-IV may represent a pharmacological target to reduce the generation of histotoxic oxidants by GM-CSF-responding neutrophils.

Neutrophil dysfunction and increased susceptibility to infection.

A critical evaluation of 3 years' experience using laboratory screening to detect neutrophil dysfunction is described. Neutrophil dysfunctions in patients with recurrent bacterial infections were investigated by using the following screening tests: (1) neutrophil chemotaxis towards N-formylmethionyl peptides (FMLP) and the complement fragment C5a; (2) neutrophil production of superoxide anions (O2-) in response to phorbol myristate acetate and opsonized zymosan particles; and (3) examination of May-Grünwald and myeloperoxidase cytochemical staining of peripheral blood smears. These tests were carried out in 100 patients suffering from infections and suspected of having altered neutrophil functional competence. A minority of patients was found to have well defined neutrophil dysfunction syndromes: chronic granulomatous disease (four cases), Chediak-Higashi disease (one case) and myeloperoxidase deficiency (one case). Of the remaining 94 patients, in whom infections localized to airways and/or skin predominated, 53 cases were found to have impaired chemotaxis (41 cases) or partial defects of the O2- production. Defects of chemotaxis toward FMLP and those towards both FLMP and C5a were the most frequent abnormalities. No defect was found in the other 41 patients. Moreover, impaired neutrophil chemotaxis was found in some patients with selective IgA deficiency (five cases) or immotile cilia syndrome (seven cases). The results suggest that (a) additional screening tests are required to ameliorate the efficiency of the diagnostic work-up of the patients suspected to have neutrophil dysfunction; and (b) further evaluation, also at the molecular level, should be considered at least in selected cases of non-classified neutrophil dysfunction in order to clarify diagnosis and plan rational therapeutic strategies.

Sulphonamides as anti-inflammatory agents: old drugs for new therapeutic strategies in neutrophilic inflammation?

1. It is well known that neutrophils act as mediators of tissue injury in a variety of inflammatory diseases. Their histotoxic activity is presently thought to involve proteinases and oxidants, primarily hypochlorous acid (HOCl). This oxidant is also capable of inactivating the specific inhibitor of neutrophil elastase (alpha 1-antitrypsin), thereby favouring digestion of the connective matrix. 2. In the present work, we found that sulphanilamide and some sulphanilamide-related anti-inflammatory drugs such as dapsone, nimesulide and sulphapyridine reduce the availability of HOCl in the extracellular microenvironment of activated neutrophils and prevent the inactivation of alpha 1-antitrypsin by these cells in a dose-dependent manner. The ability of each drug to prevent alpha 1-antitrypsin from inactivation by neutrophils correlates significantly with its capacity to reduce the recovery of HOCl from neutrophils. Five other non-steroidal anti-inflammatory drugs were completely ineffective. 3. Therefore, sulphanilamide-related drugs, i.e. dapsone, nimesulide and sulphapyridine, have the potential to reduce the bioavailability of neutrophil-derived HOCl and, in turn, to favour the alpha 1-antitrypsin-dependent control of neutrophil elastolytic activity. These drugs appear as a well-defined group of agents which are particularly prone to attenuate neutrophil histotoxicity. They can also be viewed as a previously unrecognized starting point for the development of new compounds in order to plan rational therapeutic strategies for controlling tissue injury during neutrophilic inflammation.

Triggering of respiratory burst by tumor necrosis factor in neutrophils adherent to fibronectin. Evidence for a crucial role of CD18 glycoproteins.

Human neutrophils, plated on fibronectin (FN)-coated wells, were found to release large quantities of superoxide anion (O2-) in response to tumor necrosis factor alpha (TNF-alpha). The O2- release was completely inhibited by two monoclonal antibodies (MoAbs, MHM23 and TS1/18) against CD18 glycoproteins. An independently derived anti-CD18 MoAb (60.3) was ineffective. These MoAbs failed to inhibit neutrophil adhesion to FN-coated surfaces. Moreover, neutrophils incubated for 30 min on FN and then washed to remove non-adherent cells, were responsive to TNF-alpha in the presence of anti-CD18 MoAbs MHM23 and TS1/18. Consequently, the CD18-dependent capacitation of the respiratory burst can occur before TNF-alpha triggering. Finally, neutrophils plated on FN in the presence of anti-CD18 MoAbs and then washed, i.e. adherent cells blocked in their surface CD18 molecules, released O2- after adding TNF-alpha but only in the absence of additional anti-CD18 MoAbs. This is consistent with a TNF-alpha ability to induce rapid expression and activation of new oxidative burst-capacitating CD18 molecules. The results suggest that the anchorage of neutrophils to FN surfaces depends on adherence molecules apparently unrelated to CD18, probably the so-called fibronectin receptors (FNRs), whereas the capacitation of the respiratory burst in response to TNF-alpha requires the intervention of CD18 glycoproteins, available on the membrane of "resting" neutrophils or mobilized to the cell surface by TNF-alpha.