Lymphoid cell resistance to glucocorticoids in HIV infection

ft. Steroid Biochem. Molec. Biol. Vol. 57, No. 5/6, pp. 259-263, 1996

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Mini Review L y m p h o i d Cell R e s i s t a n c e to G l u c o c o r t i c o i d s in H I V I n f e c t i o n S i m o n K. Kawa and E. Brad T h o m p s o n The University of Texas Medical Branch, Department of Human Biological Chemistry and Genetics, 603 Basic Science Building, Galveston T X 77555-0645 U.S.A.

In h u m a n s i n f e c t e d with the HIV-1 vi r us t h e r e m a y be a d i s p r o p o r t i o n a t e severi t y o f signs a n d s y m p t o m s o f illness c o m p a r e d to t he f r a c t i o n o f CD4 ÷ i nfect ed T - l y m p h o i d cells. In p a r t , this m a y be d u e to a l t e r e d i n t e r c e l l u l a r signalling s ys t em s a n d i n t r a c e l l u l a r signal t r a n s d u c t i o n . G l u c o c o r t i c o i d s a re well k n o wn f o r l:heir effects on the vitality a n d f u n c t i o n o f l y m p h o i d cells. P a t i e n t s with HIV infections often show elevated c i r c u l a t i n g levels o f cortisol, suggesting s o m e m i s f u n c t i o n in th e r e g u l a t o r y s y s te m s t h a t m a i n t a i n the levels o f this critical h o r m o n e . At t he cellular level, it is k n o w n t h a t b o t h a c u t e HIV i n f e c t i o n a n d g l u c o c o r t i c o i d s can cause a p o p t o t i c cell d e a t h in t h y m i c l y m p h o c y t e s . Howew~r, c h r o n i c a l l y H I V - i n fect ed cells a p p e a r to be r e s i s t a n t to g l u c o c o r t i c o i d - e v o k e d cell d eath . G l u c o c o r t i c o i d receptor--ligand b i n d i n g studies on p a t i e n t s ' cells have shown r e d u c e d affinity b e t w e e n t he r e c e p t o r b i n d i n g sites a n d test steroids. In vitro, c h r o n i c a l l y H IV -i nfect ed cells o f t h e l y m p h o i d C E M flue displayed r e s i s t a n c e to g l u c o c o r t i c o i d - i n d u c e d apoptosis. T h e s e cells showed r e d u c e d n u m b e r s o f b i n d i n g sites with little a l t e r a t i o n in a p p a r e n t affinity b e t w e e n llgand a n d r e c e p t o r . T h u s it a p p e a r s th~tt t h e r e m a y often be m a l f u n c t i o n o f t he n o r m a l g l u c o c o r t i c o i d r e s p o n s e in H I V- in f ected cells p r o b a b l y due to a l t e r e d i n t e r a c t i o n s b e t w e e n t he g l u c o c o r t i c o i d r e c e p t o r a n d its h o r m o n e . S u c h a l t e r a t i o n s m a y have clinical c o n s e q u e n c e s , including t he possibility o f a relatively l o n g e r life s p a n o f i n f e c t e d CD4 ÷ T - l y m p h o c y t e s , as well as syst em i c effects o f c h r o n i c a l l y elevated c o r tis o l levels. C o p y r i g h t © 1996 E l s evi er Science L t d

ft. Steroid Biochem. Motec. BioL, Vol. 57, No. 5/6, pp. 259-263, 1996

One of the major mysteries concerning the clinical manifestations of H um a n Immunodeficiency Virus type (HIV) infection is that the signs and symptoms evoked cannot be explained quantitatively by the small proportion of infected CD4-positive cells. This is particularly notable in the earlier stages of the illness. To establish infection cumulative evidence has pointed to the critical importance., of a direct interaction between the HIV viral envelope and the CD4 antigen, a transmembrane protein expressed on the surface of several cell types, but particularly on T helper/inducer lymphocytes [1-4]. The eventual result of HIV infection is a severe irrLpairment of the immune system [5-16], though early reports suggested that only a T h e University of Texas Medical Branch, D e p a r t m e n t of H u m a n Biological C h e m i s t r y and Genetics, 603 Basic Science Building, Galveston, T X 7 7 5 5 5 - 0 6 4 5 , U.S.A. Tel: 409-772-2271, Fax: 409-772-5159 Received 24 October 1995; Accepted 30 N o v e m b e r 1995. 259

minor fraction of CD4-positive ceils in patients can be demonstrated to contain virus [17]. Later studies using more sensitive techniques, such as PCR, have confirmed that the majority of cells are uninfected [18]. Nevertheless, the symptoms and signs which develop indicate that there is widespread abnormal function of the immune system, and a gradual depletion of immune cells. One possible explanation is that the complex network of interactions among cells of the immune system has been disorganized [19]. Interactions among the immune system, the central nervous system, and other major tissues are orchestrated by hormone responses and by a network of lymphokines, cytokines, and their receptors, all of which function in an endocrine or paracrine fashion [20-22]. This has lead to the study of possible interactions between cellular responses to certain hormones and hormone-like substances on HIVinfected cells, to determine if altered immune-

