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Original Studies |
Max-Planck-Institute of Psychiatry (A.S., J.M., E.F., D.M.H., F.H., T.P.), D-80804 München, Germany; and Max-Planck-Institute of Immunobiology (C.G.), D-79108 Freiburg, Germany
Address all correspondence and requests for reprints to: Andreas Schuld, M.D., Max-Planck-Institute of Psychiatry, Kraepelinstraße 10, D-80804 Munich, Germany. E-mail: schuld{at}mpipsykl.mpg.de
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Abstract |
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were
determined. Confirming earlier studies, temperature and cytokine levels
showed monophasic, dose-dependent increases in response to endotoxin.
In contrast, endocrinological effects of endotoxin showed a complex,
biphasic pattern: cortisol levels were not affected by 0.2 ng/kg but
significantly increased during the first 6 h following 0.4 and 0.8
ng/kg endotoxin, whereas ACTH and DHEA levels were
significantly enhanced during the first 6 h following 0.8 ng/kg
only. ACTH, DHEA, and cortisol secretion was blunted 6–12
h following 0.8 ng/kg. DHEA-S levels were unaffected
during the first 6 h following all dosages, but between 6–12 h
after injection they were significantly increased following 0.2 ng/kg,
unaffected by 0.4 ng/kg, and significantly decreased following 0.8
ng/kg endotoxin. The present results suggest that similarly to
glucocorticoids, the adrenal androgens DHEA and
DHEA-S play an important role during early host responses
to bacterial infections in humans. |
Introduction |
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, the major effector hormone of the HPA system,
cortisol, is released in large quantities from the adrenal gland during
ongoing host defenses. Cortisol represents a pivotal negative feedback
signal that limits the extent of the host responses by suppressing the
release of inflammatory cytokines (1, 2, 3). In contrast to
glucocorticoids, very little is known about the role of the most
abundant steroidal products of the adrenal gland,
dehydroepiandrosterone (DHEA) and
DHEA-sulfate (DHEA-S), during acute infection
and inflammation. These steroids have been shown to exert various
immunomodulatory effects in vitro and in vivo,
which are overlapping with those of glucocorticoids, but are not
identical: in vitro they partly antagonize some effects of
cortisol on carbohydrate and lipid metabolization, lymphocyte function,
and cytokine synthesis (4). Similar to the effects of
glucocorticoids, in vivo DHEA pretreatment has
been shown to result in a substantially reduced mortality in various
experimental models of infection and inflammation
(5, 6, 7). However, it still remains unknown whether these steroids play a role in the physiological regulation of early host response; the available information on circulating levels of DHEA and DHEA-S during infection and inflammation is conflicting: during experimental endotoxemia in animals, DHEA or DHEA-S levels have been found to be unchanged, decreased, or increased, depending on the experimental model used (8, 9, 10). Very recently, it has been reported in humans that the administration of Escherichia coli endotoxin increases DHEA levels within the first 6 h after injection (11). In patients, DHEA and DHEA-S levels, in general, have been found to be reduced during chronic infectious or inflammatory diseases including HIV infection, when an AIDS-defining illness was present (12, 13, 14, 15). During the early stages of HIV infection, however, DHEA levels have been reported to be increased (14, 16). Moreover, in umbilical cord blood of infants from mothers with infections, DHEA-S levels also have been found to be increased (17). Although it seems difficult to draw definite conclusions from these studies, they suggest that DHEA and DHEA-S might play a role that differs between the early and late phases of infectious diseases, respectively.
To further elucidate the role of DHEA and
DHEA-S during the early phases of primary host responses,
the experimental model of low-dose endotoxinemia in healthy volunteers
seems to be a promising tool: endotoxin, a major cell wall component of
Gram-negative bacteria, is known to play an important role in the early
phase of bacterial infection and Gram-negative sepsis (18, 19). In humans, numerous studies have shown that the injection
of low doses of endotoxin mimics many clinical signs and symptoms of
acute infection: it transiently increases body temperature and heart
rate, alters leukocyte counts, and induces flu-like symptoms lasting
several hours (19, 20, 21). The prominent monophasic increases
in plasma levels of inflammatory cytokines such as tumor necrosis
factor (TNF-) and IL-6 following endotoxin administration are
the most important mediators of all these phenomena (20, 22, 23) including the activation of the HPA system during
endotoxinemia (1, 2).
