Acta Herpetologica 9(2): 131-138, 2014 ISSN 1827-9635 (print) © Firenze University Press ISSN 1827-9643 (online) www.fupress.com/ah DOI: 10.13128/Acta_Herpetol-13188 In vitro temperature dependent activation of T-lymphocytes in Common wall lizards (Podarcis muralis) in response to PHA stimulation Roberto Sacchi1,*, Enrica Capelli1, Stefano Scali2, Daniele Pellitteri-Rosa1, Michele Ghitti1, Emanuele Acerbi1, Elena Pingitore1 1 Dipartimento di Scienze della Terra e dell’Ambiente, Università degli Studi di Pavia, Via Ferrata 9, I-27100 Pavia, Italy. *Correspond- ing author. E-mail: roberto.sacchi@unipv.it 2 Museo Civico di Storia Naturale, C.so Venezia 55, I-20121 Milano, Italy Submitted on 2013, 28th October; revised on 2014, 23rd June; accepted on 2014, 24th June Editor: Uwe Fritz Abstract. Ecological immunology attempts to explain the variability of immune response among individuals by invok- ing costs and trade-offs, which may optimize the immune defence against pathogens. In ectotherms body temperature is correlated to that of the surrounding environment, so that their entire physiology, including immune functions, is influenced by the environmental temperature. We used in vitro phytohaemoagglutinin (PHA) stimulation in order to assess the effects of temperature on cell mediated adaptive response in male and female Common wall lizards (Podar- cis muralis). Cell cultures were prepared from blood samples, inoculated with PHA and incubated at 22°C, 25°C, 32°C, and 38°C for three days. PHA stimulation caused proliferation of T-lymphocytes, but the effect depended on the incubation temperature. Lymphocyte proliferation was significantly impaired at both 22°C and 38°C compared to 32°C, which represented the highest levels of activation. Furthermore, lymphocyte activation was more variable in males while females were less immune suppressed than males at low temperatures. Differences between sexes suggest a possible influence of steroid hormones. Keywords. Ecological immunology, adaptive immune response, temperature dependent effects, phytohaemoaggluti- nin PHA, T-lymphocyte proliferation, ectotherms. INTRODUCTION Ecological immunology investigates the physiologi- cal or molecular basis of immune responses, by placing them in the context of ecology and adaptation (Shel- don and Verhulst, 1996; Norris and Evans, 1999; Sadd and Schmid-Hempel, 2009). There is extensive evidence that immune defence is costly, requiring investment of energy, nutrients, and time, during the development, maintenance, and use of the immune system (Klas- ing and Leshchinsky, 1999; Lochmiller and Deerenberg, 2000). When resources are limited, allocation of energy to immune defences may be modulated by the need to spend energy on other functions such as growth, repro- duction, and maintenance (Nelson and Demas, 1996). Thus, multiple trade-offs occurring between immune functions and other compartments and life-processes cause severe constraints on the evolution of immune response and the other fitness related traits. Costs of development, maintenance and use of immune defence have been widely investigated on homeotherm vertebrates, and especially birds (Nor- ris and Evans, 1999; Martin et al., 2008). By contrast, ecthotermic vertebrate have been less extensively studied (see Zimmerman et al., 2010), even if a large amount of researches had been done on fish (e.g., Bly and Clem, 1992; Collazos et al., 1996; Köllner and Kotterba, 2002; Ndong et al., 2007; Prophete et al., 2009). Ectotherms are particularly interesting under an eco-immunological perspective, as body temperature is correlated to that of 132 R. Sacchi the surrounding environment, so that their entire physi- ology, including immune functions, is influenced by the environmental temperature (Zimmermann et al., 2010). This offers an interesting opportunity to investigate the costs of the immune response, as the trade-offs medi- ated by the immune function are likely to have different settings at different environmental temperatures. Both endotherms and ectotherms show seasonal variation in immune-response, but ectotherms display higher vari- ability due to their dependence on environmental tem- peratures as heat source (Zimmermann et al., 2010). The immune system of ectotherms has been shown to respond across a wide range of temperatures, but strongest responses occur at species-specific temperature ranges, with impaired responses at temperatures above and below the optimal threshold (Le Morvan et al., 1998; Mondal and Rai, 2001; Merchant et al., 2003; Merchant and Britton, 2006; Raffel et al., 2006). For example, exper- iments carried out on carp (Cyprinus carpio) immunized against bovine serum albumin, revealed that the prima- ry antibody response is suppressed at low