Q J Med 1998; 91:755–766
Emergent immunoregulatory properties of combined
glucocorticoid and anti-glucocorticoid steroids in a
model of tuberculosis
R. HERNANDEZ-PANDO, M. DE LA LUZ STREBER, H. OROZCO, K. ARRIAGA,
L. PAVON, O. MARTI1, S.L. LIGHTMAN1 and G.A.W. ROOK2
From the Experimental Pathology Laboratory, Department of Pathology, Instituto Nacional de la
Nutricion ‘Salvador Zubiran’, Mexico City, Mexico, 1Department of Medicine, Bristol Royal
Infirmary, Bristol, and 2Department of Bacteriology, UCL Medical School, London, UK
Received 22 July 1998 and in revised form 4 September 1998
Summary
In Balb/c mice with pulmonary tuberculosis, there
is a switch from a protective Th1-dominated cytokine profile to a non-protective profile with a Th2
component. This switch occurs while the adrenals
are undergoing marked hyperplasia. Treatment with
the anti-glucocorticoid hormones dehydroepiandrosterone or 3b, 17b-androstenediol, during the period
of adrenal hyperplasia, maintains Th1 dominance
and is protective. We investigated the effects of
these hormones as therapeutic agents by administering them from day 60, when the switch to the
non-protective cytokine profile was already well
established. Given at this time (day 60), doses that
were protective when given early (from day 0) were
rapidly fatal. A physiological dose of the glucocorticoid corticosterone was also rapidly fatal. However
when the corticosterone and the anti-glucocorticoid
(AED or DHEA) were co-administered, there was
protection, with restoration of a Th1-dominated
cytokine profile, enhanced DTH responses, and
enhanced expression of IL-1a and TNFa. Therefore
this combination of steroids has an emergent property that is quite unlike that of either type of steroid
given alone. It may be possible to exploit the antiinflammatory properties of glucocorticoids while
preserving a Th1 bias, by combining glucocorticoids
with DHEA or suitable metabolites.
Introduction
Immunity to M. tuberculosis requires a Th1 pattern
of cytokine release, accompanied by expression of
TNFa.1–5 Balb/c mice that have been pre-immunized
using protocols that prime Th2 cells have enhanced
susceptibility to direct intratracheal infection with
M. tuberculosis. This susceptibility is much greater
than that of unimmunized control animals.4 Similarly,
in this model the appearance in the lesions of T cells
releasing Th2 cytokines heralds the appearance of
areas of pneumonia and rapid progression to death.4,6
The switch from an almost exclusively Th1 pattern
of T-cell infiltration to the mixed Th1+Th2 pattern
that characterizes disease progression in this strain,
occurs during a phase of adrenal hypertrophy.7,8 The
glucocorticoid steroids (GC) from the hypertrophic
adrenal may be a factor causing the switch to Th2.
Thus GC cause newly-recruited T cells to develop a
Th2 cytokine profile.9,10 Similarly, GC can enhance
Th2 activity and synergize with Th2 cytokines.11–14
In models of Th1-mediated autoimmune disease,
such as experimental autoallergic encephalomyelitis
and adjuvant arthritis, an initial Th1 response can
be modulated, and even switched to Th2 by GC
released via the HPA axis.15,16 These findings may
also explain why GC tend to reactivate human and
murine tuberculosis,17,18 and why restraint stress
Address correspondence to Professor G.A.W. Rook, Department of Bacteriology, UCL Medical School, Windeyer Building,
46 Cleveland Street, London W1P 6DB. e-mail: [email protected]
© Oxford University Press 1998
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reactivates latent tuberculosis in mice by a
GC-dependent mechanism.19
The effects of natural antagonists of GC are
therefore of considerable potential importance in
tuberculosis. Dehydroepiandrosterone (DHEA) and
its metabolite 3b,17b-androstenediol (AED) have
been shown to have ‘anti-glucocorticoid’ properties
in a number of systems.20–22 Similarly, they promote
Th1 cytokine production from murine and human
cells,23,24 and downregulation of Th2.24,25
In a previous study,26 DHEA or AED, when
administered at low dose three times/week, either
throughout the infection, or only for the first 3
weeks when the adrenals were enlarged, markedly
enhanced resistance to M. tuberculosis in Balb/c
mice. This effect correlated with a reduced bacterial
load, enhanced production of IL-1a, TNFa, and IL-2,
and limitation of the appearance of IL-4+ cells in
the infected tissues.
