MD Derek Humphreys 
  
MD. Liana Schlesinger 
  
PhD. Marcelo Lopez 
  
A Verónica Araya 
  
A Veronica Araya 
  
MD. A Veronica Araya 
  
  
  
  
  
  
SPONTANEOUS AND LPS-STIMULATED INTERLEUKIN-6 PRODUCTION IN PERIPHERAL BLOOD MONONUCLEAR CELLS (PBMC) AND DEREGULATION OF THE HYPOTHALAMIC-PITUITARY-ADRENAL AXIS IN PATIENTS WITH MAJOR DEPRESSIVE DISORDERS

Keywords: Major depressive Disorders, HPA axis, LPS

whether they are published or not. The documents may come  

Introduction

The state of the art in neuro-sciences establishes a clear association between Major Depressive Disorders (MDD) and hyperactivity of the Hypothalamus-Pituitary-Adrenal (HPA) axis [START_REF] Holsboer | Altered hypothalamic-pituitaryadrenocortical regulation in healthy subjects at high familial risk for affective disorders[END_REF][START_REF] Holsboer | Antidepressants and hypothalamic-pituitary-adrenocortical regulation[END_REF][START_REF] Gold | Clinical and biochemical manifestations of depression[END_REF][START_REF] Michelson | Pathophysiologic and somatic investigations of HPA axis activation in patients with depression[END_REF][START_REF] Plotsky | Psychoneuroendocrinology of depression[END_REF], main homeostatic system of the organism [START_REF] Cacioppo | Autonomic, neuroendocrine, and immune responses to psychological stress: the reactivity hypothesis[END_REF][START_REF] Ader | Psychoneuroimmunology: interactions between the nervous system and the immune system[END_REF][START_REF] Chrousos | The concepts of stress and stress system disorders. Overview of physical and behavioral homeostasis[END_REF]). An early escape of cortisol to dexamethasone suppression [START_REF] Carroll | Resistance to suppression by dexamethasone of plasma 11-O.H.C.S. levels in severe depressive illness[END_REF][START_REF] Carroll | A specific laboratory test for the diagnosis of melancholia: standardization, validation and clinical utility[END_REF], high circadian patterns for plasma cortisol and adrenocorticotropic hormone (ACTH) [START_REF] Gold | Clinical and biochemical manifestations of depression[END_REF][START_REF] Michelson | Pathophysiologic and somatic investigations of HPA axis activation in patients with depression[END_REF][START_REF] Musselman | Adrenal function in major depression[END_REF][START_REF] Gold | Corticotropin-releasing-hormone in the pathophysiology of melancholic and atypical depression and in the mode of action of antidepressant drugs[END_REF] and high levels of corticotropin realesing factor (CRF) in cerebro-spinal fluid have been described in depressive patients [START_REF] Irwin | Stress-induced immune suppression: role of brain CRH and autonomic nervous system mechanisms[END_REF]. CRF elevation is speculated to be of supra-hypothalamic origin, since depressive patients show a weak ACTH response to CRF injection. This unbalance has been interpreted as a central feedback dysfunction probably due to CRF-receptor downregulation [START_REF] Felten | Noradrenergic sympathetic neural interactions with the immune system: structure and function[END_REF]. According to these findings, it has been postulated that CRF, a neurpopeptide of endocrine, neurochemical, immunological and behavioral effects, would be inadequately down-regulated in major depression, resulting in a persistent activation of the HPA-axis [START_REF] Plotsky | Psychoneuroendocrinology of depression[END_REF][START_REF] Cacioppo | Autonomic, neuroendocrine, and immune responses to psychological stress: the reactivity hypothesis[END_REF][START_REF] Friedman | The role for CRH and the sympathetic nervous system in stress induced immunosuppression[END_REF].