To study the physiological role of adrenal androgens during early host response processes, we have administered three doses (0.2, 0.4, and 0.8 ng/kg body weight) of Salmonella abortus equi endotoxin to three groups of healthy male volunteers in a single-blind, placebo-controlled experiment. During the following 12 h, plasma levels of inflammatory cytokines, ACTH, cortisol, DHEA, and DHEA-S had been serially assessed.
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Experimental subjects |
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TopAbstractIntroductionExperimental subjectsMaterials and MethodsResultsDiscussionReferences |
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Materials and Methods |
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TopAbstractIntroductionExperimental subjectsMaterials and MethodsResultsDiscussionReferences |
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At 1630 h, the subjects entered the laboratory and were under continuous observation during the entire experimental session, while a physician was on call permanently. At 1700 h, an iv canula was inserted in an antecubital forearm vein. The blood line was kept patent with saline solution containing heparin (400 IU/L) to prevent clotting. Blood was sampled until 1100 h the next morning. After withdrawal blood was stabilized with Na-EDTA (1 mg/mL blood) and aprotinine (300 KIU/mL blood), and following immediate centrifugation and aliquotation plasma was frozen to -20 C or -80 C, respectively. Blood pressure was monitored until 2300 h with a Dinamap Vital Daten Monitor 1846SX (Critikon, Norderstedt, Germany); one lead electrocardiogram and rectal temperature (temperature monitor model 8055; S&W, Albertslund, Denmark) were monitored throughout the entire experimental session, as well as standard sleep polygraphy. The results of polygraphic sleep recordings have been already reported elsewhere (24). At 1800 h, subjects received light food, and later only mineral water was offered ad libitum. At 2300 h, light was turned off and the subjects were instructed to sleep until they woke up spontaneously. They were offered breakfast 30 min after awakening but remained in bed until 1200 h, 60 min after the last blood sample was taken. At 2300 h, vehicle or endotoxin was iv injected in a single-blind, placebo-controlled crossover design using a sterile and essentially protein-free (<0.08%) solution of Salmonella abortus equi endotoxin prepared for use in humans (for details see Refs. 20 and 25): in balanced order, 0.2 (n = 7), 0.4 (n = 5), or 0.8 ng (n = 5) per kg body weight endotoxin or 0.9% saline solution were administered at 2300 h during two experimental sessions separated by 2 weeks. The three groups of subjects did not differ significantly in mean age or body mass index.
Hormone and cytokine assays
Plasma levels of ACTH, cortisol, DHEA, and
DHEA-S were determined by coated tube RIAs (ACTH:
Nichols Institute Diagnostics, San Juan Capistrano, CA;
cortisol: ICN Biomedicals, Inc. Carson, CA;
DHEA: Biochem Immunosystems, Freiburg, Germany;
DHEA-S: DRG Instruments, Marburg, Germany), the limit of
detection was 1.0 pg/mL for ACTH, 0.3 ng/mL for cortisol, 0.02 ng/mL
for DHEA, and 1.7 µg/dL for DHEA-S; the
intra- and interassay coefficients of variation were below 8%,
respectively, for all assays. IL-6 and TNF- were determined by
enzyme-linked immunosorbent assays (Medgenix Diagnostics, Brussels,
Belgium). The limit of detection was 3.0 pg/mL for both cytokines, and
the intra- and interassay coefficients of variation were below 8%.
Statistical methods
Data analysis was performed using commercially available
personal computer software (SPSS for Windows 7.5; SPSS, Inc., Chicago, IL). For statistical analysis, the area
under the response curve (AURC) according to the trapezoid method was
computed for the various host response parameters. The response curves
were defined as the time courses of the differences between the two
experimental conditions (verum and placebo) at every time point. Visual
inspection of the data (Figs. 1
and 2) showed that the cytokine and
temperature responses were monophasic, whereas hormonal responses
showed biphasic temporal patterns characterized by an initial increase
in secretion, followed by a blunting. Therefore, we divided the
hormonal response curves into halves (from 2300–0500 h and from
0500–1100 h). To test within each dose whether endotoxin had an effect
on the AURC, one-sample t tests were used. To compare the
effects of the different doses, ANOVA was used, followed by post
hoc tests (lowest significant difference test). Because the
endotoxin-induced increases in body temperature and the levels of
inflammatory cytokines, ACTH, and cortisol are well established
(1, 2, 19, 21), we computed one sided P values
for these parameters. For DHEA and DHEA-S
levels, two-sided P values were computed because no specific
hypothesis was available. P values below 0.05 were
considered significant. All data reported in the tables represent
means ± SD; the figures show means ±
SEM.
