Roflumilast potently inhibits CXCL1 mediated neutrophil migration in COPD patients

Chronic obstructive pulmonary disease (COPD) is associated with glucocorticosteroid insensitive inflammation. Neutrophils from COPD patients migrate more in response to CXCL1 than cells from control subjects. Roflumilast, a PDE4 inhibitor recently approved for COPD in the EU, reduces neutrophil numbers in sputum from COPD patients. The mechanism of this anti-inflammatory effect is not clear, but may be due to inhibition of neutrophil migration. Thus this study investigated the effects of roflumilast N-oxide (RNO), the active metabolite of roflumilast, on neutrophil migration in comparison with another PDE4 inhibitor, rolipram. Neutrophils were isolated from whole blood samples from healthy subjects and COPD patients. Chemotaxis was measured using 48-well Boyden chambers and [Ca 2+ ] i by fluorimetry. Both RNO and rolipram completely inhibited migration of neutrophils from healthy subjects towards CXCL1 (3nM) with EC 50 values of 0.18±0.13 nM and 2.8±0.51 nM, n=4, respectively. A similar response was seen with neutrophils obtained from patients with COPD (EC 50 RNO: 0.74±0.89 nM; rolipram 42±45.9 nM, n=3). Having established that cell migration was attenuated by PDE4 inhibitors, the effects of these compounds on CXCL1 stimulated [Ca 2+ ] i was investigated. RNO (1μM) inhibited the peak of [Ca 2+ ] i and area under the curve (AUC) by 83.0±2.6% and 90.3±2.1%, n=6. Rolipram (1μM) also inhibited these responses by 80.7±3.2% and 86.3±2.1%, n=6. These data indicate that oral PDE4 inhibitors may have added anti-inflammatory effects by reducing CXCL1 stimulated neutrophil migration into the lung via regulation of [Ca 2+ ] i and this could contribute to the anti-inflammatory effects of roflumilast in the treatment of COPD.