Nicoletta Arduino

Differential regulation of spontaneous and immune complex-induced neutrophil apoptosis by proinflammatory cytokines. Role of oxidants, Bax and caspase-3

Neutrophil apoptosis represents a crucial step in the mechanisms governing the resolution of neutrophilic inflammation. Several soluble mediators of inflammation modulate neutrophil survival, retarding their apoptosis, whereas neutrophil activation by immune complexes (IC) results in the acceleration of apoptosis. To investigate neutrophil fate at the site of inflammation, we studied the effects of interleukin (IL)‐2, IL‐6, IL‐8, IL‐15, GM‐CSF, and fMLP on spontaneous and IC‐induced neutrophil apoptosis and the mechanisms regulating the survival of these cells. Spontaneous apoptosis was inhibited by GM‐CSF, IL‐6, and IL‐15, but only GM‐CSF overturned IC‐induced apoptosis. No role of oxidants on the modulation of IC‐dependent apoptosis was found. Indeed, fMLP or GM‐CSF augmented the IC‐dependent oxidative response, whereas the other compounds were ineffective. CGD neutrophils showed low levels of spontaneous apoptosis, but when exposed to IC, underwent a sharp increment of the apoptotic rate in a GM‐CSF‐inhibitable manner. Conversely, the expression of the proapoptotic protein Bax in 18‐h aged neutrophils was down‐regulated by GM‐CSF, IL‐6, and IL‐15. Furthermore, IC induced a nearly threefold Bax up‐regulation, which was completely reversed only by GM‐CSF. Accordingly, the spontaneous activity of caspase‐3 was inhibited by GM‐CSF, IL‐6, and IL‐15. Furthermore, IC induced a sharp increment of enzymatic activity, and only GM‐CSF inhibited the IC‐dependent acceleration. Our results show that apoptosis of resting and IC‐activated neutrophils is regulated differently, GM‐CSF being the most potent neutrophil antiapoptotic factor. The results also unveil the existence of an oxidant‐independent, Bax‐ and caspase‐3‐dependent, intracellular pathway regulating neutrophil apoptosis.

CCL19 and CXCL12 Trigger in Vitro Chemotaxis of Human Mantle Cell Lymphoma B Cells

Purpose: Few data are available in the literature on chemokine receptor expression and migratory capability of mantle cell lymphoma (MCL) B cells. Information on these issues may allow us to identify novel mechanisms of chemokine-driven tumor cell migration. Experimental Design: The research was designed to investigate: (a) expression of CCR1 to CCR7 and CXCR1 to CXCR5 chemokine receptors; and (b) chemotaxis to the respective ligands in MCL B cells and in their normal counterparts, i.e., CD5+ B cells. Results: Malignant B cells from MCL patients and normal counterparts displayed similar chemokine receptor profiles. MCL B cells were induced to migrate by CXCL12 and CCL19, whereas normal CD5+ B cells migrated to the former, but not the latter chemokine. Overnight culture of MCL B cells and their normal counterparts with CXCL12 cross-sensitized other chemokine receptors to their ligands in some tumor samples but not in CD5+ B cells. Conclusions: CCR7 and CXCR4 ligands may play a key role in tumor cell migration and spreading in vivo. CXCL12 may additionally contribute by sensitizing MCL B cells to respond to the ligands of other chemokine receptors.

Delayed Neutrophil Apoptosis Induced by Synovial Fluid in Rheumatoid Arthritis

Abstract: The fate of neutrophils at sites of inflammation, where these cells are likely exposed to both anti‐ and proapoptotic influences, needs to be clarified. To investigate this issue, we studied the survival of neutrophils in the presence of articular fluids from RA joints before and after immune complex activation. Eight of eleven samples of RA synovial fluid studied were found to inhibit spontaneous and immune complex‐stimulated neutrophil apoptosis. No relationships were found between GM‐CSF and TNF‐α concentrations measured on each sample of synovial fluid studied and the levels of neutrophil apoptosis detectable in the presence of the same synovial fluid. Furthermore, no activity on neutrophil survival was observed at either physiologic or pharmacologic concentrations of estradiol. On the contrary, the synovial fluid anti‐apoptotic activity correlates (r2= 0.8818, p < 0.0001) with the adenosine detected at concentrations in each sample ranging from 18.7 to 52.4 μM. Finally, synovial fluids were incapable of interfering with neutrophil activation evaluated as superoxide anion production. Our results suggest that the microenvironment of rheumatoid synovial fluid is a proinflammatory milieu responsible for the in loco persistence of activated and long‐surviving neutrophils.

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.

