Behavioral Ecology Vol. 11 No. 6: 654-662
© 2000 International Society for Behavioral Ecology
Effects of testosterone on cell-mediated and humoral immunity in non-breeding adult European starlings
Department of Psychology, Behavioral Neuroendocrinology Group, The Johns Hopkins University, Baltimore, MD 21218, USA
Address correspondence to Deborah L. Duffy. E-mail: dduffy{at}jhu.edu .
Received 20 September 1999; revised 26 March 2000; accepted 18 April 2000.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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One of the primary assumptions of the immunocompetence hypothesis is that testosterone is immunosuppressive. Although many studies in birds and mammals have supported this assumption, conflicting results have been reported in a variety of species. We investigated the effects of testosterone manipulation on both cell-mediated and humoral immunity in adult songbirds, European starlings (Sturnus vulgaris). Male and female starlings were wild-caught, housed in the laboratory, and implanted with either empty silastic capsules or capsules containing testosterone. Six weeks after implantation, humoral immunity was assessed by injecting the birds with a novel antigen, keyhole limpet hemocyanin, and measuring specific antibody responses 10 and 15 days later via an enzyme-linked immunosorbant assay. Cell-mediated immunity was assessed 7 weeks after implantation via intradermal injection of the T-cell mitogen phytohemagglutinin into the wing web and measuring the degree of swelling 24 h later. Antibody responses to antigenic challenge were significantly suppressed in testosterone-treated females 10 days post-injection and in both sexes 15 days post-injection. Furthermore, there was a significant inverse relationship between individual variability in antibody responsiveness and plasma testosterone concentrations. Cell-mediated responses to phytohemagglutinin stimulation were also significantly suppressed in testosterone-treated males compared to same-sex controls. Testosterone treatment significantly increased plasma corticosterone concentrations compared to controls, and the possibility of this effect mediating the immunosuppressive effects of testosterone is discussed. The present study is among the first to demonstrate testosterone-induced suppression of both cell-mediated and humoral immunity in a species of songbird.
Key words: immune function, cell-mediated, humoral, immunity, birds, starling, phytohemagglutinin, KLH, testosterone, corticosterone, immunocompetence.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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It is often stated that testosterone (T) suppresses immune function, based on numerous studies in mammals and birds (for reviews, see Grossman, 1985
;
Hillgarth and Wingfield, 1997;
John, 1994;
Marsh and Scanes, 1994;
McCruden and Stimson, 1991;
Nelson and Demas, 1996; and
Schuurs and Verheul, 1990).
However, reviews of the literature reveal that the relationship between T and
immune function is quite complex. While many studies in mammals demonstrate a
general pattern of T-induced immunosuppression, some researchers report either
no relationship or a positive relationship between T and immunity (see
Ansar Ahmed et al., 1985 for
review).
The majority of studies investigating the effects of androgenic steroids on
immune function in birds have used domestic chickens as a model (for reviews,
see Glick, 1984;
John, 1994;
Marsh and Scanes, 1994; and
Schuurs and Verheul, 1990). In
general, T administration appears to suppress immunity, but as with mammalian
studies, there have been some contradictory findings. For instance, T
treatment of obese strain (OS) chickens reduced the severity of spontaneous
autoimmune thyroiditis (SAT) but only when administered early in development
(Gause and Marsh, 1986). The
immunosuppressive effect of T treatment on SAT onset in OS chickens was not
observed in adults in which T administration had either no effect or even
enhanced the severity of the disease
(Gause and Marsh, 1986;
Marsh and Scanes, 1994). In
contrast, early castration of chickens has been demonstrated to suppress
cell-mediated immune responses (Mashaly,
1984).
Likewise, studies investigating the effects of T on humoral immunity have
produced contradictory findings. In chickens, T treatment induces premature
atrophy of the bursa of Fabricius, the primary lymphoid organ of humoral
immunity in birds, and significantly suppresses immunoglobulin (Ig) G antibody
responses to immunization with bacterial antigens
(Gause and Marsh, 1986;
Glick, 1984;
Hirota et al., 1976).
Similarly, implantation of male barn swallows (Hirundo rustica) with
T results in a temporary decrease in immunoglobulin concentrations
(Saino et al., 1995).