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endocrine interactions influence the course of the disease. In addition, in HIV-infected individuals several neuroendocrine disorders have been described, including: hypogonadism [23-25], low T 3 syndrome [26, 27], hyponatremia [28], hyperprolactinemia [23], alterations in the hypothalamic-pituitary-adrenal (HPA) axis [ 2 4 , 2 5 , 2 9 - 3 2 ] , and rarely, anterior hypopituitarism [27]. Among the neuroendocrine disorders that have been described in HIV-infected patients, alterations in the HPA axis and glucocorticoid hormones comprise one group that could well be associated with immunodysfunction, neurologic deterioration and weight loss, symptomatology frequently seen in Acquired Immunodeficiency Syndrome (AIDS) patients. Several studies of adrenal function in HIV-infected patients have revealed high levels of cortisol and alterations in the HPA axis. In an early report Verges et al. [29] investigated the glucocorticoid function of a group of HIV-infected men and women to determine the incidence of adrenocortical abnormalities at various stages of HIV infection, using the classifications defined by the Centers for Disease Control (CDC). Compared with control patients, groups II (asymptomatic) and III (lymphadenopathy) exhibited higher levels of A C T H and basal cortisol. No significant differences were found between group IV (clinical manifestation of AIDS) and the control group. Catania etal. [30], demonstrated that the response of the HPA axis to an antigenic challenge is impaired in AIDS patients. In their analysis of circadian rhythms of plasma hormone levels, Villette et al. [24], showed an altered adrenal hormonal state in HIV-infected male patients, even during the asymptomatic period of the infection, and Nevena et al. [25], demonstrated cortisol levels 35-55% higher than controls in HIV-infected patients in all C D C groups. Azar et al. [31], studied HPA function in non-AIDS patients with advanced HIV infection and found that about 25% of patients with no clinical evidence of pituitary or adrenal disease have reduced pituitary reserve with high basal A C T H and cortisol, and about 25% have reduced adrenal reserve with high basal cortisol and inappropriately normal basal A C T H , whereas about 50% maintain normal HPA axis activity with increased basal cortisol secretion. Finally, Coodley et al. [32] compared the endocrine function of patients suffering from the HIV wasting syndrome with other HIV-positive patients without wasting and found cortisol levels were higher in patients with wasting compared with patients with similar CD4 counts without wasting. T h e increased levels of cortisol found in asymptomatic patients argue for an early deregulation of the adrenocortical axis in HIV infection. Although adrenocortical insufficiency has also been reported during the evolution of AIDS, it occurs infrequently and is likely to be a late complication. T h e reasons for the increased A C T H secretion and deregulation of the HPA axis in

HIV-infected patients are yet to be elucidated; in any case the result is elevated circulating glucocorticoid levels. Glucocorticoids have profound effects on the immune system [33]. T h e y are often employed as immunosuppressive agents, with both direct and indirect inhibitory effects on immune cell functions [34]. T h e indirect immunosuppressive effects of glucocorticoids are very important, and are the result of glucocorticoid-evoked reduction in levels of many lymphokines [35]. Glucocorticoids also are directly or indirectly inhibitory to other classes of immune system cells as well [35]; so elevation in these steroids can be disruptive to the entire interactive immunity network. It has been proposed that one physiological role of glucocorticoids is to dampen the potential for over-activity of the immune system [36, 37]. Glucocorticoids provoke lysis in certain types of lymphoid cells; this stimulation of cytolysis requires both an adequate n u m b e r of glucocorticoid receptors (GR) and other, as yet undefined 'lysis functions' [38-43]. In addition to the effects described above, glucocorticolds are known to interact with other organs and systems. In the central nervous system, in addition to the physiological negative feedback control of hypothalamic-pituitary release of A C T H , cortisol modulates perception and emotion. In the lymphoid system glucocorticoids have dramatic catabolic effect on lymphoid tissue, manifested by lymphoid and thymic atrophy and lymphopenia in hypercortisolism states. T h e overall pattern of cortisol's physiological actions include many that are catabolic, with increased protein breakdown and nitrogen excretion. In states of cortisol excess these catabolic effects of cortisol are manifested on skeletal muscle by atrophy and muscle weakness [44]. Glucocorticoids mediate their effects on target cells by binding to a specific intracellular GR, which is 'activated' and translocated into the nucleus [45]. There the active complex binds to specific cis-active D N A sequences termed glucocorticoid response elements (GREs), initiating induction/suppression of gene expression [46-50]. Classic GREs are arranged as shown by the consensus motif G T T A C A n n n T G T T C T . Complete or partial GREs have been found in the vicinity of the coding sequences of glucocorticoidregulated genes, near or within the transcriptional enhancers of many retroviruses, and within the genome of HIV [51-55]. Although the functional nature of these putative GREs within the HIV genome has yet to be fully established, there are some data to suggest that the glucocorticoid hormone-receptor signal transduction network might interact with HIV regulatory pathways and increase the rate of viral replication [52-55]. After binding steroid, the G R can also interact with other important transcription factors, including AP-1, NFK B, CREB, and c/EBP [56], to cause altered regulation of genes, many important to immune cell function.