In the study reported here, we tested the ability
of the same anti-glucocorticoid steroids to treat
animals already in the late progressive phase of the
disease, since a therapeutic effect would be of
potential clinical value. Steroids tested were administered from day 60, when the animals in this model
of pulmonary tuberculosis already have a marked
switch to Th2, downregulated IL-1a and TNFa,4,6
and adrenals that have atrophied to 50% of their
normal size.7,8 We have found that the anti-GC
steroids AED or DHEA, used within the dose range
that is protective when given during the early phase
of adrenal hyperplasia, are rapidly toxic when administered from day 60. Since the adrenals are atrophic,
and corticosterone levels are low at this late stage
in the infection, GC supplements were also tried but
these, like the anti-GC, were also rapidly fatal.
Surprisingly, combinations of GC and either AED or
DHEA had emergent protective properties, quite
unlike the properties of either type of steroid used
alone. These findings suggest the possibility of novel
therapeutic strategies for human disease that enable
exploitation of anti-inflammatory properties of GC
without simultaneously causing immune deviation.
Methods
Experimental model of tuberculosis
infection in mice
All animal work was performed in conformity with
the Home Office regulations in the UK, or the Local
Ethical Committee for Experimentation in Animals in
Mexico. The tuberculosis model has been described
in detail elsewhere.6,7 Briefly, male Balb/c mice were
used at 6–8 weeks of age. Virulent M. tuberculosis
H37Rv was cultured in Youman’s modification of
the medium of Proskauer and Beck. After 4 weeks
of culture, the colonies were suspended in phosphate-buffered saline (PBS) containing 0.05% Tween
80 by shaking for 10 min with glass beads. The
suspension was centrifuged for 1 min at 350 g to
remove large clumps of bacilli. A preliminary bacterial count was then made by smearing the supernatant at a known ratio of volume to area, and counting
10 random fields after staining by the Ziehl-Neelsen
technique. The suspension was finally diluted to
1×106 bacteria in 100 ml PBS and aliquotted at
−70 °C. Before use bacteria were recounted, and
viability checked as described.27
To achieve intratracheal infection, mice were
anaesthetized with 18 ml of intraperitoneal pentothal,
equivalent to a dose of 56 mg/kg. The trachea was
exposed via a small midline incision, and 1×106
viable bacteria were injected in 100 ml PBS. The
incision was then sutured with sterile silk, and the
mice were maintained vertical until the effects of
the anaesthetic had worn off. Infected animals were
maintained in groups of five in cages fitted with
micro-isolators. At 60 days, surviving mice were
randomly allotted to the required experimental
groups, as described below. They were therefore a
relatively healthier subset of mice than the average,
so there are minor differences between the findings
in this study, and those in the previous one where
animals were treated from the day of infection. Mice
were exsanguinated at 1, 3, 7, 14, 21, 28, 60 and
120 days after starting steroid treatment. Data are
plotted as days since infection (i.e. days 61, 63,
67, etc.).
Determining the appropriate doses of DHEA
and its derivative AED
DHEA and its derivative AED were both obtained
from Sigma and dissolved in ultrapure olive oil
(Sigma). There are wide discrepancies in the doses
used in mice by different authors, ranging from
1 g/kg,28 to 500 mg/kg.29 Therefore the correct dose
range for these steroids in mice was obtained by
determining the dose that optimally opposed the
ability of corticosterone to cause involution of the
thymus. This assay was based on the observations
of Blauer et al.20 Briefly, the doses of AED or DHEA
to be tested were injected s.c. into five mice
on three consecutive days. Aetiocholanolone (3ahydroxy-5b-androstan-17-one), a derivative of DHEA
that is inactive in this assay, was used as a control.
The last dose was given at the same time as 1.6 mg
of corticosterone, and the thymuses were weighed
24 h later. This dose of corticosterone had been
shown previously to cause a 50% reduction in
thymus weight. The thymus protection assay suggested a dose range of 1–20 mg/mouse. Then further
DHEA and glucocorticoids in pulmonary TB
dose-response studies were done over a narrower
dose range, using 3 s.c. doses/week (i.e. Monday,
Wednesday and Friday, rather than three consecutive
doses) in the tuberculosis model itself. Histological
changes and maintenance of DTH responsiveness
were used as endpoints, and these changes have
been described in detail elsewhere.26 Selected doses
were 50 mg 3 times/week for DHEA and 25 mg 3
times/week for AED. These are the dose regimens
used throughout the experiments reported here. This
dose regimen was started on day 60, and continued
until the animals were killed. Control mice received
olive oil only on the same days.
