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Glucocorticoids and the Immune Function in the Human Immunodeficiency Virus Infection: A Study in Hypercortisolemic and Cortisol-Resistant Patients¹

Departments of Endocrinology (G.N., M.B., T.V.) and Immunology (A.T., M.C.), L. Sacco University Hospital, Milan, Italy

Address all correspondence and requests for reprints to: Prof. G. Norbiato, L. Sacco University Hospital, Via G. B. Grassi 74, 20157 Milan, Italy.

	Abstract

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Immunological studies in human immunodeficiency virus (HIV)-positive patients suggest that the disease progression is accompanied by a defective production of type 1 cytokines [interleukin-2 (IL-2) and IL-12], an increased production of type 2 cytokines (IL-4, IL-6, and IL-10), and an increased production of IgE. HIV infection is also associated with activation of the hypothalamo-pituitary-adrenal axis function and increased plasma and urinary cortisol concentrations. As cortisol is involved in the physiological regulation of cytokines, a study was conducted to examine cytokine patterns in two groups of hypercortisolemic patients, one with normal sensitivity to glucocorticoids and the other with glucocorticoid resistance.

Ten HIV-infected patients with normal receptor affinity to glucocorticoids (AIDS-C), 10 HIV-infected patients with low receptor affinity to glucocorticoids (AIDS-GR), and 20 healthy subjects were studied. Receptor characteristics of peripheral blood mononuclear cells were evaluated by [³H]dexamethasone binding. Serum cortisol and urinary free cortisol were measured by RIA. Serum ACTH and IgE were measured by immunoradiometric assay, and IL-2, IL-4, and IL-10 cytokines and interferon- were measured by enzyme-linked immunosorbent assay.

AIDS-C patients showed low IL-2 and high IL-4, IL-10, and IgE concentrations; conversely, AIDS-GR patients showed high IL-2 and low IL-4 and IgE concentrations.

Thus, in HIV infection, elevated cortisol levels suppress cell-mediated immunity and stimulate humoral immunity, whereas this response is not detected in cortisol-resistant patients. These findings indicate that cortisol and its receptors are critically involved in the regulation of immune function in HIV infection.

	Introduction

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EVIDENCE suggests that HIV infection is associated with a progressive reduction in type 1 cytokines, interleukin-2 (IL-2) and IL-12 (1), and an increase in type 2 cytokines, IL-4, IL-6, and IL-10 (2). These changes in the immune response may be due to an alteration in the immune-endocrine dialogue, which is modulated by the hypothalamic-pituitary-adrenal (HPA) axis and cortisol (3). This interpretation is based on two types of cortisol-related alterations that may affect glucocorticoid-regulated responses in human immunodeficiency virus (HIV)-infected patients: hypercortisolism (4, 5, 6, 7, 8, 9) and cortisol resistance (6, 9, 10).

The present study was formulated to investigate the relationship between cortisol and the T helper functions in HIV infection. T helper (Th) cells are divided into Th1 and Th2 subclasses based on their functions and cytokine production patterns (11, 12). In general, Th1-related cytokines promote Th1 activity (i.e. cell-mediated immunity) and inhibit Th2 activity (i.e. humoral immunity), and Th2-related cytokines do the contrary. Cell-mediated immunity is mainly stimulated by the bioactive type 1 cytokine IL-12 and interferon- (IFN), which promote the proliferation of Th1 cells (13, 14). Humoral immunity is mainly caused by the type 2 cytokines IL-4 and IL-10, the main growth factors of Th2 cells (15).

In this study, the production of IL-2, IL-4, IL-10, and IFN was examined in two groups of HIV patients with similar disease durations, extents of opportunistic infections, and CD4⁺ counts. Both groups had elevated cortisol levels; the first had normal receptor affinity to glucocorticoids in their peripheral blood mononuclear cells (PBMC), and the second had low affinity.

	Subjects and Methods

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The study was approved by the L. Sacco University Hospital ethics committee, and the subjects gave their informed consent.

Two groups of HIV-infected patients underwent the study. The diagnosis of acquired immune deficiency syndrome (AIDS) was made according to the criteria established by the Center for Disease Control (16). The first 10 patients (7 men and 3 women), referred to as AIDS-C, had high values of plasma and urinary cortisol and normal receptor characteristics. The second 10 patients (7 men and 3 women), referred to as AIDS-GR, had high serum and urinary levels of cortisol, but glucocorticoid resistance. The diagnosis of glucocorticoid resistance was based on a K_d of the PBMC glucocorticoid receptor 2 SD higher than in normal subjects. The control group (C) was composed of 20 age- and sex-matched healthy seronegative heterosexual adults. The population ages of the three groups and CD4 counts are reported in Table 1. Seven AIDS-C and six AIDS-GR patients did not receive immunomodulatory or antiviral therapy before the study; three AIDS-C and four AIDS-GR had been treated with zidovudine for an average period of 1.5 yr. Patients were not treated with antifungal agents or other drugs affecting glucocorticoid function. Both AIDS-C and AIDS-GR groups had similar disease duration, number and extent of opportunistic infections, and previous therapy.

