Functional analysis of the skin-swelling response to phytohaemagglutinin

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1 Functional Ecology 2010, 24, doi: /j x Functional analysis of the skin-swelling response to phytohaemagglutinin Michal Vinkler*,1,2, Hana Bainová 3 and Tomáš Albrecht 1,2 1 Department of Zoology, Faculty of Science, Charles University in Prague, Viničná 7, Prague, CZ , Czech Republic; 2 Institute of Vertebrate Biology, Academy of Sciences of the Czech Republic, v.v.i., Květná 8, CZ Brno, Czech Republic; and 3 Department of Ecology, Faculty of Science, Charles University in Prague, Viničná 7, Prague, CZ , Czech Republic 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. Key-words: avian innate and adaptive immunity, ecoimmunology, ecological immunology, ecotoxicology, inflammatory response, PHA-induced hypersensitivity, phytohaemagglutinin, T-cell-mediated immunocompetence Introduction The experimental induction of an immune response with a particular agent is a basic approach to standardized examination of the individual immune defence capacity (Ardia & Schat 2008). To investigate cell-mediated immunity in *Correspondence author. vinkler1@natur.cuni.cz immunoecology and ecotoxicology phytohaemagglutinin (PHA) is almost universally the agent of choice (Kennedy & Nager 2006). Results obtained by the PHA skin-swelling test are often interpreted as measurements of T-cell-mediated immunocompetence (e.g. Saino et al. 2003; Brommer 2004; Cucco et al. 2006; Mougeot 2008; Tella et al. 2008; Bonato et al. 2009). There are, however, several indications that we may misunderstand the immunological nature of Ó 2010 The Authors. Journal compilation Ó 2010 British Ecological Society

2 1082 M. Vinkler et al. the test (Kennedy & Nager 2006). As there are different predictions for adaptive and innate immunity regarding, for example, their energetic costs and developmental rates (Palacios et al. 2009), this is an important issue in ecological research. The test based on PHA intradermal application was originally developed in medical (Airo et al. 1967; Bonforte et al. 1972) and veterinary research (Goto et al. 1978; Cheng & Lamont 1988). Being later adopted in ecology (Lochmiller, Vestey & Boren 1993), it was modified to better fit the requirements of field studies (Smits, Bortolotti & Tella 1999). At present, the test compares the wing-web thickness before and after the subcutaneous application of PHA. The difference corresponding to the swelling response expressed in metrical units is then taken as an index of the immune responsiveness. Thanks to its methodological simplicity a significant number of studies have adopted the PHA skin-swelling test (Kennedy & Nager 2006). Surprisingly, despite the widespread use of this test, little is known about the immunological mechanisms underpinning the swelling response seen following the application of PHA in vivo (Kennedy & Nager 2006). Based on information obtained by the few studies that attempted to clarify these immunological processes we are aware that the response is highly complex, involving both cells of the adaptive and innate immunity (Goto et al. 1978; McCorkle, Olah & Glick 1980; Martin et al. 2006). The composition of the cellular infiltrate indicates this response is typically proinflammatory (Campbell & Ellis 2007). However, there is also some evidence suggesting that the response may be somehow specifically related to T-cell activity. First, PHA is known to possess lymphoproliferative properties (Nowell 1960). The PHA molecules bind to T-cell receptors regardless of their specifity and induce T-cell mitosis (Licastro, Davis & Morini 1993). Secondly, in thymectomized animals the inflammatory response to PHA is severely impaired (Goto et al. 1978). Recently, Tella et al. (2008) have shown that various blood-borne lymphocyte subsets may be affected by in vivo PHA treatment and that after repeated exposure to PHA, acquired immunity may develop in birds. This evidence convinced many authors to interpret their results as measurements of T-cell-mediated immunocompetence. It is, however, beyond any doubt that the inflammatory response measured following primary exposure to PHA inoculation is not acquired, and cannot be the result of T-cell proliferation per se. The leukocyte infiltration starts 30 min after PHA application (McCorkle, Olah & Glick 1980), that is, it is too rapid for lymphocyte mitosis to occur (Tokuyasu, Madden & Zeldis 1968). Alternatively, PHA could activate the few T cells present at the injection site prior to the treatment and these might in response induce inflammation. This