Higher stress protein levels are associated with lower humoral and ...

Functional Ecology 2006 20, 647–655

Higher stress protein levels are associated with lower humoral and cell-mediated immune responses in Pied Flycatcher females Blackwell Publishing Ltd

J. MORALES,*† J. MORENO,* E. LOBATO,* S. MERINO,* G. TOMÁS,* J. MARTÍNEZ DE LA PUENTE* and J. MARTÍNEZ‡ *Departamento de Ecología Evolutiva, Museo Nacional de Ciencias Naturales-CSIC, José Gutiérrez Abascal 2, E-28006 Madrid, Spain, and †Departamento de Microbiología y Parasitología, Facultad de Farmacia, Universidad de Alcalá, E-28871 Alcalá de Henares, Spain

Summary 1. The proper functioning of immune defences may be traded-off against protecting the organism from physiological stress through the induction of stress protein (HSP) synthesis. Immune function could also be negatively affected by haemoparasite infections. 2. We studied whether two induced immune responses (the humoral response to a tetanus vaccine and the T-cell-mediated response to phytohaemaglutinin (PHA) injection) were associated with the levels of two stress proteins (HSP60, HSP70), with haemoparasite infection and with condition in Pied Flycatcher, Ficedula hypoleuca Pallas, females. HSP levels, haemoparasite infection and condition were assessed on days 1 and 11 of nestling age, prior to tetanus and PHA challenges, respectively. 3. Females with higher HSP60 levels prior to tetanus challenge mounted lower humoral responses. Females parasitized by Haemoproteus showed lower humoral responses, when controlling for HSP60 levels. No association was detected for HSP70 levels. 4. Females with higher HSP60 and HSP70 levels prior to PHA challenge, independently of Haemoproteus infection, showed lower cell-mediated responses, when correcting for laying date. Female condition was not associated with immune responses. 5. These results suggest that synthesizing more HSPs to mitigate stress may be tradedoff against mounting humoral and cell-mediated immune responses, and agree with immune defences being costly. Key-words: Birds, blood parasites, HSPs, immunocompetence, life-history trade-offs Functional Ecology (2006) 20, 647– 655 doi: 10.1111/j.1365-2435.2006.01139.x

Introduction

© 2006 The Authors. Journal compilation © 2006 British Ecological Society

Immune defence is generally assumed to be costly (for review see: Sheldon & Verhulst 1996; Klasing & Leshchinsky 1999; Lochmiller & Deerenberg 2000; Norris & Evans 2000), as it elevates energy expenditure (Martin, Scheuerlein & Wikelski 2003; Martínez, Merino & Rodríguez-Caabeiro 2004) and its maintenance is associated with substantial nutritional and energetic costs (Lochmiller & Deerenberg 2000). These costs may be the basis for life-history trade-offs (Sheldon & Verhulst 1996; Zuk & Stoehr 2002). There has been growing interest in the potential trade-off †Author to whom correspondence should be addressed. E-mail: [email protected]

between investment in immune defences and other maintenance functions, modulating parasitism and life-history decisions by hosts (Sheldon & Verhulst 1996; Zuk & Stoehr 2002). Elucidating the relative importance of the causes of immunosuppression would increase our understanding of the basis of life-history trade-offs (Råberg et al. 1998). Individual expression of immunity might be heavily influenced by physiological and ecological factors, such as stress and parasitism (Wakelin 1997). Under a wide variety of stressors of an internal or an external nature, several families of proteins known as stress proteins or heat-shock proteins (HSPs) are synthesized to perform protein-stabilizing functions and to maintain cellular homeostasis (reviewed by Sørensen, Kristensen & Loeschcke 2003). Stress-induced expression of HSPs 647