Corticosterone was dissolved in drinking water at
3 mg/ml. This concentration has been shown to result
in corticosterone levels in adrenalectomized animals
that are within the physiological range.30 Moreover,
administration in this way also mimics the correct
rodent diurnal rhythm.30
Assessment of colony-forming units in
infected lungs
Half of each right and half of each left lung was
used for colony counting, while the other halves
were used for studying other parameters. Lungs were
disrupted in a Polytron homogenizer (Kinematica,
Luzern, Switzerland) in sterile 50 ml tubes containing
3 ml isotonic saline. Four dilutions of each homogenate were spread onto duplicate plates containing
Bacto Middlebrook 7H10 agar (Difco Lab code
0627-17-4) enriched with OADC also from Difco
(code 07-22-64-0). The time for incubation was 21
days. Two animals were sacrificed at each time
point, in two different experiments, so the data points
are the means of data on four animals.
Assessment of serum corticosterone levels
The procedure used will have caused some stressinduced increases in plasma corticosterone, but it
was strictly standardized for all animals. Mice were
anaesthetized with 18 ml intraperitoneal pentothal,
equivalent to a dose of 56 mg/kg, at 1000 am. They
were then immediately immobilized, and exsanguinated by cutting the brachial artery. Blood from groups
of five animals was pooled, and the corticosterone
levels were assayed using the COAT-A-COUNT kit
for corticosterone (Diagnostic Products).
Preparation of tissue for histology,
morphometry and immunohistochemistry
For histological study, the lungs were perfused via
the trachea with 100% ethanol and immersed for
24 h in the same fixative. Parasaggital sections were
taken through the hilus, and these were dehydrated
757
and embedded in paraffin, sectioned at 5 mm and
stained with haematoxylin and eosin. The following
five parameters were then measured in mm2 with a
Zidas Zeiss image analysis system; area of peribronchial infiltration; area of perivascular infiltration;
area of granuloma; area of interstitial inflammation;
% of lung affected by pneumonia. Measurements
were done blind, and data are expressed as the
mean of 4–6 animals ±SD.
For immunohistochemistry, lung sections were
mounted on silane-coated slides, deparaffinized, and
the endogenous peroxidase quenched with 0.03%
H O in absolute methanol. Lung sections were
2 2
incubated overnight at 4°C with biotin-labelled polyclonal goat antibodies against IL-2 or IL-4 (R&D
Systems), or with polyclonal rabbit antibodies to
TNFa or IL-1a (Genzyme) diluted 1/50 in phosphatebuffered saline (PBS). Bound antibodies were
detected with avidin biotin peroxidase (Vector), and
counterstained with hematoxylin. Immunohistochemistry was quantified blind. Three random fields
of each pulmonary compartment were evaluated at
400× magnification in 4–6 mice, and expressed as
mean±SD. Thus each point is based on 12–18 fields.
Immunohistochemically positive and negative cells
located in the alveolar-capillary interstitium, perivascular and peribronchial inflammation, granulomas and
pneumonic areas were counted and the number of
positive cells was expressed as a percentage of the
total number of cells present.
In situ hybridization for CRH
Expression of mRNA encoding corticotrophin releasing hormone (CRH) in the paraventricular nuclei of
Balb/c mice with pulmonary tuberculosis was examined by in situ hybridization essentially as described
elsewhere,31 using an oligonucleotide probe for
exonic sequence 496–543. The probe was labelled
using terminal deoxytransferase to add 35S-labelled
deoxy-ATP (1000 Ci/mmol) to the 3∞ end of the probe.
