The role of endogenous steroid hormones in the generation of T helper 2‐mediated autoimmunity in mercuric chloride‐treated Brown–Norway rats

Abstract

Injection of Brown–Norway rats with mercuric chloride (HgCl₂) activates a T helper type 2 (Th2) autoimmune response, with production of a number of autoantibodies and vasculitis primarily affecting the gut. Glucocorticoids have been shown to suppress Th1 and to promote the development of Th2‐type responses. Conversely dehydroepiandrosterone (DHEA) promotes Th1 responses with suppression of Th2 responses. This study set out to define the role of these hormones in this animal model. Rats were adrenalectomized (Adx) with no steroid replacement (n = 11), Adx with basal steroid replacement given by a 25 mg corticosterone pellet inserted subcutaneously (n = 13), or sham‐Adx (n = 14) prior to administration of HgCl₂. In both groups of Adx animals there was a delay in the production of immunoglobulin E (IgE) and serum concentrations on day 9 were marginally lower (P = 0·035, repeated measures anova). All of the animals Adx with no steroid replacement and two Adx animals with steroid replacement died between 10 and 14 days after HgCl₂ challenge. There was no difference in the severity of caecal vasculitis between the groups. A significant increase in adrenal size was noted following administration of HgCl_2. Administration of subcutaneous DHEA implants (100 mg and 200 mg) had no significant effect on IgE concentrations or severity of vasculitis. These observations do not support the hypothesis that corticosterone and DHEA play a central role in setting the Th1/Th2 balance in this experimental Th2‐mediated autoimmune disease; in contrast with the Th1‐mediated autoimmune disease experimental allergic encephalomyelitis where corticosterone plays a key role in immunoregulation.

Introduction

The functional split of T‐helper (Th) lymphocytes into cells primarily secreting interleukin‐2 (IL‐2) and interferon‐γ that induce classical cell‐mediated immune responses (Th1) and cells secreting IL‐4 that direct the immune system towards antibody production, in particular immunoglobulin E (IgE), (Th2), probably plays an important role in the regulation of autoimmunity. ¹ There is a regulatory balance between these subsets of Th cells, with Th2 suppressing the Th1 response and vice versa. Excessive activation of either population can result in damaging autoimmunity. The Th1/Th2 balance can be influenced by a number of hormones and it has been proposed that the neuroendocrine system plays a key role in determining the pattern of Th response. ^2,3

When Brown–Norway (BN) rats are given mercuric chloride (HgCl₂) subcutaneously, a marked Th2 response develops, with increased serum IgE concentrations, production of a number of auto‐antibodies and vasculitis primarily affecting the gut. This response peaks at 2–3 weeks, and then resolves spontaneously, with the development of a more Th1‐type response. ^4,5

There is evidence from both in vivo and in vitro experiments in mice and rats that glucocorticoids both suppress Th1 responses and divert T lymphocytes towards a Th2‐type response. ^6,7 However, IL‐4 production was found to be suppressed by glucocorticoids in humans. ^8,9 This apparent paradox may be explained by the suppression of IL‐4 by glucocorticoids in primed but not un‐primed lymphocytes. ¹⁰ Physiological stress in humans has been noted to reduce Th1‐type responses with enhancement of Th2 responses (reviewed in ref. 11). The susceptibility of BN rats to HgCl₂‐induced autoimmunity declines with age, ¹² as does the magnitude of their stress corticosterone response. ¹³ The susceptibility of certain rat strains to Th1 autoimmune diseases is based on a hypothalamic defect resulting in a blunted stress steroid response. ¹⁴ BN rats appear to have a normal hypothalamic–pituitary axis. ¹³

Glucocorticoids can act as a co‐factor for class‐switching to IgE production in vitro in both normal human B lymphocytes ¹⁵ and in chronic lymphocytic leukaemia cells ¹⁶ but it is unclear whether they are essential for class‐switching to IgE. This is clearly an important issue as glucocorticoids are often used as anti‐inflammatory agents in Th2‐mediated diseases and this treatment, while reducing the degree of organ damage, may be propagating the underlying immunological process.

In contrast to glucocorticoids, the androgen dehydroepiandrosterone (DHEA) enhances Th1‐type responses in mice, with augmentation of IL‐2 secretion ¹⁷ and suppression of antibody production. ¹⁸ DHEA usually circulates in sulphated form (DHEAS), being cleaved to the active molecule by a sulphatase in the target tissues. The level of DHEA sulphatase in murine lymphoid tissues correlates with the level of Th1 or Th2 responsiveness, with high levels promoting Th1 responses and low levels promoting Th2 responses. ¹⁹ Serum levels of DHEAS differ significantly between species, being the most abundant adrenal androgen in humans, with serum levels in the adult male of around 2000 ng/ml, ²⁰ whereas serum levels in rats are less than 1 ng/ml. In rodents, DHEA is not made in the adrenals and is probably mostly gonadal in origin.