648 J. Morales et al.

© 2006 The Authors. Journal compilation © 2006 British Ecological Society, Functional Ecology, 20, 647–655

contributes dramatically to tolerance of otherwise lethal conditions (Parsell & Lindquist 1994). However, an over-expression of HSPs is known to have deleterious consequences (Feder & Hofmann 1999). Their response usually results in a concomitant reduction in the synthesis of other proteins, presumably including antibodies (Parsell & Lindquist 1994). Therefore, it is hypothesized that their synthesis represents a significant energetic cost (Hamdoun, Cheney & Cherr 2003), which can even lead to reduced fecundity (reviewed by Parsons 1996). Furthermore, they are often ascribed a role in autoimmunity (reviewed by Råberg et al. 1998), because HSPs of a host and its pathogen can be similar owing to the evolutionarily conserved structure of these proteins (Kaufman 1990). Considering these associated costs, we could expect that the up-regulation of the protective HSP system in stressed individuals would lead to a down-regulation of immune function. HSPs have become accurate and reliable indicators of physiological or environmental stress in avian populations in the wild (Merino et al. 1998; Eeva et al. 2000; Moreno et al. 2002; Martínez-Padilla et al. 2004; Morales et al. 2004; Tomás et al. 2005; Garamszegi et al. 2006). Nevertheless, their association with induced immune responses has been little studied in wild birds. Haemoparasites, especially malarial parasites such as Haemoproteus, can be immunosuppressive (Wakelin 1994). They may result in increased metabolism or reduced assimilation ability of hosts, thus affecting the allocation of resources that could otherwise be used for maintenance and reproduction (Møller 1997; Merino et al. 2000a). Furthermore, they are known to produce chronic infections (Valkiûnas 2005), which might be especially harmful during their relapse in spring, coinciding with hormonal changes during reproduction of their hosts (Folstad & Karter 1992; Apanius 1998). Therefore, the negative effects of blood parasitaemias are expected to impair their hosts’ immune function (Roitt, Brostoff & Male 2001). We know of only two studies in captive birds that have reported a negative relationship between infection by blood parasites and an induced immune response (González et al. 1999; Navarro et al. 2003). Only direct inductions of immune responses would allow the study of trade-offs involving immune defence (Norris & Evans 2000). To explore such trade-offs, the assessment of various aspects of immunity is required (Norris & Evans 2000). Birds have a relatively complex immune system and are ideal subjects for manipulating immunocompetence and life-history decisions in wild populations (Norris & Evans 2000). The aim of this study was to address the association of two experimentally induced immune responses (the humoral response to a tetanus vaccine and the T-cell-mediated response to PHA injection) with the levels of two stress proteins (HSP60 and HSP70), haemoparasite infection and physical condition in breeding Pied Flycatcher females. HSP levels, haemoparasite infection and condition were assessed on days 1 and 11 of the nestling

period, just prior to challenge with the vaccine and PHA, respectively. We predict that high levels of HSPs and the presence of haematozoa would be negatively associated with immunocompetence. Conversely, a higher physical condition would be positively associated with immunocompetence. We specifically focused on females, because previous studies in this (Moreno, Sanz & Arriero 1999; Moreno et al. 2001) and in other Pied Flycatcher populations (Cichoñ, Dubiec & Chadzinska 2001) have reported that female immunocompetence can be affected by enhanced reproductive effort (and presumably, by higher physiological stress levels), but this has not been found in males (Ilmonen et al. 2003).

Methods      The Pied Flycatcher is a small (12–13 g) hole-nesting passerine of European woodlands. For details about its biology see Lundberg & Alatalo (1992). Egg-laying in the population under study typically begins in late May and clutch sizes in our population ranged from four to seven eggs with a mode of six eggs. This study was conducted during the 2004 breeding season in a deciduous forest of Pyrenean oak, at an elevation of 1200 m in Segovia province, central Spain (40°48′N, 4°01′W). A study of nestbox breeding birds has been conducted in this area since 1991 (Sanz et al. 2003). All birds are individually ringed with numbered aluminium bands (DGCN bands, ringing permit by regional authorities).