Briefly, after killing by exsanguination, mice were
decapitated, the skin was removed and the skull was
opened with scissors taking care to not damage the
brain. The olfactory nerve and the spinal cord were
severed and the brain was removed, avoiding anatomical deformation, and immediately frozen by
immersion in liquid nitrogen. Frozen sections of
12 mm thickness were obtained from the paraventricular nuclei, mounted on gelatin-coated slides and
fixed in 4% formaldehyde in PBS for 5 min at room
temperature. After washing, slides were placed for
10 min at room temperature in 0.9% NaCl containing
0.25% acetic anhydride and 0.1 M triethanolamine
hydrochloride. Next the slides were transferred
through ethyl alcohol washes: 70% (1 min), 80%
(1 min), 95% (2 min) 100% (1 min), followed by
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R. Hernandez-Pando et al.
chloroform 100% (5 min), alcohol 100% (1 min), and
alcohol 95% (1 min). The slides were then hybridized
in a humid chamber at 37 °C overnight with 50%
formamide; 4×SSC; 500 mg/ml sheared DNA; 250 mg
yeast tRNA; 1×Denhards (0.02% Ficoll; 0.02% polyvinylpyrrolidone and 0.02% BSA); 10% Dextran
sulphate (molecular mass 500 kDa) and 200 dpm per
slide of 35S-labelled CRH oligonucleotide DNA probe
in 10 mM DTT. A slide labelled with different concentrations of 14C provided standards. Each slide
was covered with 50 ml of hybridization mixture and
small squares of parafilm were used as coverslips.
Posthybridization washes were: 4×15 min washes in
1×SSC at 55 °C; 2×30 min washes in 1×SSC at
room temperature; and two brief distilled water dips.
The sections were then exposed to Hyperfilm
(Amersham International) at room temperature and
quantified using a computer-assisted image analysis
system (Image 1.41; Wayne Rasband).
Measurement of delayed hypersensitivity
(DTH)
Culture filtrate was harvested from M. tuberculosis
H37Rv grown as described above for 5–6 weeks.
Then culture filtrate antigens were precipitated with
45% (w/v) ammonium sulphate, washed and redissolved in PBS. For evaluation of DTH, each mouse
received an injection of 20 mg antigen in 40 ml PBS
into the hind foot-pad. In order to achieve very low
non-specific background swelling, the needle perforated the skin near the ‘heel’. Then the needle
travelled under the skin so that antigen was injected
over the ‘palm’, where the readings of swelling were
also made. We have found that ensuring the absence
of skin puncture wound at the site where the DTH
is read reduces background and variability. The
swelling at the ‘palm’ was measured with an engineer’s micrometer before and 24 h after the injection.
larger peak between 14 and 28 days. These weight
changes correlated with changes in serum corticosterone levels (Figure 1). The adrenal weights and
corticosterone levels fell between 28 days and 60
days, at which time these values plateaued at
approximately 50% of the starting values. These
changes in adrenal weight also correlated with
changes in expression of CRH in the paraventricular
nuclei (Figure 1, lower panel), indicating that the
initial phase of adrenal hypertrophy is secondary to
activation of the HPA axis.
Effects of GC (corticosterone) or of anti-GC
steroids (AED and DHEA) on survival and
bacterial numbers when administered from
day 60 in tuberculous Balb/c mice.
Previous studies had shown that either AED or DHEA
is protective when administered from the start of
infection and during the early period of adrenal
hypertrophy. In contrast, when these steroids were
administered from day 60, at a time when the
adrenals were atrophic, they resulted in accelerated
death (Figure 2) ( p<0.005 relative to controls for
both steroids, log rank test). In all repeat experiments,
animals treated in this way died too quickly for
meaningful immunological analysis, so they are not
included in the further immunological studies
described below.
Since the adrenals are atrophic at day 60, we
considered the possibility that the anti-GC effects of
AED and DHEA were exacerbating a state of adrenal
insufficiency. However mice with pulmonary tuber-
Statistical analysis
Student’s t test, 2-tailed for unpaired data, was used
to compare morphometric and immunohistochemical
data. A p value of 0.05 or less was regarded as
significant. Survival data were compared at individual
points by Fisher’s exact test, and survival curves
were analysed from Kaplan-Meier plots, using the
log rank test.
Results
Changes in adrenal size and in
corticosterone levels in Balb/c mice with
pulmonary tuberculosis
After intratracheal infection, there was a small
increase in adrenal weight at 3 days, followed by a
Figure 1. The hypothalamo-pituitary adrenal axis in Balb/c
mice during the course of pulmonary tuberculosis.
a %, Serum corticosterone; #, weight of left adrenals;
6, weight of right adrenals. b Expression of CRH in the
paraventricular nuclei (%). The day 0 value for CRH
expression, difficult to see on the scale used, was
157±41. Means±SD are shown.