Pro-inflammatory cytokines have endocrine, electrophysiological and behavioral effects [START_REF] Felten | Noradrenergic sympathetic neural interactions with the immune system: structure and function[END_REF][START_REF] Maes | Relationships between interleukin-6 activity, acute phase proteins and function of the hypothalamicpituitary-adrenal axis in severe depression[END_REF][START_REF] Blalock | The syntax of immune-neuroendocrine communication[END_REF]. They are able to activate the HPA-axis [START_REF] Ader | Psychoneuroimmunology: interactions between the nervous system and the immune system[END_REF] at the central level, where they can act on the hypothalamus or the pituitary stimulating CRF or ACTH secretion, and they can stimulate steroidogenesis directly on the adrenal cortex, where interleukin receptors have been identified [START_REF] Path | The role of interleukin-6 in the human adrenal gland[END_REF][START_REF] Haddad | Cytokines and neuro-immuneendocrine interactions: a role for the hypothalamic-pituitary-adrenal revolving axis[END_REF]. Based on this evidence, and considering that the acute administration of cytokines may reproduce many of the physical symptoms of depression, it has been suggested that cytokines play an important role in the pathophysiology of depression (16,20,21). But, although high levels of IL-6, IL-1 and acute phase proteins have been reported in depressive disorders, a typical cytokine profile has not yet been determined and the correlation between cytokine production and HPA axis function is still subject of investigation.

Since progress in neuroscience has associated depression to a situation of physical or psychological chronic stress, in the formulation of the present study we considered an integrated view including endocrine, immune and psychiatric aspects of depression. Our aim is to determine if pro-inflammatory cytokines production is related to HPA axis dysfunction in MDD.

Patients and Methods

Patients.

A psychiatrist selected 9 patients, mainly of hispanic origin, 2 males and 7 females, aged between 19 to 46 years, with Major Depressive Disorder diagnosed according to DSM -IV criteria [START_REF]Diagnostical and statistical Manual of Mental Disorders[END_REF]. The 21-item Hamilton Depression Rating Scale (HDRS) [START_REF] Hamilton | Development of a rating scale for primary depressive illness[END_REF] and the Montgomery-Asberg

Rating Scale for Depression (MADRS) [START_REF] Montgomery | A new depression scale designed to be sensitive to change[END_REF] assessed severity of depression. Patients with other axis I diagnosis, with major medical illness or endocrine disorders, high suicide risk, and those who had taken anti-depressants, lithium salts, anticonvulsants or neuroleptics during the last 12 months were excluded from the study. All patients were free of drugs known to interfere with immune or endocrine function, they had never taken major psychotropic drugs (except benzodiazepines), and

were not regular drinkers.

Eleven healthy controls, 3 males and 8 females, in the same range of age, were selected from volunteers with no lifetime history of axis II or I disorders and no history of axis I disorder in first-degree relatives. They were in good physical health and had not been exposed to any psychotropic medication in the last year.

For at least three weeks prior the study, all the participants did not receive non-steroidal antiinflatory drugs or glucocorticoids and they were free of any infectious disease.

Subjects with twenty-four hour free urinary cortisol (FUC) over 100 g/24 hour were excluded from the study.

HPA Assessment

Twenty-four hour urine collections for measurement of FUC excretion were obtained during the first study day. Subjects were then demanded to come to the Endocrinology Unit of the University of Chile Clinical Hospital at 8 a.m., 3 p.m. and 11 p.m. for three separate plasma cortisol determinations. After the last baseline cortisol sampling, 1 mg. of oral dexamethasone (Oradexon) was administered to all subjects at 11p.m and plasma cortisol levels were determined 9 hours later, according to Carroll's protocol for the dexamethasone suppression test (DST) [START_REF] Carroll | Resistance to suppression by dexamethasone of plasma 11-O.H.C.S. levels in severe depressive illness[END_REF].

Patients were required to return at 8 a.m. to the same hospital a week later to perform the Ovine-Corticotropine-Releasing Factor (oCRF, Bachem, Torrance, CA) stimulation test. An intravenous heparinized cannula was then inserted in each arm. After 30 minutes of supine rest period, baseline samples were obtained (-15 and 0) for basal cortisol and ACTH assessment.

oCRF was administered as an intravenous bolus injection of 1g per kilogram of body weight.

