Figure 5.

To study the effects of stress and asthma on function and expression of the glucocorticoid receptor (GR), splenocytes and whole lung tissue was investigated. Naïve mice (white bar) and mice subjected to stress (SDR, hatched bars), allergen (Af, grey bars) or allergen and stress (Af+SDR, grey hatched bars) were studied. On day 15 (48 h after Af challenge) spleens for GR nuclear translocation and lungs for GR mRNA and protein expression were harvested. (A): SDR inhibited nuclear translocation and GRE binding of the GR. Splenocytes were isolated and incubated in the presence of dexamethasone (10-7M). GR nuclear translocation was quantitated by an ELISA based TransAM GR kit with GRE-coated wells. Mean±STDEV of n=3 (duplicate experiments). Densitometric data are corrected for values obtained from HeLa cell positive controls. (B, D): Combination of Af and SDR reduced expression of GR protein. Total protein was isolated from spleens (B) and lung tissue (D). Western blot analysis was performed using an anti mouse GRα monoclonal antibody. Expression was assessed using densitometric analysis and GAPDH as control. Mean±SEM of n=6 (duplicate experiment). (C): Combination of SDR and Af reduced expression of GR mRNA in the lung tissue. Total RNA was isolated from lungs of mice. Reverse transcription and realtime PCR were performed. β2 macroglobulin mRNA was used as control. Data were analyzed using the ΔΔ Ct Method. Mean±SEM of n=4–6 (duplicate experiments) (A–D): *p<0.05 Af vs. Af+SDR