DHEA and glucocorticoids in pulmonary TB
759
combinations). The increased survival caused by
AED+corticosterone (Figure 2) did not achieve statistical significance relative to the controls ( p=0.07,
log rank test) but it was highly significant relative to
corticosterone alone or AED alone ( p<0.0003 in
both cases). This treatment regimen, and a regimen
where DHEA was used instead of AED, was therefore
repeated in further groups of mice, and subjected to
morphometric and immunohistochemical analysis.
Figure 2. Survival curves for Balb/c mice that had been
infected by the intratracheal route with M. tuberculosis.
On day 60, surviving animals were allotted randomly to
the following treatment groups. #, Controls (vehicle
only); %, DHEA only; 6, AED only; d, corticosterone
only; +, AED+corticosterone.
culosis that were treated from day 60 with a replacement dose of corticosterone also died rapidly
(Figure 2) ( p<0.005 relative to controls, log rank
test).
Since GC and anti-GC were both toxic when used
alone, but must act through different receptors and
via different mechanisms, it was hypothesized that
the immunological benefits of the anti-GC might be
manifested without toxicity if given at the same time
as the GC. Therefore AED or DHEA was given to
animals that were also receiving corticosterone. The
result was an emergent property, unlike that of either
type of steroid administered alone, resulting in
increased survival (Figure 2), and a reduction in
bacterial load of approximately 1 log (Figure 3),
detected as CFU on days 88 and 120, (i.e. 26 and
60 days after starting the treatment with steroid
Figure 3. CFU from the lungs of Balb/c mice that had
been infected via the trachea with M. tuberculosis on day
0, and then treated from day 60 with GC, anti-GC, or
both together. #, Controls (vehicle only); d, corticosterone only; &, DHEA+corticosterone. Animals given
DHEA only died too quickly for analysis of CFU at these
time-points. The combination of corticosterone and DHEA
caused a 1 log reduction in CFU by day 88, that was
sustained, though not lowered further, at day 120.
The effects of corticosterone alone, or in
combination with DHEA or AED, on lung
pathology when administered from day 60
in tuberculous Balb/c mice
Morphometric analysis of the zones of inflammation
revealed that the combination of AED+corticosterone caused a progressive increase in granuloma
formation (Figure 4a). Corticosterone alone also
appeared to transiently increase the area of granuloma, but the cellular composition of the granulomata
was different in these animals (see below) and it was
accompanied by a simultaneous increase in the
percentage of the lung involved in pneumonia
(Figure 4b). No animals treated with corticosterone
alone survived beyond 120 days.
Although DHEA+corticosterone, unlike AED+
corticosterone, did not result in enhanced granuloma
formation (Figure 4a), DHEA was as effective as AED
in stopping the enhancement of pneumonia caused
by corticosterone given alone (Figure 4b).
Immunohistochemical analysis of
expression of IL-1a and TNFa
TNFa is essential for immunity to tuberculosis in
mice,1 and perhaps for the formation of granulomata,32 but its expression has declined by day 60
in the model used here.4,6 We therefore investigated whether the beneficial effects of AED+
corticosterone on granuloma formation could be
related to restoration of TNFa expression. The percentage of cells expressing IL-1a or TNFa was
assessed by immunohistochemistry in the granulomatous and pneumonic areas.
Figure 5 shows that the combination of
AED+corticosterone caused a significant increase
in cells that were positive for IL-1a (Figure 5a) or for
TNFa (Figure 5b) in the areas of granuloma. This is
illustrated in representative immunohistochemical
examples shown in Figure 8a and b. Data were
similar using DHEA instead of AED (data not shown).
The effect was less striking, using either steroid
combination, in areas of pneumonia. This is illustrated for DHEA+corticosterone in Figure 5c and d.
There was a trend towards increased expression of
IL-1a and TNFa in the areas of pneumonia, but this
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R. Hernandez-Pando et al.
Figure 4. Morphometric analysis of tuberculosis-infected lungs in treated and untreated Balb/c mice. a Area of granuloma.
b Percentage of the lung affected by pneumonia. AED+corticosterone increased the area of granuloma, while corticosterone
alone increased the area of pneumonia. #, control animals; +, treated with AED+corticosterone; &, treated with
DHEA+corticosterone; d, treated with corticosterone alone. ¶, p<0.05; *, p<0.025; **, p<0.005.
was not significantly different from control animals
at most time points (Figures 5c and d). However, the
addition of the DHEA or AED did totally reverse the
depression of TNFa expression caused by corticosterone.
