Transgenic Animals with Impaired Type II Glucocorticoid Receptor Gene Expression

Transgenic Animals with Impaired Type I1 Glucocorticoid Receptor

Gene Expression A Model to Study Aging of the

Neuroendocrine-Immune System

BIANCA MARCHETTI,~ ANDY PEIFFER.~ MARIA C. MORALE," NUNZIO BATTICANE,"

FRANCESCO GALLO," AND NICHOLAS BARDEN* UDepartment of Pharmacology

Medical School University of Catania Viale Andrea Doria, 6 95125 Catania, Italy

and bLaboratory of Molecular Psychogenetics

Laud University Hospital Research Center and

Department of Physiology Lava1 University

Quebec GI V 4G2, Canada

INTRODUCTION

Glucocorticoids control a wide range of membrane and genomic effects on cells, including those of the immune system. In the central nervous system glucocorticoids influence neuronal excitability and neurotransmitter metabolism, alter protein synthesis and regulate hypothalamic-pituitary-adrenal (HPA) axis activity.',* In the immune system, glucocorticoids inhibit the cascade of immune and inflammatory responses at multiple levels, both directly via an action on immunocompetent cells, and indirectly via effects on leukocyte migration and release of inflammatory mediator^.^.^ Intracellular glucocorticoid receptors (GR) confer tissue responsiveness to circulating glucocorticoids and mediate their genomic and posttranslational effect^.^ Two types of corticosteroid receptor, distinguishable by their ability to bind corticosterone, have been identified6,7 as classical mineralcorticoid (type I) and glucocorticoid (type 11) receptors by cloning their comple- mentary DNAs. The type I receptor controls the basal circadian rhythm of corticosteroid secretion. Both receptors are involved in negative feedback, but the type I1 receptor may be more important for terminating the stress response.

The glucocorticoids secreted during stress act upon the hippocampus, the hypothalamus, and the pituitary to shut off the secretion of corticotropin releasing factor (CRH) and adrenocorticotropic hormone (ACTH) and to terminate the stress response (see Ref. 8). At the immune system level, corticosteroids constitute

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the final effector end point in the afferent arm of the immune system-HPA axis loop. Their physiologic role can best be viewed as the rapid induction of immune tolerance and suppression of inflammation in response to antigenic or pro-inflammatory stimuli (see Ref. 9).

Another important aspect in the relationships between glucocorticoid, the immune system and aging is the sex steroid background and the profound hormonal (especially estrogenic) sensitivity of the HPA axislo-" and the immune a x i ~ . ~ ~ , ~ ~ For example, one major immune compartment where glucocorticoids exert their effects is the thymus gland, the key organ of the immune system, and sex steroids have been shown to alter the expression of type I1 GR,I6 thymocyte responsiveness and thymus morphology.14 The sex steroid background is also known to modulate immune responsiveness, and a sexual dimorphism of the HPA has also been described.

When glucocorticoid elevation persists in different situations including para- physiological (such as aging) or frankly pathological (such as chronic stress secondary to mental disorders) situations, glucocorticoid receptors are down-regulated. l 7

Paradoxically, the decreased number of receptors and resulting glucocorticoid hypersecretion lead to a loss of neurons within the h ippocamp~s . '~- '~ The impairment of pituitary-adrenal shut-off may also result in impairments of the behavioral role of the hippocampus.20 On the other hand, the interruption of the immune system-HPA axis loop is known to concur in the pathogenesis of autoimmune disease^,^ and persistent elevated glucocorticoid levels of the aged rat might participate in aging-induced immune abnormalities, including the development of autoimmune diseases. In this context, it seems important to mention that immunopatho- logical mechanisms are believed to exist in a number of neurological disorders, and evidence supporting the role of the immune system includes lymphocytic infiltration in viral encephalitis and inflammatory demyelinating diseases, such as multiple sclerosis.21 Interestingly enough, a sex-linked predisposition and a specific impairment of stress hormone (glucocorticoids and catecholamines) negative feedback effects on immune system reactivity are apparent in autoimmune diseases.

A transgenic mouse with impaired corticosteroid receptor function by partially knocking out gene expression with type I1 glucocorticoid receptor antisense RNA was recently created.?? These transgenic animals with impaired type I1 glucocorticoid receptor function have increased HPA axis activity?? reminiscent of that observed during the process of aging and/or pathological aging. These animals could thus be useful for studying neuroendocrine and immune changes in aging and age-associated pathologies, as well as for evaluating new therapies of aging- induced neuroimmune degenerative diseases.

In this article, we shall first briefly discuss the role of glucocorticoids in the regulation of immunity, with a particular emphasis on the interplay between sex steroids and adrenal steroids in the outcome of the immune response, and then focus on the alteration of the HPA-immune loop in transgenic mice.

