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First published online June 13, 2008
Journal of Experimental Biology 211, 2155-2161 (2008)
Published by The Company of Biologists 2008
doi: 10.1242/jeb.017178
Cell-mediated immune activation rapidly decreases plasma carotenoids but does not affect oxidative stress in red-legged partridges (Alectoris rufa)
1 Instituto de Investigación en Recursos Cinegéticos, IREC (CSIC,
UCLM, JCCM), Ciudad Real, Spain
2 School of Biological Sciences, University of Aberdeen, Aberdeen, UK
3 Estación Biológica de Doñana (CSIC), Seville, Spain
4 Department of Biology, University of Saskatchewan, Saskatoon, Canada
* Author for correspondence (e-mail: lorenzo.perez{at}uclm.es)
Accepted 6 April 2008
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Summary |
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 TOP Summary INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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Key words: antioxidants, immune response, phytohaemagglutinin, red-legged partridge, sexual selection, TAS, TBARS
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INTRODUCTION |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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;
Hill and McGraw, 2006). To
disentangle how and why carotenoid-based ornaments may have evolved and are
maintained as honest sexual or social signals, the mechanisms and costs
associated with ornament expression must be understood
(Andersson, 1994).
Apart of their potential value as signals of foraging capacity (they cannot
be synthesized de novo by animals but must be acquired through the
diet), it has been suggested that carotenoids may have antioxidant and
immune-enhancing properties that may confer another way of honesty to
carotenoid-based signals (Lozano,
1994; Olson and Owens,
1998; Møller et al.,
2000; Blas et al.,
2006). Because of their molecular composition, carotenoids may act
as effective scavengers of free radicals, such as reactive oxygen and nitrogen
species (Krinsky, 1989). These
free radicals are released during immune responses and help to counter
invading pathogens (Halliwell and
Gutteridge, 1999). However, the toxicity of free radicals is not
restricted to the pathogen and their overproduction can damage host tissues,
leading to a situation of oxidative stress. Carotenoids used for antioxidant
protection would no longer be available for signal expression, and therefore
carotenoid-based ornaments have been hypothesized to act as honest individual
signals of health, oxidative stress or antioxidant defences
(Lozano, 1994;
Olson and Owens, 1998;
Møller et al., 2000;
Hartley and Kennedy, 2004).
However, there is scarce evidence supporting that carotenoids have important
antioxidant function in vivo (see
Costantini and Møller,
2008), at least as compared with other antioxidants such as
vitamins A, C and E or uric acid (Hartley
and Kennedy, 2004). In fact, studies assessing whether immune
system activation leads to oxidative damage in birds have yielded
contradictory results (Alonso-Alvarez et
al., 2004; Bertrand et al.,
2006; Costantini and Dell'Omo,
2006; Hõrak et al.,
2006; Hõrak et al.,
2007).
Recent studies have shown that a humoral immune challenge (intraperitoneal
injection of sheep red blood cells, SRBC) decreased circulating carotenoids
(McGraw and Ardia, 2003;
Peters et al., 2004;
Aguilera and Amat, 2007) (but
see Hõrak et al., 2006)
and carotenoid-based coloration (Faivre et
al., 2003; McGraw and Ardia,
2003; Peters et al.,
2004). Similarly, the intraperitoneal injection of
lipopolysaccharide (LPS) of Escherichia coli, which promotes an
inflammatory response followed by antibody production, decreases plasma
carotenoids (Koutsos et al.,
2003; Alonso-Alvarez et al.,
2004) and carotenoid-based coloration
(Alonso-Alvarez et al., 2004).