In contrast to reported immunosuppressive effects of T, previous studies
report either no effect or enhancing effects of T on IgG and IgM antibody
responses to antigenic challenge in birds
(Hasselquist et al., 1999;
Leitner et al., 1996;
Ros et al., 1997). Due to
these conflicting results, it is necessary for more studies to be conducted
using adult wild birds in order to determine whether the effects of gonadal
steroids on immunity in young chickens can be generalized to bird species in
the wild (Hasselquist et al.,
1999; Ros et al.,
1997; Saino et al.,
1995; Zuk et al.,
1995). Furthermore, studies measuring both cell-mediated and
humoral immunity are necessary because it is possible that T may
differentially affect different components of the immune system
(Norris and Evans, 2000;
Zuk and Johnsen, 1998).
Based on the general assumption that T suppresses immune function,
hypotheses regarding the interaction between immunocompetence and reproductive
fitness have been developed (Folstad and
Karter, 1992; Hamilton and
Zuk, 1982). The immunocompetence hypothesis
(Folstad and Karter, 1992), in
particular, has recently gained attention among behavioral ecologists
investigating sexually-selected characters in passerines
(Hasselquist et al., 1999;
Møller et al., 1996;
Saino and Møller, 1996;
Saino et al., 1995; Saino et
al.,
1997a,c).
However, to date, few studies have investigated the effects of T on both
cell-mediated and humoral immunity in any species of passerine. Thus, in the
present study, we sought to investigate the effects of high physiological
doses of T on both cell-mediated and humoral immunity in adult songbirds,
European starlings (Sturnus vulgaris), using specific and direct
measures of immune function in vivo. The assessment of the humoral
response to antigenic challenge was performed using an enzyme-linked
immunosorbant assay (ELISA) developed by us specifically for use with
starlings. This assay is a highly quantitative and direct measure of antibody
response to a specific antigen. Cell-mediated immunity was measured via the
subcutaneous injection of the T cell mitogen phytohemagglutinin (PHA) using a
technique that has widely been used in birds (e.g,
Johnsen and Zuk, 1999;
Zuk and Johnsen, 1998;
Lochmiller et al., 1993;
Mashaly, 1984;
Parmentier et al., 1993;
Saino et al., 1997b;
Soler et al., 1999).
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Adult (
2 years old) male (n = 8) and female (n = 13)
reproductively inactive (photorefractory) starlings were caught in Maryland
(39°18' N lat.) in the summer of 1998. They were housed in
single-sex cages on a light:dark schedule of 18:6 and received food (Purina
turkey starter crumbs) and water ad libitum. This photoperiod
maintained the birds in a reproductively inactive state in which their gonads
were completely regressed (Nicholls et
al., 1988), thus ensuring that their endogenous concentrations of
gonadal steroids were at a minimum. The birds all had black beaks, a sensitive
external indicator of the absence of T in the plasma of starlings
(Ball and Wingfield, 1987;
Dawson, 1983;
Witschi and Miller, 1938). The
birds were allowed to complete their feather molt prior to T treatment or
assessment of immunity in order to control for any energetic expense that may
be associated with molting
(Lindström
et al., 1993). Once all birds completed molt, they were implanted
with either testosterone-filled (four males and seven females) or empty
silastic implants (four males and six females). Under secobarbital anesthesia
(.07 ml of 50 mg/ml solution injected intramuscularly), two 10 mm silastic
implants (1.47 mm i.d., 1.96 mm o.d.) were placed within the peritoneal cavity
via a small incision in the left flank. This dose has previously been
demonstrated to result in T concentrations within the high normal
physiological range for adult males
(Bernard and Ball, 1997). Another group of reproductively inactive adult birds (12 males and seven females) were caught in the fall of 1998 and exposed to the same experimental procedures described herein. The only substantial difference between the two cohorts was that the implants for the second group were placed subcutaneously over the left flank rather than intraperitoneally in order to provide slower absorption of the hormone. Again, one half of the birds received T-filled implants (six males and three females) while the other half received blank implants (six males and four females).
Hormone analysis
Blood samples were taken at zero, two, four, six, and eight weeks after
implantation by puncturing the alar vein with a 25-g needle and collecting
approximately 500-1000 µl of blood into heparinized microcaraway tubes. The
blood was then transferred into 1.5 ml microcentrifuge tubes that were
centrifuged at 1387.5 g for 15 min at 4°C. The plasma fraction
was pipetted off and stored in 0.5 ml centrifuge tubes at -70°C until
assayed for T concentrations via radioimmunoassay (RIA). The T RIA for the
first group of birds was performed using a Coat-A-Count Total Testosterone
125I kit (Diagnostic Products Corporation, Los Angeles, CA, USA)
following the manufacturer's protocol. This assay was highly sensitive (i.e.,
100 pg/ml) and specific (i.e., cross-reactivity to
5
-Dihydrotestosterone (DHT) is 3.3%, to 17ß-estradiol is 0.02%,
and to corticosterone is 0.002%).