Glucocorticoids and the Immune System

Glucocorticoid hormones are among the most potent anti-inflammatory, anti- allergic and immunosuppressive agents known, and they act in a very complex way at various steps of the immune response. Corticosteroids inhibit the cascade of immune and inflammatory reactions by preventing antigen- and mitogen-induced lymphocyte proliferation, accessory cell antigen presentation, antigen expression, and accessory cell amplification of the antigenic ~ i g n a l . ~ . ~ Such wide-ranging of

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immunosuppressive effects are exerted directly on immunocompetent cells, and indirectly via effects on leukocyte migration and release of inflammatory medi- a t o r ~ . ~ , ~

Glucocorticoids have been shown to inhibit the production of a number of lymphokines including interleukin 1 (IL-I) and its mRNA,23 IL-2 and its cognate receptor (see Refs. 24, 25), gamma interferon (yIFN), granulocyte-macrophage- colony stimulating factor (GM-CSF), IL-3,2s or IL-6.26,27 Glucocorticoids also induce the synthesis of a group of proteins, known collectively as lipocortins.28 Moreover, glucocorticoid response elements are present in the 5’-flanking region of the IL-6 gene,27 and inhibition of both LPS- or mitogen-induced accumulation of IL-6 mRNA has been observed following corticosteroid t~-eatment.~~.~’

While most studies concur in demonstrating that glucocorticoids suppress cell-mediated immune responses, their effects on B cell function are more variable. An increase in the in vivo production of IL-4 by glucocorticoids has been reported.29 Moreover, a synergism has been demonstrated between IL-4 and glucocorticoids in triggering the activation and differentiation of B cells into IgE-producing cells.3n

Interestingly, glucocorticoids exert a cytolytic effect on immature T cells, which include the totality of thymocytes and a number of tumor T cells, inducing a suicide program in IL-2 dependent cells, called programmed cell death, or a p o p t ~ s i s . ~ ~ , ’ ~ The influence of glucocorticoids could play some role in the dramatic alteration of thymus architecture and function during sexual maturation (see Ref. 14). These changes suggest that an interplay exists between glucocorticoids and sex steroids in mediating the alterations of thymus-dependent immune functions. In fact, the impact of the sex steroid hormone background in immune responsiveness and hypothalamic-pituitary-adrenal (HPA) axis activity is well illustrated by the presence of a sexual dimorphism in both the HPA axis1n-13 and the immune response (see Refs. 14, 15).

Glucocorticoid Receptors in the Immune System

In rat tissues, two receptor systems for corticosteroids have been found, i.e., type I and type I1 receptor^.'^,^^ Type I receptors from a variety of tissues bind aldosterone and corticosterone in vitro with equivalent binding affinity, as do recombinant DNA-derived human mineralcorticoid receptors. Type I1 GR, the classical dexamethasone-preferring binding site’ has much lower affinity for corticosterone than do type I receptors, and bind dexamethasone with high affinity. Type I1 GR are present in nearly all mammalian tissues and have a multitude of physiological functions. In both the brain and anterior pituitary, one of the many actions of this receptor is its involvement in the feedback regulation of the HPA axis during the stress response (see Ref. 1).

Mapping of the type I1 GR mRNA has revealed highest concentrations in thymus, spleen, liver, lung, hypothalamus, pituitary gland and cerebellum, while testis and parathyroid tissues contained low concentrations of type I1 mRNA.34

The effects of corticosteroids on cells of the immune system, as in other corticosteroid-responsive cells, are mediated through both soluble and nuclear glucocorticoid receptors (GR).35-4n These receptors are present in greater number in activated, compared to resting lymphocytes.36 Within the thymus gland, the key organ of the immune system, and regarded as the primary site for T cell lymphopoiesis, GR have been reported to occur in greater numbers before thymocyte mat~ration.’~ Miller and c o - ~ o r k e r s ~ ~ recently showed that GR binding mea-


sures in lymphoid tissues and pituitary are less responsive to varying levels of endogenous hormone than are binding measures in hippocampus. However, other investigators have reported an up-regulation of GR in lymphoid compartments after adrenale~tomy,~' or treatment with metyrapone, mimicking the effect of adrenalectomy. A down-regulation of lymphocyte GR has been reported in conditions associated with elevated cortisol levels, such as d e p r e ~ s i o n , ~ ~ or after treatment with de~amethazone.~' Studying the intracellular localization of the GR in the male rat thymus, McGymsy r t u I . ~ ~ have reported that reticulo-epithelial cells and thymocytes displayed nuclear GR immunoreactivity in immunostained thymus sections.

We recently confirmed the expression of a gene coding for a type I1 GR. Using a 2.2-kb probe, which corresponds to the 3'-end of rat GR mRNA, we detected in the thymus a transcript of the same size as that revealed in liver and anterior pituitary gland t i s s ~ e s . ~ ~ - ~ ~ It thus appears that the GR gene is constitutively expressed in the rat thymus, and this finding accords with the detection of GR in thymus gland revealed by radioreceptor and with the identification of type I1 GR mRNA recently reported in lymphoid organs.

