Synovial fluid from patients with rheumatoid arthritis inhibits neutrophil apoptosis: role of adenosine and proinflammatory cytokines.

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.

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.

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.

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.