Functional analysis of the skin‐swelling response to phytohaemagglutinin

Summary 1. The phytohaemagglutinin (PHA) skin‐swelling test is widely used in immunoecology and ecotoxicology to estimate cell‐mediated immunity. Although often presumed, the involvement of T cells in generating an immune response to PHA in vivo remains unclear. 2. To investigate the mechanism triggering this response we have compared in zebra finch ( Taeniopygia guttata ) the immune responses to two PHA isolectins differing in their biological properties and one control protein. 3. In Experiment I, we applied PHA‐P (the commonly used L and E isolectin mixture) into one wing‐web and bovine serum allbumin (BSA) into the other. The swelling response to PHA‐P was significantly stronger than to BSA confirming that the reaction is governed by PHA‐specific properties. 4. In Experiment II, purified PHA‐L (the T‐cell‐stimulating isolectin) and PHA‐E (the erythroagglutinating isolectin) were compared. Contrary to our expectations, PHA‐E induced a significantly stronger swelling response than PHA‐L. Histological analysis revealed significantly higher quantities of erythrocytes and thrombocytes in PHA‐E‐treated patagia. Nevertheless, there was a positive correlation between the magnitudes of these swellings. 5. In Experiment III, we tested whether the results obtained by the application of PHA‐P and pure PHA‐L differ. Here, we failed to find any significant difference between these two preparations in their immunostimulatory activity. Magnitudes of the PHA‐L‐ and PHA‐P‐induced swellings were positively correlated. 6. To the best of our knowledge this is the first study to compare the biological activity of purified PHA fractions in vivo and also the first to show the importance of erythroagglutination in the development of an inflammatory response to PHA‐P. 7. Our results indicate that the skin‐swelling test using PHA‐P reliably mirrors the individual general proinflammatory potential. However, the immunological background of the test is highly complex and the test results cannot be interpreted as measurements of the adaptive immunity or T‐cell activity. This interpretational change importantly alters our view on the test results regarding the costs of the response or the evolutionary immunological adaptations.