S. Dogné

Mechanisms of Persistent Nf-Kappa B Activity in the Bronchi of an Animal Model of Asthma

In most cells trans-activating NF-κB induces many inflammatory proteins as well as its own inhibitor, IκB-α, thus assuring a transient response upon stimulation. However, NF-κB-dependent inflammatory gene expression is persistent in asthmatic bronchi, even after allergen eviction. In the present report we used bronchial brushing samples (BBSs) from heaves-affected horses (a spontaneous model of asthma) to elucidate the mechanisms by which NF-κB activity is maintained in asthmatic airways. NF-κB activity was high in granulocytic and nongranulocytic BBS cells. However, NF-κB activity highly correlated to granulocyte percentage and was only abrogated after granulocytic death in cultured BBSs. Before granulocytic death, NF-κB activity was suppressed by simultaneous addition of neutralizing anti-IL-1β and anti-TNF-α Abs to the medium of cultured BBSs. Surprisingly, IκB-β, whose expression is not regulated by NF-κB, unlike IκB-α, was the most prominent NF-κB inhibitor found in BBSs. The amounts of IκB-β were low in BBSs obtained from diseased horses, but drastically increased after addition of the neutralizing anti-IL-1β and anti-TNF-α Abs. These results indicate that sustained NF-κB activation in asthmatic bronchi is driven by granulocytes and is mediated by IL-1β and TNF-α. Moreover, an imbalance between high levels of IL-1β- and TNF-α-mediated IκB-β degradation and low levels of IκB-β synthesis is likely to be the mechanism preventing NF-κB deactivation in asthmatic airways before granulocytic death.

Enhanced survival of lung granulocytes in an animal model of asthma: evidence for a role of GM-CSF activated STAT5 signalling pathway

BACKGROUND As granulocyte/macrophage colony stimulating factor (GM-CSF) mediated delay of granulocyte apoptosis contributes to the accumulation of inflammatory cells at the site of inflammation in many diseases, we sought to determine whether asthma is also associated with a GM-CSF dependent increase in lung granulocyte survival. Moreover, because GM-CSF mediates its effects through activation of signal transducer and activator of transcription 5 (STAT5), we also investigated the potential role of STAT5 in allergic inflammation. METHODS Blood granulocytes were recovered from six healthy and six heaves affected horses, a model of asthma. Lung granulocytes were obtained by bronchoalveolar lavage (BAL) from the same horses. Granulocytes were cultured in the presence or absence of anti-GM-CSF receptor antibodies for different times and apoptosis was determined using the Annexin-V/propidium iodide detection method. Nuclear protein extracts from cultured granulocytes were analysed for STAT5 binding activity by electrophoretic mobility shift assay. RESULTS BAL fluid granulocytes from heaves affected horses demonstrated a significant delay in apoptosis compared with blood granulocytes from the same horses and blood and BAL fluid granulocytes from healthy horses. Conversely, the rate of apoptosis in blood granulocytes from healthy and heaves affected horses was comparable. The enhanced survival of BAL fluid granulocytes from affected horses was suppressed in the presence of antibodies directed against GM-CSF receptors. Increased levels of active STAT5 were found in BAL fluid granulocytes from heaves affected horses and were markedly reduced after treatment with anti-GM-CSF receptor antibodies. CONCLUSIONS These data indicate that granulocyte survival is enhanced in the lung of heaves affected horses and suggest a role for a GM-CSF activated STAT5 pathway in delaying apoptosis of lung granulocytes in this model of asthma.

p65 Homodimer activity in distal airway cells determines lung dysfunction in equine heaves

Nuclear factor-kappaB (NF-kappaB) activity, which is a key regulator of inflammatory gene expression, is increased in bronchial epithelial cells from horses suffering from heaves (a hypersensitivity-associated inflammatory condition of the lung). To determine whether this increased activity extends to distal airways and to other pulmonary cells, cells recovered by broncho-alveolar lavage (BAL) in healthy and heaves-affected horses were assessed for NF-kappaB activity. NF-kappaB activity was much higher in BAL cells from heaves-affected horses, especially during crisis (disease exacerbation), than in cells from healthy horses. Moreover, the level of NF-kappaB activity found in BAL cells was positively correlated to total lung resistance and to the proportion of neutrophils present in BAL fluid. Finally, prototypical p65-p50 NF-kappaB heterodimers were absent from BAL cells, which mostly contained p65 homodimers. These results (1) show that increased NF-kappaB activity is a general feature of heaves lung; (2) demonstrate the importance of p65 homodimers in neutrophilic inflammation; and (3) suggest that the use of specific NF-kappaB inhibitors could improve lung function in heaves-affected horses.

IMPAIRED ACCUMULATION OF GRANULOCYTES IN THE LUNG DURING OZONE ADAPTATION

Respiratory alterations induced by an acute exposure to ozone (O(3)) paradoxically resolve during multiday exposure. This adaptation is characteristically accompanied by a gradual attenuation of lung neutrophilia. As maintenance of neutrophilia at the site of inflammation is due to cytokine-mediated delayed neutrophil apoptosis, which is associated with reduced levels of Bax, a proapoptotic protein, we sought to determine whether defects in these mechanisms could account for O(3) adaptation. Lung granulocytes obtained at different time points from calves exposed to 0.75 ppm O(3) for 12 h/d for 7 consecutive days neither showed enhancement of survival nor Bax deficiency, when compared to blood granulocytes. To further investigate the effects of an exogenous oxidative stress on neutrophil survival, human granulocytes were treated with hydrogen peroxide alone, or in combination with granulocyte/macrophage colony-stimulating factor, an antiapoptotic cytokine. Both treatments led to rapid apoptosis associated with downregulation of Bcl-x(L) and Bcl-2, two antiapoptotic proteins. This study shows that O(3) adaptation is associated with a failure in the mechanisms leading to accumulation of neutrophils at the site of inflammation, and suggests that this defect is due to direct proapoptotic effects of exogenous oxidative stress on granulocytes.