The expression of type I1 GR gene in rat thymus is consistent with several observations suggesting a role of glucocorticoids in the regulation of thymus- dependent immune functions.

The Hypothalamus-Pituitary-Adrenal (HPA)-Immune Axis Loop

With the recent elucidation of a negative feedback loop between the brain and the immune system, the HPA axis has emerged as a major component in the complex circuitry permitting dialogue between neuronal and immune c e l k 9 Acti- vation of the immune system following antigenic or mitogenic challenge triggers the release of soluble immune products (including IL-I , tumor necrosis factor, TNF, IL-2, platelet activating factor, PAF and IL-6) that act at different levels the brain-pituitary-immune axis as signals of the afferent arm of the immune response (see Ref. 9). The products released from activated immunological cells can affect not only the activity of the HPA axis, but also trigger a sympathetic reflex mechanism contributing to the final dynamic regulation of the immune response (see Refs. 46-48).

Generally speaking, cytokine activation of the HPA axis causes the adrenals to secrete corticosterone, which represents the efferent arm of the immune re- s p o n ~ e . ~ ~ ~ ~ ~ For instance, IL-6 has been implicated as part of the host response that leads to an increase in plasma glucocorticoid level^.^'^^^ The increased circulating levels of plasma ACTH and glucocorticoids then provide a mechanism whereby the resultant immunosuppression serves to contain and to control immune reactivity in response to antigenic or pro-inflammatory stimuli (see Refs. 9, 49). Cortico- tropin releasing factor (CRF) has been implicated as a mediator of IL-1-induced HPA axis a c t i v a t i ~ n . ~ ~ ~ ~ ~

To underline the complexity of steroid-immune interactions, cytokines and glucocorticoids are also known to act synergistically in the regulation of certain acute phase inflammatory proteins. Thus, glucocorticoids have been reported to potentiate the ability of IL-6 to stimulate the production of the protective, positive acute phase proteins (see Ref. 5 2 ) . An absence, or a deficiency in this negative feedback loop has been clearly demonstrated to favor the development of inflammatory diseases. 9.50s

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FIGURE 1. Alterations of glucocorticoid receptor (GR) mRNA transcript levels in female thymus gland during the rat estrous cycle. Total RNA was extracted from thymus of adult cycling Sprague-Dawley female rats at different phases of the estrous cycle (diestrous 1, Dl; diestrous 2, D2; proestrous, P; and estrus, E) and hybridized on Northern blots. Results are the mean t SEM (6 animals/group), expressed as percentages of GR mRNAIP-actin mRNA ratio relative to control (Dl) values normalized to 100%. ANOVA shows a significant decrease of GR transcript levels in proestrous and a marked increase at estrus when values are compared to those measured on the diestrous (D1 and D2) phases of the cycle. * p < 0.01 vs D1 and D2 (by Dunkan-Kramer test).

Sex Hormones, the Thymus, and Type II Glucocorticoid Receptor

One of the major immune compartments where glucocorticoids exert their effects is represented by the thymus gland. Since the observation that glucocorticoids induce atrophy of the cortical area of the thymus there have been many studies on the morphologic changes and cellular events that lead to loss of cell viability. This cell death induced by glucocorticoids acting on the thymus gland is known as a p o p t o s i ~ . ~ ~ ~ ~ ~ The hormonal susceptibility of this process and the dramatic alterations of thymus architecture and function accompanying sexual maturation (see Refs. 14, 15), prompted us to study the effect of physiological as well as pharmacological variations in the sex steroid hormonal milieu in type I1 GR gene expression within the thymus gland. Our recent results clearly showed that thymic GR mRNA concentration is under the control of gonadal and adrenal hormones.

Thus, physiological fluctuations of circulating sex steroid hormones during the rat estrous cycle induce important changes in GR mRNA concentrations (FIG. 1). We observed an up-regulation of GR mRNA content during the luteal ( i .e. , estrous) phase, and a down-modulation of GR transcript on proestrous, corresponding to the phase of maximal estrogenic stimulation. The fact that sex steroids regulate GR mRNA content in the thymus is further substantiated by the 3- to 4-fold increase in GR transcript observed after castration and by the complete reversal of the castration-induced rise in GR mRNA concentration following physiological replacement with 17-fi estradiol (FIG. 2). Endogenous adrenal steroids also appear to down-regulate GR mRNA transcript within the thymus, and the concomitant

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ablation of both adrenal and ovarian glands further increases the GR mRNA content to levels even higher than those measured after ADX alone (FIG. 3).

