Expression of pituitary adenylate cyclase-activating polypeptide in the primary lymphoid organs of the duck Anas platyrhynchos

Abstract

The expression of pituitary adenylate cyclase-activating polypeptide (PACAP) was studied in the thymus and bursa of Fabricius of the duck Anas platyrhynchos, at different ages, using immunohistochemistry, Western blotting, RT-PCR and sequencing. In the thymus, PACAP immunoreactivity (-ir) was found in lymphoid cells. CD68/ and PGP 9.5/PACAP38 double labelling showed that PACAP was not expressed either in macrophages or in epithelial cells, suggesting that the PACAP-positive cells observed were lymphoid cells. Immunoreactive lymphocytes were observed in the interlobular septa. They increased in number with ageing. In the bursa, PACAP-ir was found in nerve fibres and in a few lymphoid cells. RT-PCR revealed PACAP mRNA expression in the thymus but not in the bursa. These results suggest that PACAP plays a role in the functions of the immune system in birds.

Fig. 1

Detection of PACAP38 immunoreactivity by Western blot analysis. Lane A, synthetic PACAP38; lane B, tissue extract of duck thymus (140 days). Protein markers are expressed in kDa.

Fig. 2

Immunohistochemical localization of PACAP in the duck thymus (A–F) and bursa of Fabricius (G,H) of different ages. In the thymus, PACAP-positive lymphoid cells (arrows) increased in number with ageing (30 days, A; 50 days, B; 140 days, C). PACAP-positive lymphoid cells were located in close vicinity or within blood vessels (70 days, D,E). They seem to migrate towards the interlobular septa (110 days, F). In the bursa of Fabricius, PACAP-ir was found in nerve fibres (arrowhead) and a few lymphoid cells (arrows) in the cortex of the bursal follicles (110 days, G; 140 days, H). bf, bursal follicles; bv, blood vessels; c, capillary; Ep, epithelium; tl, thymic lobules. Scale bar=25µm.

Fig. 3

Immunohistochemical localization of CD68 in the bursa of Fabricius (A) and CD68/PACAP38 (B,C) and PGP 9.5/PACAP38 (D,E) co-localization studies in the thymus. In the bursa of Fabricius, CD68-positive cells (arrows) were observed in the zone separating the cortex from the medulla and within the medulla (A). In the thymus, CD68/PACAP38 co-localization studies revealed that CD68 and PACAP38 were not co-localized (arrows; B,C). Single PGP 9.5-positive cells (arrowheads) were observed within a thymic corpuscle (D). These PGP 9.5-positive cells did not express PACAP38 (D,E). c, cortex of the bursal follicles; m, medulla of the bursal follicles; tc, thymic corpuscle; tl, thymic lobule. Scale bar = 25µm.

Fig. 4

Agarose gel electrophoresis with ethidium bromide staining of PCR products amplified from thymus and bursa of Fabricius with primers specific for the duck PACAP gene. Total RNA was used as template for first-strand synthesis and cDNA was then amplified by PCR using PACAP primers. Sizes are estimated from DNA ladder (100-bp fragments) co-electrophoresed as markers. Lane 1, thymus, 170 days; lane 2, thymus, 140 days; lane 3, thymus, 110 days; lane 4, thymus, 90 days; lane 5, thymus, 70 days; lane 6, thymus, 50 days; lane 7, thymus, 30 days; lane 8, thymus, 10 days; lane 9, bursa of Fabricius, 110 days; lane 10, bursa of Fabricius, 140 days; lane 11, control – total RNA sample without reverse transcriptase; M, markers.