mechanism has, however, never been functionally tested and verified. For in vivo applications, PHA-P, lectin extracted from Phaseolus vulgaris (Rigas & Johnson 1964; Licastro, Davis & Morini 1993), is universally used. Concerning its biological activity, PHA-P has dual function: haemagglutination (Rigas & Osgood 1955) and lymphoproliferation (Nowell 1960). The potential difficulty in interpreting results to differentiate between the relative strength of these two activities in vivo has been recently pointed out by Kennedy & Nager (2006). The commercially available PHA-P is a mixture of five isolectins that are tetramers composed of varying proportions of two PHA subunits, L and E (Yachnin et al. 1972). Thus, PHA-P is formed by PHA-L (tetramer L4), PHA-E (tetramer E4) and mixed tetramers (L3E1, L2E2 and L1E3; Felsted et al. 1977). Within any native tetramer, the L-subunit is a potent leukoagglutinin and mitogen that lacks erythroagglutinating properties, whereas the E-subunit is a potent erythroagglutinin with no mitogenic activity (Miller et al. 1975). This results in different biological activities of the PHA-L and PHA-E isolectins (Leavitt, Felsted & Bachur 1977; see also Although most of the current knowledge regarding the biological properties of PHA isolectins is based on research in mammals, the activity of the subunits in birds seems to be similar (for erythroagglutination confirmed by M. Vinkler & H. Bainova, unpublished). This study aimed to test the functional importance of T-cell activation in the process of induction of the swelling reaction to PHA subcutaneous treatment in zebra finch (Taeniopygia guttata; Fig. 1). First, we questioned whether the reaction is dependent on the specific biological properties of PHA. To test this we compared the immune response to PHA-P with the response to a non-mitogenic and non-erythroagglutinating (control) protein, bovine serum albumin (BSA; Carlson & Allen 1969). Secondly, to investigate the relative contribution of T-cell activation to the immune response, we assessed the effect of PHA-L and PHA-E. We predicted that following the application of PHA-E, no swelling response would take place if the reaction is dependent on initial T-cell activation. Histological analysis was performed to describe differences in cellular infiltration into the swelling. Finally, we compared the in vivo action of PHA-L with PHA-P to find out to what extent these different treatment inocula altered the test results. Fig. 1. The zebra finch (Taeniopygia guttata). Photograph by Hana Bainová.

3 Functional analysis of PHA skin-swelling response 1083 Materials and methods ANIMALS A total of 64 young zebra finch males in their second calendar year (c. 7 months of age) were obtained from hobby breeders. In April 2008, these birds were randomly assigned into three experimental groups (Experiment I: 10, Experiment II: 26, Experiment III: 28 individuals). Prior to any experimental treatment zebra finches were housed for 3- day acclimation in indoor cages (three to four individuals per cage) with access to food and water ad libitum. TREATMENT INOCULA PHA-P (no. L8754) as well as the purified PHA-E and PHA-L isolectins (nos L8629 and L2769) and BSA (no. A3902) were obtained from Sigma-Aldrich, St Louis, MO, USA. All proteins in each experiment were dissolved in Dulbecco s phosphate-buffered saline (Sigma-Aldrich; D5652) in a treatment dosage of 0Æ1 mgper20ll of the buffer. Republic). Three sections were made from the middle part of each biopsy sample. Later histological analysis was performed according to Martin et al. (2006) with the following modifications. In each sample, two independent places were photographed in each section (i.e. six photographs per sample) under 40 objective magnification (microscope Olympus BX51 with digital camera Olympus DP71, Tokyo, Japan; acquisition software QuickPHOTO Industrial version 2Æ3; Promicra, Praha, Czech Republic). Into all images (obtained in scale) a frame of 0Æ09 0Æ09 mm was inserted later in CorelPHOTO-PAINT X3 software (Corel Corporation, Ottawa, Canada) at a random position (but away from blood vessels). In this frame, all cells were counted using IMAGEJ 1Æ40 g software ( Six categories of cells were recognized: lymphocytes, monocytes, heterophils, basophils, erythrocytes and thrombocytes. In summary, we obtained a total cell count and individual cell type counts per 0Æ0486 sq mm. Although this method does not provide data suitable for absolute quantification of cells in the tissue it provides reliable data for amongsample comparative analysis. EXPERIMENTAL DESIGN After the acclimation period, the