The T RIA for the second group of birds was performed using an
125I double-antibody kit purchased from ICN Biomedicals, Inc.
(Costa Mesa, CA, USA) following the manufacturer's protocol. This assay was
also highly sensitive (i.e., 100 pg/ml) and specific (cross-reactivity to
5-DHT is 3.4%, and to 17ß-estradiol and corticosterone is <
0.01%). Prior to the second RIA, some samples from the first group of birds
were run using the double-antibody kit and the values were compared to those
previously obtained using the Coat-A-Count kit. The coefficient of variation
between the two assays was 11.0%. The intra-assay coefficient of variation for
the double-antibody RIA was 7.6%.
Because T implants in dark-eyed juncos (Junco hyemalis) can
elevate corticosterone concentrations
(Ketterson et al., 1991;
Klukowski et al., 1997),
plasma samples from the second group of birds taken 6 weeks after implantation
were also assayed for corticosterone via RIA. Blood samples were obtained
within 2 min for each of the birds. The corticosterone RIA was performed using
an 125I double-antibody kit available from ICN Biomedicals, Inc.
This assay is highly sensitive (i.e., 625 pg/ml) and specific (i.e.,
cross-reactivity to testosterone is 0.10%, to 5-DHT is 0.01%, and to
17ß-estradiol is < 0.01%). The only deviation from the manufacturer's
protocol was that we extended the lower limit of the standard curve by
diluting the lowest standard control (25 ng/ml) twice to yield additional
standard controls of 12.5 ng/ml and 6.25 ng/ml.
Assessment of humoral immunity
Six weeks after implantation, humoral immunity was assessed via injection
of the novel T-cell dependent antigen, keyhole limpet hemocyanin (KLH; 300
µg in 0.1 ml sterile saline, i.m.). Presumably starlings have not
previously been exposed to KLH; therefore it is assumed that the primary
antibody response is being measured (see also
Hasselquist et al., 1999). Ten
and 15 days following injection, blood samples were collected by puncturing
the alar vein with a 25-g needle and collecting approximately 500-1000 µl
of blood into microcaraway tubes. The blood was then transferred into 1.5 ml
microcentrifuge tubes and allowed to clot for 1 h. The clot was removed and
the samples were centrifuged at 1387.5 g for 15 min at 4°C. The
serum fraction was then pipetted off and stored in 0.5 ml centrifuge tubes at
-70°C until assayed for KLH-specific antibodies.
ELISA development
The KLH-specific antibody titers of the serum were measured using an ELISA.
In order to perform this assay, a secondary antibody specifically recognizing
starling IgG was custom-made for us by ICN Biomedicals, Inc. To prepare this
antibody, we provided ICN with 100 ml of starling serum from which they
isolated the IgG fraction. Two rabbits were then inoculated with purified
starling IgG, receiving a total of 4 injections spaced 1 week apart. Both
rabbits were bled once per week for 7 weeks beginning 3 weeks after the
initial inoculation. Portions of all antisera samples taken from the two
rabbits were provided for preliminary testing in our lab.
To develop and optimize our ELISA protocol using the custom secondary
antibody, we modified a protocol previously demonstrated to work well in
mammals (Demas and Nelson,
1998; Klein and Nelson,
1998). Briefly, microtiter plates that were coated with KLH were
incubated with sera from starlings previously inoculated with KLH. For each
individual serum sample, the anti-KLH antibodies bound to the KLH coating the
plate and the remaining sample was washed away. Next, the custom secondary
anti-starling IgG was added followed by a third antibody made in goat to
recognize rabbit IgG. The goat anti-rabbit IgG was purchased from Jackson
Immuno-Research Laboratories, Inc. (West Grove, PA, USA catalog #111-055-003)
as an affinity purified whole molecule conjugated to the enzyme
alkaline-phosphatase (AP). The use of the goat anti-rabbit antibody was the
major deviation from the mammalian protocol and was necessary because the
conjugation of the custom secondary antibody would not have been
cost-effective. The subsequent addition of the substrate,
p-nitrophenyl phosphate (Sigma Chemical, St. Loius, MI, USA) resulted
in a colorimetric reaction which was read using a plate reader (Bio-Rad:
Benchmark model, Richmond, CA, USA).
