title: Application of Concanavalin A during immune responsiveness skin-swelling tests facilitates measurement interpretation in mammalian ecology.
abstract: The skin-swelling test is a simple and widespread method used in field ecological research to estimate cellular immune responsiveness in animals. This immunoecological test is based on measuring the magnitude of tissue swelling response at specific times following subcutaneous application of an experimental pro-inflammatory stimulant. In the vast majority of studies across vertebrate taxa, phytohemagglutinin (PHA) is used as a universal stimulant. Given the complexity of immune response activation pathways of PHA, however, interpretation of test results can be ambiguous. Goal of this study was to improve methodology of the skin-swelling test to decrease this ambiguity. Here, we present an alternative protocol aimed at facilitating interpretation of skin-swelling data for mammals. Based on previous evidence suggesting that mammalian T cells are readily activated by Concanavalin A (ConA) in vitro, we compared cellular immune responses in vivo to PHA and ConA as an alternative pro-inflammatory stimulant in mice. We measured magnitude of tissue swelling and compared it with intensity of blood cell infiltration into tissue over a 72-hour interval. Our results corroborate that PHA and ConA show important differences in both dynamics and response amplitude in rodents. ConA induces stronger swelling with a distinct leukocyte activity pattern and higher pro-inflammatory cytokine (interleukin 6 [IL-6] and interferon gamma[IFN-γ]) expression than PHA during peak response (24-h post-treatment). Furthermore, unlike PHA, magnitude of swelling was positively associated with cellular activity (number of neutrophils infiltrating tissue) following ConA injection. We conclude that ConA is the more suitable stimulant for skin-swelling tests in mammals. This is because of the molecular binding specificity in the two lectins, that is, ConA specifically activates T cells while PHA also triggers erythroagglutination. We propose that ConA be used in all future ecological testing in mammals as it exhibits better performance and its application facilitates immunological interpretation of skin-swelling test results.
authors.affiliation: Department of Zoology Faculty of Science Charles University in Prague Viničná 7 128 44 Praha Czech Republic, EU.
authors.forename: Barbora
authors.initials: B
authors.lastname: Bílková
authors.affiliation: Department of Zoology Faculty of Science Charles University in Prague Viničná 7128 44 Praha Czech Republic, EU; Research Facility Studenec Institute of Vertebrate Biology Czech Academy of Sciences Květná 8603 65 Brno Czech Republic, EU.
authors.forename: Tomáš
authors.initials: T
authors.lastname: Albrecht
authors.affiliation: Institute of Experimental Medicine Czech Academy of Sciences Vídeňská 1083 142 20 Praha 4 Czech Republic, EU.
authors.forename: Milada
authors.initials: M
authors.lastname: Chudíčková
authors.affiliation: Institute of Experimental Medicine Czech Academy of Sciences Vídeňská 1083 142 20 Praha 4 Czech Republic, EU.
authors.forename: Vladimír
authors.initials: V
authors.lastname: Holáň
authors.affiliation: Research Facility Studenec Institute of Vertebrate Biology Czech Academy of Sciences Květná 8 603 65 Brno Czech Republic, EU.
authors.forename: Jaroslav
authors.initials: J
authors.lastname: Piálek
authors.affiliation: Department of Zoology Faculty of Science Charles University in Prague Viničná 7 128 44 Praha Czech Republic, EU.
authors.forename: Michal
authors.initials: M
authors.lastname: Vinkler
keywords: Concanavalin A; PHA‐induced hypersensitivity; T‐cell‐mediated immunocompetence; cytokine; ecoimmunology; evolutionary immunology; field immunoecological research; histology; leucocytes; phytohemagglutinin