The effects on cytokine expression were already
apparent in some animals, though not significant
overall, at the first time point studied, 24 h after the
first administration of the steroids.
The effect of DHEA and AED on delayed
type hypersensitivity responses (DTH)
Granuloma formation correlates with resistance in
this model, and pneumonia with rapid progression,4
but the role of DTH is unclear. The DTH responsiveness to soluble antigens of M. tuberculosis of
mice that received AED+corticosterone increased
steadily (Figure 6). The increase was significant relative to control animals at days 120 and 180. (This
parameter was not tested in animals treated with
DHEA+corticosterone).
Immunohistochemical analysis of
expression of IL-2 and IL-4
In this model of pulmonary tuberculosis, the cytokine
profile is biased towards Th2 by day 60.4,6
To investigate whether the beneficial effects of
AED+corticosterone and DHEA+corticosterone
were related to changes in Th1/Th2 balance,
immunohistochemical analyses of cells staining positive for IL-2 and IL-4 were performed (Figure 7).
When combined with corticosterone, the effects of
AED and DHEA were very similar. In each case
there was an increased percentage of cells expressing
IL-2 and a decreased percentage of cells expressing
IL-4. Like the effects on TNFa and IL-1a, changes in
expression in IL-2 and IL-4 were already apparent
in some animals, though not significant overall, at
the first time point studied, 24 h after the first
administration of the steroids. Representative
immunohistochemical examples are shown in Figures
8c and d.
In contrast, when corticosterone was used alone,
the trend was always the reverse, with decreased
IL-2 and increased IL-4. Relative to untreated controls, the increased expression of IL-4 achieved
significance in the areas of granuloma (Figure 7b),
the interstitial zones (Figure 7d) and the areas of
pneumonia (Figure 7f ), whereas the downregulation
of IL-2 expression following treatment with corticosterone alone was significant only in the areas of
pneumonia (Figure 7e).
The animals given AED or DHEA without corticosterone died too rapidly to permit analysis.
DHEA and glucocorticoids in pulmonary TB
761
Figure 5. Immunocytochemical analysis of cells positive for IL-1a (a and c) and TNFa (b and d) in the areas of granuloma
(a and b) and pneumonia (c and d) of the lungs of Balb/c mice infected with M. tuberculosis H37Rv, and treated with the
indicated regimen from day 60. Data are means±SD of 12–18 random fields (three fields from each of 4–6 mice).
Treatment with AED+corticosterone increased the percentage of both IL-a and TNFa-positive cells. The effect was
apparent, though not significant, at the first time point (day 61), 24 h after initiation of treatment. Corticosterone used
alone decreased expression of both IL-1 and TNF, and this effect could be abrogated by either AED or DHEA. #, control
animals; +, treated with AED+corticosterone; &, treated with DHEA+corticosterone; d, treated with corticosterone
alone. ¶, p<0.05; *, p<0.025; **, p<0.005.
Discussion
Figure 6. Delayed hypersensitivity responses (DTH) to
soluble antigens of M. tuberculosis in tuberculous mice.
Swelling was measured 24 h after challenge. Treatment
with AED+corticosterone from day 60 enhanced DTH
responses at days 120 and 180. This parameter was not
studied with the other treatment regimens. #, control
animals; +, treated with AED+corticosterone. ¶,
p<0.05; *, p<0.025; **, p<0.005.
The crucial finding in this study is that a therapeutic
effect accompanied by a switch towards a Type 1
cytokine profile, and increased expression of IL-1a
and TNFa, can be achieved in a very late phase of
tuberculosis in a murine model, by giving a combination of GC and anti-GC, while either type of steroid
used alone is rapidly fatal. This emphasizes the
importance of the neuroendocrine system in this
disease. Tuberculosis infection first stimulates the
HPA axis by an effect at the hypothalamic level, and
later turns off the HPA axis, as has been seen in
other models of chronic inflammation.33 At the
adrenal level, this downregulation of the HPA axis
had been noted in previous studies of the tuberculosis
model studied here.7,8 Manipulations of the neuroimmune response to infection by this organism can
influence cytokine expression in a therapeutically
useful way.