Taken together, these studies support the hypothesis that mounting an immune
response may drain carotenoids from the blood stream. However, rarely are the
potential effects of immune challenge on plasma carotenoids, antioxidant
defences and oxidative stress-induced damage measured concurrently [see
Hõrak et al. (Hõrak et al.,
2007) for a discussion of the importance of this point], which
makes it difficult to ascertain the sequence of immune response activation,
oxidative stress, decrease in circulating carotenoids and subsequent decrease
in carotenoid-based ornamentation. In addition, the above-mentioned SRBC or
LPS challenge protocols promote a systemic response that may be activated for
weeks. These are only specific types of a wide array of immune responses that
vertebrates activate to prevent and combat an infection. In most cases,
pathogens are fought at the site of entrance into the body via local
inflammatory and cellular responses with scarce systemic implications
(Roitt et al., 2001). However,
the possible effect of such a local immune response on circulating carotenoids
and coloration remains poorly known
(Costantini and Dell'Omo,
2006; Hõrak et al.,
2007).
The phytohaemagglutinin (PHA) skin test is probably the most popular and
widespread measure of immunocompetence used by avian ecologists
(Smits et al., 1999;
Martin et al., 2006;
Kennedy and Nager, 2006). A
subcutaneous injection of PHA promotes an inflammatory response encompassing
T-cell mediated infiltration of granulocytes, macrophages and lymphocytes, in
a complex and dynamic process that involves both innate and acquired
components of the immune system (Martin et
al., 2006). As a result, a small but measurable swelling is
produced during the following hours at the site of injection, the magnitude of
which is used as an index of cell-mediated immunocompetence. Although
PHA-induced skin swelling is not a cost-free reaction (e.g.
Alonso-Alvarez and Tella,
2001), it seems to elicit a more local response (compared with LPS
or SRBC intraperitoneal injection) with few relevant systemic effects
(Merino et al., 1999;
Hõrak et al.,
2000).
In this study, we analyzed the effect of a local cell-mediated immune response (promoted by the intradermal injection of PHA) on plasma carotenoids, total antioxidant status (TAS), oxidative damage (TBARS, a measure of lipid peroxidation) and carotenoid-based coloration in male red-legged partridges (Alectoris rufa Linnaeus), a species with conspicuous carotenoid-based ornaments (red coloration of eye rings and bill). We assigned males to one of two treatments: subcutaneous injection of phosphate-buffered saline (PBS), as a control, or injection of PHA to elicit a cell-mediated immune response. We hypothesized that immune challenge would lead to oxidative damage (i.e. increase in TBARS) and possibly also a decline in TAS. Alternatively, the immune challenge could promote the mobilization of antioxidant defences and cause an increase in TAS. In addition, if carotenoids have an important antioxidant function, we predicted that plasma carotenoids would change in the same way as TAS does. Given a possible trade-off between carotenoid allocation to coloured signals or to antioxidant defences, we also predicted that carotenoid-based ornamentation would decrease in the PHA-challenged group as compared with the control group. We also explored correlatively the relationships between TAS, TBARS and carotenoids in order to assess the relative contribution of these pigments to antioxidant defences of the individual. Finally, we evaluated whether carotenoid-based ornaments in the red-legged partridge may act as honest signals of immunocompetence or antioxidant defences, by exploring the correlations between carotenoid-based coloration, cell-mediated immunity, TAS and TBARS.
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MATERIALS AND METHODS |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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)] and fed ad libitum with commercial food pellets
containing 5.26 µg carotenoids (96% lutein) per gram of food. Although the
carotenoid content of the diet of wild red-legged partridges is unknown, this
concentration in food is routinely used and recommended for captive reared
partridges (Blas et al., 2006;
Pérez-Rodríguez et al.,
2007;
Pérez-Rodríguez,
2008). On 11 December, after 2 weeks of adaptation to cages, we took a blood sample (0.8 ml) from the brachial vein of the right wing of each male using a heparinized syringe. Blood was stored at 4°C and centrifuged at 7000 g within 4 h of collection. Plasma and pellets were then separated and stored at –80°C until analysis. We also took a high resolution (2272x1704 pixels) digital photograph of the left side of the head to measure bill and eye ring redness. All pictures were taken under fluorescent light and against a grey standard background. The distance from the camera (Nikon Coolpix 4500) to the bird was held constant (40 cm) and a grey standard reference (Kodak Gray Scale, Kodak, New York, USA) was placed in all pictures next to the head of the bird.