Lymphoid Cell Resistance Both HIV and glucocorticoids evoke programmed cell death in certain lyraphoid cells [57, 58]. Thus, the interaction between glucocorticoid action and HIV may be clinically relevant. Glucocorticoid resistance in mononuclear leukocytes (MNL) from HIV-infected patients has been reported [59]. In this report, Norobato et al. studied a group of AIDS patients that in spite of exhibiting high serum and urinary cortisol levels, had a n u m b e r of signs and symptoms consistent with adrenal insufficiency, a clinical picture consistent with the diagnosis of peripheral resistance to cortisol. Competitive binding studies revealed that the affinity of the M N L G R for [3H]dexamethasone was reduced and the n u m b e r of receptors increased in these patients, suggesting that a receptor abnormality was responsible for the glucocorticoid resistance. T h e cause of the glucocorticoid receptor abnormality in these HIVinfected patients has yet to be identified. In another study, Vagnucci and Wiinkelstein [60], reported changes in the dissociation constant of G R in HIV-infected individuals, showing that in more than half of the patients studied, GR-ligand affinity was reduced . We described glucocorticoid resistance in vitro associated with a reduced n u m b e r of G R sites in cells infected with HIV-1 [61], without dramatic change in affinity. Because glucocorticoids are a classic cause of apoptosis in lymphoid cells, and HIV infection also has been said to cause apoptosis [57, 58], we had initially hypothesized that the h o r m o n e plus the virus would

261

synergize to cause even greater kill. But when we tested this hypothesis directly in C E M cells, a well known HIV-sensitive CD4 ÷ cell line, our results proved that rather than synergism, there was antagonism between HIV infection and glucocorticoids. This was suggested initiaUy by experiments during the acute phase of infection. At this time cells are being killed by HIV. Uninfected cell cultures were killed even more by dexamethasone. But combined treatment resulted in cell survival between that seen with either treatment alone (Fig. 1). This clearly was not synergism and suggested that the HIV infection reduced cellular susceptibility to the steroid. To have better control over conditions, we turned to chronically infected cells. C E M cells chronically infected with HIV-1 and no longer experiencing HIV-induced lysis were treated with various concentrations of cortisol or dexamethasone and examined for viability. At fully lethal concentrations of steroid virtually all the sensitive cells have been killed after 4-7 days. At both times however, the infected cells showed significantly increased survival in the presence of either [61]. T h e chronically infected, uncloned C E M cells contained fewer receptor binding sites without a significant change in their affinity for steroid [61]. This correlation between lowered receptors and lowered sensitivity is consistent with data in hepatoma cells correlating gene regulation to quantity of G R [55]. While the quantity of cellular vglucocorticoid receptors does not seem to predict the

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Days post i n f e c t i o n / t r e a t m e n t Fig. l . A c u t e H I V infection r e d u c e s C E M cell s e n s i t i v i t y to d e x a m e t h a s o n e - e v o k e d cell death. Starting at t i m e z e r o , C E M cells w e r e e x p o s e d to HIVe~13 and 10 ~ M d e x a m e t h a s o n e ( D E X ) , s i n g l y o r in c o m b i n a t i o n . Control ceils r e c e i v e d neither the v i r u s o r s t e r o i d . A l i q u o t s o f e a c h c u l t u r e w e r e c o u n t e d d a i l y f o r v i a b l e cells; non-viable ceils not yet lysed were e x c l u d e d b y the T r y p a n blue dye uptake m e t h o d 161]. T h e m e a n s ( N - - 3 ) a r e s h o w n f o r a n i n d i v i d u a l e x p e r i m e n t ( o f t h r e e s e p a r a t e fully r e p l i c a t e e x p e r i m e n t s ) .

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extent of response from one cell type or culture to the next, within a single cell type responsiveness to glucocorticoid may be proportional to the quantity of G R [62-67]. Alterations in ligand affinity and/or in quantity of GR in cells of infected patients therefore may be relevant to the corticoid resistance and abnormalities of immune function observed clinically. Lowered G R numbers or lessened affinity for cortisol could not only lower the sensitivity of genes controlled by simple GR:GRE site interactions but also affect a much wider array of genes regulated by complexes of the GR with other transcription factors. The altered balance of G R and transcription factors could result in very complicated changes in regulatory networks, difficult to predict. Elevated cortisol levels in HIV + individuals may have important consequences. If their HIV-infected C D 4 + T cells are somewhat less sensitive to the lyric effects of cortisol than uninfected T cells, the infected cells may on average survive longer in the elevated cortisol environment, and this may hasten expansion of the HIV ÷ cell population. Other cells of the immune system besides lymphocytes contain G R [35, 68], and therefore also may have altered viability or function due to the elevated cortisol. Thus there may be widespread and cumulative consequences derived from the change in steroid responsiveness. These questions should be addressed experimentally. HIV-infected cultured cells may provide informative systems in which to examine these issues.

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