View this table: [in this window] [in a new window]   Table 1. Serum and urinary cortisol, ACTH, IFN, and IL-10 in AIDS-C, AIDS-GR patients, and healthy controls

Serum samples were obtained on 2 consecutive days from patients and subjects who had been fasting since midnight; on the first day, blood was sampled for plasma ACTH, serum cortisol, IL-2, IL-4, IL-10, IFN, and IgE in supine patients at 0800 h. At the same time on the second day, blood was sampled for PBMC. Urine samples were collected under the same conditions on both days for the assessment of 24-h urinary free cortisol.

Serum cortisol and urinary free cortisol were measured by RIA methods, and serum ACTH was determined by immunoradiometric assay (Allegro, Nichols Institute, San Juan CA). Serum concentration of IL-2, IFN, IL-4, and IL-10 were evaluated with commercially available enzyme-linked immunosorbent assays (Genzyme, Cambridge, MA) as follows: IL-2, human IL-2 Intertest-2 (detection limit of the assay, 4 pg/mL; range, 4 to >1024 pg/mL); IFN, human IFN Intertest (detection limit of the assay, 3 pg/mL; range, 3 to >1024 pg/mL); IL-4, human IL-4 Intertest-4 (detection limit of the assay, 6 pg/mL; range, 6 to >1024 pg/mL); IL-10, human IL-10 Intertest-10 (detection limit of the assay, 5 pg/mL; range, 5 to >512 pg/mL) following the procedures suggested by the manufacturer. Values for all cytokines were calculated from a standard curve of the corresponding recombinant human cytokine according to the instructions of the manufacturer. IgE was measured by the CAP system (Pharmacia, Uppsala, Sweden). All analyses were performed twice.

PBMC preparation

PBMC isolated from heparinized peripheral blood of patients and control subjects by Ficoll-Paque sedimentation (17) were washed and suspended in DMEM (Life Technologies, Grand Island, NY) containing 50 µg/mL gentamicin and 2 mmol/L-glutamine. More than 95% of PBMC were viable, as determined by the trypan blue exclusion test and counting with a hemocytometer.

[³H]Dexamethasone binding

The binding capacity of PBMC was determined by incubating the cells in 0.5 mL DMEM with various concentrations of [³H]dexamethasone (45 Ci/mmol; New England Nuclear, Boston, MA) with and without a 200-fold molar excess of cold dexamethasone (Sigma Chemical Co., St. Louis, MO).

Each tube contained 1 million cells/0.4 mL medium, and incubation was performed at 4 C overnight. The cells were harvested on glass fiber filters (GF/C, Whatman, Clifton, NJ), washed twice with 10 mL cold phosphate-buffered saline, and analyzed for radioactivity in a Packard Tri-Carb scintillation spectrometer (Packard, Downers Grove, IL). Binding capacity, expressed as femtomoles of receptors per million cells, and the apparent dissociation constant were calculated according to Scatchard’s method (18) using the Ligand program (19).

Statistical analysis

Hormonal and receptor data are expressed as the mean and SD. Data relevant to cytokines are expressed as the mean and SE in tests and figures. The statistical analysis procedures were based on a t test for samples with equal variance assumed. Values were log transformed to stabilize the variance, and t tests were performed on the log-transformed values. The values shown are derived from the results of a two-tailed statistical analysis.

	Results

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The population ages and CD4 counts are reported in Table 1.

Glucocorticoid receptors in PBMC cells

Analysis of [³H]dexamethasone binding to PBMCs from AIDS-C, AIDS-GR, and control subjects showed a single class of binding sites. Mean values of the affinity constant (K_d) were 3.0 ± 1.1 nmol/L in AIDS-C, 11.2 ± 3.6 nmol/L in AIDS-GR, and 2.3 ± 0.8 nmol/L in C subjects. The difference was significant for the K_d of the AIDS-GR group vs. those of other groups (P < 0.01), but not between AIDS-C and C groups. The receptor number (B_max) was 6.8 ± 2.5 fmol/million cells (1E⁶) in AIDS-C, 17.3 ± 7.2 fmol/1E⁶ in AIDS-GR, and 5.9 ± 1.6 fmol/1E⁶ in C subjects. The B_max in AIDS-GR was significantly higher than those in the other groups (P < 0.01)

Hormones and cytokines

Plasma cortisol and urinary free cortisol values were higher in AIDS-C and AIDS-GR groups than in controls. ACTH concentrations were highest in the AIDS-GR group, whereas ACTH concentrations were similar in AIDS-C and C groups (Table 1).