To determine the optimal concentrations of the samples and antisera, we conducted a series of five pilot studies using sera from an additional 15 birds that had been injected (i.m.) with three different doses of KLH (200 µg, 300 µg, or 400 µg of KLH in 0.1 ml sterile saline). Blood samples were obtained 10 and 15 days post-injection and the individual samples were used in the pilot studies at dilutions of 1:20, 1:40, 1:80, 1:160, 1:320, 1:640, 1:1280, and 1:2560. In addition, pooled sera samples from five birds never exposed to KLH (i.e., negative controls) were included in the same dilutions to determine the degree of serum-dependent non-specific binding. On each plate, one well received no starling serum in order to assess the degree of serum-independent non-specific binding.
In the initial pilot studies, portions of the individual anti-starling sera samples from the two rabbits were pooled giving us one pooled sample from each rabbit. Both anti-starling sera pools were run separately in the initial pilot assay. Antisera from one rabbit provided much less serum-dependent non-specific binding and was used in all subsequent assays. For this antiserum, serum-dependent non-specific binding was on average 86% higher than the no serum control values and 52% lower than the positive control values. The remaining pilots used combinations of different dilutions of the anti-starling IgG and the AP-conjugated goat anti-rabbit. It was determined that dilutions of 1:500 for each antibody provided the best results. A final pilot comparing the results among all of the anti-starling sera samples (one each from the seven blood samples) from the rabbit that provided optimal results did not reveal a substantial difference among the samples. Furthermore, a dose of 300 µg of KLH yielded the highest antibody responses.
ELISA protocol
For the results reported here, the following protocol was used. Microtiter
plates were coated with KLH (0.5 mg/ml in sodium bicarbonate buffer, pH = 9.6)
and blocked with 10% nonfat dry milk in phosphate-buffered saline containing
0.05% Tween 20 (PBS-T). Thawed serum samples were diluted 1:40, 1:80, 1:160
and 1:320 with PBS-T and 150 µl of each sample were added in duplicate to
the wells of the microtiter plates. Positive control samples (pooled sera from
the starlings used in the pilot studies that exhibited high concentrations of
anti-KLH IgG) and negative control samples (pooled sera from starlings that
were not exposed to KLH) were added in duplicate to each plate. One well on
each plate had no starling sera added to assess the level of serum-independent
non-specific binding. The plates were sealed, incubated at 37°C for 3 h,
then washed five times with PBS-T.
The plates were then incubated with the secondary anti-body (diluted 1:500) that was raised in rabbit to recognize specifically starling IgG for 1 h at 37°C and washed five times with PBS-T. The AP-conjugated goat anti-rabbit was added to the wells (dilution 1:500) and incubated for 1 h at 37°C. The plates were washed three times with PBS-T and 150 µl of the enzyme substrate p-nitrophenyl phosphate (1 mg/ml in diethanolamine substrate buffer) was added to each well. The plates were protected from light during the enzyme-substrate reaction that was terminated after 10 min by adding 50 µl of 1.5 M NaOH to each well.
The optical density (OD) of each well was determined using a plate reader
equipped with a 405 nm wavelength filter. The average OD for each set of
duplicate wells was calculated and the OD for each sample was expressed as the
ratio of the sample to the negative control (P/N) for the same dilution to
minimize intra- and inter-assay variability
(deSavigny and Voller, 1980;
Leitner et al., 1996). All
sera samples for both days 10 and 15 were run in one assay. Ten samples from
both days were run in a second assay and the inter-assay coefficient of
variation was 3.1%.
Assessment of cell-mediated immunity
Seven weeks after implantation, cell-mediated immunity was assessed via
intradermal injection of the T-cell mitogen PHA (Sigma, St. Louis, MO). The
thickness of the site of injection, the wing web, was measured to the nearest
0.01 mm using a pressure-sensitive thickness gauge (Mitutoyo Corporation,
Model # ID-S1012E) immediately prior to and 24 h following injection. The
right wing web was injected with 0.5 mg of PHA in 0.1 ml PBS and the left wing
web was injected with 0.1 ml of the vehicle alone
(Lochmiller et al., 1993;
Saino et al., 1997b). The
degree of the immune response was assessed as the percent increase in wing web
thickness (calculated as the degree of swelling of the PHA-injected wing
divided by the swelling of the vehicle-injected wing)
(Dhabhar and McEwen, 1996;
Mashaly, 1984).
Data analyses
Serum anti-KLH antibody titers were analyzed using two 2 (sex)x2 (T
treatment) analyses of variance (ANOVAs), one for each day post-injection (10
and 15). The percentage change in wing web thickness was log transformed to
correct for heterogeneity of variance and analyzed using a 2 (sex)x2 (T
treatment) ANOVA. The effects of T implantation on corticosterone
concentrations were analyzed using a 2 (sex)x2 (T treatment) ANOVA.
Planned comparisons were performed using Fisher's PLSD and differences were
considered significantly different if p <.05.