Sexual Dimorphism of Immune Response and HPA Axis: Role of Type II Glucocorticoid Receptor

It is an accepted notion that both humoral and cell-mediated immune responses are more active in females than in males, except during pregnancy when this difference is abrogated. 14,46,47,56s7 The existence of a sexual dimorphism in the immune response, the marked alteration of immune function induced by gonadec- tomy and hormone replacement and the well documented immune alterations during pregnancy, strongly support the hypothesis that gonadal steroids regulate immune functions (see Refs. 14, 15). Moreover, when compared with males, females of many species including human, demonstrate alterations in both T and B cell immune responses. These include higher Ig levels, increased antibody production after immunization, decreased susceptibility to a variety of infections, and differences in graft rejection time. In particular, estrogen has been shown to have an immunostimulatory effect on B cell differentiation and a suppressive effect on regulatory cell activity (see Refs. 14, 15).

The benefit of immunological dimorphism is not clear at present, but it may relate to the ability of females to withstand the stress of reproduction, and thus increase the probability of species perpetuation. 1533 Sex hormones have been

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FIGURE 2. Effect of ovariectomy (OVX) or treatment with 17-/3 estradiol (E,) of ovariecto- mized rats on thymus GR mRNA content. After OVX or sham OVX on the day of diestrous 1, animals received for 3 weeks daily S.C. injections of the vehicle (OVX control) or E2 at a regimen (0.1 pg/250 g body weight) known to maintain plasma concentration of E2 in the physiological range. Results are the mean f SEM (6 animals/group), expressed as percentages of control. ANOVA shows that OVX sharply increases GR mRNA content, while treatment with estradiol reversed the castration-induced GR mRNA rise to levels even lower than those measured in sham-operated control animals sacrificed on the day of diestrous. * p < 0.01 experimental vs sham; ** p < 0.01 experimental vs OVX (by Dunkan-Kramer test).


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FIGURE 3. Effect of adrenalectomy (ADX) alone or in combination with ovariectomy (OVX) on GR mRNA concentration in the rat thymus gland. Eight days after the rats underwent ADX alone or in combination with OVX, or sham operations on the day of diestrous 1, total RNA from thymus glands from each animal was extracted and hybridized on Northern blots. Results are the mean ? SEM for each experimental group (6 ratdgroup), expressed as percentages of sham control. * p < 0.01 vs sham control; ** p < 0.01 vs ADX alone.

indicated as the main factors responsible for the development, regulation and maintenance of dimorphism, although other hormones including GH, PRL, glucocorticoids and neural influence^^^-^^ have been suggested to possibly play a role.

In humans, a significant elevation in the circulating levels of IgM in females as compared to males is first seen at six years of age,59 and in juvenile rheumatoid arthritis, onset frequently occurs before 5 years of age. There is also an HLA genetic component and certain forms of disease are clearly more prevalent in females than in males.I5 Together these studies support the hypothesis that during maturation of effector lymphocytes, the sex hormone type and concentration in the microenvironment plays a key role.I5 The molecular mechanism(s) involved in such sexually dimorphic responses are not completely understood.

In the light of the presence of GR on devdoping and mature thymocytes, and peripheral lymphocytes, changes in mRNA levels induced by estradiol might form part of the mechanisms involved in sex-related changes in immune responses. A cyclic variation of cell-mediated immune response can be measured at the different stages of the rat estrous cycle (see Ref. 14), and this is accompanied by a cyclic variation in GR transcript during the phase of maximal estrogenic stimulation. In addition, such cyclic changes could have some pathological implications in sexually related immune diseases. For example, in mouse and humans, lupus erythematosus is more prevalent in females and estrogen accelerates the disease process, while menstruation is known to exacerbate idiopathic thrombocytopenia purpura. The sex steroid hormone milieu might also have a role in controlling stress response through immunomodulation. Estrogens have been shown to inhibit IL-6 production in IL-1 stimulated cells.60 Since IL-6 in turn appears to enhance cortisol levels

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by activating the HPA a ~ i s , ~ . ~ ~ it would then follow that the estrogenic status might influence glucocorticoid-lymphokine interactions. Therefore, it seems highly possible that the degree of susceptibility to and severity of inflammatory disease in response to a given pro-inflammatory trigger, may depend on the influence of sex steroids.

However, it is possible that sexual dimorphism of immune system activity is related to differential regulation of HPA axis activity by estrogens, since a sexual dimorphism has also been described in HPA axis regulation. In fact, higher circulating levels of corticosterone are found in females, and greater variations in plasma corticosterone both diurnally and in response to stress have been described in female animals.'0 Transcortin concentrations of female rat are at least double those found in male rat plasma.'* Sex-related differences have also been found for glucocorticoid receptor affinity, binding capacity and nuclear translocation in rat brain (see Ref. 43).

Type I1 GR mRNA concentrations in the hypothalamus and amygdala appear to be significantly influenced by circulating estrogen levels, and such regions have been suggested to represent integration sites at which gonadal steroids are able to alter stress hormone secretion.43 Moreover, OVX increases type I1 GR mRNA levels in both anterior pituitary gland and neurointermediate lobe of rats.44,45 Female rats have also been shown to adapt differently to stress in an animal model of depression.