Serum IL-2 concentrations were significantly higher in AIDS-GR than in AIDS-C (P < 0.0001) and normal controls (P < 0.02). IL-2 concentrations were very low in AIDS-C patients, even lower than those in healthy controls (P < 0.0008). IL-4 concentrations were significantly lower in AIDS-GR than in AIDS-C patients (P < 0.03). Healthy subjects had the lowest IL-4 concentrations compared to those in AIDS-C (P < 0.0003) and AIDS-GR patients (P < 0.001; Fig. 1). Serum concentrations of IFN and IL-10 were elevated in both AIDS-C and AIDS-GR patients (Table 1) compared to those in normal subjects. IgE levels were high in AIDS-C patients and low in AIDS-GR patients (P < 0.001; Fig. 2).

View larger version (19K): [in this window] [in a new window]   Figure 1. Serum concentrations of IL-2 and IL-4 in AIDS-GR (), AIDS-C (), and C ().

View larger version (13K): [in this window] [in a new window]   Figure 2. Serum concentration of IgE in AIDS-GR (), AIDS-C (), and C ().

	Discussion

Top Abstract Introduction Subjects and Methods Results Discussion References

In general, it is agreed that cortisol inhibits the production of many type 1 cytokines, such as IL-2, IL-12, and IFN (21, 22). There are conflicting reports regarding type 2 cytokines. In vivo and in vitro studies with the glucocorticoid analog dexamethasone showed an increase in IL-4 synthesis by murine lymphocytes (3, 23), whereas studies on human lymphocytes reported the inhibition of IL-4 production (24, 25). A recent in vivo study (26) demonstrated that dexamethasone has no effect on LPS-induced IL-10 production, whereas another study on rat CD4⁺ cells (27) showed that pretreatment with dexamethasone increased the production of IL-10, IL-14, and IL-13. The contemporaneous administration of IL-4 and glucocorticoids stimulates IgE production (28).

In HIV disease, the normal interaction between the HPA axis and cytokines is altered, thus producing an oversecretion of cortisol, resulting in immune suppression. In most patients, this trend continues throughout the course of the disease, whereas in a small subgroup of cortisol-resistant patients the cytokine response is reactivated, as demonstrated by the results.

In the present study, AIDS-C patients (cortisol-sensitive) had low levels of IL-2 and high levels of IL-4, whereas the opposite was found in AIDS-GR patients (cortisol resistant), i.e. high IL-2 and low IL-4. The levels of IL-10 were increased in both AIDS-C and AIDS-GR compared to those in the control group. AIDS-C had the highest levels of serum IL-10. IgE concentrations were high in AIDS-C patients and low in AIDS-GR patients. The above data confirm the remarkably different cytokine patterns in cortisol-sensitive and cortisol-resistant patients. The inhibition exerted by glucocorticoids on type 1 cytokines and the simultaneous stimulation of the major Th2 cytokines IL-4 and IL-10 may be critical in the Th1-Th2 shift in AIDS patients. The imbalance in cytokine production may also be responsible for the altered glucocorticoid receptor found in HIV-infected patients; IL-2 has been shown to synergize with IL-4 to reduce the hormone binding affinity of the nuclear glucocorticoid receptor fraction (29, 30). The vpr gene in the HIV type 1 virus may also have a role in the receptor defect (31, 32).

In conclusion, there is evidence that in the majority of patients with HIV infection, there is a chronic activation of the HPA axis system. This ongoing reaction results in elevated levels of cortisol, which inhibit the correct functioning of the immune system, manifested in a shift from a Th1 to a Th2 cytokine profile. This compromised immune responsiveness promotes virus replication (33, 34) and leads to a poor prognosis. Only if and when resistance to the immune-suppressive effects of cortisol arises, does the immune system return to the type 1 cytokine profile. At the moment, immunological studies on cortisol resistance have been performed only on patients with full-blown AIDS, and despite the recovery of their immune reaction, cortisol resistance has not been observed to change the course of their disease.

The points worthy of further consideration and study are the following: 1) a method to counteract the effects of elevated cortisol, i.e. with natural hormones known to inhibit the immunological effects of cortisol; 2) an investigation of early cortisol resistance in HIV-infected subjects and its effects on the course of HIV disease; 3) research on the immune system in patients with non-HIV low receptor affinity to cortisol; and 4) trends in immune suppression in other chronic diseases or stresses and their eventual application to HIV disease.

	Footnotes

 
¹ This work was supported by grants from Istituto Superiore di Sanità, AIDS Projects VII and VIII (to A.T. and M.C.).

Received March 19, 1997.

Revised June 19, 1997.

Accepted June 30, 1997.

	References

Top Abstract Introduction Subjects and Methods Results Discussion References

 

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