A NOVEL MURINE MODEL OF ALLERGIC INFLAMMATION TO STUDY THE EFFECT OF DEXAMETHASONE ON EOSINOPHIL RECRUITMENT

1 We have developed a novel model of allergen-induced eosinophil extra vasation into mouse air-pouches following sensitization and challenge with ovalbumin (Ova). This model was used to investigate the mechanism (s) underlying the anti-inflammatory action of the glucocorticoid horm one dexamethasone (Dex). 2 Injection of 10 mu g Ova into 6-day-old dor sal air-pouches of mice sensitized to the same antigen provoked an int ense cell accumulation as early as 6 h post-challenge (0.08+/-0.03 and 4.0+/-1.0x10(5) leucocytes in saline and Ova-treated air-pouches, res pectively), maximal at 24 h (0.02+/-0.01 and 6.0+/-0.8x10(5) leucocyte s in saline and Ova-treated air-pouches, respectively) and persisted u p to 48 h. At the 24 h time-point, the cellular infiltrate consisted o f 37% eosinophils, 18% neutrophils and 45% mononuclear cells, as asses sed by histological examination. The same ratio of eosinophil/neutroph il was obtained by fluorescence-activated cell sorting (FACS) analysis , since 72% of the polymorphonuclear (PMN) population was positive for very-late antigen-4, (VLA-4) expression. 3 Subcutaneous (s.c.) admini stration of Dex (50 or 100 mu g per mouse, -1 h) inhibited eosinophil accumulation into Ova challenged air-pouches by about 70% (P<0.05) and 75% (P<0.05), respectively, when compared to controls. Cell accumulat ion measured at 48 h after Ova injection was also significantly reduce d (-75%) by Dex administration at the 24 h time-point (n=12, P<0.05). 4 Eosinophil numbers in the bone marrow and blood were quantitated. We found that the sensitization protocol induced a 3 fold increase in eo sinophil numbers in the bone marrow (naive mice: 2.7+/-0.3x10(5); sens itized mice: 8.7+/-1.7x10(5), P<0.05) and blood (naive mice: 0.5+/-0.2 x10(5); sensitized mice: 1.5+/-0.3x10(5), P<0.01). However, 24 h follo wing Ova challenge, the eosinophil numbers in the bone marrow had drop ped (3.7+/-0.8x10(5)) with no change in the circulating pool, suggesti ng an equilibrium within the eosinophil pools had been reached. 5 Dex administration provoked a profound eosinopaenia in the blood of naive (5.2+/-1.5 to 0.9+/-0.6x10(4)) and sensitized mice (1.5+/-0.3 to 0.08/-0.02x10(5)) at 4 h. This effect was reversed within 24 h. Dex also i nhibited the release of eosinophils from the bone marrow in response t o Ova challenge. 6 We show for the first time that eosinophils express the steroid-inducible protein lipocortin 1 (LC1). FAGS analysis of eo sinophils emigrated into the Ova challenged air-pouches revealed detec table LC1-like immunoreactivity (373x10(3)). These data were also subs tantiated by LC1 detection in circulating eosinophils of interleukin-5 transgenic mice (strain: CBA/Ca). However, s.c. injection of Dex (50 mu g) did not alter LC1 levels in blood eosinophils, such that 235+/-2 1x10(3) LC1-like molecules per cell were measured after vehicle treatm ent (n=5), and 224+/-8x10(3) LC1-like molecules per eel were associate d with this cell type 1 h after steroid treatment (n=5, not significan t). Finally, resident eosinophils (in the pleural cavity) were found t o have much higher LC1 levels than that found in the blood circulation (2 fold increase. P<0.05). 7 Passive immunization of mice against LC1 with a validated antiserum (termed LCS3) and protocol failed to modif y the anti-migratory activity exerted by Dex towards eosinophil extrav asation into Ova-challenged air-pouches. The steroid (50 mu g s.c., -1 h) produced a similar degree of inhibition of eosinophil accumulation both in control animals (treated with a non-immune sheep serum) and L CS3-treated mice (-56% and 59%, respectively, n=15-21, not significant ). 8 In conclusion, the air-pouch provides a novel and convenient cavi ty to study allergen-induced cell recruitment which is sensitive to gl ucocorticoid hormone treatment. The effect of Dex on eosinophil distri bution in these experimental conditions has been studied in detail and we failed to find an important role for endogenous LC1 in these actio ns of the steroid.