Regulation of expression and function of dipeptidyl peptidase 4 (DP4), DP8/9, and DP10 in allergic responses of the lung in rats

Abstract

The expression of dipeptidyl peptidase 4 (DP4, CD26) affects T-cell recruitment to lungs in an experimental rat asthma model. Furthermore, the gene of the structural homologous DP10 represents a susceptibility locus for asthma in humans, and the functional homologous DP8/9 are expressed in human leukocytes. Thus, although several mechanisms may account for a role of DP4-like peptidases in asthma, detailed information on their anatomical sites of expression and function in lungs is lacking. Therefore, bronchi and lung parenchyma were evaluated using immunohistochemistry and histochemical/enzymatic activity assays, as well as quantitative real-time PCR for this family of peptidases in naïve and asthmatic rat lungs derived from wild-type F344 and DP4-deficient F344 rat strains. Surprisingly, results show not only that the induction of experimental asthma increases DP4 enzymatic activity in the bronchoalveolar lavage fluid and parenchyma, but also that DP8/9 enzymatic activity is regulated and, as well as the expression of DP10, primarily found in the bronchial epithelium of the airways. This is the first report showing a differential and site-specific DP4-like expression and function in the lungs, suggesting a pathophysiologically significant role in asthma.

Figure 1

Detection of DP4-like activities with a histochemical activity assay. (A–C) Lung section of a DP4-positive control rat after an incubation period of 25 min with a slight staining of the lung parenchyma. (D–F) Lung section of a DP4-negative control rat after an incubation period of 25 min with no visible staining. (G–I) Lung section of a DP4-positive control rat after an incubation period of 20 hr with a strong staining of the lung parenchyma and a weaker staining of the bronchi. (J–L) Lung section of a DP4-negative control rat after an incubation period of 20 hr with hardly any staining of the lung parenchyma and a weak staining of the bronchi. (M) Lung section of a DP4-positive control rat after 20 hr incubation in the presence of a DP4-specific inhibitor. (N) Lung section of a DP4-negative control rat after 20 hr incubation in the DP4 inhibitor solution.

Figure 2

Comparison of the DP4-like peptidases in the lungs. (A) DP4-like activity of the bronchoalveolar lavage (BAL) fluid measured by the enzymatic activity assay in vitro (*p<0.05; ###p<0.0001). Histochemical activity assay of a DP4-positive control lung (B), of a DP4-positive asthma lung (C), of a DP4-negative control lung (D), and of a DP4-negative asthma lung (E). (F,G) Staining with a DP4 antibody (red) and a T-cell antibody (blue) of a DP4-positive control lung at different magnifications. (H) The same staining of a DP4-positive asthma lung. Staining with a DP8 antibody of a control lung without BAL (I) and after BAL (J), and of an asthma lung after BAL (K). Staining with a DP9 antibody of a control lung without BAL (L) and after BAL (M), and of an asthma lung after BAL (N). Staining with a DP10 antibody of a control lung without BAL (O) and after BAL (P), and of an asthma lung after BAL (Q).

Figure 3

PCR analyses of DP4, DP8, DP9, and DP10. (A) Overview of the PCR products resulting from different primer pairs. HKG, housekeeping gene. (B) Mean normalized expression (MNE) of DP4 in DP4-positive (DP4^pos) and DP4-negative (DP4^neg) lungs of control groups and groups after asthma induction. (C) MNE of DP8 in the same groups (*p<0.05). (D) MNE of DP9 in the same groups (*p<0.05). (E) Detection of a DP10 PCR product only in cDNA from brain, trachea, and bronchi of control lungs and lungs after asthma induction. br, bronchi; par, lung parenchyma; tra, trachea; L, sample after BAL; no L, sample without BAL.