    On day 1 of the nestling period (hatching day = day 0), females were captured at the nest, weighed to the nearest 0·1 g and sampled for blood by venipuncture. On day 11, females were recaptured, blood sampled, weighed and measured (tarsus and wing length according to Svensson 1984). As a measure of body condition we used mass divided by the cube of tarsus length. They were subsequently subjected to the singlewing PHA injection protocol (Moreno et al. 1999, 2005). Pre- and postinjection measurements were highly repeatable (see Moreno et al. 2005). In both captures, after a blood smear was obtained, the blood sample was centrifuged in the field (Labnet, Cat. No. 1201–220 V, Woodbridge, NJ, USA). Cellular and serum components were separated and cold-stored until being frozen on the same day for later analyses. To estimate humoral immune response, females were injected intraperitoneally with 100 µl of diphtheriatetanus vaccine (National Public Health Institute, Helsinki, Finland) on day 1 of the nestling period, which contains two antigens novel to the birds (diphtheria 38Lf and tetanus 10Lf, mixed with the adjuvant aluminium phosphate at 1·0 mg ml−1). By using killed

649 Stress proteins and immune response

pathogens (human diphtheria-tetanus vaccine), the direct negative effects of parasites are excluded and thus, only the effects of activating the host immune defence are tested for (Ilmonen, Taarna & Hasselquist 2000). In previous studies on Blue Tits (Parus caeruleus) there is no evidence that diphtheria-tetanus vaccination would have any toxic effects on survival (Råberg et al. 2000). The females were blood sampled just prior to injection and on day 11 of the nestling period, when peak primary antibody levels were expected (i.e. 10– 14 days postimmunization for the primary response) (Svensson et al. 1998; Roitt et al. 2001). Female humoral immune response was measured as the antigen-specific antibody levels in the sera by means of an enzyme-linked immunosorbent assay (ELISA) described by Kilpimaa, Alatalo & Siitari (2004), with the only difference that we have measured the specific humoral immune response to the tetanus toxoid only (Berna Biotech España SA, Madrid), as the diphtheria toxoid was unavailable. For details see Kilpimaa et al. (2004) and Moreno et al. (2005). The preinjection serum samples from each female were run to measure the background level of each individual, and thus, in further analyses, the effect of this initial background level was controlled for (Ilmonen et al. 2000; Moreno et al. 2005).

the method does not allow the levels of each form to be distinguished separately. The levels of HSP60 and HSP70 were highly and positively associated both on day 1 (r = 0·58, P < 0·001) and day 11 of the nestling period (r = 0·83, P < 0·001). However, the inducibility of these two stress proteins can differ drastically (Voellmy 1996). This means that HSP60 and HSP70 could indicate different kinds of physiological stress, and therefore the association of these two stress proteins with the immune responses measured was analysed separately. The synthesis and stability of stress proteins depend on many aspects such as the intensity of stressors or the previous exposure to stressors (Lindquist 1986), which we cannot control under natural conditions. However, we know from studies on laboratory animals that their synthesis may start within a few minutes after induced stress (Lindquist 1986) and that their levels may last some days or even weeks (Martínez et al. 1999a,b). Although we cannot predict how stress proteins fluctuated in the days between 1 and 11, we have explored how they changed between the two captures. This will allow us to know whether HSP60 and HSP70 differ in their stability and therefore, in their association with immune responses.

 

    

Blood smears obtained on days 1 and 11 of the nestling period were air-dried, fixed in absolute ethanol and stained with Giemsa (1/10 v/v) for 45 min, in order to detect the presence of the two most common haemoparasites infecting this Pied Flycatcher population, Trypanosoma and Haemoproteus (Merino & Potti 1995; Merino, Potti & Fargallo 1997). One-half of each smear was scanned under 200× magnification in search of the extra-erythrocytic parasite Trypanosoma. In the other half, 50 fields (i.e. a mean of 595 erythrocytes per field) were scanned using 1000× magnification in search of the intracellular parasite Haemoproteus (Merino et al. 1997). Only two females showed infection by trypanosomes either on day 1 or 11 of the nestling period and consequently only the presence/absence of Haemoproteus has been analysed. Eleven females were infected by Haemoproteus on day 1 and 5 on day 11 (from 48 and 38 females, respectively).

