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-Inducible Chemokine-10/CXC Chemokine Ligand-10 in Autoimmune Primary Adrenal Insufficiency and in Vitro Expression in Human Adrenal Cells Primary Cultures after Stimulation with Proinflammatory Cytokines
Department of Clinical and Experimental Medicine and Surgery F. Magrassi, A. Lanzara, Second University of Naples (M.R., A.D., A.Be.), 80131 Naples, Italy; Department of Internal Medicine, University of Perugia (A.F., S.L., F.S.), 06126 Perugia, Italy; and Department of Clinical Pathophysiology, Endocrinology Unit, University of Florence (P.F., P.R., A.Bu., E.L., C.C., M.M., M.S.), 50139 Firenze, Italy
Address all correspondence and requests for reprints to: Dr. Mario Serio, Department of Clinical Pathophysiology, Endocrinology Unit, University of Florence, V. le Pieraccini 6, 50139 Firenze, Italy. E-mail: m.serio{at}dfc.unifi.it.
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Abstract |
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 Top Abstract IntroductionPatients and MethodsResultsDiscussionReferences |
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and synergistically increased by TNF-
addition. Hydrocortisone or ACTH alone had no effect on CXCL10 secretion in hZFC, but they both significantly inhibited cytokine-induced CXCL10 secretion. Taken together, these data suggest a potential role of hZFC, through the production of CXCL10, in regulating the recruitment of specific subsets of activated lymphocytes in autoimmune AD. |
Introduction |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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Despite high rates of positivity for adrenal cortex autoantibodies (ACA) and 21-hydroxylase autoantibodies (21OHAb) in AD-affected patients, autoantibodies probably play a minor role in tissue damage and more likely reflect an ongoing immune response (6, 7, 8). The histological picture of an affected gland shows diffuse cortical atrophy and focal lymphocytic infiltration, similar to the histological changes commonly observed in other endocrine autoimmune conditions, such as thyroiditis (6).
Both experimental and clinical evidence have sustained the concept that a cell-mediated immune response may represent the pathogenetic mechanism leading to glandular destruction (9, 10, 11). Chemokines are a recently identified family of cytokines that direct normal leukocyte migration and are involved in signaling of leukocyte development, angiogenesis, tumor growth, and metastasis (12, 13, 14). Among chemokines of the CXC family, CXC chemokine ligand 10 (CXCL10) has been recently identified to play an important role in several endocrinological autoimmune diseases, such as Hashimoto’s thyroiditis (HT), Graves’ disease (GD), and type 1 diabetes mellitus (type 1 DM) (15, 16, 17, 18, 19).
Although the mechanisms leading to glandular autoimmune process may be at least in part shared by different endocrine organs, no attempt has been made to date to extend previous observations, mainly of thyroid glands, to other endocrine organs, such as adrenals (20). Although there is extensive literature regarding cytokines in the adrenal cortex (21, 22), little is known about chemokine presence in adrenal disease, with only one clinical observation in an adrenal carcinoma (23). The aim of this study was to evaluate the possible pathogenetic role of CXCL10 in AD as well as to test the validity of the serum CXCL10 assay for the clinical management of such patients.
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Patients and Methods |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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The study group encompassed 93 patients with clinical or subclinical AD. In detail, 64 patients (18 men and 46 women) were affected by clinically evident autoimmune primary adrenal insufficiency, and 9 (seven men and two women) by clinically evident nonautoimmune AD, as diagnosed according to recently revised criteria (2). Six of these latter nonautoimmune AD patients had X-linked adrenoleukodystrophy, and three had postsurgical adrenal insufficiency. The median age and age range of our AD patients were 42 (15–76) and 49 (21–79) yr, for the 64 autoimmune and the nine nonautoimmune cases, respectively. They had been affected by the disease for a median of 5 (0.5–33) yr. They were all treated with corticosteroids and mineralocorticoid replacement therapy. Twenty patients (two men and 18 women) had subclinical autoimmune AD (SAD) and were identified by screening a large cohort of patients with extraadrenal autoimmune endocrine diseases (such as thyroid autoimmune diseases, type 1 DM, premature ovarian failure, autoimmune hypophysitis) for the presence of adrenal cortex autoantibodies (21OHAb and ACA) (24, 25, 26). All 20 SAD patients were positive for both 21OHAb and ACA. Adrenocortical function was evaluated by measuring basal plasma levels of ACTH, cortisol, aldosterone, and PRA; plasma cortisol levels were also evaluated 60 min after iv infusion of 0.25 mg synthetic ACTH (normal peak response, >550 nmol/liter). All 20 SAD patients showed high PRA, normal/low aldosterone levels, and normal basal ACTH (ranging from 9–18 pmol/liter) and cortisol levels (ranging from 260–470 nmol/liter), but impaired cortisol response to ACTH (range, 300–390 nmol/liter), in the absence of clinical signs and symptoms of AD in 12 cases.