On 14 December, birds of the PHA group (N=23) were injected
subcutaneously in the patagium of the left wing with 0.5 mg of PHA
(Sigma-Aldrich, Steinheim, Bayern, Germany; ref L-8754) suspended in 0.1 ml of
PBS (Smits et al., 1999).
Control birds (N=17) were injected with the same volume of PBS.
Before injection, the thickness of the patagium was measured with a digital
spessimeter (Mitutoyo Absolute 547-315, Kawasaki, Kanagawa, Japan) to the
nearest 0.01 mm. After 24 h, patagium thickness was again measured at the
point of injection and the difference between initial and final measurements
in PHA-injected birds was considered as the cell-mediated immune response
(Smits et al., 1999). In both
cases (before and 24 h after injection) three measures of patagium thickness
were taken. Both initial and final measurements were repeatable (intraclass
correlation coefficients: r=0.99, F23,48=510.3,
P<0.001, and r=0.99, F23,48=336.2,
P<0.001, respectively) and therefore we used average values of the
three measurements in the analyses. At the time of post-injection measurement
(15 December), we collected a second blood sample from the brachial vein of
the right wing as described above. Finally, a second set of digital
photographs was taken on 16 December, 48 h after PHA or PBS injection, in a
random subsample of 29 birds (18 PHA and 11 control birds).
Although plasma carotenoids do not show diel variation
(Pérez-Rodríguez et al.,
2007), other blood metabolites and cell-mediated immune responses
may (Martínez-Padilla,
2006;
Pérez-Rodríguez et al.,
2008). For this reason, all blood samples and immune measurements
were taken at the same time (1200-1500 h).
Laboratory analysis
TAS is a measure of the capacity of the plasma to quench free radicals, and
is primarily the result of the pooled effect of all extracellular antioxidant
compounds of the blood (i.e. uric acid, vitamin E, vitamin C, carotenoids).
TAS of plasma was assessed by means of commercial kits (Randox Laboratories
Ltd, Crumlin, Northern Ireland, UK; ref. NX2332) in an A25 BioSystems
spectrophotometer autoanalyzer (BioSystems S.A., Barcelona, Spain). Plasma
samples were incubated for 15 s with a chromogen composed of metmyoglobin and
ABTS® [2,2-azino-di-(3-ethylbenzthiazoline sulphonate). Then, hydrogen
peroxide (H2O2) was added and the samples incubated for
195 s. H2O2 addition induces the production of the
radical cation ABTS®, which generates a blue-green colour. Colour change
was determined by measuring at 600 nm before and after
H2O2 addition.
TAS values are frequently related to the level of circulating uric acid
(Cohen et al., 2007;
Hõrak et al., 2007)
which may confound interpretation of results. Uric acid is the main form of
nitrogen excretion in birds and high levels of TAS could be an indication of
incidental amino acid catabolism (i.e. high uric acid levels) rather than
regulated antioxidant protection (Cohen et
al., 2007). Therefore, we measured uric acid in all plasma samples
in the same autoanalyzer and by means of commercial kits (Biosystems SA,
Barcelona, Spain; ref. 11521), following the uricase/peroxidase procedure
(Fossati et al., 1980).
Lipid peroxidation in red blood cells was assessed following the method of
Aust (Aust, 1985). The
principle is based on the fact that most tissues contain a mixture of
thiobarbituric acid reactive substances (TBARS), including lipid
hydroperoxides and aldehydes, which increase as a result of oxidative stress.
Blood pellets were thawed and immediately diluted (1:10) and homogenized in a
stock buffer (0.01 mol l–1 PBS and 0.02 mol
l–1 EDTA), always working on ice to avoid oxidation. A sample
(1 m) of that homogenate was mixed with 2 ml of a solution sensitive to TBARS
(trichloroacetic acid 15%, HCl at 0.25 mol l–1 and
thiobarbituric acid 0.375%) and 1 ml of 2% BHT
(2,6-di-tert-butyl-4-methylphenol) in closed glass tubes. Tubes were then
warmed for 30 min at 90°C and afterwards cooled with ice-cold water. The
absorbance of the supernatant was then determined by spectrophotometry at 535
nm after centrifugation (2025 g, 15 min). Concentrations of
peroxidized lipids were determined by comparing results with those obtained
from a curve with different malondialdehyde concentrations [i.e. the end
products of lipid peroxidation (Aust,
1985)]. TAS and TBARS measurements were significantly repeatable
(r=0.94 and r=0.76, respectively; C.A.-A., unpublished).