All variables were normally distributed except for the immune response to the tetanus vaccine and its background levels, which were log-transformed. The GLM (General Linear Models) module of STATISTICA (version 2001) was used. Four GLMs were performed in order to associate the two immune responses (humoral or cell-mediated) with the two stress proteins (HSP60 and HSP70) separately. The immune responses were included as dependent variables and the stress indicators as predictor variables. Also, the presence/absence of Haemoproteus, condition and laying date were included as predictor variables to correct for their effect in the four models. In the GLM model for the humoral immune response, the initial background level of response to the tetanus toxoid was also accounted for (see above). In this model, HSP levels, the presence/absence of Haemoproteus and female condition were those values obtained on day 1 of the nestling period (just prior to immunization with the tetanus toxoid), while in the GLM model for the cell-mediated immune response, were those obtained on day 11 (just prior to immunization with PHA). Interaction terms between the presence/absence of Haemoproteus and HSP levels in both the initial and the final model were originally included but subsequently removed, as none of them was significant (both P > 0·6). All data available have been included, which explains the different sample sizes in the GLM models. Both immune responses (humoral and cell-mediated) were obtained from 33 females. However, from 15

     


The levels of HSP60 and HSP70 were determined from the blood cellular fraction of females by means of a western blot, on days 1 and 11 of the nestling period. For details of the protocol see Moreno et al. (2002) and Tomás et al. (2005). With this method we assess the levels of both the inducible and the constitutive components of HSPs, as the primary anti-HSPs antibodies available recognize both forms. Nevertheless,


additional females, only the humoral response was obtained, as they could not be trapped for a third time on the day following PHA injection, and from five other females only the cell-mediated response was obtained, as they could not be trapped on day 1 of the nestling period. Although these five females had not been previously injected with the tetanus vaccine, their PHA response did not differ from that of vaccinated females (F1,36 = 0·04, P = 0·84). Therefore, both vaccinated and non-vaccinated females were included in the GLM for the cell-mediated response to PHA.

Results Females showing a higher HSP60 level on day 1 of the nestling period mounted a lower humoral immune response to the tetanus toxoid, when correcting for laying date, condition, the presence/absence of Haemoproteus and the background level of response to the tetanus toxoid on day 1 (Fig. 1, Table 1). Also, females

Fig. 1. Association between the level of the stress protein HSP60 on day 1 of the nestling period with the level of the humoral immune response to the tetanus toxoid (log-transformed).

Table 1. The two GLM analyses with the humoral immune response to the tetanus toxoid as the dependent variable in relation to the two stress proteins (HSP60 and 70). In both GLMs, the presence/absence of Haemoproteus, condition and laying date on day 1 of the nestling period were entered as predictor variables. In bold significant results at P < 0·05 Humoral response to tetanus†


HSP60 Haemoproteus Laying date Condition Tetanus background level† HSP70 Haemoproteus Laying date Condition Tetanus background level† †Log-transformed.