The control group constituted 48 healthy volunteers of similar age and sex distribution, mainly recruited among hospital staff and their relatives. Inclusion criteria for the control group were negativity for serum thyroid [thyroglobulin antibodies (Ab) and thyroperoxidase Ab] and adrenal autoantibodies and no evidence of clinical or subclinical autoimmune disease. Serum CXCL10 levels were assayed in all patients and controls. All study subjects gave their informed consent to the study, which was approved by the local ethical committee.
Materials
DMEM and Ham F-12 (1:1 mixture), PBS, BSA, glutamine, antibiotics, insulin/transferrin/selenium, and collagenase type IV were obtained from Sigma-Aldrich Corp. (St. Louis, MO). Fetal bovine serum was obtained by Unipath (Bedford, UK). Synthetic ACTH [ACTH-(1–24), Synacthen] was purchased from Ciba (Wehir, Germany). Mouse antihuman monoclonal antibody against vimentin (clone V9) was purchased from DakoCytomation (Carpinteria, CA). The ACS:Centaur kit was obtained from Chiron Diagnostics (Rome, Italy). Plasticware for cell cultures was purchased from Falcon (Oxnard, CA). Disposable filtration units for growth medium preparation were purchased from PBI International (Milan, Italy).
Tissue specimens
A total of eight tissues specimens were used in this study. Normal adrenals were removed during expanded nephrectomy due to renal carcinoma or from organ donors (age, 32–72 yr). Approval for the use of human material was given by the local ethical committee. Adrenocortical fragments, collected immediately after surgery, were processed for cell preparations. Part of the adrenocortical fragments was paraffin-embedded for morphological analysis, performed by hematoxylin-eosin staining; the typical structure of the zona fasciculata (ZF), characterized by cells organized in columns surrounded by a tight capillary network, was evidenced (not shown).
Cell cultures of human ZF cells (hZFC)
Primary cell cultures of hZFC were established from six normal adrenals, within 1 h after surgery, as described by Munari-Silem et al. (27) with some modifications (28). Briefly, normal adrenals were freed of fat and decapsulated, and the subcapsular zone as well as the medulla were discarded. The remaining tissue was minced and incubated for 20 min at 37 C in PBS containing 2 mg/ml collagenase. To facilitate dispersion, tissue was minced with a Pasteur pipette with a fine heat-polished tip, and the cell suspension was filtered through a cell strainer (80 µm pore size mesh; Sigma-Aldrich Corp.) and centrifuged 10 min at 1400 rpm. The pellet was resuspended in a culture medium consisting of a 1:1 (vol/vol) mixture of DMEM/F-12 with 10% fetal bovine serum, 2 mM glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin, enriched with a mixture of insulin/transferrin/selenium. Isolated cells were plated onto 35-mm diameter culture dishes at density of 50–70 x 103 cells/dish and cultured at 37 C in 95% air/5% CO2 in a fully humidified environment.
In vitro cortisol secretion studies
For metabolic characterization of ZF cells, cortisol was tested along with 17-hydroxyprogesterone and aldosterone secretion, after 24 h of growth in their growth medium, in five preparations of ZF cells. Cortisol was by far the main secretory product of the cultured adrenocortical cells, thus supporting their ZF origin (data not shown). In our experimental conditions, cell proliferation was negligible. Mouse antihuman monoclonal antibody against vimentin (clone V9) was used to characterize cell cultures. Cells showed positive staining for vimentin (>85% for all preparations), supporting their mesodermal origin (data not shown).
