Cytokine expression in phytohaemagglutinin‐induced skin inflammation in a galliform bird

Abstract

Researchers interested in ecological immunology face substantial methodological problems: 1) most immunological approaches are difficult to perform in free‐living animals, 2) in some of the applicable methods the immunological background of the test remains unclear. The latter is also true for the phytohaemagglutinin (PHA) skin‐swelling test, a trait of cell‐mediated immunity commonly measured in ecology. A lack of direct evidence documenting the immunological processes in the tissue limits our understanding of the mechanism triggering the response to PHA. Understanding of this mechanism is, nonetheless, crucial for us to uncover the nature of ecological costs and benefits of investments into the response. As knowledge of cytokine signalling in the tissue may clarify the response mechanism, in our study we investigated the association between the PHA‐induced skin‐swelling and tissue cytokine expression in males of grey partridge Perdix perdix. In PHA‐challenged birds we assessed expression of nine cytokines (IL‐1β, IL‐2, IL‐4, IL‐6, IL‐10, IL‐12, IL‐17, TGF‐β, IFN‐γ) in wing‐web skin during an early stage of the immune response. We examined the relationship between the magnitude of tissue swelling and cytokine expression. Contrary to some earlier expectations we did not find any differential expression of T‐cell growth factor, IL‐2, in the tissue. Hence, T‐cell proliferation at the time of the swelling measurement is unlikely. We detected differential expression in Th17 pro‐inflammatory (IL‐1β, IL‐6) and anti‐inflammatory (TGF‐β) cytokines. The PHA‐induced swelling response was only weakly linked to the expression of TGF‐β. We also found relationships between the PHA‐induced swelling response and phenotypic traits of the birds; the PHA swelling was positively associated with the extent of melanin‐based breast ornamentation and negatively related to body size. Our results might suggest that variation in swelling is influenced by total numbers of responding cells rather than by differences in signalling. Moreover, we revealed significant correlations in expression of IL‐1β, IL‐6 and TGF‐β. These findings are the first to show on the molecular level that the PHA skin‐swelling test actually measures inflammation process which is part of innate immune defence and not the adaptive immune response (as assumed if the test was the reflection of T‐cell proliferation).