Summary
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 α1‐antitrypsin (α1‐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 α1‐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 μmol/L) dose‐dependently prevented HOCl‐induced inactivation of α1‐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 α1‐AT in a cell‐free assay. Accordingly, chlorhexidine reversed HOCl‐mediated inhibition of α1‐AT capacity to complex with PPE.
4. These data suggest that chlorhexidine prevents neutrophil‐induced α1‐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.
Summary
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).
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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 μmol/L SSZ and 25 μg/mL IC. In some experiments, cells were co‐incubated with 20 μmol/L Z‐IETD‐fmk (a caspase 8 inhibitor) or 20 μmol/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.
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At 100 μmol/L, SSZ significantly accelerated IC‐induced neutrophil apoptosis. Treatment of neutrophils with 20 μmol/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 μmol/L SSZ increased caspase 3 activation.
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In conlusion, 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.
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-kB/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-kB pathway governing the expression of XIAP. Both the pathways are susceptible to inhibition by monocyte treatment with pharmacologic concentrations of dexamethasone.
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.
Human neutrophils, plated in fibronectin‐coated wells and stimulated with N‐formyl‐methionyl‐leucyl‐phenylalanine (fMLP), were found to undergo a massive and prolonged respiratory burst, as measured ...by monitoring superoxide production. The β2‐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 β‐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 β2‐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.
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.
Leptin is a pleiotropic molecule involved in energy homeostasis, hematopoiesis, inflammation, and immunity. Hypoleptinemia characterizing starvation has been strictly related to increased ...susceptibility to infection secondary to malnutrition. Nevertheless, ESRD is characterized by high susceptibility to bacterial infection despite hyperleptinemia. Defects in neutrophils play a crucial role in the infectious morbidity, and several uremic toxins that are capable of depressing neutrophil functions have been identified. Only a few and contrasting reports about leptin and neutrophils are available. This study provides evidence that leptin inhibits neutrophil migration in response to classical chemoattractants. Moreover, serum from patients with ESRD inhibits migration of normal neutrophils in response to N-formyl-methionyl-leucyl-phenylalanine with a strict correlation between serum leptin levels and serum ability to suppress neutrophil locomotion. Finally, the serum inhibitory activity can be effectively prevented by immune depletion of leptin. The results also show, however, that leptin by itself is endowed with chemotactic activity toward neutrophils. The two activities-inhibition of the cell response to chemokines and stimulation of neutrophil migration-could be detected at similar concentrations. On the contrary, neutrophils exposed to leptin did not display detectable Ca(2+)(i) mobilization, oxidant production, or beta(2)-integrin upregulation. The results demonstrate that leptin is a pure chemoattractant devoid of secretagogue properties that are capable of inhibiting neutrophil chemotaxis to classical neutrophilic chemoattractants. Taking into account the crucial role of neutrophils in host defense, the leptin-mediated ability of ERSD serum to inhibit neutrophil chemotaxis appears as a potential mechanism that contributes to the establishment of infections in ERSD.
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.
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.
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