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Figure 7. Immunocytochemical analysis of (a, c and e) IL-2-positive and (b, d and f) IL-4-positive cells in the granulomas
(a and b), interstitial compartment (c and d) and areas of pneumonia (e and f ) of the lungs of Balb/c mice with pulmonary
tuberculosis. Treatment with the indicated steroid regimen was initiated on day 60. Data are means±SD of 12–18 random
fields (three fields from each of 4–6 mice). Treatment with either AED+corticosterone or DHEA+corticosterone caused
a marked increase in cells staining for IL-2 and a corresponding decrease in cells staining for IL-4. Corticosterone used
alone had the reverse effect. #, control animals (treated with vehicle only); d, treated with corticosterone only; +, treated
with AED+corticosterone; &, treated with DHEA+corticosterone. ¶, p<0.05; *, p<0.025; **, p<0.005.
DHEA opposes a wide range of GC-mediated
effects in many organ systems and cell types. Thus
DHEA will oppose the activation of hepatic enzymes
such as tyrosine aminotransferase, by GC in both
the rat21 and the mouse,34 and it increases sensitivity
to insulin in diabetic rats.35 Similarly, the effects of
DHEA in contextual fear conditioning closely
resemble those of adrenalectomy, and suggest that
in the central nervous system the effects are broadly
anti-glucocorticoid.36 DHEA also opposes the mitogenic effect of GC for Schwann cells, and in this
respect mimics RU 486, though DHEA is not itself a
receptor antagonist.37
The ability of DHEA to oppose the effects of GC
on the immune system are of particular relevance to
this study. DHEA will oppose dexamethasone-induced
apoptosis and involution in the thymus.20 DHEA was
also protective against acute viral infections.38
Although these experiments did not necessarily indicate an anti-glucocorticoid effect, it was shown
recently that DHEA can reverse susceptibility to infection caused by prednisone in Balb/c mice,39 which
was the strain used in the experiments reported here.
A series of ex vivo and in vitro experiments in the
mouse suggested that DHEA tends to increase Th1
and decrease Th2 cytokine production,24,40 and that
has been borne out by studies of infection models.26,41
Data from human studies are scanty, but DHEA
DHEA and glucocorticoids in pulmonary TB
763
Figure 8. Representative histopathology and immunohistochemistry at 180 days in Balb/c mice with pulmonary tuberculosis.
(a and c) control mice; (b and d) mice treated with AED+corticosterone. a Lung granulomas from control animals 180
days after intratracheal infection with M. tuberculosis show few IL-1 immunostained macrophages. b In contrast, numerous
IL-1 immunostained cells are seen at 180 days in lung granulomas from a similarly infected mouse that had been treated
with AED+corticosterone from day 60. c Abundant cells staining positive for IL-4 in lung granulomas of control animals
examined at 180 days. Few such cells were seen in the granulomas of lungs from mice treated with AED+corticosterone (d).
has been claimed to promote IL-2 production,23
and to downregulate IL-4 production from human
peripheral blood lymphocytes.25 Similarly, some
increases in immune function, including mitogendriven IL-2 production, were seen when DHEA was
given to elderly subjects,42 in whom DHEA levels
are usually low.43 The effects of DHEA in the murine
model of pulmonary tuberculosis reported here
strongly imply that it causes not only enhanced
expression of Th1 cytokines, but also of the proinflammatory cytokines IL-1a and TNFa.
Pilot dose-response studies, and the results of the
experiments with optimal doses reported here and
in the previous study,26 suggest that 3b,17b-androstenediol is even more active than DHEA. This
possibility had been suggested by the work of Padgett
and colleagues studying T-cell proliferation in vitro,44
and agrees with the known role of the five or more
17b-hydroxysteroid dehydrogenases (17b-HSD) and
reductases, which convert 17-keto oestrogens
and androgens to the more active 17b-hydroxy
derivatives, or inactivate them by converting them
back to 17-ketosteroids.45,46 These enzymes constitute a reversible shuttle system analogous to the
cortisol/cortisone shuttle, which regulates local GC
levels in an organ-specific way. The distribution of
the 17b-hydroxysteroid enzymes was found to be
ubiquitous in the Balb/c mouse.47 However, these
studies used cell lysates, and the direction of action
of these enzymes is affected by lysis and by cofactor
concentrations,45 so the physiological equilibrium
points between DHEA and its 17-hydroxy derivative
764
R. Hernandez-Pando et al.
AED in individual mouse tissues are not known, and
little attention has been paid to the immunological
role of this shuttle system.