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

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 (O−2) production and adherence. Results, expressed as mean ± 1 SEM, 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 O−2 production. Among eleven agents, tumor necrosis factor-alpha (TNF, 3.0 ± 0.3 nmoles O−2/5 × 104 neutrophils/180 min, p < 0.001), granulocyte-macrophage colony stimulating factor (GM-CSF, 2.1 ± 0.3 nmoles O−2/5 × 104 neutrophils/180 min, p < 0.05) and formyl-peptides (fMLP, 2.5 ± 0.5 nmoles O−2/5 × 104 neutrophils/180 min, p < 0.01) caused massive O−2 production by neutrophils adherent to FN. None of the mediators was capable of triggering O−2 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 μg, mean ± 1 SEM), GM-CSF (IC50 LM: 0.36 ± 0.16 μg, mean ± 1 SEM) and fMLP (IC50 LM: 0.54 ± 0.008 μg, 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 O−2 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.

Effect of clotting factors concentrates on lymphocyte and neutrophil function in vitro.

: Immunological abnormalities have been reported in haemophiliacs. Although infections with HIV, hepatitis and other viruses may contribute to these abnormalities, immune defects are detectable also in HIV seronegative haemophiliacs. It is likely that chronic exposure to extraneous proteins in clotting factor concentrates (CFCs) may play a role in immunomodulation, but the underlying mechanisms remain unclear. The results of the present paper show that: a) soluble HLA class I (sHLA-I), soluble Fas-ligand (sFas-L) and transforming growth factor beta 1 (TGF-beta1) are detectable in plasma derived but not in recombinant CFCs; b) the level of sHLA-I and sFas-L is proportional to the grade of CFCs purity whereas TGF-beta1 showed very variable levels; c) soluble molecules detected in CFCs exert immunomodulatory effects in vitro like apoptosis induction in Jurkat cells and inhibition of mixed lymphocyte reaction response, antigen-specific lymphocyte cytotoxic activity and neutrophil chemotaxis.

In vitro inhibition of human neutrophil histotoxicity by ambroxol: evidence for a multistep mechanism

Neutrophils are major culprits for the protease/antiprotease imbalance during various lung diseases, that is, chronic obstructive pulmonary disease, cystic fibrosis, idiopathic pulmonary fibrosis and adult respiratory distress syndrome. Thus, these cells are presently considered an ideal target for the pharmacologic control of tissue injury during these diseases. This study was planned in order to investigate if ambroxol and its precursor bromhexine are actually capable of preventing alpha‐1‐antitrypsin (A1AT) inactivation by stimulated neutrophils and possibly to look into the mechanisms underlying this event. Ambroxol inhibited the production of superoxide anion by activated neutrophils, whereas bromhexine had no inhibitory effect. Ambroxol decreased the production of hypochlorous acid (HOCl) from activated neutrophils with high efficiency, whereas bromhexine had a modest activity. Ambroxol and bromhexine were capable of limiting the chlorination of monochlorodimedon by HOCl, displaying the capacity of directly scavenging the oxidant. Ambroxol decreased the release of elastase and myeloperoxidase from activated neutrophils, whereas bromhexine was ineffective. Ambroxol prevented the A1AT inactivation by neutrophils, whereas bromhexine was completely ineffective. Among drugs currently available for in vivo use in humans, ambroxol is unique by virtue of its ability to prevent neutrophil‐mediated A1AT inactivation via inhibition of HOCl production as well as HOCl scavenging. Also taking into account its capacity for curbing elastase release, the drug displays the potential to lessen the burden of oxidants/proteases and to increase the antiprotease shield at the site of inflammation. Thus, ambroxol appears to be a good candidate for raising attempts to develop new therapeutic histoprotective approaches to inflammatory bronchopulmonary diseases.

6-amino-4-oxo-1,3-diphenyl-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-carbonyl derivatives as a new class of potent inhibitors of Interleukin-8-induced neutrophil chemotaxis.

A series of 6-amino-4-oxo-1,3-diphenyl-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-carbonyl derivatives was synthesized. The compounds demonstrated to be novel, potent and selective inhibitors of Interleukin-8-induced human neutrophil chemotaxis. A SAR study was performed by varying the carbonyl function at position 5 and the chain linked to the amino group at position 6 of the scaffold. All the compounds of the series displayed inhibitory activity at nano- or picomolar concentrations against Interleukin-8-driven migration and no activity against fMLP- and C5a-induced chemotaxis. The binding tests of selected compounds on CXCR1 and CXCR2 receptors were negative. The most potent derivative showed in vivo efficacy in a mouse model of Zymosan-induced peritonitis.