F

df

P

9·06 5·46 1·03 3·62 1·26 1·12 2·63 2·24 2·78 0·19

1, 42 1, 42 1, 42 1, 42 1, 42 1, 42 1, 42 1, 42 1, 42 1, 42

0·004 0·024 0·32 0·064 0·27 0·30 0·11 0·63 0·10 0·66

that were infected by Haemoproteus on day 1 mounted a lower humoral immune response (means ± SE: −1·13 ± 0·08 and −1·40 ± 0·10, for non-infected and infected females, respectively; Table 1). The presence/absence of Haemoproteus was not associated with HSP60 (P = 0·38). The whole model was significant (adjusted r2 = 0·18, P = 0·018). After removing the prevalence of Haemoproteus from this model, the negative association between HSP60 levels and the humoral response remained significant (P = 0·01). Females did not differ in their humoral immune response according to the levels of the other stress indicator, HSP70 (Table 1). Females showing a higher level of HSP60 on day 11 of the nestling period mounted a lower cell-mediated immune response to PHA, when correcting for laying date, condition and the presence/absence of Haemoproteus on day 11 (Table 2). This model was marginally significant (adjusted r2 = 0·14, P = 0·066). The same was true for HSP70 on day 11 (Table 2). In this case, the whole model was significant (adjusted r2 = 0·18, P = 0·034). The presence/absence of Haemoproteus was not associated with either HSP60 or HSP70 on day 11 (P = 0·25 and P = 0·39, respectively). The humoral immune response to the tetanus toxoid was not associated with the cell-mediated response to PHA, when correcting for the initial background level of response to the toxoid (PHA: F1,30 = 1·01, P = 0·32; background level of response: F1,30 = 0·16, P = 0·69). HSP60 level on day 1 was positively associated with that on day 11 (r = 0·45, P = 0·005), indicating that those females that were more stressed on day 1 were also more stressed on day 11. The same was true for HSP70 levels (r = 0·35, P = 0·033). HSP60 levels on day 1 were not significantly different from HSP60 levels on day 11, although they tended to increase (t = −1·90, P = 0·066; mean ± SE: HSP60 on day 1 = 5715·16 ± 191·14, HSP60 on day 11 = 6299·48 ± 344·69). HSP70 levels on day 1 were significantly lower than those on day 11 (t = −2·32, P = 0·026; mean ± SE: HSP70 on day 1 = 9837·76 ± 280·58, HSP70 on day 11 = 10935·83 ± 493·42). Table 2. The two GLM analyses with the cell-mediated immune response to PHA as the dependent variable in relation to the two stress proteins (HSP60 and 70). In both GLMs, the presence/absence of Haemoproteus, condition and laying date on day 11 of the nestling period were entered as predictor variables. In bold significant results at P < 0·05 Cell-mediated response to PHA

HSP60 Haemoproteus Laying date Condition HSP70 Haemoproteus Laying date Condition

F

df

P

5·10 0·71 2·67 0·62 6·96 0·60 2·72 1·48

1, 33 1, 33 1, 33 1, 33 1, 33 1, 33 1, 33 1, 33

0·031 0·41 0·11 0·44 0·013 0·44 0·11 0·23



Discussion Females with higher HSP60 levels that were infected by Haemoproteus prior to tetanus challenge mounted lower humoral immune responses. Those with higher HSP60 and HSP70 levels prior to PHA challenge, independently of infection, showed lower cell-mediated immune responses. This supports the idea that a high level of physiological stress may lead to a general impairment of immune responses (Apanius 1998) and is in accordance with previous experimental studies reporting a stress-induced immunosuppression after either increased reproductive effort (Nordling et al. 1998; Moreno et al. 1999; Cichoñ et al. 2001; Saino et al. 2002; Pap & Márkus 2003; Ardia 2005; but see Ilmonen et al. 2003) or cold temperatures (Svensson et al. 1998). However, the association of HSPs and either reproductive or thermal stress in birds remains to be elucidated. Our results concurs with a previous study in nestling Red-Rumped Swallows Hirundo daurica, in which the cell-mediated response to PHA was negatively associated with the levels of HSP60, measured on the following day after immunization (Merino et al. 2001). Our results also agree with the general idea that immune defences are costly and suggest that there could be a trade-off between mounting immune responses (either humoral or cell-mediated) and activating the synthesis of HSPs to mitigate stress. This trade-off may agree with the three main hypotheses proposed to explain immunosuppression (reviewed by Schmid-Hempel 2003), which may not be mutually exclusive. First, oxidative stress induced by different stressors could be reflected in higher levels of HSPs (reviewed by Sørensen et al. 2003; see also Martínez et al. 1999c), leading to an unavoidable suppression of the humoral response (Jenkins 1993). This non-adaptive explanation of immunosuppression suggests that the inevitable damage induced by these stressors would have impaired the capacity of the immune system to respond to different antigens. According to this explanation, Hall (1994) found that the induction of HSP70 by heat-shock led to an inhibition of cytokine production. Second, immunosuppression could be caused by resource limitation (Sheldon & Verhulst 1996; Norris & Evans 2000). Thus, the enhanced synthesis of HSPs in highly stressed individuals could have withdrawn essential metabolic components necessary for mounting efficient immune responses, leading to the negative association between HSP levels with both immune responses. This could occur because immune cells have to trade off resources allocated to HSP synthesis and to antibody or cytokine production. According to this idea, we would expect immune responses to be condition-dependent (e.g. Moreno et al. 1999, Merino, Møller & De Lope 2000b; Fargallo et al. 2002; Lifjeld, Dunn & Whittingham 2002; but see for instance Westneat, Hasselquist & Wingfield 2003). However, condition was not associated with any of the immune responses. Despite this absence of obvious