Blood samples were obtained at 5, 15, 30, 45 and 60 minutes for measurements of ACTH and cortisol responses [START_REF] Chrousosgp | The corticotropin releasing factor test. An aid in the evaluation of patients with Cushing´s syndrome[END_REF]. An additional blood sample was drawn before the injection of oCRF for cytokine assessment. Plasma was stored at -20C and processed within the first thirty days from sampling.

Cortisol and ACTH determinations

Cortisol and ACTH were determined by commercial chemoluminescent enzyme immunometric assays (Immulite, Diagnostic Products Corporation, Los Angeles, CA). The intraassay variation coefficient was 7%, with an interassay coefficient of 10% for both hormones.

Cytokine measurements

Induction of IL-1, TNF and IL6

Peripheral blood mononuclear cells (PBMC) were isolated by Hypaque gradient (Sigma Diagnostics, St Louis MO), washed twice in PBS, and 2.5 x 10 6 cells/ml were resuspended in RPMI 1640 culture medium supplemented with gentamicin, glutamine 0.5%, heat-inactivated AB plasma and 1 ug/ml indomethacin. Two hundred L of cell suspension were dispensed in 96-well flatbottom microtiter plates in the absence (spontaneous cytokine release) and presence of endotoxin: lipopolysaccharide (LPS, Sigma) per well for 24 hours at 37C in 5% CO2/95% air. Cell viability after incubation was over 90%. Supernatants were collected and stored at -20ºC until assay for pro-inflammatory cytokines.

Cytokine Determinations

All cytokine determinations were performed using specific, commercial enzyme-linked immuno-absorbent assays (ELISA): (RD Systems, Minneapolis, MN), the assay sensibility being 3.6 pg/ml).

Statistical Analysis

Results are expressed as meanSE. Parametric results were assessed by analysis of variance (ANOVA). The total and integrated ACTH and cortisol responses to oCRF were calculated by the trapezoid method and expressed as the area under the concentration-time curve (AUC) from 0-60 min. The Mann-Whitney statistic test was used to assess the significance of mean differences among both groups with 95% confidence interval. Relationships between variables were assessed by means of Spearman's rank order correlation coefficient. A p-value of less than 0,05 was considered as statistical significant.

Ethical Considerations

This study was approved by the Ethics in Human Research Committee of the Institute of Nutrition and Food Technology, University of Chile, in Santiago de Chile. Informed written consent form was obtained from each patient and control after the aims of the project had been fully explained to them.

Results

There were no significant differences in age, gender, 24-hour FUC, or plasma cortisol circadian variations (8 a.m., 3 p.m. and 11 p.m) between depressive patients and control subjects (Table 1), but, depressive patients, had higher 11 p.m. cortisol level than controls. Patients and controls were both suppressors to dexamethasone under 4 g/dl, but we observed a suppressive response to undetectable levels of cortisol only in controls.

HPA assessment

The responsiveness of the pituitary and adrenal components of the HPA axis to administration of oCRF is shown in fig A positive correlation between ACTH AUC after oCRH injection and LPS-activated IL-6 secretion was observed in major depressives, with a Spearman R of 0,75 (p <0,005) (Fig. 3). We found no correlation between these variables in control subjects (Fig. 3b).

No differences in LPS-stimulated IL-1 and TNF production by PBMC between major depressives and controls could be determined. Spontaneous production was undetectable for both cytokines.

Discussion

The most consistent finding of our study was higher spontaneous IL-6 secretion by PBMC in depressive patients as compared to controls. Conversely LPS stimulated IL-6 secretion was lower in depressives. No differences in IL-1 or TNF- production between depressives and controls were found. An association between altered IL-6 secretion and HPA axis disorder could be established according to the criteria defined for this study.

The alteration of immune parameters in depressive disorders has been widely discussed elsewhere [START_REF] Plotsky | Psychoneuroendocrinology of depression[END_REF][START_REF] Maes | Relationships between interleukin-6 activity, acute phase proteins and function of the hypothalamicpituitary-adrenal axis in severe depression[END_REF][START_REF] Maes | Negative immunoregulatory effects of antidepressants: inhibition of interferon  and stimulation of interleukin 10 secretion[END_REF][START_REF] Leonard | Stress and the immune system in the etiology of depression and anxiety[END_REF][START_REF] Connor | Depression, stress and immunological activation: the role of cytokines in depressive disorders[END_REF][START_REF] Anisman | Endocrine and cytokine correlates of major depression and dysthymia with typical or atypical features[END_REF]. Pro-inflammatory cytokine production currently represents the main subject of interest regarding this association: high levels of IL-6, IL-1 and acute phase proteins have been reported in depressive disorders. Nevertheless, no characteristic cytokine profile for depression has been determined.