The presence of receptors for estrogens and testosterone on the reticulo- epithelial matrix of the thymusI4 strongly argues in favor of a direct effect of these steroids at the thymus gland level. Therefore, it seems tempting to speculate that estrogen-induced decrease in GR mRNA levels in the thymus results in a decreased sensitivity of this tissue to circulating glucocorticoids, and sexually dimorphic immune responses. A schematic representation of the possible interactions between glucocorticoids, sex steroids and soluble immune products is given in FIG. 4. It is, however, not possible to clarify at present whether the observed changes in mRNA concentrations represent, indeed, modifications at the level of gene transcription that lead to decreased receptor protein levels.

Estrogens have been shown to regulate mRNA levels of cellular oncogenes such as c-fos.62 This transcriptional regulation occurs via estrogen-response elements which have been shown to mediate estrogen regulation of the vitellogenin gene63 and are similar to but distinct from glucocorticoid-response elements. Both the carboxy- and amino-terminal regions of the estrogen receptor have been shown to inhibit glucocorticoid response.64 This negative action of steroid receptors has been suggested to involve competition of the receptor for functionally limiting transcription factors.65

Thus, it is possible that estrogens may function as negative regulatory elements contributing to repress GR transcriptional activity in specific tissues including some neuronal and peripheral structures. The tissue-specific sensitivity to estrogen of type I1 GR mRNA concentration observed in particular subsets of neurons such as the hypothalamic neurons, and at peripheral integration sites such as the pituitary gland and the thymus, point to their possible modulatory role in glucocorticoid feedback regulation and immune homeostasis (FIG. 4).

The expression and estrogenic regulation of the GR gene in the thymus might provide a mechanism by which estrogens modify thymocyte sensitivity to circulating glucocorticoids, and thus the immune responsiveness. This phenomenon may have clinical implications insofar as sexual dimorphism for certain immune diseases is concerned.


FIGURE 4. Schematic representation of the possible interactions between the hypothalamus-pituitary-adrenal (HPA) axis, the soluble products of stimulated immune cells, the thymus and gonadal hormones. The complex circuitry involves feedback effects at the central (hypothalamus), and peripheral sites (pituitary gland, adrenals, thymus, gonads, lymphocytes, mast cells and macrophages). Increased production of cytokines during immune system stimulation activates the HPA circuit, responsible for the subsequent shut-off of the immune response. On the other hand, the pre-existing sex steroid milieu may modulate glucocorticoid effects via an action on different immune compartments, possibly at the level of GR 11 receptor gene expression.

Transgenic Animals with Impaired Type II Glucocorticoid-Receptor Function Bearing Antisense R N A Transgene

A transgenic animal with impaired type I1 glucocorticoid receptor gene expression was recently created to study the glucocorticoid feedback effect on the HPA axis.22 To achieve this goal, a type I1 glucocorticoid receptor antisense RNA construct previously shown to decrease functional type GR in cell cultures66 was used. The mechanism of action of antisense RNA is not clear and so far there is but an example of antisense RNA inhibition of gene expression (myelin basic protein) in transgenic animal^.^' When injected directly into the cytoplasm of cells, antisense RNA must complex with the 5’ region of the endogenous mRNA, where ribosomes bind and initiate translation, resulting in RNA duplex formation. The translation block prevents the synthesis of the gene product and phenocopies mutation of the gene of interest.


Using amplification by reverse transcriptase with the polymerase chain reaction (PCR), we demonstrated the presence and uniqueness of antisense mRNA in tissue extracts from transgenic animals.22 Northern blot analysis of endogenous type I1 glucocorticoid receptor messenger RNA indicates a maximal 50-70% decrease in the hypothalamus and frontal cortex of the three different transgenic lines ana- lyzed; while a smaller 30-55% decrease was seen in liver.22

The decreased type I1 glucocorticoid receptor mRNA concentrations are re- flected by decreased glucocorticoid binding activity in tissues where the transgene is expressed. In transgenic animals, glucocorticoid binding sites are diminished in the frontal cortex, hypothalamus, pituitary and liver.