condition-dependence, we cannot exclude the possibility that limiting dietary substances that directly enhance immune capacity, rather than food as a source of energy in general (Møller & Saino 1998), were crucial for both the synthesis of HSPs and for producing an immune response. In this case, we would not necessarily expect a condition-dependence of immune responses. Third, females showing high HSP levels could have down-regulated immune responses adaptively to avoid autoimmunity (Råberg et al. 1998) or oxidative stress (von Schantz et al. 1999). This seems reasonable because of the commonly ascribed role of HSPs in autoimmunity, because of the evolutionarily conserved structure of HSPs (Kaufmann 1990; Pockley 2003). The immunosuppressive response to stress has been well studied in humans and in laboratory animals. It is generally accepted that the influence of psychological and physical stress leads to a general suppression of immune responses (Glaser et al. 1998; Pedersen & Hoffman-Goetz 2000; Pedersen & Steensberg 2002). As Pied Flycatcher females invest more energy in reproduction than their mates, for instance during incubation and brooding (Moreno et al. 1995), they may suffer more from immunsuppression than their mates as a result of working stress. This could be one reason why in a recent study in male Collared Flycatchers Ficedula albicolis, no significant associations between stress proteins levels and total immunoglobulin levels were found (Garamszegi et al. 2006). Also, only for females was a negative association between field metabolic rate and immune response found (Moreno et al. 2001). Stress induces a complex neuroendocrine response with an activation of the hypothalamus– pituitary–adrenal gland axis that suppresses specific immunity (reviewed by Berczi 1998). However, stress may boost in turn non-specific immunity (such as the activation of acute-phase proteins), which can help to fight infection or injury without exposing the organism to the risk of autoimmunity (Berczi 1998). The physiological mechanisms leading to the immunosuppressive response to stress involve elevation in plasma of glucocorticoids, which alter mononuclear cell trafficking in lymph nodes and inflammatory sites (Dohms & Metz 1991; Sapolsky 1992; Sheridan et al. 1998). Other neuroendocrine products may also modulate immunity, such as catecholamines (Sheridan et al. 1998). However, unravelling the mechanisms of stress-induced immunosuppression is extremely difficult owing to methodological and technical problems in comprehensively evaluating these phenomena (Dohms & Metz 1991). Indeed, El-Lethey, HuberEicher & Jungi (2003) showed that chronic stress in chickens leads to the impairment of humoral and cellmediated immunity, but that depending on the antigen tested, there are stress-resistant and stress-susceptible antigen responses. The negative association between HSP levels and immune responses, however, may not necessarily imply



a trade-off. An alternative interpretation could be that individuals with higher immune responses are stronger (because of high-quality genes or phenotype) and are thus more tolerant to stress or have a lesser need to respond to stress. In our study, this could explain why females with low levels of HSPs induced higher immune responses. Indeed, it has been proposed that if the fitness costs associated with inducing high HSPs levels are high, an increased resistance to stress can evolve without the need of simultaneous increase in HSP levels (Bettencourt, Feder & Cavicchi 1999; Sørensen, Dahlgaard & Loeschcke 2001). Also, it could be possible that the negative associations found between HSP levels and the immune responses measured are due to adaptive physiological regulations that do not entail deleterious effects for the organism. The implications of immunosuppression after induced HSP synthesis deserves further investigation. The two stress indicators measured, HSP60 and HSP70, could differ in their inducibility (Voellmy 1996). HSP70 clearly increased from day 1 to day 11, so it could be more affected by the stress resulting from reproductive activities. This could explain why the cell-mediated response was associated with both HSPs levels on day 11, while the humoral immune response was associated only with HSP60 levels on day 1. On the contrary, HSP60 levels did not vary significantly between captures. Although it has been reported that the majority of genes responding to stress return to normal prestress levels within some hours after stress (Sørensen et al. 2005), it could be that HSP60 reflects a more persistent stress. Besides, previous studies in wild avian populations have found that HSP60 levels, but not HSP70, were negatively associated with nestling growth and positively with another stress indicator (the heterophil : lymphocyte ratio) (Moreno et al. 2002), predicted the stress resulting from parasitism in adults (Merino et al. 2002) or that resulting from sibling competition (Martínez-Padilla et al. 2004). This may suggest that HSP60 is a more accurate indicator of certain environmental types of stress. However, we are unable to infer the type of stressor that most affects each HSP, owing to the plethora of uncontrolled factors influencing HSPs under natural conditions. Actually, in a previous study, HSP60 and HSP70 decreased between captures (Morales et al. 2004), which emphasizes the complex dynamics of the stress response. In addition, the cascade mechanisms that are involved in the induction of the two induced immune responses differ greatly (Roitt et al. 2001) and, thus, they might be differently related to the two stress indicators. It could be that a certain cell line, for instance B lymphocytes, which are mainly involved in the induction of humoral immune responses, could contribute to a higher degree to the absolute levels of a certain HSP (HSP60 in this case). This could explain why the humoral immune response was associated only with HSP60 levels. Unfortunately, the assay used to measure the levels of HSPs does not allow us to evaluate the

proportional contribution of different cell lines to the absolute levels of different HSPs. Although it is well known that blood parasites can exert a negative effect on immune responses (Roitt et al. 2001), we only know of two studies that have found a negative association between infection by haemoparasites and an induced immune response. González et al. (1999) and Navarro et al. (2003) reported a negative relationship between the cell-mediated response to PHA and infection by Haemoproteus in captive birds. We did not find an association between the presence of Haemoproteus and the cell-mediated response, which concurs with a previous study in the same population (Morales et al. 2004), but we found that females initially infected with Haemoproteus showed reduced humoral immune responses. The fact that parasites affected the humoral and not the cell-mediated response could be due to the timing at which both responses were assessed. As the prevalence of Haemoproteus decreases over the course of the breeding season (Sanz et al. 2002; Morales et al. 2004; present study), this could explain why no association was found between the final presence of Haemoproteus and the cell-mediated response induced at the end of the nestling period, while a negative association was found between the presence of the parasite and the humoral response induced at an earlier stage. The negative association between HSP60 levels and the humoral response remained significant after removing Haemoproteus prevalence from the initial model, which suggests that other stressors apart from haemoparasite infection might be mediating the previous association. Nevertheless, when including Haemoproteus prevalence, the association between HSP60 and the humoral response was much stronger, which agrees with the idea that haemoparasites might play an important role in stress-induced immunosuppression. In conclusion, we have found that higher levels of HSP60 were negatively associated with the induction of humoral and cell-mediated immune responses in breeding females, while the levels of HSP70 were only associated with the cell-mediated response. This suggests a trade-off between the synthesis of HSPs, presumably induced to mitigate physiological stress, and the induction of immune responses. We discuss that two adaptive hypotheses on immunosuppression are likely to explain our results, i.e. the resourceallocation (Sheldon & Verhulst 1996; Norris & Evans 2000) and the autoimmunity-avoidance hypotheses (Råberg et al. 1998). Alternatively, HSP levels may be an expression of oxidative stress at the cellular level that unavoidably damages the efficiency of the immune system (Jenkins 1993).

Acknowledgements The study received financial support from projects CGL2004-00787/BOS to J. Moreno and BOS200305724 to S.M. (DGI-Ministerio de Educación y Ciencia).


We thank T. Calvo for help in the field and M. Artiles for haemoparasite detection. E. Virtanen provided invaluable advice during lab work. J. Morales thanks R. V. Alatalo and H. Siitari for the invitation to their laboratory in Jyväskylä to learn immunological techniques. Rodrigo Vásquez stimulated this enquiry through his accurate questions. We were authorized by J. Donés, Director of ‘Centro Montes de Valsaín’ (Ministerio de Medio Ambiente) to work in the study area. Dirección General del Medio Natural (Junta de Castilla y León) authorized the capture, ringing and sampling of birds in the study area. This paper is a contribution from the field station ‘El Ventorrillo’.

References


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