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.

Immunoregulatory Effects of Imuno TF® (transfer factors) on Th1/Th2/Th17/Treg Cytokines

“…Additionally, two mitogens were used to evaluate the immunomodulatory effects of the Imuno TF ® : LPS and Con A. Both LPS and Con A are widely used in in vitro and in vivo tests to assess the ability of cells to secrete various types of cytokines [29][30][31].…”

Immune responsiveness to phytohemagglutinin displays species but not sex differences in three anuran species

“…Among several immunological assays, the phytohemagglutinin (PHA) skin-swelling test is one of the most widely used techniques to evaluate the magnitude of vertebrate immune response ( Sheldon & Verhulst, 1996 ; Boughton, Joop & Armitage, 2011 ; Vinkler & Albrecht, 2011 ; Brock, Murdock & Martin, 2014 ). The assay consists of a subcutaneous injection of PHA that induces infiltration of leukocytes and where swelling is interpreted as an integrative immune response ( Christe, Arlettaz & Vogel, 2000 ; Kennedy & Nager, 2006 ; Martin et al, 2006 ; Demas et al, 2011 ; Brock, Murdock & Martin, 2014 ) or as an inflammation in endotherms ( Vinkler et al, 2012 ; Bílková, Vinklerová & Vinkler, 2015 ; Bílková et al, 2016 ). However, inferences using this assay should be carefully evaluated because not always a greater immune response or inflammation means better defenses against infections or higher survival probabilities ( Graham, Allen & Read, 2005 ; Vinkler et al, 2012 ).…”

“…PHA assay has been also used to assess the immune responsiveness under different ecologically relevant conditions, such as when amphibians are forced to accelerate the metamorphosis by exposition to environment desiccation ( Gervasi & Foufopoulos, 2008 ), treated with protein restricted diets ( Venesky et al, 2012 ), and exposed to density stress ( Clulow, Harris & Mahony, 2015 ). In different vertebrate species, PHA response peaks in a wide time window, from 3 to 48 h ( De Bellocq et al, 2006 ; Martin et al, 2006 ; Turmelle et al, 2010 ; Xu & Wang, 2010 ; Brown, Shilton & Shine, 2011 ; Finger et al, 2013 ; Josserand et al, 2015 ; Zhang, Huang & Zhao, 2015 ; Bílková et al, 2016 ), with no apparent sex differences in some small mammals ( Zhang, Qiu & Wang, 2011 ; Merlo, Cutrera & Zenuto, 2014 ) and amphibians ( Brown et al, 2015 ; Clulow, Harris & Mahony, 2015 ). Most up-to-date information available on the time course and temporal dynamics of the PHA responses within and among populations or species are for birds ( Navarro et al, 2003 ) and small mammals ( De Bellocq et al, 2006 ; Zhang, Huang & Zhao, 2015 ).…”

Peer Review #2 of "Immune responsiveness to phytohemagglutinin displays species but not sex differences in three anuran species (v0.1)"

“…Among several immunological assays, the phytohemagglutinin (PHA) skin-swelling test is one of the most widely used techniques to evaluate the magnitude of vertebrate immune response (Sheldon & Verhulst, 1996;Boughton, Joop & Armitage, 2011;Vinkler & Albreeht, 2011;Broek, Murdoek & Martin, 2014). The assay consists of a subcutaneous injection of PHA that induces infiltration of leukocytes and where swelling is interpreted as an integrative immune response (Christe, Arlettaz & Vogel, 2000;Kennedy & Nager, 2006;Demas et al, 2011;Broek, Murdoek & Martin, 2014) or as an inflammation in endotherms (Vinkler et al, 2012;Bílková, Vinklerová & Vinkler, 2015;Bílková et al, 2016). However, inferences using this assay should be carefully evaluated because not always a greater immune response or inflammation means better defenses against infections or higher survival probabilities (Graham, Allen & Read, 2005;Vinkler et al, 2012).…”