Effects of GCs on gene expression in BAL and lung parenchymal cells

The effects of GC therapy on inflammation are thought to strongly depend on the regulation of gene expression. The GR is known to modulate the expression of many genes either by transactivation or transrepression. In order to investigate differences in gene regulation in the absence of the DNA-binding dependent mode of GR action, samples from wild type and GR^dim mice were analyzed via RT-qPCR. Since a variety of cytokines, chemokines and other inflammatory mediators are involved in asthmatic reactions but may be differently regulated dependent on the cell type, the analysis was done with BAL samples and lung samples separately. These independent analyses of gene expression provided the opportunity to investigate the influence of GCs on the infiltrating cells as well as inflamed tissue. The

allergic response in the lungs led to an increased expression of many pro-inflammatory genes in BAL and lung tissue samples.

The only genes with nearly unaltered gene expression in the BAL were MIP-1α and CXCR4 while in the lung samples these genes also showed increased expression. Interestingly, expression of the important mediators of monocytes and macrophages migration MCP-1 and MIP-1α was strongly increased in lung samples from AAI mice and GC therapy led to further elevation. The expression of CXCR4 remained unchanged without any observable effect of AAI induction and GC therapy. This result was surprisingly because of the reported role for this chemokine receptor in the mobilization of hematopoietic stem cells into the bloodstream during an inflammatory response and its expression on TH2 cell subset. iNOS and IL-10 expression in BAL samples was nearly the same independent of the treatment. However, because the iNOS is a characteristic marker of the inflamed epithelium and activated macrophages it was interestingly to analyze its expression in the lung tissue. Based on the obtained data, the regulation of these two genes in the inflamed tissue was more critical for therapeutic efficiency of AAI as their regulation in the cellular infiltrate. The results of the iNOS gene expression analysis in lung tissue samples revealed a successful suppression of iNOS expression in samples of wild type mice while the expression of this inflammatory enzyme was even upregulated in GC-treated asthmatic GR^dim mice. IL-10 showed a very similar regulation as iNOS. In contrast to the expression of this cytokine in the BAL samples, the IL-10 expression in the lung samples was strongly increased after AAI induction. This overexpression of IL-10 in the inflamed lungs could be strongly suppressed in wild type mice by Dexamethasone treatment while the IL-10 mRNA levels in the lung samples of GC-treated GR^dim mice was slightly upregulated. These data demonstrate that these two inflammatory mediators are more important in the inflamed tissue than in the infiltrating cells. Additionally, the expression of these two genes was strongly dependent on the DNA-binding dependent mode of GR action in the lungs.

Figure 63: Effects of GC therapy on gene expression of inflammatory mediators in BAL and lung samples of wild type and GR^dim mice. The data represent an overview of all results obtained by RT-qPCR analysis.

The induction of AAI led to an elevated expression of IL-5 and Eotaxin-2, the prominent markers of eosinophilia. This upregulation could be strongly suppressed by GC therapy in BAL cells from wild type and GR^dim mice. Although GCs could reduce the expression of these genes in infiltrating cells in GR^dim mice, the expression of IL-5 and Eotaxin-2 in lung parenchymal cells was not altered by Dexamethasone treatment. On the one hand, these results show that the observed lung infiltration with eosinophils (see FACS data) is more dependent on the cytokine and chemokine expression in lung cells. On the other hand these data demonstrate the differences in gene regulation by GCs between individual cell types.

This ambivalent effect of GC therapy was also seen in the regulation of CXCR3 expression.

While its expression in the BAL samples was nearly unaltered after GC therapy in both genotypes, the expression in lung tissue samples was reduced in wild type and GR^dim mice.

CXCR3 was preferentially reported to be expressed on activated T_H1 cells, NK cells but also on epithelial and endothelial cells of the lungs. It was also reported, that the expression of this chemokine receptor can influence the migration of T_H2 cells into the lungs and the activation

of effector T cells. However, based on this result, the reduction of CXCR3 in the inflamed lung tissue by GCs did not prevent the strong infiltration seen in GR^dim mice.

The expression of a number of key mediators of asthmatic reactions was influenced by GC therapy in a DNA-binding dependent mode of action. The elevated expression of IL-4 and IL-13 was proposed to play a crucial role in the pathogenesis of asthma by activating and recruiting a variety of inflammatory cells involved in inflammation. Increased IL-4 and IL-13 levels were observed in BAL and lung samples of AAI mice of both genotypes. The expression of IL-13 was suppressed in a DNA-binding dependent mode of action in BAL and lung samples. The expression of IL-4 in the lungs but not the BAL of wild type mice was increased because of GC therapy and suppressed in the BAL but not lung samples. These confusing effects could be based on a variety of factors including differences in the cell composition between BAL and lung tissue and possible dissimilarity in T_H2/T_H1 balance regulation. The inflammatory mediator RANTES was strongly increased after AAI induction.

RANTES was reported to be able to induce eosinophilia in the lungs independent of the presence of the allergen thereby increasing asthma severity in patients. The expression data demonstrated a strong suppression of RANTES after GC-therapy in samples of wild type mice with AAI. While the expression in allergic wild type mice was significantly downregulated, GC-therapy of GR^dim mice did neither affect RANTES mRNA expression in BAL nor lung tissue samples. This observation showed a strong dependency of RANTES regulation on the DNA-binding dependent mode of GR action.