temperatures (Avtalion, 1969), particularly that mediated by T-helper cells (Le Morvan et al., 1996). Similarly, Mondal and Rai (2001) reported that the highest levels of phagocytosis and cytotoxicity of splenic macrophages from Hemidac- tylus flaviviridis occurred at 25°C, with impaired mac- rophage function at both higher and lower temperatures. In reptiles, the studies on temperature effects on immune system chiefly focused on the seasonal varia- tion of immune response in both innate (Mondal and Rai, 2001; Merchan et al., 2003, 2004) and adaptive immunity (Muñoz and De la Fuente, 2001; but see Zim- mermann et al., 2010 for a review). Surprisingly, no one has specifically investigated the response of the immune system to short time variation of temperature such that occurring daily or with weather instability. Short time effects of temperature are particularly interesting under an eco-immunological perspective, since pathogen ability to infect ectotherms can be heavily affected by fast tem- perature-dependent fluctuations of host immune system (Wright and Cooper, 1981), with direct consequences on the behavioural, ecological and physiological strategies that individuals adopt to optimize fitness. In the present study we measured the T-cell medi- ated immune response of Common wall lizards Podarcis muralis under different temperatures, covering the typical thermal-activity range of the species. To do this, we set up cell cultures from blood samples that were activated with phytohaemagglutinin (PHA) and incubated at four different temperatures. PHA is a lectin found in plants, especially legumes, which causes local swelling and oedema, driven by mitogenesis and infiltration of T-lym- phocytes into the injected tissue (Goto et al., 1978). The Common wall lizard is a small lizard (snout-vent length, SVL, 45-75 mm) widespread in southern and central Europe, which mates multiply and produces two clutch- es per year on average (Sacchi et al., 2012) during its life (max lifetime 5 years, Barbault and Mou, 1988; pers. obs.). Breeding season starts from late February and ends in July (Sacchi et al., 2012), and body temperature dur- ing activity is near 33°C, being slightly higher (33-36°C) in warmer regions (e.g. Central Italy) and lower (32°C) in mountain areas (Avery, 1978; Braña, 1991; Tosini and Avery, 1994). The immune-system has been previously investigated in relation to polymorphic ventral coloura- tion (Sacchi et al., 2007; Galeotti et al., 2010) or immuno- competence handicap hypothesis (Oppliger et al., 2004), but earlier study have not examined the influences of the environmental factors on the immune system response. MATERIALS AND METHODS Individual collection From 6 July to 4 August 2012, we collected by noosing 24 (12 males and 12 females) adult Common wall lizards (snout- vent length, SVL > 50 mm, Sacchi et al., 2012) in a farm in the surrounding of Pavia (Northern Italy, 45°11’31”N, 9°9’11”E). We carried out five sampling sessions in which at least one male and one female were collected. After capture, lizards were measured by a digital calliper (accuracy ± 0.1 mm) for SVL and transferred in cloth bags to the laboratory at the University of Pavia. Blood sampling and cell cultures Blood samples (15-20 μl) were collected in heparinized capillary tubes from the postorbital sinus (MacLean et al., 1973) and inoculated in 15 ml of RPMI 1640 medium supplemented with 10% bovine serum. Cell suspension was then subdivided into two 7 ml sub-cultures, one of which was inoculated with 1% PHA solution (PHA-P Sigma L-8754, 50 mg in 10 ml phos- phate buffered saline (Oppliger et al., 2004; Sacchi et al., 2007). The remaining solution (1 ml) was used to assess starting cell concentration (SCC) using a Neubauer chamber, and only live lymphocytes were counted. Each sub-culture was then dis- tributed in four 1.5 ml culture tubes, and incubated at 22°C, 25°C, 32°C, and 38°C for 3 days. Afterwards, cell were collected, re-suspended and newly counted. This second count involved only proliferating lymphocytes. Stimulation of T-cell after incu- bation was evaluated by determining the colony-forming units per ml (CFU) and the ratio between the total cell recovery to SCC (growth index, GI). Unfortunately, 30 samples incubated at 32°C were lost because the stove broke up during experiment. So our final sample included 48 cultures for 22°C, 25°C and 38°C, but only 18 for 32°C. 133Temperature effects on T-cells in P. muralis Statistical analyses Preliminarily, we ran a t-test for matched pairs to check for differences in lymphocyte stimulation between the PHA- treated and the control cultures at each of the four incubation temperatures. The CFU in the full sample showed a Poisson- like distribution and thus its relationship