The BN rat/HgCl₂ model provides a tool to study the effect of endocrine manipulation, specifically of corticosterone and DHEA levels, in a highly polarized Th2‐type autoimmune response.

Materials and methods

Animals

Male BN rats weighing 250–350 g were purchased from Harlan‐Olac UK Ltd, Bicester, UK. All procedures were performed under halothane anaesthesia.

Treatment with mercuric chloride

HgCl₂ (Sigma, Poole, UK) was dissolved at a concentration of 1 mg/ml in phosphate‐buffered saline and was injected subcutaneouly at a dose of 1 mg/kg for a total of five doses given on alternate days. ²¹

Adrenalectomy and steroid replacement

Animals were bilaterally adrenalectomized (Adx) or sham‐adrenalectomized (sham‐Adx) by blunt dissection through a single dorsal incision. Completeness of adrenalectomy was confirmed by histological examination of tissue removed and by checking for residual adrenal tissue at post‐mortem. Adx animals (and the controls) were given 0·9% saline to drink. Replacement steroid therapy was given by inserting implants of corticosterone 25 mg mixed with cholesterol (Sigma), subcutaneously in the flank. ²² All other animals were given 100% cholesterol implants. All of the steroid/cholesterol implants used weighed 100 mg at the start of the experiment and had uniform shape. This dose has been shown to replace normal rat basal corticosterone levels. ²³ Surgery and steroid implant insertion were performed 3 days prior to the first HgCl₂ injection.

DHEA treatment

Implants containing 100 mg DHEA were made as described above and inserted 3 days prior to the first HgCl₂ injection, with cholesterol implants being used as a control.

Hormone assays

Serum DHEAS was measured using a radioimmunoassay kit (Pantex, Santa Monica, CA) with 68% cross‐specificity for DHEA. Estradiol and testosterone were assayed following ether extraction by enzyme immunoassay kits (DRG Instruments GmbH, Marburg, Germany) which do not cross‐react with DHEAS.

IgE enzyme‐linked immunosorbent assay (ELISA)

Serum was prepared from blood collected from a cut in the tail vein. Total IgE was measured by ELISA as described. ²⁴ Briefly, 96‐well plates (Dynex Technologies Ltd, Billingshurst, Sussex, UK) were coated with monoclonal anti‐rat IgE heavy chain (Serotec Ltd, Oxford, UK) in carbonate buffer. Unoccupied binding sites were blocked with powdered milk. Known concentrations of rat IgEκ myeloma protein (Serotec) or serum samples were added in duplicate to coated wells and singly to anti‐IgE‐free wells. Binding was detected with alkaline phosphatase‐conjugated monoclonal anti‐rat κ‐ and λ‐light chain antibodies (Sigma) followed by p‐nitrophenyl phosphate substrate. The optical density (OD) at 405 nm was read after 20 min using a Dynatech multiplate reader (Dynex Technologies). A standard curve created from the OD of the known concentrations of IgE on each plate was used to calculate the IgE concentration from the mean OD of the test samples.

Caecal vasculitis

The severity of caecal vasculitis was scored by an experienced observer (DBGO), as described previously, ²⁵ who was blind as to the experimental treatment of each animal. The score combines the severity of mucosal and serosal vasculitis with 0 being normal and 10 being the most severe vasculitis seen.

Histology

Tissues were fixed in buffered formalin, and paraffin‐embedded sections were stained with haematoxylin and eosin. Histology was reported by an experienced pathologist (DRT) who was blind as to the experimental treatment of each animal.

Results

Effect of adrenalectomy on serum IgE concentrations

These data are pooled from three separate experiments. Rats were Adx with no steroid replacement (n = 11), or Adx with steroid replacement given by a 25‐mg corticosterone/75‐mg cholesterol pellet inserted subcutaneously (n = 13). A further group underwent sham‐adrenalectomy (n = 14) prior to administration of HgCl₂. In both groups of Adx animals there was a delay in the production of IgE. Serum concentrations of IgE on day 9 were marginally lower (0·21 ± 0·09 mg/ml; mean + SD) for sham‐Adx animals versus Adx with steroid replacement (0·14 ± 0·06 mg/ml) and Adx with no replacement (0·16 ± 0·08 mg/ml) (P = 0·035, repeated measures anova) as shown in Fig. 1.