The transgene incorporated into the mouse genome was designed to disrupt normal HPA axis activity. The increased plasma concentrations of ACTH of transgenic animals is suggestive of a failure of glucocorticoids to inhibit HPA axis activity at either, or both, central and pituitary sites.22 Plasma corticosterone concentration is also raised, but not to the same extent ofACTH, suggesting that the adrenalgland, which appears slightly hyperplastic, may become refractory to continuous glucocorticoid stimulation. The most remarkable feature of these transgenic mice is greatly increased weight and fat deposition (see Ref. 22) . Steroid hormones govern a large range of processes affecting development and physiological homeostasis and may influence feeding and behavioral patterns.68 On the other hand, glucocorticoids are beneficial for normal in utero and postnatal maturation of tissues such as the lung and pancreas.69 For example, corticosterone promotes the development of the pan- creatic islet during the perinatal period, but high concentration of the steroid may attenuate p-cell responsiveness to glucose during this period .70

Glucocorticoids have a role in the development of obesity, caused by different e t i o l ~ g y . ~ ~ ? ~ ~ It seems interesting to mention that the obese fa/fa Zucker rat shows a decreased number of GR with raised serum ACTH and corticosterone.70

Glucocorticoids can decrease the number of receptors in both hippocampus and pituitary gland, and the high corticosterone levels in transgenic mice, secondary to the decreased type I1 glucocorticoid receptor levels, could be an additional factor in down-regulating the type I1 glucocorticoid receptor.

Patients suffering from different forms of mental illness (including severe depression, pathological aging) often show an increased activity of the HPA, as indicated by hypersecretion of CRF and cortisol, and a premature escape from the cortisol-suppressant action of dexamethasone (see Refs. 2 2 , 7 2 ) . On the other hand, marked dysregulations of immune system function appear with age, including the ability of glucocorticoids to shut-off the immune response, with consequent increased susceptibility to the development of autoimmune diseases. Interestingly enough, in patients suffering from inflammatory diseases, such as rheumatoid arthritis, the disease is frequently associated with a behavioral syndrome similar to that of atypical d e p r e s ~ i o n . ~ ~

It is possible that this lack of sensitivity to glucocorticoids could be due to an abnormality of type I1 glucocorticoid receptor regulation at the level of the limbic- hypothalamic-immune system. Thus we evaluated immune system function during the ontogeny of neuroendocrine-immune system function as well as under conditions of immune system stimulation, such as challenge with complete Freund adjuvant and BSA.

Development of Neuro-tmmune Connections in Transgenic Animals with Impaired Type II Glucocorticoid Receptor Function

Because of the profound modulation of immune homeostasis exerted by glucocorticoids and in view oftheir crucial role during the agingprocess, we investigated


the alterations of immune functions secondary to HPA axis dysfunction in transgenic mice bearing a type I1 antisense RNA transgene. The interest in studying neuro-immune connections in these animals stems from the fact that deficiencies in the HPA-immune system loop are known to participate in the development of a number of immune abnormalities, including autoimmune diseases, which are often present in the elderly population. Moreover, the process of aging is known to result from a gradual impairment of the organism’s ability to cope with environ- mental (including stressful) stimuli, as a result of HPA malfunction. Although it is not possible to determine direct cause-effect relationships between the perturba- tion of HPA axis with the neuro-immune abnormalities of aging individuals, the interruption of the immune system-HPA axis loop may undoubtedly participate in this phenomenon.

Another aspect which merits consideration is the marked hormonal (especially estrogenic) sensitivity of the HPA-immune axis. The degree of susceptibility to and severity of inflammatory diseases in response to a given pro-inflammatory or antigenic signal, besides the effects of the organism’s immune response genes, depends on: I . the integrity of the HPA axis; 2. the efficient down-regulation exerted by the sympathetic system; and 3. the pre-existing sex steroid background, as discussed in the previous sections.

Ontogenesis of Cell-Mediated Immune Response

To study possible alterations in the development of neuro-immune connections, the postnatal maturation of the cell-mediated immune response was followed in both thymocyte and splenocyte cell cultures at different time intervals. As observed in FIGURES 5 and 6, and in agreement with our previous data obtained in rats,46 intact male and female mice display a gradual age- dependent increase in the capacity to respond to a T-dependent mitogen, such as concanavalin A, or phytohemoagglutinin (PHA, not shown). Maximal splenocyte blastogenic potential is reached before sexual maturity, while the establishment of gonadal hormone feedback results in a marked decrease in the incorporation of 3H-thymidine from splenocytes of both males and females. In the thymus, a different pattern is observed for males and females. In fact, a higher and more robust response is observed when splenocytes are prepared from female rats at each time interval studied (not shown).

Transgenic mice bearing type 11 GR antisense RNA show a characteristic pattern of cell-mediated immune response maturation characterized by a reduc- tion in the lymphocyte’s responsiveness from 5 to 15 days after birth, and a marked stimulation of blastogenic potential between 25 and 60 days (FIGS. 5 and 6). Then, mice bearing the antisense RNA transgene failed to show the physiological inhibition of immune responsiveness normally occurring at the onset of puberty. These results suggest a possible delay in sexual maturation of transgenic mice and/or the inability of glucocorticoids to modulate this phenomenon.