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RESULTS |
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Humoral immunity
The data from the birds caught in the summer and fall of 1998 were pooled providing us with a total of 40 subjects (20 males and 20 females). One male that received blank implants was removed from analysis due to leakage of the antigen during injection. Testosterone treatment significantly reduced IgG antibody response to KLH at both 10 and 15 days post-injection [F(1,35) = 8.518, p <.05 and F(1,35) = 11.209, p <.05, respectively]. Planned comparisons revealed T-induced suppression of antibody responses in females 10 days post-injection and in both sexes 15 days post-injection (see Figure 1).
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Hormone analysis revealed that T treatment elevated plasma T concentrations
in both sexes to within the high normal range for adult breeding males (see
Figure 2;
Bernard and Ball, 1997). Closer
examination of the individual T concentrations indicated variability within
each of the T-treated groups. Therefore, two regressions were performed, one
for each day that antibody responses were analyzed (10 and 15 days
post-injection), using the data from the T-implanted birds to investigate
whether the individual variability in T concentrations could predict
individual variability in antibody responses to KLH. For these analyses,
plasma T concentrations from samples taken 6 weeks post-implantation were used
as the independent variable because it was at that time that the birds
received antigenic challenge. A significant negative correlation was observed
between plasma T concentrations and antibody responses at 15 days
post-injection [see Figure 3;
F(1,18) = 7.921, p <.05, r2
=.318].
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Cell-mediated immunity
As for humoral immunity, the data from both sets of birds were pooled. Due
to leakage of the mitogen during injection, 13 birds were removed from the
analysis to yield a final number of 27 birds.
Figure 4 illustrates the effect
of T manipulation on the response to in vivo mitogenic stimulation.
Planned comparisons revealed that males who received T-filled capsules
exhibited a significantly smaller swelling response to mitogenic stimulation
than males that received blank implants
(Figure 4). This effect was not
observed in females. Unlike that observed in the humoral response, regression
analyses revealed no correlation between individual variations in T
concentrations and wing web swelling.
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Hormone analysis
As stated above, hormone implantation significantly raised T concentrations
to within the high physiological range for adult breeding males
(Figure 2;
Bernard and Ball, 1997).
Further analysis revealed that T treatment significantly elevated plasma
corticosterone concentrations in both males and females
[Figure 5; F(1,12) =
11.996, p <.05]. Three birds were omitted from the corticosterone
analysis due to an insufficient quantity of sample and one bird was omitted
because its concentration of corticosterone fell below the limits of
detectability of the assay.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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The present experiment demonstrates that T concentrations within the high normal physiological range suppress humoral and cell-mediated immunity in European starlings. Cell-mediated immunity in males (Figure 4) and humoral immunity in both males and females (Figure 1) were suppressed by T manipulation. Furthermore, a strong negative relationship exists between T concentrations and IgG response to antigenic challenge. Because only the highest testosterone concentrations suppressed antibody responses, one possible interpretation is that humoral immunosuppression acts as a constraint on the magnitude of testosterone elevation associated with the breeding season for this species.
Immunosuppression is only one of several ways that high T concentrations
have been demonstrated to be costly to males. In various taxa, high T
concentrations associated with the breeding season are known to facilitate
courtship and aggressive behaviors which can be energetically expensive and
may result in injury (Dufty,
1989; Emerson and Hess,
1996; Johnsen,
1998; Moore and Marler,
1987; Wingfield et al.,
1987,
1990). Furthermore, elevated T
concentrations are associated with a decrease in paternal care in some
biparental species of birds, thus imposing a reproductive fitness cost
(Cawthorn et al., 1998;
Hegner and Wingfield, 1987;
Ketterson and Nolan, 1992;
Ketterson et al., 1992;
Oring et al., 1989,
Silverin, 1980;
Wingfield et al., 1990). Also,
prolonging the period of peak T concentrations beyond what is normally
experienced can increase mortality in some bird species
(Dufty, 1989;
Nolan et al., 1992). Thus, in
addition to immunosuppression, there are numerous constraints on the magnitude
and duration of high T concentrations, only a sample of which have been
discussed here.