The high spontaneous secretion of IL-6 in depressives found in our study agrees with the report from Maes et al [START_REF] Maes | The effects of psychological stress on humans: increased production of proinflammatory cytokines and a Th1-like response in stress induced anxiety[END_REF]. In another report, that included depressive inpatients under antidepressant therapy, Maes et al ( 16) described high IL-6 secretion in PHA-stimulated PBMC; and

Seidel et al [START_REF] Seidel | Cytokine production and serum proteins in depression[END_REF] have reported slightly elevated IL-1 and IL-6 levels in activated PBMC from depressive inpatients. We think, however, that skew problems should be considered in the interpretation of these studies, since inpatients represent a particular group of depressives, including chronic, treatment-resistant depressive disorders, patients with suicidal ideas, major sleep disorders and significant weight loss [START_REF] Maes | The relevance of the in-versus outpatient status for studies on the HPA axis in depression[END_REF]. To avoid this situation, our study design considered strict inclusion and exclusion criteria, resulting in a reduced number of patients and controls. Only healthy subjects, free of any antidepressive or hormonal therapy and within an age range normally not affected by the beginning or decline of the gonadal function were included in our study.

Antidepressants are known to interfere with CRF receptor balance and are able to modify cytokine production [START_REF] Holsboer | Antidepressants and hypothalamic-pituitary-adrenocortical regulation[END_REF][START_REF] Cacioppo | Autonomic, neuroendocrine, and immune responses to psychological stress: the reactivity hypothesis[END_REF][START_REF] Maes | Negative immunoregulatory effects of antidepressants: inhibition of interferon  and stimulation of interleukin 10 secretion[END_REF]. Oral contraceptives can increase pro-inflammatory cytokine production in LPS stimulated PBMC, and also with plasma cortisol determination through an increase in Cortisol Binding Globulin levels [START_REF] Rohleder | Impact of oral contraceptive use on glucocorticoid sensitivity of pro-inflammatory cytokine production after psychosocial stress[END_REF]. Both are widely used drugs in our country. Amongst patients, only major depressives with no personality disorders were included. Moreover, two depression scales were used as a means of homogenization of the study group, considering that the severity of depression has been associated to HPA axis unbalance [START_REF] Maes | Negative immunoregulatory effects of antidepressants: inhibition of interferon  and stimulation of interleukin 10 secretion[END_REF][START_REF] Brown | The 24-hour dexamethasone suppression test in a clinical setting: relationship to diagnosis, symptoms, and response to treatment[END_REF]. Other studies on whole blood production of cytokines have also reported altered serum levels of IL-6 in depressives [START_REF] Steptoe | Lack of association between depressive symptoms and markers of immune and vascular inflammation in middle-aged men and women[END_REF][START_REF] Musselman | Higher than normal plasma interleukin-6 concentrations in cancer patients with depression: preliminary findings[END_REF][START_REF] Kagaya | Plasma concentrations of interleukin-1beta, interleukin-6, soluble interleukin-2 receptor and tumor necrosis factor alpha of depressed patients in Japan[END_REF][START_REF] Kubera | Plasma levels of interleukin-6, interleukin-10, and interleukin-1 receptor antagonist in depression: comparison between the acute state and after remission[END_REF][START_REF] Haack | Plasma levels of cytokines and soluble receptors in psychiatric patients[END_REF][START_REF] Sluzewska | Interleukin-6 serum levels in depressed patients before and after treatment with fluoxetine[END_REF], but unfortunately, disaccording inclusion criteria and methodological difference with our design do not allow direct comparisons with those results. We think that serum cytokine determinations can not be directly compared with the spontaneous production obtained from blood cells in culture.