with sex, treatment (PHA vs control) and temperature (analysed as a four levels factor) was investigated by a GLMM with Poisson error distri- bution and log link function. The number of colonies counted after the three day incubation was the dependent variable, while the individual identity entered the model as random factor. All main effects and two-way interactions sex × temperature, sex × treatment and temperature × treatment were included in the models as fixed effects. We also included the SVL as covariate in order to account for possible effects of individual size on immune-response. The growth index GI was slightly skewed, and achieved normality after four root transformation. The rela- tionship between GI and sex, treatment and temperature was thus analysed using a linear mixed model including individual identity as random factor, and the same fixed factors, covariate and their interactions used in the analysis of CFU. Both mod- els were then optimized by removing all non-significant terms (using likelihood-ratio tests) until only significant terms were retained (Zuur et al., 2009). All tests were performed using R 2.13.1 statistical package (R Development Core Team, 2010), and unless otherwise stated, values reported are means ± SE. RESULTS Peripheral blood lymphocytes of both sexes were actually stimulated by PHA at all incubation tempera- tures, as both CFU and GI were significantly far higher in activated cultures than in controls. However, the differ- ence between the GI values of females in PHA-activated and control recorded at 32°C was marginally not signifi- cant, probably because of the small sample (see Table 1 for statistics). PHA-activated lymphocytes showed the characteristic activated morphology with enlarged size (2-3 times) and spikes and cell aggregates (clones) con- taining from few to several dozen cells (Fig. 1). The GLMM confirmed the capacity of PHA to acti- vate lymphocytes proliferation, but the intensity of the response was highly affected by both incubation tem- peratures and sex (Fig. 2a). Indeed, the CFU significantly increased in PHA treated cultures with respect to con- trols, but strictly depending on the incubation temper- ature (temperature × treatment: LRT-χ² = 29.34, df = 3, P < 0.0001, Fig. 2a). The highest activation was recorded at 32°C (8056 ± 1197 colonies/ml), while the minimum occurred at both 25°C (2760 ± 489 colonies/ml) and 38°C (2617 ± 531 colonies/ml). Moreover, males showed on average a significant higher activation than females, irrespective of the incubation temperatures (males: 3583 ± 523 colonies/ml, females: 3482 ± 452 colonies/ml; sex × treatment: LRT-χ² = 13.74, df = 1, P = 0.00021), even though PHA-activation was higher in females than in males when cultures were incubated at 22°C (sex × temperature: LRT-χ² = 40.97, df = 3, P < 0.0001, Fig. 2a, Table 1). Finally, the size of individuals did not sig- nificantly affect the activation of lymphocytes (p-value at removal > 0.66). The final model for GI included only the interac- tion temperature × treatment (LRT-χ² = 13.24, df = 3, P = 0.0041), confirming that cell proliferation was higher at 32°C (GI = 13.27 ± 2.26) than in all other temperatures (GI22°C = 8.56 ± 1.93, GI25°C = 7.69 ±1.65, GI38°C = 5.13 ± 0.88, Fig. 2b). The GI was higher in males than in females at 32°C (Fig. 2b), but the effect of sex was not significant (P > 0.81), and was removed from the final model. As for CFU, the size of individuals had no signifi- cant effects on immune-response. Finally, the intensity of the lymphocyte activation was highly variable among individuals, as both models revealed a highly significant effect of the random factor Table 1. In vitro effects of PHA administration on T-lymphocyte proliferation assessed by colony-forming units (CFU) and growth index (GI) in male and female Common wall lizards at four differ- ent temperatures. PHA- activated Controls N t P CFU Males 22°C 2643 ± 694 26 ± 26 12 3.774 0.0031 25°C 2890 ± 692 0 12 4.176 0.0015 32°C 8489 ± 1767 1484 ± 1484 6 3.000 0.030 38°C 2760 ± 787 0 12 3.505 0.0049 Females 22°C 4414 ± 791 0 12 5.581 0.00016 25°C 2630 ± 721 26 ± 26 12 3.570 0.0044 32°C 7187 ± 1005 0 3 7.155 0.019 38°C 2473 ± 746 195 ± 195 12 2.970 0.013 GI Males 22°C 5.85 ± 1.16 1.09 ± 0.22 12 3.906 0.0024 25°C 6.17 ± 1.24 1.25 ± 0.45 12 6.578 0.00039 32°C 16.7 ± 2.18 1.28 ± 0.67 6 3.416 0.019 38°C 4.95 ± 0.93 0.91 ± 0.29 12 4.958 0.00043 Females 22°C 11.26 ± 3.58 1.58 ± 0.67 12 5.745 0.00012 25°C 9.20 ± 3.07 1.56 ± 0.57 12 5.437 0.00020 32°C 6.32 ± 1.05 0.82 ± 0.81 3 3.237 0.084 38°C 5.30 ± 1.53 1.47 ± 0.54 12 3.487 0.0051 134 R. Sacchi (CFU: LRT-χ² = 406.38, df = 1, P < 0.0001, GI: LRT-χ² = 22.01, df = 1, P < 0.0001). DISCUSSION In this paper we analysed the effect of short-time