In vitro CXCL10 secretion studies
Primary cultures were used within 3–4 d; cells were washed once with PBS, trypsinized, and counted with a hemocytometer. For CXCL10 secretion assays, 3000 cells were seeded onto 96-well plates in growth medium. After 24 h, the growth medium was removed, and the cells were accurately washed in PBS and incubated in phenol red- and serum-free medium. After 24-h starvation, different stimuli were added in phenol red- and serum-free medium containing 0.1% BSA (200 µl/well). Cells were incubated for 24 h with proinflammatory cytokines [1000 U/ml interferon- (IFN-); R&D Systems, Minneapolis, MN], 8 ng/ml TNF- (R&D Systems), and 1 nmol ACTH, alone or in combination. According to previous studies (15), these concentrations were selected after preliminary studies to yield the highest responses. After 24 h, the medium (adrenocortical cell-conditioned medium) was kept frozen at –20 C until performing the CXCL10 assay. Cells in phenol red- and serum-free medium containing 0.1% BSA were used as the basal control. All experiments were repeated three times with three different cell preparations. To differentiate direct effects of ACTH from secondary effects mediated by glucocorticoids (GC), a second set of experiments was performed. Cells were incubated for 24 h with proinflammatory cytokines [1000 U/ml IFN- and 8 ng/ml TNF- alone or in combination in the presence of 1, 10, and 100 nmol hydrocortisone (ICN Biomedicals, Inc., Aurora, OH)]. The second set of experiments was repeated three times with two different cell preparations.
Steroid hormone measurement
Steroid hormone secretion into adrenocortical cell-conditioned medium was measured by direct chemiluminescent assay kit (ACS:Centaur, Chiron Diagnostics, Emeryville, VA) with specific anticortisol, anti-17-hydroxyprogesterone, and antialdosterone antibodies. Results for steroid hormones were calculated for 106 cells.
Serum assays
Serum CXCL10 levels were assayed by a quantitative sandwich immunoassay using a commercially available kit (R&D Systems), with a sensitivity ranging from 0.41–4.46 pg/ml and a mean minimum detectable dose of 1.67 pg/ml. The intra- and interassay coefficients of variation were 3% and 6.9%, respectively. Samples were assayed in duplicate. Quality control pools at low, normal, and high concentrations were present in each assay.
Immunofluorescence and laser confocal microscopy
Human zona fasciculata cells were cultured for 24 h in the presence or absence of IFN- and TNF- in chamber slides, fixed with acetone for 10 min, and then incubated with a rabbit antihuman CXCL10 polyclonal antibodies (PeproTech Europe, London, UK) and a goat antirabbit 488 (1:1000; Molecular Probes, Eugene, OR) as secondary antibody. For double-label immunofluorescence, sections were incubated with monoclonal anticytokeratin antibodies (clone C-11, Sigma-Aldrich Corp., St. Louis, MO) for 30 min, followed by goat antimouse IgG1 546 (1:1000, Molecular Probes). After appropriate washings, a second incubation was performed with rabbit antihuman CXCL10 polyclonal Ab (PeproTech Europe), followed by goat antirabbit 488 (1:1000; Molecular Probes) as secondary antibody. Nuclei were counterstained with Topro-3. Slides were mounted in antifading mounting media (Vectashield, Vector Laboratories, Inc., Burlingame, CA) and examined by conventional confocal microscopy on an LSM 510 META microscope system (Carl Zeiss, Inc., Jena, Germany).
Statistical analysis
Statistical analysis was performed using SPSS software (SPSS, Inc., Evanston, IL). The comparison of serum IP-10/CXCL10 levels among different groups was performed by Mann-Whitney U test for unpaired data. Correlation between two variables was ascertained by linear regression analysis and Spearman’s correlation test. Frequencies between groups were compared by 
2 test. P < 0.05 was considered statistically significant. To test the independent effects of different variables independently of a covariate, multiple regression analysis was used, and partial correlation coefficients were computed. Data are reported as the median and ranges unless otherwise noted.