Spontaneous cytokine production could thus hypothetically explain high plasma levels of IL-6 reported in depression, but this remains a subject of speculation [START_REF] Maes | Increased plasma concentrations of interleukin-6, soluble interleukin-6, soluble interleukin-2 and transferrin receptor in major depression[END_REF].

Based on the differences between spontaneous and stimulated cytokine production observed in our study, it could be argued that lower IL-6 secretion under conditions of stimulation with LPS are due in part to the chronic hyperactivity of the HPA axis. Considering the fact that no intracellular storage system exists for cytokines, we assume that the chronic stress underlying the depressive disorder could lead to a continuous spontaneous maximal IL-6 secretion, and a decreased LPS response.

An association between altered pro-inflammatory cytokine production and the modulation of the stress system in depressive disorders has been postulated. Our study showed a clear association between the functional HPA axis dysfunction and altered cytokine secretion, as manifested by the correlation between blunted ACTH AUC [START_REF] Kathol | Consistent reduction of ACTH responses to stimulation with CRH, Vasopressin and hypoglycaemia in patients with major depression[END_REF][START_REF] Holsboer | Blunted corticotropin and normal cortisol response to human CRF in depression[END_REF] and secretion of IL-6 by activated PBMC in major depressive patients but not in controls. It has been established in animal models that IL-6, a pleiotropic cytokine of immune and non-immune origin, is able to produce an increase in ACTH and corticosterone in the acute response, but in the chronic state might decrease the ACTH response, suggesting that high levels of this cytokine could modulate the activity of the HPA axis [START_REF] Holsboer | Antidepressants and hypothalamic-pituitary-adrenocortical regulation[END_REF][START_REF] Arborelius | The role of corticotropinreleasing factor in depression and anxiety disorders[END_REF][START_REF] Oshima | The differential ACTH responses to combined dexamethasone/CRH administration in major depressive and dysthymic disorders[END_REF]. IL-6 affects then CRH, ACTH and cortisol secretion [START_REF] Maes | Major depression and activation of the inflammatory response system[END_REF] permanently, leading to a more global unbalance of the regulatory mechanisms evidenced as an attenuated ACTH response to oCRH injection, as seen in our patients.

These findings support the cytokine theory for depression, which establishes a proinflammatory cytokine disorder resulting in the HPA dysfunction that underlies the unbalance of neuro-transmission systems traditionally associated to the manifestations of depressive disorders [START_REF] Maes | Relationships between interleukin-6 activity, acute phase proteins and function of the hypothalamicpituitary-adrenal axis in severe depression[END_REF][START_REF] Maes | Immunoendocrine aspects of major depression[END_REF]. An alteration in the contra-regulatory mechanism of cytokines is assumed in the formulation of this theory; glucocorticoids should normally inhibit IL-6 production by PBMC, while in depressives there is a coexistence of increased spontaneous cytokine production and HPA hyperactivity [START_REF] Anisman | Endocrine and cytokine correlates of major depression and dysthymia with typical or atypical features[END_REF]. This theory is further supported by the fact that increased IL-6, leading to acute phase protein synthesis, is associated to a sickness behaviour syndrome that reproduces many of the somatic and vegetative symptoms of the mood disorders. Accordingly, our results suggest that chronic increase of basal IL-6 production could affect the normal regulatory process of HPA function, thus maintaining high cytokine levels in spite of the regulatory effects of cortisol, which are at maximum functional levels, as represented by the blunted ACTH response to oCRH.

Although there were not significant differences in cortisol levels between patients and controls, depressives tend to show slightly higher plasma cortisol levels at 11 pm in depressives.

The outpatient sampling conditions must be considered in the evaluation of this variable. Since healthy controls were exposed to daily stress, including that of coming to the hospital three times a day, one would expect higher cortisol levels in this group.