thickness of both wings was measured in all birds in the centre of the patagium (wing-web) region using a pressure-sensitive digital micrometer (Mitutoyo 7301, Mitutoyo Corporation, Kanagawa, Japan; accuracy 0Æ01 mm). Immediately thereafter the treatment inocula were injected into these spots. Then, each individual was placed back into its cage and kept in quiet until the response measurement 24 h (±1 h) later. As the dynamics of the response to PHA-P is known (including the cellular infiltration; Martin et al. 2006) we decided for a 24-h period, which is commonly used in ecological studies (Smits, Bortolotti & Tella 1999). All treatments were performed between 13:00 and 18:00 h to minimize the effect of diurnal variation in the swelling response (see Navarro et al. 2003). All measurements were made in triplicate and for further calculations the average values were used. Repeatabilities of the measurements were as follows: Experiment I: r BSA0 =0Æ90, r BSA24 =0Æ96, r PHA-P0 =0Æ89, r PHA-P24 =0Æ98; Experiment II: r PHA-L0 =0Æ91, r PHA-L24 =0Æ99, r PHA-E0 =0Æ96, r PHA-E24 =0Æ97; Experiment III: r PHA-P0 =0Æ93, r PHA-P24 =0Æ98, r PHA-L0 =0Æ93, r PHA-L24 =0Æ99 (calculated according to Lessells & Boag 1986). Wing-web swelling index was later calculated as the patagium s average thickness measurement 24 h after the injection minus the average thickness prior to the injection (Smits, Bortolotti & Tella 1999). Three experiments were conducted: (i) the right wing-web of each bird was treated with PHA-P and the left one with BSA (Experiment I); (ii) the right wing-web of each bird was treated with PHA-E and the opposite one with PHA-L (Experiment II); and finally (iii) the right wing-web of each bird was treated with PHA-P and the left one with PHA-L (Experiment III). STATISTICAL ANALYSES For comparisons of PHA skin-swelling responses, the paired t-test was performed. In all cases, the approximation of the sample distribution to the Gaussian distribution was tested by one-sample Kolmogorov Smirnov goodness-of-fit test. As some of the individual cell type samples in patagium biopsy sections did not show Gaussian distribution (owing to small sample size), exact Wilcoxon rank-sum test was used for the comparisons of individual cell type infiltration. Correlations were tested using Pearson s product-moment correlation. The significance level was set to P = 0Æ05. All statistical analyses were performed using S-PLUS 6.0 software (Lucent Technologies, Inc, Murray Hill, NJ, USA). ETHICAL NOTE This research was approved by the Ethical Committee of the Department of Science, Charles University in Prague (reference no.: ). Results EXPERIMENT I The swelling response was significantly greater in PHA-Ptreated wings than in BSA-treated wings (paired t-test: t = )13Æ63, d.f. = 9, P << 0Æ001). In fact, no measureable swelling was induced by the injection of BSA (paired t-test, post- vs. pre-bsa: t = )2Æ56, d.f. = 9, P =0Æ985). HISTOLOGICAL ANALYSIS After metric measurement of the swelling response in Experiment II a tissue sample (2 mm in diameter) was punched in 12 randomly chosen individuals from the centre of the inoculation site. The samples were immediately fixed with 10% buffered formalin in which potential edge contamination was washed out. Later they were embedded into paraffin, sectioned and stained with haematoxylin and eosin (see Goto et al and Martin et al. 2006; performed by BIOLAB Praha Laboratory, Praha, Czech EXPERIMENT II The swelling response was significantly stronger in PHA-Etreated wings than in wings to which PHA-L was applied (paired t-test: t = )7Æ41, d.f. = 25, P << 0Æ001; Fig. 2). Also the absolute cell infiltration into the tissue investigated in the 12 individuals from histological sections tended to be greater in PHA-E-induced sites (paired t-test: t = )1Æ95, d.f. = 11, P = 0Æ077). Pairwise comparison also revealed

4 1084 M. Vinkler et al. Fig. 2. Swelling responses to inoculation of wing-webs with PHA-L and PHA-E (n = 26). Swelling characterized as the wing-web thickness 24 h after PHA treatment minus the thickness prior to the treatment. Mean indicated by dot and SD by whiskers. PHA indicates phytohaemagglutinin. Fig. 3. Relationship between measurements of the swelling responses to inoculation of wing-webs with PHA-L and PHA-E (n =26, r = 0Æ483). PHA indicates phytohaemagglutinin. Table 1. Cell counts recorded in histological sections of inoculated tissues in Experiment II (n = 12) expressed in cells per 0Æ0486 sq mm PHA-L PHA-E Cell type Range Mean ± SD Range Mean ± SD Lymphocytes Æ08 ± 20Æ Æ42 ± 27Æ68 Heterophils Æ58 ± 31Æ Æ67 ± 59Æ53 Basophils Æ91 ± 31Æ Æ25 ± 9Æ93 Macrophages 1 6 2Æ75 ± 1Æ Æ50 ± 5Æ04 Erythrocytes Æ08 ± 48Æ Æ67 ± 131Æ10 Thrombocytes Æ67 ± 14Æ Æ00 ± 26Æ41 Total cell count Æ08 ± 77Æ Æ50 ± 165Æ45 PHA, phytohaemagglutinin. differences in the quality of the cellular composition (see Table 1). We have found significantly higher erythrocyte (Wilcoxon rank-sum test: Z = )2Æ08, P = 0Æ038) and thrombocyte numbers (Wilcoxon rank-sum test: Z = )2Æ66, P = 0Æ008) in the PHA-E-exposed wing-webs compared with the wing-webs injected with PHA-L. In contrast to PHA-L-treated wings, the PHA-E samples frequently contained erythrocyte and thrombocyte clots. However, both in PHA-L and PHA-E treatments some samples contained massive concentrations of lymphocytes and thrombocytes (with accidental admixture of other cell types) along the blood vessels. There was a significant positive correlation between the magnitude of responses induced by PHA-L and PHA-E at the metric level (Pearson s product-moment correlation: r =0Æ483, n =26, P =0Æ013; Fig. 3). Fig. 4. Swelling responses to inoculation of wing-webs with PHA-L and PHA-P (n = 28). Swelling characterized as the wing-web thickness 24 h after the PHA treatment minus the thickness prior to the treatment. Mean indicated by dot and SD by whiskers. PHA indicates phytohaemagglutinin. EXPERIMENT III There was no significant difference between the magnitudes of the swelling in wings inoculated with PHA-L and PHA-P (paired t-test: t =0Æ57, d.f. = 27, P =0Æ576; Fig. 4). The swelling response to PHA-P was significantly correlated with Fig. 5. Relationship between measurements of the swelling responses to inoculation of wing-webs with PHA-L and PHA-P (n =28, r = 0Æ530). PHA indicates phytohaemagglutinin. that induced by PHA-L (Pearson s product-moment correlation: r =0Æ530, n =28,P =0Æ004; Fig. 5).

5 Functional analysis of PHA skin-swelling response 1085 Discussion Our experiments are the first to assess the importance of the two independent functional properties of PHA (lymphocyte mitogenic and erythrocyte agglutinating; Kennedy & Nager 2006) in induction of the swelling response in vivo. There are three key findings revealed by this study. First, as BSA failed to induce any response comparable with that induced by PHA-P, the skin-swelling response to PHA is caused by the specific biological properties of PHA. Secondly, both PHA-L and PHA-E subunits are able to stimulate the swelling response. This response is significantly stronger in the case of PHA-E (erythroagglutinative subunit) and the cellular composition of the infiltration caused by the different isolectins is distinct. Thirdly, the metric measurements of the swelling responses to PHA-E and PHA-L as well as PHA-P and PHA-L are positively correlated and there is no significant difference between experimental treatment with PHA-L and PHA-P. Based on knowledge that PHA possess lymphoproliferative properties (Nowell 1960) and that in vivo response to PHA is impaired by thymectomy (Goto et al. 1978), many authors presume that the PHA-induced swelling results from T-cell proliferation or infiltration. Thus, the test results were often interpreted as measurements of T-cell-mediated immunocompetence (e.g. Saino et al. 2003; Brommer 2004; Cucco et al. 2006; Mougeot 2008; Bonato et al. 2009). Nevertheless, as shown already by McCorkle, Olah & Glick (1980) and more recently by Martin et al. (2006) this is not the case and other leukocyte types also infiltrate the tissue (leading to a cellular composition that greatly resembles other inflammatory processes; Carlson & Allen 1969; Campbell & Ellis 2007). Although it is thus clear that T-cell mitosis is not responsible for the swelling response, one could argue that the intensity of the immune response is dependent on the efficiency of initial T-cell activation at the treatment site. Most recently, Tella et al. (2008) attempted to restore the original view on the PHA skin-swelling test as a reliable evaluator of acquired T-cell-mediated immunocompetence. Although their results indicate the ability of the immune system to respond in an adaptive manner to repeated PHA challenges (which is uncommon in ecological studies) they lack any power to demonstrate that these processes are also involved in the primary response (adopted in virtually all immunoecological and ecotoxicological research). The changes found by Tella et al. (2008) in lymphocyte subsets in the circulation very likely reflect a general proinflammatory immune response. The results of Goto et al. (1978) support this view. Neonatal thymectomy seriously unbalances vertebrate immunity in general (Rose 1994), influencing various