Carotenoids were quantified by diluting 60 µl of plasma in acetone (1:10
dilution). The mixture was vortexed and centrifuged at 11 000
g for 10 min to precipitate the flocculent proteins. The
supernatant was examined in a ShimadzuUV-1603 spectrophotometer (Kyoto, Japan)
and the optical density at 446 nm [the wavelength of maximal absorbance for
lutein (Mínguez-Mosquera,
1993)] was determined. Finally, plasma carotenoid concentration
(µg ml–1) was calculated using a lutein standard curve
(Sigma-Aldrich, ref. 95507).
Carotenoid-based coloration
We analysed digital photographs using Adobe Photoshop v 7.0. For each male,
we calculated the RGB (red, green, blue) components of the eye ring, nostril,
upper mandible and lower mandible, separately. The same components were
calculated for the grey reference. Following previous studies with this
(Villafuerte and Negro, 1998)
and other carotenoid-pigmented species
(Pike et al., 2007), the
intensity of carotenoid-based red coloration (redness hereafter) was
calculated as R divided by the average of R, G and B. `Redness' values of the
grey reference were used to standardize all colour measurements and correct
for possible subtle differences in luminance between pictures. The average
redness of the grey reference in the entire picture set was calculated. For
each photographs, the difference between the redness of the standard and the
average was subtracted from the redness of the ornaments in order to control
for any subtle variation in illumination between photographs.
There exists a considerable variability between birds in the relative
proportion of the bare white skin around the eye that has red (carotenoid)
pigmentation. This is related to sexual dimorphism and is condition-dependent
in this species
(Pérez-Rodríguez,
2007). Therefore, for each male, we calculated the percentage of
pixels of the eye lore skin pigmented by carotenoids (hereafter referred to as
eye ring pigmentation) also using Adobe Photoshop v 7.0.
Statistical analyses
For simplification, and to adjust the analysis to the possible biological
meaning of the coloured structures considered, we separated colour variables
of the eye ring (eye ring redness and eye ring pigmentation) from those
measured on the bill (nostril and upper and lower mandible redness), which are
more keratinized structures. This separation is meaningful since eye rings are
soft tissues and therefore may change colour more rapidly than bill. To
evaluate overall bill redness, we conducted a principal component analysis on
bill colour variables (nostril, upper mandible and lower mandible). The first
principal component (bill PC1) explained 68% of variance, with nostril, upper
and lower mandible redness all having positive loadings (0.61, 0.60 and 0.51,
respectively). We thus used PC1 scores as an index of overall bill redness. We
explored relationships between carotenoids, ornamentation and cell-mediated
immune response using Pearson correlations. The effect of PHA-induced response
on carotenoids or colour variables was tested using general linear mixed
models, with sampling time (before or 24 h after injection) and treatment (PHA
or PBS) as a fixed factors and individual as a random variable. All variables
were normally distributed (Shapiro–Wilk tests) and all tests were
two-tailed.
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RESULTS |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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6% of the variance in TAS after controlling for uric acid concentration.
TBARS of red blood cells were not significantly explained by TAS (alone or
after controlling for uric acid concentration), uric acid or by plasma
carotenoids (all P>0.75).
Plasma carotenoids, carotenoid-based coloration and cell-mediated immunity
Eye ring redness positively correlated with eye ring pigmentation
(Table 1). All other
correlations among colour variables were non-significant. Plasma carotenoid
levels before experimental treatment were significantly and positively related
to eye ring pigmentation and bill redness, but not with eye ring redness
(Table 1). TAS and TBARS before
experimental treatment were not related to any colour trait (all
P>0.25).
|
In PHA-injected birds, cellular immune response (wing web swelling at 24 h) showed a positive association with plasma carotenoids before immune challenge (Table 1, Fig. 1). Furthermore, both eye ring redness and pigmentation before the experiment, but not bill redness, were significantly related to PHA response (Table 1, Fig. 1). TAS and TBARS before immune challenge were not related to the strength of the response (both P>0.93).