That the processes subserving the maturation and differentiation of the thymus gland are somehow impaired in transgenic mice is supported by the marked alterations in the lymphocyte subsets of thymus and spleen, especially concerning the populations of the CD4-t CD8- (T-helper), CD4- CD8+ (T- suppressor), as well as the double positive CD4t CD8+. Indeed, transgenic mice show a significantly higher CD4/CD8 ratio, and a marked increase in the


J Male intact

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FIGURE 5. Maturation of cell-mediated immune response in the spleen of intact and transgenic male mice. Proliferative activity was monitored by the incorporation of 3H-thymidine following addition of the T-dependent mitogen, concanavalin A (Con-A), to cultures of splenocytes of the different age groups. Results are the mean 2 SEM of 3 splenic preparations (3 animalsiexperimental age-group).

population of immature T-subsets, indicating impairment in the processes of T-cell differentiation.

Ontogenesis of Type I1 Glucocorticoid Receptor

The HPA axis of mammals is not fully functional throughout an animal’s life cycle. During the first few weeks of postnatal life the system exists in a relatively dormant state, becomes fully activated by puberty, and then declines with age.73 In the rat, and to a less certain extent in the human newborn and other mammals, the initial period just after birth is characterized by an absence of pituitary and adrenocortical secretory responses to stressors that normally produce robust responses in the adult. This period of time is known as the stress hyporesponsive period (SHRP). On the other hand, although relatively unresponsive to certain stimuli, the HPA axis responds in varying degrees to other types of stressors including e n d ~ t o x i n . ~ ~ The teleological explanation for the existence of such a stress hyporesponsive period has been suggested for the potentially detrimental effects of high glucocorticoid levels on neuronal maturation and differentiation and on immune function.

It was, thus, of interest to follow the development of type I1 GR gene expression in the key organ of the immune system, in view of the possible role of glucocorticoids during the differentiation of the different T-cell subsets and in T-dependent immune functions. Moreover, as already mentioned, glucocorticoids, acting

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FIGURE 6. Maturation of cell-mediated immune response in the spleen of intact and transgenic female mice. Proliferative activity was monitored by the incorporation of )H-thymidine following addition of the T-dependent mitogen, concanavalin A (Con-A), to cultures of splenocytes of the different age groups. Results are the mean 2 SEM of 3 splenic preparations (3 animals/experimental age-group).

through a specific suicide program (apoptosis) in the thymus, might be responsible for the selection and turnover of thymocytes, as well as for their possible interactions with the thymic microenvironment. FIGURE 7 illustrates the expression of type I1 GR in central and peripheral male tissues. As shown, in analogy to what we observed at the level of the hippocampus, transgenic mice show a 40-60% loss of GR gene expression in the thymus.

A specific developmental pattern of type I1 GR mRNA concentration was observed in mice during postnatal development. As observed in FIGURE 8, type I1 GR mRNA concentration is very low in two-day-old mice and gradually increases to reach maximal levels at approximately 25 days in both males and females. A sexual dimorphism in the postnatal development of GR gene expression within the thymus is clearly observed during the course of maturation. At 25 days of age, GR mRNA concentration is approximately 2.5 times higher in males than in females. On the other hand, transgenic mice show a significant loss of type I1 GR mRNA content with levels being some 40-60% reduced when compared to intact mice. Interestingly enough, GR gene expression in the thymus is low during the first two weeks of life, corresponding to the SHRP, indicating a reduced influence and/or inability of the stress hormone to exert its influence in the thymus. Previous studies on CRH expression during ontogeny in outbred rats have indicated that hypothalamic CRH mRNA levels, determined by in situ hybridi~ation’~ and CRH-like immunoreactivity, decrease substantially just prior to and immediately after birth, and increase again by postnatal day 3, plateauing at the end of the first week of life. Plasma corticosterone falls within hours after birth, reaches a nadir at postnatal day

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8, and increases only minimally in response to stress. Plasma corticosterone levels increase gradually and respond to stress by postnatal day 14. Also in the thymus, GR mRNA sharply increases after 14 days of age, suggesting the establishment of a marked glucocorticoid influence in the thymus at that age. It is also of interest to note a clear inhibition of GR mRNA activity around sexual maturity, suggesting the possibility of an interplay between sex steroids and glucocorticoids in the control of GR mRNA concentration in the develop- ing thymus.

Type 11 Glucocorticoid Receptor Gene Expression and Immune Reactivily to Pro-Inflammatory Stimuli

Infection and inflammation are potent stimulators of HPA axis activity; conversely, a variety of stressors have been shown to impair mitogen-induced lymphocyte proliferation. Injection of a mycobacterial Freund's type adjuvant in the rat leads to an inflammatory disease of the joints. Adjuvant-induced arthritis (AA) represents a T-lymphocyte-dependent, strain-specific autoimmune disease, which has been reported to trigger the release of pituitary hormone^.^ This polyarthritis might be expected to cause chronic activation of the HPA axis. Attention has recently focused on the roles of various immune mediators

2

GR mRNN 6-actin mRNA

1

0

Intact

Transgenic

Hippocampus Liver Thymus

T i s s u e s

FIGURE 7. Type I1 glucocorticoid receptor (GR) gene expression in the brain and peripheral tissues. Total RNA (20 pg) extracted from mouse hippocampus, liver and thymus were separated on 1 .O% agarose-formaldehyde denaturing gel, blotted onto nylon filters, and hybridized with glucocorticoid receptor cDNA probe. Under these stringency conditions, a single strong band, corresponding to the 6.7-kb glucocorticoid receptor mRNA, is identified. Simultaneous measurement of the p-actin mRNA content by hybridization of filters with a p-actin cRNA produced a 2.2-kb band corresponding to B-actin mRNA.