variation of temperature on the lymphocyte activation in male and female Common wall lizards, assessed by PHA injection in blood cultures. Despite the fact that temperature is expected to affect the immune response more extensively in ectotherms than in endotherms, the susceptibility to sudden changes of environmental tem- perature by immune response of the same individual, to our knowledge, had never been performed in a terres- trial reptile. Previous immunological studies in reptiles used PHA injection to examine the negative effect of steroids on cell-mediated immunity (Belliure et al., 2004; Oppliger et al., 2004; Berger et al., 2005; Huyghe et al., 2009), morph-specific differences in immune response (Sacchi et al., 2007; Huyghe et al., 2009), or female pref- erence for males with better immune response (López and Martín, 2005). Several components of the defence mechanisms of reptiles including phagocyte bactericidal activity, leuko- cyte mobilization and humoral mediators of inflamma- tion have been reported to be temperature-dependent (Farag and El Ridi, 1984; Muñoz and De la Fuente, 2001; Merchant et al., 2003, 2004, Keller et al., 2005). However, most studies did not analysed the response of the same individual to different temperatures, rather compared the responses of different groups of individuals to differ- ent temperatures (Farag and El Ridi, 1984; Muñoz and De la Fuente, 2001). So, these findings cannot actually be considered a demonstration of a direct effect of tem- perature on the immune system, since several other fac- tors may affect immune-response (e.g., hormones Mondal and Rai, 2002; Belliure et al., 2004; Huyghe et al., 2009). The in vitro activation of lymphocytes enabled us to set up an experimental design with repeated measures within individual, in which every lizard has been exposed to all the incubation temperatures. This design allowed us to actually evaluate the change in the immune function of a given individual in response to the variation of thermal condition. Thus any significant change in lymphocytes proliferation can be attributed to the direct effect of envi- ronmental temperature. In this study we found that the lymphocytes pro- liferation following PHA stimulation was significantly impaired in cultures kept at both 22°C and 38°C com- pared to that kept at 32°C, which represented the high- est levels of activation in both sexes. Since the ability of lymphocytes to proliferate following a mitogenic stimu- lus in vitro is an indication of immune response in vivo Fig. 1. Morphology of lymphocytes of Common wall lizards in blood cultures: a) a single lymphocyte (black arrow) among eryth- rocytes (white arrow); b) a lymphocyte with spikes and a small (c) and a large (d) cell aggregates. Fig. 2. The in vitro effect of temperature on colony-forming unit (CFU, a) and growth index (GI, b) of T-lymphocytes of male (white) and female (gray) Common wall lizards incubated with (PHA-activated) or without (Controls) phytohaemoagglutinin. 135Temperature effects on T-cells in P. muralis (Clem et al., 1984), our data suggest that Common wall lizards keep a temperature range in which immune response is optimal, while temperatures above and below this range exert a suppressive effect. Our data agree with past researches on thermal activity, which indicated that Common wall lizards reach the maximum efficiency near to 33°C (Avery, 1978; Braña, 1991; Tosini and Avery, 1994), and suggest that physiological processes in this species are optimized near this temperature. This conclu- sion agrees also with previous findings in other species of reptiles: the highest levels of phagocytosis and cytotoxic- ity of splenic macrophages of Hemidactylus flaviviridis occurred at 25°C, with impaired macrophage function at both higher (37°C) and lower temperatures (7°C and 15°C, Mondal and Rai, 2001). In Alligator mississippiensis antimicrobial and amoebacidal activity occurred between 5°C and 40°C, and was significantly reduced at tempera- tures below 15°C and above 30°C (Merchant et al., 2003, 2004). Accordingly, most physiological processes are opti- mized near 30-31°C (Merchant et al., 2003, 2004). Earlier reports on the effects of temperature on the immune system were aimed to sustain the hypothesis that reptiles might be immunosuppressed during the win- ter months, when body temperatures are far below the optimum. In our study, however, we found that immune suppression may already occur during the breeding sea- son, in coincidence with cold periods, or even during night time. Indeed, body temperatures of lizards remain remarkably constant during the day, but decrease sub- stantially both in the early morning and in the late even- ing (Braña, 1991). These findings have relevant conse- quences