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Despite the clear effect of PHA-induced immune response on circulating carotenoids, none of the colour variables was affected by experimental treatment (Table 2). However, eye ring and bill redness (PC1) increased during the study in control and PHA-challenged birds.
Among PHA-injected birds, neither absolute nor relative (percentage of initial plasma carotenoids) change in plasma carotenoids was related to the intensity of cell-mediated immune response (rP=–0.19, N=23, P=0.35 and rP=–0.10, N=23, P=0.62, respectively). Thus, we had no evidence that the greater cellular immune responses consumed more carotenoids than weaker ones.
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DISCUSSION |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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;
von Schantz et al., 1999;
Møller et al., 2000;
Blas et al., 2006). The
underlying hypothesis is that carotenoids have antioxidant properties and that
the activation of an immune response leads to a situation of oxidative stress
that may force a reallocation of available carotenoids for quenching free
radicals rather than for ornament pigmentation. As a result, ornament
expression is expected to decrease in challenged individuals
(Lozano, 1994;
von Schantz et al., 1999;
Møller et al., 2000).
Therefore, apart from foraging efficiency, carotenoid based ornaments might
indicate immunocompetence, health and antioxidant status. This study gives
mixed support for this hypothesis.
We found that male red-legged partridges with more intense carotenoid-based
coloration (specifically, redder and more extensively pigmented eye rings)
mounted stronger cellular responses after intradermal injection of PHA.
However, carotenoid-based coloration, although positively associated with
higher levels of circulating carotenoids, was not related to TAS or TBARS.
Furthermore, cell-mediated immune response correlated with circulating
carotenoids, but not with TAS or TBARS. These results are consistent with the
hypothesis that carotenoid-based ornaments may honestly signal
immunocompetence (Blount et al.,
2003; McGraw and Ardia,
2003; Peters et al.,
2004; Mougeot,
2008), but not antioxidant defences, as recently suggested
(Hartley and Kennedy, 2004).
In addition, we found that the contribution of plasma carotenoids to TAS was
small and they were not related to TBARS. An endogenous metabolite, uric acid,
explained 36% of the variance in TAS, similar to the findings of recent
studies (Cohen et al., 2007;
Hõrak et al., 2007).
Only when uric acid was statistically controlled for, were plasma carotenoids
weakly (marginally) related to TAS, explaining a further 6% of variation.
Thus, the hypothetical role of carotenoids as powerful antioxidants in
vivo is questionable and should not be assumed "a
priori" (Costantini et al., 2006;
Costantini et al., 2007;
Isaksson et al., 2007;
Costantini and Møller,
2008).
We found that T-cell-mediated immune activation reduced circulating
carotenoids. This decrease was marked (average 13% of initial circulating
levels) and fast, as it was detected as soon as 24 h after PHA injection. A
decrease in plasma carotenoids (and, eventually, carotenoid based
ornamentation) as a result of mounting a systemic immune response
(intraperitoneal injection of SRBC or LPS) has been reported by previous
studies (Faivre et al., 2003;
McGraw and Ardia, 2003;
Peters et al., 2004;
Alonso-Alvarez et al., 2004;
Aguilera and Amat, 2007).
However, the effect of a more local challenge, such as the intradermal
injection of PHA in the wing patagium, is much less known. Recently, a study
on zebra finches (Taeniopygia guttata), found a similar decrease in
circulating carotenoids after PHA injection
(McGraw and Ardia, 2007),
although no control (PBS injected) birds were used in that study. By contrast,
an increase in circulating carotenoids have been found in Eurasian kestrel
(Falco tinnunculus) nestlings 24 h after PHA injection
(Costantini and Dell'Omo,
2006), whereas no change in circulating carotenoids was detected
72 h after PHA challenge in adult greenfinches (Carduelis chloris)
(Hõrak et al., 2007).