Levels of DHEA and its sulphate derivative DHEAS
are much lower in rodents than in man, where there
are large quantities (~4 mg/ml of plasma in young
adults) of strongly albumin-bound DHEAS. In rats,
there are <1 ng/ml of DHEAS in plasma, and
DHEAS and fatty acid esters of DHEA were found
in the adrenals of this species at 5.1±2.6 and
16.1±6.4 ng/g tissue, respectively.48 The study
reported here used optimal doses selected from
pilot experiments, of 150 mg/week of DHEA, or
75 mg/week of AED, which may be more appropriate
than the very high doses used in some previous
studies (discussed in detail in reference 49).
The mode of action of DHEA as an anti-GC
remains unknown. Its effects as a neurosteroid are
mediated by the sulphate which causes allosteric
changes in the GABA receptors, and the recent
A
hypothesis that it acts as an anti-GC by binding
to peroxisome-proliferator-activated receptor alpha
(PPARa) also implies that the sulphate is active.50
However, there is strong evidence that inhibition of
steroid sulphatase activity eliminates the immunological effects of DHEAS but not of DHEA, so removal
of the sulphate is clearly essential at some stage,
though it might be reversed after entry into certain
target cells.51 A single claim for the presence of
specific receptors for DHEA in T cells has yet to be
confirmed.52 Similarly, the claim that the activity is
due to 7-hydroxylated derivatives of 3b,17b androstenediol requires thorough confirmation.44
In this study the steroids were not administered
until day 60, when in Balb/c mice with pulmonary
tuberculosis there is a well-established switch
towards a Th2 cytokine profile,4,6,8 and atrophy of
the adrenals.7 We do not know why this adrenal
atrophy occurs, though it occurs earlier if the animals
are manipulated so that the Th2 component appears
earlier, suggesting a link with the presence of a Th2,
or mixed Th1+Th2 cytokine profile,7,8 and in the
studies reported here, adrenal atrophy was accompanied by decreasing expression of CRH in the
paraventricular nuclei, raising the possibility that
central pathways are involved. Nevertheless, CRH
expression remained higher than in control animals,
so the atrophy of the adrenals down to 50% of their
normal weight cannot be entirely attributable to
decreased CRH expression, and a direct effect of the
Th2 response on the adrenals is a possibility.
Surprisingly, administration of a very low replacement dose of corticosterone from day 60 not only
failed to exert a therapeutic effect, but actually
caused a further loss of IL-1a and TNFa, a further
shift towards IL-4 expression, and accelerated death.
Thus the reduced adrenal weight, reduced corticos-
terone and low CRH expression in the PVN may
reflect a physiological necessity at this stage of the
infection. We have shown that in order to raise
corticosterone levels, it is necessary to raise levels
of AED or DHEA as well. This implies that there is
a critical ratio of these two types of steroid.
There are a number of infections, such as tuberculosis, syphilis, and AIDS in which a fall in the DHEA/
cortisol ratio is a correlate of severity or progression
of disease.53–55 Similarly, we have pointed out elsewhere that the large changes in serum DHEAS levels
between early childhood (0–5 years; low DHEAS),
the period of adrenarche (5–10 yrs; rising DHEAS)
and early postpubertal life (10+; high DHEAS)
correlate with striking changes in susceptibility to
tuberculosis, and in the nature of the disease that
occurs.49,56 These purely circumstantial correlations
can be interpreted in other ways, but the study
presented here provides direct evidence for a synergistic emergent property of appropriate combinations
of GC and anti-GC. The only GC dose studied was
a physiological replacement dose, and further studies
will be needed to discover whether the Th1 switching
effect can be increased by more precise adjustment
of the ratio, or conceivably by the use of still lower
doses of AED or DHEA without any corticosterone.
Acknowledgements
Dr R. Hernandez-Pando would like to thank the
National University of Mexico and the CONACYT
for financially supporting his work, and The Royal
Society of Great Britain for supporting a visit to
the UK. The authors are also grateful for funding
from the INCO-DC programme of the European
Community.
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