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Results |
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TopAbstractIntroductionExperimental subjectsMaterials and MethodsResultsDiscussionReferences |
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Administration of endotoxin evoked prominent,
monophasic, and dose-dependent increases in the plasma levels of
TNF- and IL-6, and in rectal temperature (Fig. 1
and Table 1). The peak in the temperature response
lagged 
2 h behind the maximum of the two cytokines plasma levels we
measured herein. Statistical analysis of the AURCs confirmed the dose
dependency of these responses. Whereas TNF- and IL-6 secretion
increased in response to all doses that were administered, rectal
temperature showed no significant change following the lowest dose of
0.2 ng/kg, but was significantly increased following both higher
dosages.
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Compared with the monophasic, dose-dependent increases in cytokine
plasma levels that were induced by the injection of endotoxin, the
endocrine responses were considerably more complex (Fig. 2 and Table 2). Following the injection of 0.2 ng/kg
endotoxin, ACTH, cortisol and DHEA plasma levels were not
affected significantly, but DHEA-S levels increased
significantly between 6 and 12 h after injection. Administration
of 0.4 ng/kg endotoxin induced a prominent increase in cortisol levels
during the first 6 h following injection, when the plasma levels
of ACTH, DHEA, and DHEA-S remained constant.
During the second half of the experiment, the plasma levels of ACTH,
cortisol, and DHEA, but not those of DHEA-S
were slightly suppressed following 0.4 ng/kg endotoxin; however, this
blunting of secretion was significant only for ACTH. The highest dose
of endotoxin administered (0.8 ng/kg) induced prominent and significant
increases in the plasma levels of ACTH, cortisol, and DHEA
that were followed by a significant blunting of secretion of all these
hormones. In contrast, DHEA-S secretion did not increase,
but was significantly reduced later on between 6 and 12 h after
injection.
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Discussion |
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TopAbstractIntroductionExperimental subjectsMaterials and MethodsResultsDiscussionReferences |
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Effects of endotoxin on inflammatory cytokines, body temperature, ACTH, and cortisol
In line with numerous earlier studies, we found that endotoxin
induces a rapid and dose-dependent increase in rectal temperature and
in plasma levels of IL-6 and TNF-, followed by a rise in rectal
temperature, which was dose-dependent as well
(19, 20, 21).
The lowest dose of endotoxin had no effects on cortisol or ACTH
release. However, following 0.4 ng/kg, there was a considerable
increase in cortisol secretion that occurred without a concomitant
increase in ACTH plasma levels. The injection of 0.8 ng/kg endotoxin
induced prominent surges in both cortisol and ACTH levels during the
first 6 h after the injection, followed by a blunting of secretion
during hours 6–12. These results confirm earlier studies reporting a
stimulatory effect of endotoxin on the HPA system, which is mediated by
inflammatory cytokines such as IL-6 and TNF- at the hypothalamic,
pituitary, and adrenal levels (20, 22, 26, 27). Whereas
the highest dosage of endotoxin was followed by an increase of cortisol
and ACTH in parallel, we show here for the first time that endotoxin at
a dose of 0.4 ng/kg may induce cortisol release without concomitant
increases in ACTH release. This finding supports in humans the concept
based on animal and in vitro studies (28) that
inflammatory cytokines are able to directly stimulate cortisol release
from the adrenal gland. For IL-6 this recently has been shown in
vitro with human adrenal cells (29). However, in the
present study, a quite small increase in IL-6 levels was observed in
response to 0.4 ng/kg endotoxin, whereas the increase in TNF- levels
was somewhat more prominent. Because TNF- in vitro has
been shown to inhibit rather than to stimulate cortisol release from
human fetal adrenal cells (30), ACTH-independent cortisol
release following endotoxin might involve other, yet unidentified
cytokines and neuroendocrine factors (2).
Effects of endotoxin on the levels of DHEA and DHEA-S
In a recent study, it has been shown that a single dose of E. coli endotoxin in humans is able to increase DHEA levels (11). In addition to confirming this finding, we show here that it is dose dependent, and we add information on concomitant changes in DHEA-S levels, as well as the complex temporal pattern of these endocrine changes.
In the present study, we found that the pattern of endotoxin-induced changes in DHEA secretion is qualitatively similar to the ACTH response, supporting the hypothesis that ACTH plays an important role in stimulating DHEA-release from the adrenals (31): 0.8 ng/kg endotoxin induced a significant increase in ACTH and DHEA levels during the first 6 h after the injection of endotoxin, followed by a blunting of secretion.