These inconsistencies between studies may be due to differences in timing of
sampling (Hõrak et al.,
2007) or to differences among taxa, or age groups in carotenoid
allocation priorities or in the ability to buffer the impact of cell-mediated
immune challenge. The growing body of literature on this subject will allow
assessing the relative importance of these factors in the future.
Despite an important decrease in plasma carotenoids, we did not find any
effect of cell-mediated immune activation on TAS or TBARS. Intradermal
injection of PHA rapidly induces an inflammation and local infiltration of
several cell types (thrombocytes, basophils, eosinophils, heterophils,
lymphocytes, macrophages) (Martin et al.,
2006). Once activated, heterophils and macrophages produce
reactive nitrogen and oxygen species
(Koner et al., 1997;
Nathan and Shiloh, 2000;
Coleman, 2001) that are
expected to affect the antioxidant machinery of the individual. In fact,
unlike our study, the above mentioned recent studies reported an increase in
lipid peroxidation and free radical production accompanied by a decrease or
increase, respectively, in total antioxidants after PHA injection
(Costantini and Dell'Omo,
2006; Hõrak et al.,
2007).
How can the marked reduction in available carotenoids, despite the absence
of any effect on TAS or TBARS, be explained? One possible explanation is that
the antioxidant properties of carotenoids buffered the free radical production
associated with the activation of a local immune response, thus preventing any
oxidative damage. However, as stated above, the relative contribution of
plasma carotenoids to TAS was weak. Alternatively, if carotenoids were
significant antioxidants, the 13% decrease in circulating levels after PHA
injection should have negatively affected TAS. Instead of such a decrease, we
found that TAS tended to increase during the study, in all birds (a pattern
attributable to the weak generalized tendency to increase uric acid levels
during the experiment). The absence of any effect of carotenoid depletion on
TAS or TBARS may be explained by a compensatory increase in circulating levels
of non-measured antioxidants (such as vitamin E, vitamin C, enzymes, etc)
(Vider et al., 2001;
Tauler et al., 2006;
Hõrak et al.,
2007).
Another possible explanation for the effect of cell-mediated immune
challenge on plasma carotenoids irrespective of oxidative stress is that
plasma carotenoids are poor antioxidants in vivo
(Hartley and Kennedy, 2004;
Costantini and Møller,
2008) but are particularly sensitive to the negative effect of
free radicals (that is, free radicals have a deeper impact on plasma
carotenoids than vice versa). Carotenoids can be attacked at many
different sites at their long conjugated aliphatic chains, so it is likely
that even low concentrations of free radicals (with no significant effect on
TAS or TBARS) may have an important impact on circulating carotenoids, which
lose their pigmentary properties after being attacked by oxidants. If so,
carotenoids would not be signalling antioxidant ability, as our result
suggest, but could be short-term indicators of changes in oxidative
stress.
A third possibility is that plasma carotenoids are required for mounting an
immune response, but perform functions other than antioxidant protection. In
fact, carotenoid supplementation usually enhances immune response
(Blount et al., 2003;
McGraw and Ardia, 2003;
Aguilera and Amat, 2007) and,
conversely, immune response depletes available carotenoids
(McGraw and Ardia, 2003;
Alonso-Alvarez et al., 2004;
Aguilera and Amat, 2007) (this
study). By contrast, carotenoid supplementation does not always increases
antioxidant protection (for a review, see
Costantini and Møller,
2008), and the effect of immune activation on oxidative stress has
yielded contradictory results
(Alonso-Alvarez et al., 2004;
Bertrand et al., 2006;
Costantini and Dell'Omo, 2006;
Hõrak et al., 2006;
Hõrak et al., 2007). It
seems therefore that, with the information currently available, the link
between carotenoids and immunity is sounder than that between these two
factors and oxidative stress. Therefore, alternative links between immunity
and carotenoids, not necessarily based on the antioxidant properties of these
pigments, should be explored and considered in future studies. For instance,
induction of acute phase response in chickens depletes carotenoids in plasma
but increases their concentration in thymocytes
(Koutsos et al., 2003), whose
activation requires carotenoid derivatives as cofactors
(Garbe et al., 1992).
