Inhibition of airway hyperresponsiveness and pulmonary inflammation by roflumilast and other PDE4 inhibitors.

Roflumilast is an oral, once-daily phosphodiesterase 4 (PDE4) inhibitor with anti-inflammatory activity. We compared the anti-inflammatory effects of roflumilast with those of PDE4 inhibitors rolipram, piclamilast, and cilomilast in ovalbumin (OVA)-sensitized and challenged Brown-Norway rats. Animals were treated orally 1h before OVA challenge with roflumilast (0.3, 1.0, and 3.0mg/kg), rolipram (0.8, 2.8, and 8.3mg/kg), piclamilast (10.0, 20.0, and 30.0mg/kg), or cilomilast (10.3, 34.3, and 103.0mg/kg). Airway hyperresponsiveness (AHR) against adenosine was investigated by measuring airway resistance 200min after OVA challenge. Subsequently, neutrophil influx and tumor necrosis factor-alpha (TNF-alpha) release in the lungs were determined by bronchoalveolar lavage. Direct bronchodilation at the time point of AHR assessment by PDE4 inhibitors was examined in serotonin-challenged animals. Evaluation of neutropenic animals or treatment with anti-TNF-alpha antibody revealed that AHR was independent of neutrophil accumulation or TNF-alpha release. Roflumilast (50% inhibitory dose [ID(50)]=1.5mg/kg) inhibited AHR 3-, 16-, and 27-fold more potently than rolipram, piclamilast, and cilomilast, respectively. Likewise, roflumilast was a more potent inhibitor of neutrophil influx (ID(50)=0.9mg/kg) than rolipram (ID(50)=6.9mg/kg), piclamilast (ID(50)=28.1mg/kg), or cilomilast (ID(50)=37.7mg/kg). Roflumilast, rolipram, and piclamilast-but not cilomilast-suppressed OVA-induced TNF-alpha release in a dose-dependent manner. Roflumilast (ID(50)=0.9mg/kg) exhibited 9- and 23-fold more potent inhibition of TNF-alpha release than rolipram and piclamilast, respectively. Roflumilast did not inhibit serotonin-induced bronchoconstriction 4.5h after administration, suggesting that inhibition of AHR by roflumilast results from anti-inflammatory, not bronchodilatory, effects. This study suggests that roflumilast has anti-inflammatory action and provides rationale for the investigation of roflumilast in asthmatic patients.

The early allergic response in small airways of human precision-cut lung slices.

To study the role of small airways in the early allergic response (EAR), the method of human precision-cut lung slices (PCLS) was developed and used to examine the bronchoconstriction elicited by passive sensitisation and allergen provocation. Viable human PCLS of 250-microm thickness containing airways <1.5 mm in outer diameter were prepared from lung lobes obtained from lung resection and taken into culture. According to the low release of lactate dehydrogenase and the constant ciliary beat frequency, human PCLS were viable for at least 3 days. Following overnight passive sensitisation with serum from allergic individuals, administration of grass-pollen extract or activating immunoglobulin E antibody resulted in immediate airway contraction that was quantified by videomicroscopy. The extent of the EAR increased with decreasing airway size (outer airway diameter), with the strongest response occurring in the terminal bronchioles. Histamine receptor antagonism was ineffective, and leukotriene or thromboxane receptor antagonism attenuated the early allergic response only in some cases. However, simultaneous blockade of leukotriene and thromboxane receptors almost completely prevented the early allergic response in the precision-cut lung slices from all individuals, suggesting such a dual treatment as a potential future asthma therapy.

Immediate allergic response in small airways.

The role of small airways in the immediate allergic response is largely unknown. We therefore used the model of precision-cut lung slices (PCLS) in combination with quantitative videomicroscopy to study the early allergic response to allergen in airways ranging from 50 to 900 microm. After PCLS from untreated Wistar rats had been passively sensitized for 16 h with serum from sensitized Brown Norway rats, exposure to 0.1% ovalbumin resulted in an immediate allergic response. Both extent (r = 0.74, p < 0.0001) and velocity (r = 0.49, p < 0.0001) of the allergen-induced bronchoconstriction increased with decreasing airway size. In addition, we observed that smaller airways not only contracted stronger and quicker, but that they also relaxed faster, suggesting that smaller airways are more reactive in principle. The allergen-induced bronchoconstriction in PCLS was prevented by the serotonin receptor antagonist ketanserin (IC(50) 6 nM), but not by antagonists directed against histamine, acetylcholine, PAF, or endothelin receptors, or by cyclooxygenase or lipoxygenase inhibitors. Like allergen, serotonin provoked responses that were stronger in smaller airways. These findings suggest that the immediate allergic response in rat PCLS depends largely on serotonin and that this response can occur in nearly all airway generations, but is most pronounced in the smallest airways, that is, the terminal bronchioles.

Changes in airway resistance by simultaneous exposure to TNF-alpha and IL-1beta in perfused rat lungs.

Tumor necrosis factor (TNF)-alpha and interleukin (IL)-1beta are formed simultaneously under inflammatory conditions such as asthma and acute respiratory distress syndrome. Here we investigated the effects of TNF-alpha (10 ng/ml) and/or IL-1beta (10 ng/ml) in isolated blood-free perfused rat lungs. In lungs precontracted with methacholine, IL-1beta alone and IL-1beta/TNF-alpha decreased airway resistance 10 min after administration, whereas TNF-alpha alone had no effect. In untreated lungs, airway resistance was unaltered by either cytokine alone but started to increase 40 min after treatment with both cytokines together, indicating bronchoconstriction. The bronchoconstriction was accompanied by a steroid-sensitive increase in cyclooxygenase (COX)-2 mRNA expression and thromboxane formation. The cytokine-induced bronchoconstriction was blocked by the thromboxane receptor antagonist SQ-29548, indomethacin, the selective COX-2 inhibitor NS-398, and the steroid dexamethasone. We conclude that IL-1beta has an early bronchodilatory effect (after 10 min) that is unchanged by TNF-alpha. However, at later time points (after 40 min), IL-1beta and TNF-alpha in concert cause a COX-2- and thromboxane-dependent bronchoconstriction. Our findings show that TNF-alpha and IL-1beta exert complex and time-dependent effects on lung functions that cannot be predicted by studying each cytokine alone.