322 ANNALS NEW YORY ACADEMY OF SCIENCES

GR m R " B-actin mRNA

1

2 .

0

Male

Female

2 5 9 1 4 2 5 36 4 8

D a y s

FIGURE 8. Ontogenesis of type I1 glucocorticoid receptor (GR) gene expression and presence of a sexual dimorphism in the thymus. Intact male and female mice were sacrificed at different time intervals after birth, and thymic specimens were processed as described in legend for FIGURE 7. Note the significant increase of type 11 GR mRNA within the male thymus at 25 days, with respect to female mice.

in the regulation of the HPA axis during the stress of inflammation. Because of the profound dysregulation of HPA axis activity of the transgenic mice, it was of interest to analyze the immune capability to respond to inflammatory stressor, such as Freund complete adjuvant. For this aim, animals were injected with the adjuvant in BSA, and sacrificed at 3 and 7 days following the first challenge, or at 14 and 21 days after the second challenge of BSA alone.

At the thymus gland level, and in intact mice type I1 GR mRNA and the immune response were characterized by well-defined alterations. A significant decrease of type I1 GR mRNA concentration was observed at 3 days post-injection, and corresponded to a significant stimulation of immune reactivity. An approximately 70% loss of GR mRNA content followed at 15 days, while a return to normal was observed at 21 days post-injection. Immune response was gradually down-regulated, with levels returning to normal by 21 days. In transgenic mice, a significantly higher cell-mediated immune response was present in basal conditions, while, injection of antigenic trigger resulted in a potent down-regulation of immune reactivity already at 3 days post-injection.

On the other hand, in the spleen of transgenic mice, immune responsiveness still remained higher, and failed to show the inhibitory phase, suggesting that in these animals the HPA axis dysfunction (and possibly a catecholaminergic malfunction) coupled to an altered sex-steroid background may result in inappro- priate down-regulation of immune function leading to autoreactivity. In this con- nection it seems interesting to recall that the Lewis rat is the prototype strain for the study of a number of experimental autoimmune diseases, including experimental


autoimmune encephalomyelitis (EAE), uveitis (EAU), orchitis (EAO), adjuvant- induced arthritis (AA), and streptococcal cell wall (SCW)-induced arthritis. This strain of rats has recently been shown to have a significant disturbance in endocrine immune r eg~ la t ion .~ The Lewis rat appears deficient in its ability to generate marked elevations of ACTH and corticosterone in response to a number of external challenges (e.g., rIL-la, serotonin agonists, and streptococcal petidoglycan poly- saccharide).

Sternberg et al. traced the origin of this deficiency to depressed synthesis and secretion of hypothalamic CRH.9-.55 It has thus been proposed that a dysfunction in the HPA axis may contribute to the Lewis rat’s susceptibility to multiple inflammatory diseases, relative to histocompatible, but resistant (e.g. , Fisher 344, PVG) rat strains.

This study clearly supports a primary role of the HPA axis in the down- regulation of the immune response and suggests that type I1 GR is an integral part of the HPA-immune loop.

CONCLUSION

The present article underlines the possible connections between the effects of a dysregulation of the HPA axis activity with: a. glucocorticoid-mediated immuno- depression of senescence and b. glucocorticoid-sex steroid interactions in sex- linked autoimmune diseases, as well as in aging-induced reproductive-immune failure.

With the use of a transgenic mouse bearing a type I1 GR antisense RNA we have observed specific alterations of the HPA-immune system loop, which are reminiscent of the alterations observed during the process of aging. The failure of transgenic mice to display the physiological down-regulation of the immune response after puberty and after immunogenic or pro-inflammatory challenges clearly indicates the inability of the HPA axis to efficiently exert its physiologic inhibitory role. Moreover, type I1 glucocorticoid receptor expression in the thymus appears to be importantly involved in the processes of both maturation and differentiation of the different lymphocyte subsets.

These transgenic mice with impaired type I1 glucocorticoid receptor gene expression may represent a good model to study neuro-immune connections during the establishment of aging and aging-associated pathologies, as well as for evaluating new therapies of aging-induced neuroimmune degenerative diseases.

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Transgenic Animals with Impaired Type II Glucocorticoid Receptor Gene Expression

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