Furthermore, carotenoids and some of their derivatives are involved in the
expression of immune-related genes
(Geissmann et al., 2003), up
regulation of proteins involved in cell-to-cell communication
(Basu et al., 2001) or increase
membrane fluidity (Chew and Park,
2004), functions of vital importance when mounting an immune
response.
The clear reduction in plasma carotenoids induced by PHA injection did not
translate into rapid changes in carotenoid-based coloration. In fact, we found
that eye ring and bill redness significantly increased during the 5-day period
between measurements, to a similar extent in both control and PHA-treated
birds. This increase is consistent with the seasonal variation of
carotenoid-based coloration described for this species
(Pérez-Rodríguez,
2008), so a possible explanation for the lack of effect of the
treatment is that individuals were experiencing an allocation trade off in the
use of available carotenoids (ornamentation vs immunity) and
prioritized investment in sexual signalling because of the proximity to the
reproductive season. Another possible explanation is that 48 h is too short a
period to observe any such effect on coloration, or that the decrease in
circulating carotenoids was too small to exert an impact. Although such a
rapid response in carotenoid based coloration has been observed in other
species (Velando et al., 2006;
Rosen and Tarvin, 2006), the
ornaments of the red-legged partridge might require more time to reflect
changes in circulating carotenoids. In addition, as the red coloration of our
study species is mainly based on asthaxanthin (R. Mateo, C.A.-A., L.P.-R. and
J.V., unpublished data), ingested carotenoids cannot be used directly but have
to be metabolized, thus implying a greater delay in colour responses. It is
also possible that the local response elicited by PHA injection did not last
long enough to have impact on coloration (the PHA stimulated swelling usually
disappears after 2 days) (Smits et al.,
1999). It should also be noted that subcutaneous injection of PHA
only mimics a local attack of a potential pathogen. In case of a real
infection, the cell-mediated immune response would be extended over time (and
surely other arms of the immune system would be also activated) thus
increasing the magnitude and duration of the drop in circulating carotenoids
and its potential impact on coloration. For instance, in red grouse
Lagopus lagopus scoticus, experimental reductions of nematode
parasites were shown to increase cell-mediated immunity
(Mougeot and Redpath, 2004) as
well as circulating carotenoids and ornamental coloration
(Martinez-Padilla et al.,
2007; Mougeot et al.,
2007), indicating that recurrent infections negatively affect
cellular immunity and carotenoid availability and use.
In conclusion, our results suggest that carotenoid-based ornamentation may honestly indicate immunocompetence. By contrast, it does not appear to signal antioxidant capacity. In fact, the relative contribution of circulating carotenoids to plasma antioxidant defence was weak. Mounting a cell-mediated immune response depleted circulating carotenoids, but no effect on oxidative damage or antioxidant defences was detected. This suggests that the link between carotenoid ornamentation and immunity may not necessarily rely on the antioxidant properties of carotenoids or the oxidative stress associated with mounting a cellular immune response. Alternative mechanisms to explain the immunoenhancing properties of carotenoids are needed and should be considered in future studies. Given the complexity of the interactions between individual antioxidants, total antioxidant status, oxidative damage and immunity, further research is required to fully understand the relative roles of carotenoids and antioxidants in honest sexual signalling.
|
Acknowledgments |
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References |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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Aguilera, E. and Amat, J. A. (2007). Carotenoids, immune response and the expression of sexual ornaments in male greenfinches (Carduelis chloris). Naturwissenschaften 94,895 -902.[CrossRef][Medline]
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Alonso-Alvarez, C., Bertrand, S., Devevey, G., Gaillard, M., Prost, J., Faivre, B. and Sorci, G. (2004). An experimental test of the dose-dependent effect of carotenoids and immune activation on sexual signals and antioxidant activity. Am. Nat. 164,651 -659.[CrossRef][Medline]
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