Figure 1.

Serum IgE concentrations in adrenalectomized HgCl₂‐treated BN rats. Animals were adrenalectomized (Adx) with basal steroid replacement [25 mg corticosterone (Cort.), n = 13] or no steroid replacement (n = 11). A further group underwent sham‐Adx (n = 14) prior to administration of HgCl₂. Serum IgE concentrations were determined by ELISA. For differences between the groups repeated measures anova gave P < 0·035.

Effect of adrenalectomy on survival

All 11 animals undergoing Adx with no steroid replacement and two of 13 Adx animals with replacement died between 10 and 14 days after HgCl₂ challenge. These data are pooled from three separate experiments where animals were killed on day 9, day 10, or day 20 after the first HgCl₂ injection to inspect caecal vasculitis. Kaplan–Meier survival curves for the experimental groups noted above are shown in Fig. 2. The difference between the groups was highly significant by the log‐rank test (P = 0·0001).

Figure 2.

Mortality in adrenalectomized animals treated with HgCl₂. Animals were adrenalectomized (Adx) with basal steroid replacement [25 mg corticosterone (Cort.), n = 13] or no steroid replacement (n = 11). A further group underwent sham‐Adx (n = 14) prior to administration of HgCl₂. Kaplan–Meier survival is shown with P = 0·0001 for a difference between the groups (log‐rank).

Caecal vasculitis

Caecal vasculitis scores were recorded for Adx animals with and without basal corticosterone replacement, and for sham‐Adx animals treated with HgCl₂. In one experiment, animals were killed 9 days after the first HgCl₂ injection and in another on day 10. There was no difference in score between the groups (Fig. 3).

Figure 3.

Caecal vasculitis in adrenalectomized animals. Caecal vasculitis scores were recorded for adrenalectomized animals with and without basal corticosterone replacement, and from sham‐adrenalectomised animals treated with HgCl₂. In one experiment, animals were killed 9 days (•) after the first HgCl₂ injection and in another on day 10 (○).

Four animals were adrenalectomized and not treated with HgCl₂. They were killed on day 14 after adrenalectomy and had no evidence of caecal vasculitis and no increase in serum IgE concentration.

Adrenal weights

A significant increase in adrenal weight was noted 9 or 10 days following administration of HgCl₂ (Table 1). On histological examination there was hypertrophy of the adrenal cortex, with an increase in the proportion of large pale cells present.

Table 1.

Adrenal hypertrophy after treatment with HgCl₂; adrenal weight/rat weight (mg/g), median (range)

	Unchallenged	HgCl2‐treated	P‐value*
Experiment 1	0·138 (0·114–0·202) n = 8	0·174 (0·166–0·191) n = 4	0·04
Experiment 2	0·167 (0·152–0·201) n = 11	0·291 (0·284–0·294) n = 5	0·002

^*P values calculated using (Mann–Whitney U)

DHEA treatment

Large doses of DHEA were given by subcutaneous implants, a method known to give stable hormone levels over a number of weeks. ²² Serum concentration of DHEA was assayed using a radioimmunoassy kit for DHEAS that has 68% cross‐reactivity with DHEA. Results in Table 2 show that 100‐mg DHEA subcutaneous implants raised serum concentrations to 10–20 times the normal range and 200‐mg implants to 30–40 times the normal range. Serum testosterone levels were also increased in DHEA‐treated animals (Table 2) but there was no increase in serum estradiol, concentrations being < 25 pg/ml in all animals tested. Doses of 100 mg and 200 mg had no significant effect on IgE concentrations (Fig. 4). The data shown are pooled from three separate experiments which gave similar results. Similarly there was no difference in the severity of caecal vasculitis either macroscopically (Fig. 5, Mann–Whitney U P > 0·05) or histologically (data not shown).

Table 2.

Serum concentrations of DHEAS and testosterone

	Days after first HgCl2 injection
Treatment	0	4	9	14
DHEAS (ng/ml)
Cholesterol 100 mg	< 1	< 1	< 1	< 1
	< 1	< 1	< 1	< 1
DHEA 100 mg	15	20	12	11
	22	22	22	16
DHEA 200 mg	32	40	42	52
	31	31	35	35·2
Testosterone (ng/ml)
Cholesterol 100 mg	0·80 9·82	0·95 7·37	0·77 1·14	ND
DHEA 100 mg	10·13 8·60	5·37 3·68	8·60 9·21	ND
DHEA 200 mg	18·42 15·04	9·52 11·05	17·81 17·81	ND

Results shown are for individual animals.