In the interpretation of these results, it must be noted that the antibody
responses measured in the present experiment are a composite of both IgM and
IgG isotypes due to the likelihood of cross-reactivity of the anti-starling
IgG secondary antibody. The primary antibody response, presumably as assessed
here, consists primarily of IgM in the initial stages of the response followed
by a switch to IgG. It is possible that administration of T reduced IgG
synthesis without decreasing the IgM response. Also, the antibody response to
KLH is dependent on T-helper lymphocytes, a component of the cell-mediated
immune system, for antigen recognition and isotype switching. Thus, it is
possible that the suppression of the antibody response to KLH by T was the
result of an interference with the switching from IgM to IgG. Some evidence in
domestic poultry supports this alternative mechanism (see
Schuurs and Verheul, 1990 for
review). In chickens, embryonally administered T results in a decrease in IgG,
but not IgM antibody responses which appears to be due to an impairment of
isotype switching (Glick and Sadler,
1961; Hirota et al.,
1976,
1980;
Lerner et al., 1971;
Schurrs et al., 1987;
Verheul et al., 1986).
Comparison with previous studies
These data are in agreement with previous studies in some avian species
suggesting suppressive effects of T on immune function
(Al-Afaleq and Homeida, 1998;
Leitner et al., 1996;
Verhulst et al., 1999;
Zuk et al., 1995). For
instance, exogenous administration of T to broiler chicks resulted in a
decrease in the total number of leukocytes, lymphocytes and the weight of the
bursa of Fabricius (Al-Afalaq and Homeida,
1998). Likewise, IgM responses to sheep red blood cells (SRBC) in
white leghorn chickens treated embryonically with T were significantly
impaired compared to untreated controls
(Hirota et al., 1976). Other
indirect evidence further supports the hypothesis of T-induced humoral
immunosuppression. For example, in domestic chickens selectively bred to
produce either high or low antibody responses to E. coli or SRBC, low
responders had elevated testosterone concentrations compared to high
responders (Leitner et al.,
1996; Verhulst et al.,
1999). However, in these studies the direction of causality is
unknown and it is possible that enhanced immunity suppresses T concentrations
in selectively bred chickens (Verhulst et
al., 1999).
Studies in wild species have also provided evidence for T-induced
immunosuppression. Barn swallows treated with T in the field exhibited a
temporary decrease in total immunoglobulin levels
(Saino et al., 1995). In red
jungle fowl, endogenous testosterone concentrations were negatively correlated
with lymphocyte counts (Zuk et al.,
1995). A recent study in male dark-eyed juncos (Junco
hyemalis) has also demonstrated that artificial elevation of T suppresses
cell-mediated and humoral immunity (Casto JM and Ketterson ED, personal
communication).
The data presented here differ from the findings of some other recent work
examining the effects of physiological concentrations of T on humoral immunity
in another species of songbird, red-winged blackbirds (Agelaius
phoeniceus). In that study, naturally-occurring increases in T
concentrations in males did not lead to suppression of a composite of IgG and
IgM antibody responsiveness to KLH injections
(Hasselquist et al., 1999).
Possible reasons for these different results include species and
methodological differences. One potentially important methodological
difference is that in the present experiment the primary response to KLH
stimulation was assessed whereas Hasselquist and colleagues measured secondary
responses to KLH injection.
The secondary antibody response involves a different population of B cells
known as memory B cells which may respond differently to the presence of T.
Also, while the primary antibody response is first dominated by the IgM
isotype prior to switching to IgG, the secondary antibody response is IgG
dominated (Janeway and Travers,
1997). Thus, it is possible that only the primary response to a
given antigen is influenced by elevated T concentrations
(John, 1994;
Wunderlich et al., 1992).
There is some evidence in a mammalian species that the ability of T to disrupt
immune responses to a given pathogen may depend on previous exposure to that
pathogen. Testosterone-induced attenuation of resistance to Plasmodium
chabaudi malaria in female laboratory mice (strain C57BL/10) was
eradicated if the mice had previously overcome infection with the same
parasite (Wunderlich et al.,
1992).
Finally, Hasselquist et al.
(1999) inoculated their birds
with a mixture of KLH and Freund's incomplete adjuvant which is simply an
oil-in-water emulsion that facilitates the uptake of the antigen by
macrophages in the initial stages of the response
(Janeway and Travers, 1997).
In addition to measuring the secondary response to KLH, the use of an adjuvant
by Hasselquist et al. may also have contributed to differences in the
proportion of IgM to IgG isotypes in the sera. In preliminary testing,
Hasselquist et al. observed a peak primary antibody response (a composite of
IgG and IgM) 12 days after the first inoculation with KLH. Therefore, in our
study we assumed that the peak primary antibody response would occur around 10
to 15 days post-injection (Klein and Nelson,
1998,
1999). However, in the former
study, an increased rate of isotype switching due to the use of an adjuvant
may have resulted in a shift of the peak response. Thus, we cannot conclude
that our time points of 10 and 15 days post-injection represent the peak
antibody response. A time course study in which blood samples are taken every
week after inoculation would need to be performed in order to ascertain with
certainty the peak response time as well as the duration of the response.