immune reactions. As Goto et al. (1978) admit, some individuals responded to PHA even after thymectomy. This would be hardly possible if the response was solely based on T-cell activation. Thus, there is, to our knowledge, no clear evidence showing that the PHA skin-swelling test mirrors T-cell function. On the contrary, in Experiment II we demonstrated that the erythroagglutinative subunit of PHA (PHA-E), which lacks the ability to activate T cells, triggers a stronger swelling response than the T-cellbinding subunit (PHA-L). Based on the current knowledge, this finding disproves the former interpretation that the PHA-induced skin-swelling test indicates T-cell activity. In PHA-E-induced swelling, erythrocytes and thrombocytes were much more prevalent than in swelling induced by PHA-L. Erythrocytes do not penetrate through the blood vessel walls. Their presence in the sections (apart from blood vessels) thus likely results from their release into the tissue following minor (visually undetectable) tissue damage, which occurs during any treatment application (see Kennedy & Nager 2006). We propose that because of its biological properties, PHA-E promotes erythrocyte agglutination in the damaged tissue as well as in the nearby vessels. This might stimulate an immune response directed to wound healing. In this process thrombocytes play a prominent role (Lucas & Jamroz 1961; Campbell & Ellis 2007), explaining their increased numbers detected in PHA-E-induced swelling. The inflammatory response induced by PHA treatment is highly complex (McCorkle, Olah & Glick 1980; Martin et al. 2006; present data). Results from Experiment III advocate the idea that the involvement of many different immunity components in the resultant immune response may to a great extent surpass the effect of dissimilarity in stimulation via PHA-L and PHA-E. Furthermore, the positive correlation between the magnitude of the swelling response to PHA-L and PHA-E (Experiment II) also indicates that the PHAinduced skin-swelling is not completely dependent on the kind of initial stimulation. These findings suggest that the PHA skin-swelling test very likely measures the general inducibility of proinflammatory signalling leading to cellular infiltration at the site of inflammation. This signalling forms an intricate web of interactions which is regulated at different levels (Vaday et al. 2001). There is no doubt that lymphocytes (including T cells) are also involved (as shown by Tella et al. 2008). However, regardless of this presumed involvement, the test does not provide any explicit information about T-cell responsiveness. Conclusion The PHA skin-swelling test is the most widely used test available for estimation of the vertebrate immune response in vivo (Kennedy & Nager 2006). Although it is clear that the test is able to reveal severe immunodeficiency (Goto et al. 1978), its convenience for more subtle immunological testing has been questioned (Kennedy & Nager 2006; Owen & Clayton 2007). Our results indicate that the PHA skin-swelling test is a robust tool useful for quick screening of an individuals proinflammatory potential (i.e. the ability to mount an inflammatory response). However, the complexity of the response does not allow any closer characterization of the nature of immunological variance detected by this method. The test cannot be used as a clear indicator of T-cell responsiveness. As different costs and benefits may be predicted for adaptive and innate immunity this interpretational shift may alter our view on the ecological meaning of the test results.

6 1086 M. Vinkler et al. Acknowledgements The authors are very grateful to Jana Břehova for her attentive involvement in the experimental work and to Mark Gibson, Martina Pokorna and Dagmar Vinklerova for their comments on the manuscript. Grants from the Czech Science Foundation (project nos: GACR and ), the Ministry of Education, Youth and Sport of the Czech Republic (project no.: MSMT ) and the Academy of Sciences of the Czech Republic (project no.: AV0Z ) formed a framework for this study. T. Albrecht was partially supported by the Research Centrum project no. LC References Airo, R., Mihailes, E., Astaldi, G. & Meardi, G. (1967) Skin reactions to phytohaemagglutinin. Lancet, 1, Ardia, D.R. & Schat, K.A. (2008) Ecoimmunology. Avian Immunology (eds F. Davison, B. Kaspers & K.A. Schat), pp , Academic Press, Elsevier, London. Bonato, M., Evans, M.R., Hasselquist, D. & Cherry, M.I. (2009) Male coloration reveals different components of immunocompetence in ostriches, Struthio camelus. 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