in the perspective of ecological-immunology, since short-time fluctuations of temperature might severely affect the trade-offs between immune func- tion and other compartments and life-processes, with remarkable effects on behavioural and physiological fitness-related traits. For example, temperature-depend- ent suppression of immune function might increase the susceptibility to parasite infections during the breeding season and consequently reduce the reproductive efforts of less resistant individuals. Furthermore, the immuno- competence handicap hypothesis (Folstad and Karter, 1992) states that androgens have a dual effect in stimu- lating the expression of the secondary sexual characters while suppressing the immune functions. The reduction in the immune response following unpredictable fluctua- tions of temperature below or above the optimum, might amplify the suppressive action of androgens, with nega- tive effects on the expression of secondary sexual traits of males. However, these additional “thermal” costs might be paid differentially by the different individuals; for example lizards keeping less quality territories including low quality basking sites might suffer the highest levels of temperature-dependent immune suppression. Actually, both individuals settled in marginal territories and subor- dinate individuals might spend much more time to reach optimal body temperature than dominant individuals occupying the best basking sites, and therefore might be exposed for longer time to the consequences of tempera- ture-dependent immune suppression. The effects of tem- perature on the immune functions in ectotherms should, ultimately, increase the variance of male quality, and con- sequently amplify the difference in the expression of the secondary sexual traits among them. In this scenario, endotherms are not expected to pay additional costs in immunocompetence due to fluctuations of environmental temperatures, since they use metabolic heat to maintain body temperature within the optimal physiological range. So, under high variable temperature regimes (e.g. in tem- perate climates) endotherms might reveal a lower vari- ability in male secondary sexual traits than ectotherms, and this difference should be reduced in more stable cli- matic conditions (e.g. in tropical regions). A second relevant result of this study was the dif- ference in temperature-dependent immune suppression between males and females. Earlier studies in mam- mals have well established sex differences in cell-medi- ated immune responses, with females generally having enhanced immune-reactivity than males (Ansar Ahmed et al. 1985; Grossman, 1989; Cannon and St. Pierre, 1997; Klein, 2004). Among reptiles, sex differences in immune response have been reported in the Striped sand snake Psammophis sibilans and in the Yellow-bellied house gecko H. flaviviridis, in which females have higher response than males (Saad, 1989; Mondal and Rai, 1999, 2002). In both cases, sex-associated immune differences have been related to sex steroids, even if experimental studies have shown that immunosuppressive effects of sex hormones act mainly on innate immunity (Mondal and Rai, 1999, 2002). Indeed, gonadectomy of both males and females causes a considerable increase in percent- age phagocytosis, while in vitro administration of dihy- drotestosterone (DHT) and 17β-estradiol (E2) suppresses phagocytosis, cytotoxic activity of splenic macrophages and IL-1 secretion (Mondal and Rai, 1999, 2002). On the other hand, Con A and PHA stimulations did not cause significant differences between sexes in the T-lym- phocytes proliferation in both Caspian pond turtle and loggerhead sea turtles (Muñoz and De la Fuente, 2001; Keller et al., 2005), suggesting that adaptive response is similar in males and females. Contrary to these latter, we showed that PHA stimulation in Common wall liz- ards causes dependent activation of T-lymphocytes, sug- gesting that adaptive immune response might be actually 136 R. Sacchi affected by sex steroids. However, this difference between sexes varied at different temperatures, and immuno- suppression was more severe on males at temperatures below the optimum and more severe on females at tem- peratures above the optimal. This different response by sexes might be explained by different and opposite effects of male and female hormones on the immune function, but future researches are needed to highlight the spe- cific effect of hormones on lymphocyte activation in this species. In conclusion, in vitro activation of immuno- response is a powerful and effective tool to investigate the effect of biotic and abiotic factors on the immune func- tion in natural populations of reptiles. 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