Effects of testosterone in females
Importantly, the females in the present experiment experienced T
concentrations that were within the normal physiological range for adult
breeding males (Bernard and Ball,
1997). The purpose here was to determine whether a given dose of T
would have equal effects in both sexes. If immunity in females was suppressed
to a greater extent than that observed in males, it may indicate that a
mechanism has evolved in males to cope with the high T concentrations
associated with the breeding season.
Although adult females in the wild do have detectable concentrations of T
during the breeding season, the dose of T that these females received was
supraphysiological (Dawson,
1983; Kessel,
1951). Therefore, it is not yet clear whether a physiological dose
of testosterone suppresses humoral immunity in females. Unlike males,
cell-mediated immunity was not suppressed by high T concentrations in females.
It is possible that this sex difference is a result of the difference in T
concentrations between males and females 2 weeks after implantation (see
Figure 2). The high peak in T
concentrations in males at week 2 could have affected the cell-mediated
response to PHA 4 weeks later. The duration of T-induced immunosuppression is
not yet clear in adult birds and requires further investigation.
Role of reproductive condition
Because the birds in the present experiment were in a reproductively
inactive condition, it could be argued that the T treatment used was not truly
physiological. It could reasonably be expected that other variables associated
with reproductive condition interact with high T concentrations to suppress
immunity further in starlings. It is known in some cases that biological
responses to gonadal steroids do change with season in birds and mammals
(e.g., Hinde and Steele,
1978). Exogenous gonadal steroids are more effective at
facilitating reproductive behaviors in seasonally-breeding species when
administered during day lengths associated with the breeding season than when
given during day lengths associated with the non-breeding season. Also,
photoperiod-dependent effects of T have been identified in starlings. The
effects of T on the neural circuit controlling song production appear to be
attenuated in reproductively inactive (i.e., photorefractory) male starlings
compared to birds in a reproductively active condition
(Bernard and Ball, 1997).
Photoperiodic manipulation affects in vitro cell-mediated immune
responses to mitogenic stimulation in starlings, irrespective of gonadal
secretions (Bentley et al.,
1998). Birds in a reproductively active condition exhibit
decreased proliferative responses of splenocytes to mitogenic stimulation
compared to birds in a reproductively inactive condition. Therefore, it is
possible that the suppressive effects of T would be intensified if the birds
in the present experiment were in a reproductively active condition.
In general, there are a number of possible mechanisms that could be
responsible for the seasonal changes in responsiveness to T administration,
including changes in the number and/or activity of androgen receptors
(Moeller et al, 1988;
Nastiuk and Clayton, 1994;
Slater and Scheck, 1998;
Soma et al., 1999;
Tahka et al., 1997;
Wood and Newman, 1993).
However, to date no study has reported photoperiod-induced fluctuations in
androgen receptors within the avian immune system.
Role of corticosterone
The mechanism through which T affects immunity is not completely
understood. Androgen receptors have been localized within the thymus in
mammals and within the bursa of Fabricius in chickens
(Grossman, 1985;
Marsh and Scanes, 1994;
McCruden and Stimson, 1984;
Schuurs and Verheul, 1990;
Sullivan and Wira, 1979).
Another possible mechanism though which T could be acting involves
corticosterone. This hormone, typically secreted in response to stress,
modulates immune function in a variety of mammalian and avian species (see,
e.g., Dunn, 1989;
Fowles et al., 1993;
Trout and Mashaly, 1994).
As with androgen receptors, glucocorticoid receptors have been localized in
a variety of lymphoid tissues, including the bursa of Fabricius in chickens
and the thymus in chickens and rodents
(Bakker and Kendall, 1997;
Coulson et al., 1982;
Fassler et al., 1986;
Miller et al., 1998;
Sullivan and Wira, 1979).
Similar to reports in dark-eyed juncos
(Ketterson et al., 1991;
Klukowski et al., 1997), T
treatment in the present study raised basal corticosterone concentrations
above those observed in untreated same-sex controls. Therefore, it is possible
that T-induced corticosterone elevation is responsible for the attenuation of
humoral and cell-mediated immunity observed in the present study. However,
endogenous increases in T are not necessarily associated with increases in
basal corticosterone concentrations. Despite this, T mediates many behaviors
that may increase the likelihood that an individual will experience a
stressful situation (e.g., aggressive interactions;
Balthazart, 1983; Wingfield et
al., 1987,
1990); natural increases in
glucocorticoids caused by these stressors may influence immunocompetence
concomitant with T concentrations associated with the breeding season.