Considering the criteria defined by Carroll et al. for the 1 mg dexamethasone suppression test in depression, our study did not find Non-suppressors (NS) amongst depressives. Recently, different groups have proposed a cut-point lower than 4 g/dl as suppression criteria for normal subjects, varying from 1.8 to 3 g/dl [START_REF] Wood | Evidence for the low dose dexamethasone suppression test to screen for Cushing´s syndrome-recommendation for a protocol for biochemistry laboratories[END_REF][START_REF] Contreras | Sindrome de Cushing. Revisión a propósito de una casuística[END_REF][START_REF] Arana | The dexamethasone suppression test for diagnosis and prognosis in psychiatry: commentary and review[END_REF][START_REF] Ribeiro | The DST as a predictor of outcome in depression: a meta-analysis[END_REF]. In agreement with this, we found that almost all the control subjects suppressed cortisol to undetectable levels and depressives showed cortisol levels over 1 g/dl after dexamethasone. Ethnical differences in biodisponibility and pharmacokinetics of dexamethasone should also be considered. Other reports have even suggested the need to consider sub-types of depression in the interpretation of the test, since only 14% of depressive outpatients, 36% of major melancholic inpatients, and 41% of depressives with psychotic symptoms were NS in recent reports [START_REF] Zyah | The lower limits of the dexametasone window in Chinese depressives[END_REF][START_REF] Yanovski | Etiology of the differences in corticotropin-releasing-hormone-induced adrenocorticotropin secretion in black and white women[END_REF].

No difference was observed between patients and controls for the cortisol AUC , but the difference for ACTH AUC, decreased in patients, indicates a tendency (p= 0,106), though not significant, considering the reduced number of cases. As it should be expected, the difference in response appears earlier for ACTH (5 min.) than for cortisol (45 min.). This could point to the direct action of IL-6 on the steroidogenic activity in the adrenal cortex.

In conditions of chronic stress such as the model of major depression proposed in this study, HPA axis regulation is altered. The blunted response of ACTH to oCRF, the increase spontaneous IL-6 production and the decrease Il-6 production after LPS challenge established in our study, is a manifestation of the chronic hyperactivity of this axis, main stress regulatory system of the organism. Interleukin-6 determination could be considered a marker of severity of the deregulation of this system, considering that cortisol levels and dexamethasone suppression test show a great variability, are difficult to interpret or are in the case of a CRH response test too expensive to be used in ordinary practice.

Aknowledgments

We wish to thank the precious collaboration of Egardo Caamaño, who kindly processed the blood assays, as well as Daniza Tolosa and Angelica Romero, who were always present during the challenge tests. 

  1. The ACTH and cortisol response curve to oCRF was flatter and delayed in major depressives compared to controls. The response difference was significant at 5 minutes post stimulus for ACTH (Fig 1a) and at 45 min. for cortisol (Fig 1b) (p < 0,05).The AUC ACTH was decreased in major depressives compared with controls; 83413389.9 pg/ml/min versus 186104104.8 pg/ml/min but, the difference was not significant (p=0.06). The AUC cortisol was similar in both groups, 1146.8.2113.6 g/dl/min in major depressives, and 1391.81123 g/dl/min in controls (NS).

	Cytokine

measurements

PBMC spontaneous IL-6 production was significantly higher in major depressives with mean values of 3541.2726.8 pg/ml as compared to a mean value of 380.477.5 pg/ml in control subjects (p<0.001). LPS-induced IL-6 production by PBMC was significantly lower in major depressives 19867.73649.2 pg/ml versus 33142.21547.2 pg/ml in healthy controls, difference that was significant (p < 0,005) (Fig

2

).

Table 1 .

 1 Demographic, clinical and baseline laboratory profile of major depressive patients and control subjects

		Depressive Patients	Control Subjects
		(n = 9)	(n = 11)
	Gender (M/F)	2/7	3/8
	Age	31	33
	HDRS-21	35  3,8	5,3  1,8
	MADRS	35  4,6	5,3  2,1
	FUC g/24 h)	54,9  21,9	55,618,3
	Plasma cortisol (g/dl)		
	8 am	20,1  3,7	19,5  7,7
	3 pm	9,9  2,3	9,6  4,0
	11 pm	4,8  2,1	3,6  1,9
	Suppressors to Dexamethasone	9/9	11/11
	(< 4 g/dl)		

HDRS: Hamilton Depression Rating Scale; MADRS: Montgomery-Asberg Rating Scale; FUC: Free Urinary Cortisol