Figure 4.

IgE concentrations in DHEA‐treated animals. Three days prior to the first HgCl₂ injection either one or two subcutaneous implants containing 100 mg DHEA or cholesterol were inserted. (a) Groups: 100 mg cholesterol (n = 16), 100 mg DHEA (n = 17). (b) Groups: 200 mg cholesterol (n = 10), 200 mg DHEA (n = 17). Serum IgE was determined by ELISA.

Figure 5.

Caecal vasculitis in DHEA‐treated animals. Caecal vasculitis scores were recorded for animals treated with HgCl₂ and either 200 mg DHEA or 200 mg cholesterol. The animals were killed 14 days after the first HgCl₂ injection. There was no significant difference between the groups (Mann–Whitney U P > 0·05).

Discussion

This series of experiments was designed to test the hypothesis that endogenous steroid hormones and DHEA play a critical role in the development and regulation of Th2‐induced autoimmunity in HgCl₂‐treated BN rats. Previous work from our group demonstrated that high doses of methyl prednisolone given intravenously suppressed caecal vasculitis but had no effect on serum IgE levels. ²⁶ In a previous study of the effects of adrenalectomy in this model no difference was found in the proportion of animals producing IgG antibodies to laminin or renal IgG deposits. However, no specific markers of a Th2 response were examined and the antibody response was not quantified. There was some reduction in spleen size in adrenalectomized animals which may have been due to a reduction in the lymphoproliferation that is seen in this condition. ²⁷ Adrenalectomy was shown here to delay the IgE response but certainly did not suppress it completely, suggesting that in this model glucocorticoids may control the rate of evolution of the Th2 response, but certainly do not play an essential role in IgE class‐switching.

The finding of 100% mortality in adrenalectomized rats within 2 weeks of treatment with HgCl₂ contrasts with one death in 14 similarly treated animals observed until day 16. ²⁷ There are several possible reasons for this discrepancy. The previous study used female rats, which tend to get less severe manifestations of the disease, ²⁴ and also used younger animals (75–99 g). The severity of caecal vasculitis is dependent on the gut flora of a rat colony ⁵ and this may also have been a factor. The mortality observed was likely to be due to inability to control the lethal effects of cytokines such as tumour necrosis factor and IL‐1. ²⁸ There was certainly no observable difference in the severity of tissue damage as manifested by caecal vasculitis. This observation reinforces the essential role of endogenous glucocorticoid hormones in the regulation of systemic inflammatory responses, which has been noted previously in Th1‐mediated conditions such as experimental allergic encephalomyelitis ²³ but has not been described previously for a Th2‐mediated process.

The adrenal hypertrophy may have been a marker of increased adrenal hormone secretion. However, a similar observation was made in Sprague–Dawley rats treated with 1 mg/kg methyl mercury on alternate days for 6 weeks that developed hyperplastic adrenal glands with enlargement of the zona fasciculata, and suppression of stress‐induced corticosterone production. ²⁹ Serum corticosterone levels were not measured in the current study.

DHEA given subcutaneously in large doses made no difference to IgE production or to the severity of caecal vasculitis. In rats DHEA is not normally present in large amounts in the serum and the doses given resulted in serum concentrations 10–40‐fold higher than in normal animals. By giving DHEA the need for cleavage of the pro‐hormone DHEAS to active DHEA was bypassed. This observation suggests that DHEA is unlikely to play a significant role in the regulation of HgCl₂‐induced autoimmunity in BN rats. A possible confounding factor in giving large doses of DHEA is its metabolism to testosterone and estradiol. There was some increase in serum testosterone concentration over the normal range for BN rats of 0·7–2·5 ng/ml reported by Banerjee et al. ³⁰ Estradiol was less than 25 pg/ml in all animals tested which is the upper end of the normal range for male BN rats. ³⁰ Given that testosterone inhibits the production of IL‐4 ³¹ the increased serum testosterone concentrations do not confound the observation that DHEA did not inhibit the Th2 response.

In conclusion, adrenal steroids probably play a contributory role in the evolution of the Th2 response but in this model were not essential. No effect was seen with large doses of DHEA. The endocrine system does seem to play a key role in setting the balance between Th1‐ and Th2‐type responses in some autoimmune processes, such as experimental allergic encephalomyelitis. ³² The negative findings here do not allow the generalization of this hypothesis ^2,3 to the pathogenesis and regulation of Th2‐induced autoimmunity.

Abbreviations

Adx

adrenalectomy

BN

Brown Norway

DHEA

dehydroepiandrosterone

HgCl₂

mercuric chloride