Functional significance
In the present study, T treatment resulted in an average reduction of 15.5%
(17% in females and 14% in males) in the humoral immune response 10 days after
immunization with KLH and a 22.5% reduction (25% in females and 20% in males)
in antibody responses 15 days post-immunization. Furthermore, a 50% reduction
in the cell-mediated response to PHA injection was observed in males. It is
not known what impact this degree of immune function impairment would have on
the survival of starlings in the wild. In order to address this issue, field
studies investigating the effects of T manipulation on immunocompetence and
long-term survival are needed.
Experiments assessing long-term survival of individuals in wild populations
are uncommon due to difficulties in tracking individuals from one breeding
season to the next (however, see Dufty et al., 1989;
Ketterson et al., 1992; Saino
et al., 1995,
1997a). One noteworthy
exception is the work in barn swallows (Hirundo rustica)
demonstrating that immunocompetence is a reliable predictor of long-term
survival in the wild (Saino et al.,
1997a). Furthermore, artificial elongation of tail feathers of
barn swallows decreases IgG antibody responses following SRBC inoculation and
decreases the probability of surviving to the next breeding season
(Saino et al, 1997a). Thus,
factors affecting immune responses to antigenic stimulation can have an impact
on long-term survival. Administration of T implants to male barn swallows also
reduced the probability of survival to the next breeding season by 50%,
although T-induced immunosuppression was not detected
(Saino et al., 1995). Whether
the degree of impairment observed in the present study would have notable
effects on survival remains an open question.
The data presented here provide an important contribution to the field by
demonstrating a T-induced reduction of responses to immunogenic challenge
(Norris and Evans, 2000).
Recently, Braude and colleagues
(1999) proposed the
redistribution hypothesis as an alternative to the immunosuppression model.
According to their hypothesis, elevations in T may result in a redistribution
of lymphocytes, rather than a reduction in their numbers. The authors point
out that a reduction in the number of circulating leukocytes may be due to
immune cells being relocated to other components of the immune system, such as
the lymph nodes, rather than immunosuppression as often claimed. It has
therefore been noted that multiple measures of immunity are necessary when
investigating the effects of T on immunocompetence because a shift from one
component of the immune system to another may otherwise be overlooked
(Braude et al., 1999;
Norris and Evans, 2000; and
Zuk and Johnsen, 1998).
In the present study, we observed suppression of both cell-mediated and
humoral immunity using direct methods to assess immune function. These data
suggest that rather than a T-induced shift from one component of the immune
system to another, T suppresses both the cell-mediated and humoral components.
However, because KLH is a T-cell dependent antigen, it is possible that
T-induced suppression of antibody response to KLH occurs indirectly via
suppression of cell-mediated immunity. Also, we did not assess innate
immunity, therefore the effects of T on this component of immune function and
possible consequences for adaptive immune responses remains unclear
(Norris and Evans, 2000).
Nevertheless, because we assessed immunocompetence directly by measuring
inclusive responses to immunogenic challenge, we have taken any possible
redistribution of immune cells into account. Thus, these data support the
hypothesis that T is immunosuppressive. Further study using a variety of
immunogenic challenges assessing cell-mediated and humoral immunity in
addition to innate immune measures would provide insight into the extent of
T-mediated immunosuppression.
Summary
In summary, the data presented here are in support of one of the primary
assumptions of the immunocompetence hypothesis
(Folstad and Karter, 1992).
These data provide empirical evidence that testosterone is indeed
immunosuppressive in starlings. Furthermore, the present study demonstrates a
sex difference in the effects of testosterone on cell-mediated immunity as
measured by in vivo mitogenic stimulation of T cells. While
testosterone suppressed the subcutaneous response to PHA in males, no effect
was observed in females. On the other hand, testosterone treatment suppressed
antibody responses to antigenic challenge in both sexes in a dose-dependent
fashion. Testosterone treatment also increased corticosterone concentrations
and the possibility that this effect is the mechanism through which T
manipulation suppresses immune function requires further investigation.
|
ACKNOWLEDGEMENTS |
|---|
We would like to thank Dr. R. J. Nelson and Dr. S. L. Klein for providing helpful comments on the manuscript and assistance in developing the immunological measures. We would also like to thank Dr. D. Hasselquist for his helpful advice concerning the ELISA and J. M. Casto for invaluable suggestions regarding the data and hormone analyses. We acknowledge support by NSF (IBN 9514525) and NIH (NS 35467) grants to G.F.B. and by a NSF Graduate Fellowship to D. L. Duffy.
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