Lower doses had no effects on ACTH and DHEA release
despite marked effects on IL-6 and TNF- levels. Hence, inflammatory
cytokines released during endotoxemia are unlikely to directly
stimulate DHEA release from the adrenals in contrast to
their stimulating effect on cortisol release, which has been discussed
above. Another ACTH-independent mechanism possibly involved in
endotoxin-induced adrenal DHEA release has recently been
observed by Wolkersdörfer et al. (32),
who described direct cellular interactions between lymphocytes and zona
reticularis cells in vitro, resulting in DHEA
release. In the present study, the increase in DHEA levels
induced by the highest dose of endotoxin (0.8 ng/kg) was considerably
smaller than reported following 4 ng/kg E. coli endotoxin
(11). This difference might be due to the fact that
endotoxin was injected in the evening in our study and in the morning
by Bornstein et al. (11). However, because
temperature and ACTH and cortisol responses to endotoxin are much
stronger in the evening (33), one would expect the
opposite difference (a more prominent DHEA increase in the
present study). It is also possible that the different endotoxin
preparations used have different biological activities. Although such
differences are likely, they have never been systematically
investigated.
Interestingly, the effects of endotoxin on the secretion of
DHEA-S differed markedly from those on the secretion of
DHEA: 0.2 ng/kg endotoxin, an amount that did not affect
cortisol, ACTH, and DHEA levels, induced a slight but
significant increase in DHEA-S secretion during hours
6–12 after injection; a dose of 0.4 ng/kg had no significant effect,
and 0.8 ng/kg considerably blunted DHEA-S secretion.
Obviously, the mechanisms underlying the effects of endotoxin on
DHEA-S differ from the regulation of those responsible for
the changes in DHEA levels: the reduced
DHEA-S levels 6–12 h after the injection of 0.8 ng/kg
endotoxin could be explained by a negative feedback mechanism of the
previously increased cortisol and DHEA secretion.
Alternatively, increased cortisol synthesis may result in a reduced
DHEA-S synthesis directly at the level of the 17,20 lyase
in the adrenal gland (15). The late increase in
DHEA-S levels following 0.2 ng/kg possibly may be caused
by changes in the activity of the sulfatase enzyme, which catalyzes the
conversion of DHEA-S into DHEA: it has been
shown in vitro that endotoxin itself and TNF- are potent
inhibitors of this enzyme (34). Hence, a slight increase
in DHEA-S levels might be a very sensitive marker of
neuroendocrine activation during infection and inflammation.
DHEA and DHEA-S during infection and inflammation
The dose-dependent changes in DHEA and DHEA-S levels following the administration of endotoxin in humans suggest that these steroids may play a role in the regulation of host responses to infection, like it is already well-established for cortisol (35). DHEA plasma levels have been observed to be unchanged following the injection of endotoxin in rats (10). During circulatory shock in pigs endotoxin decreased DHEA levels (9), whereas in pregnant rhesus monkeys an intrauterine streptococcal infection increased the levels of DHEA in fetal blood (8). Unfortunately, none of these studies reported androgen levels across time, so the temporal dynamics of androgen secretion are not known in animals.
Most of the previous studies in patients suffering from chronic inflammatory or infectious diseases report cross-sectional results only; in most of these studies, DHEA or DHEA-S levels were found to be reduced during chronic inflammatory processes (12, 13, 15). In contrast, studies reporting androgen levels during early phases of infection observed increased levels of adrenal androgens: DHEA was found to be increased during the early stages of HIV infection (14, 16), and increased DHEA-S levels were measured in the umbilical cord blood of infants whose mothers suffered from infections at the time of delivery (17). Hence, the regulation of DHEA and DHEA-S release during host response may differ between the early and advanced stages of infectious diseases.
In summary, DHEA and DHEA-S levels are altered in a complex and dose-dependent manner by the administration of endotoxin to humans, suggesting an important role of these steroids during acute bacterial infection. Future detailed research in this field might yield useful new therapeutic approaches to the treatment of acute and chronic infectious diseases.
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Acknowledgments |
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Footnotes |
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Received July 6, 2000.
Accepted September 6, 2000.
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References |
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, 0.2 ng/kg, n = 7; 
, 0.4 ng/kg, n =
5; •, 0.8 ng/kg, n = 5; means ± SEM are shown.
For statistical analysis see Table 1