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Results |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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Serum CXCL10 levels were assessed in 64 patients affected by proven autoimmune AD, 20 patients with SAD, nine patients with AD due to nonautoimmune cause (NAAD), and 48 healthy volunteers without any clinical or biochemical evidence of autoimmune disease. As shown in Fig. 1
, serum CXCL10 levels were significantly increased in both clinically evident [119.9 pg/ml (39.8–427.6)] and subclinical AD [124.0 pg/ml (37.0–384.7)] groups (P < 0.001 for both groups) and were comparable in NAAD patients [89.4 (56.3–149.6); not significant] vs. healthy controls [75.6 pg/ml (22.4–164.0)].
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2 = 22.677; P < 0.0001).
To evaluate the roles of other autoimmune conditions often associated with adrenal insufficiency, all AD patients were assigned to two groups on the basis of IAD (n = 25) or AD with concomitant autoimmune diseases (APS; n = 39; Table 1). As shown in Fig. 2, comparable serum CXCL10 levels were found between the two groups [117.8 pg/ml (39.8–427.6) and 121.9 pg/ml (43.6–368.9) for IAD and APS, respectively; not significantly different], whereas serum CXCL10 levels were significantly higher in both IAD and APS patients compared with controls (P < 0.001 for both comparisons). Comparable serum CXCL10 levels were found between the 20 male and 64 female patients with both clinical and subclinical AD [91.3 pg/ml (39.8–427.6) and 137.3 pg/ml (37.0–386.2) for males and females, respectively; not significantly different], indicating no influence of gender on serum CXCL10 levels in AD. Similarly, no correlation between serum chemokine levels and age or duration of disease was observed. To assess possible relationships between serum CXCL10 and commonly assayed serum markers of autoimmunity in AD, anti-21OHAb and ACA were taken into account. No relationship between serum CXCL10 levels and autoantibody titers was found.
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The evaluation of chemokine secretion in normal adrenal cells was performed by assaying CXCL10 levels in culture supernatants from hZFC. As shown in Fig. 3, although CXCL10 was absent in cultured adrenal cells under basal conditions, significant secretion of CXCL10 was induced in hZFC by stimulation with IFN- or IFN- plus TNF (P < 0.00005 for both comparisons). Stimulation of hZFC with TNF- alone was not able to induce chemokine secretion (data not shown); nevertheless, TNF- had a relevant synergic effect with IFN- in determining CXCL10 production. In fact, the combination of IFN- and TNF- significantly increased CXCL10 secretion compared with IFN- alone (P < 0.00005).
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To evaluate the effect of IFN- on CXCL10 secretion in the presence of the major physiological regulator of adrenal homeostasis, combinations of IFN- and IFN- plus TNF- with ACTH have been used. ACTH alone had no effect on CXCL10 secretion in these cells, but it significantly influenced cytokine-mediated CXCL10 secretion. In fact, stimulation with cytokines in the presence of ACTH resulted in CXCL10 levels in supernatants significantly higher than control values (P < 0.005 and P < 0.0001 for ACTH in combination with IFN- and IFN- plus TNF-, respectively), but significantly lower than those observed in the absence of ACTH (P < 0.0001 and P < 0.01 for IFN- and IFN- plus TNF- with or without ACTH, respectively; Fig. 3
). To differentiate direct effects of ACTH from secondary effects mediated by GC, experiments were repeated in the presence of 1, 10, and 100 nmol hydrocortisone (ICN Biomedicals, Inc.). As shown in Fig. 4, increasing concentrations of hydrocortisone progressively and significantly inhibited IFN--induced and IFN-- plus TNF--induced CXCL10 secretion (P < 0.001 and P = 0.001 for IFN- and IFN- plus TNF-, respectively).
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The expression of CXCL10 protein was assessed using immunofluorescence and was analyzed by laser confocal microscopy. High immunoreactivity was widely present in hZFC 24 h after stimulation with IFN- and TNF- and was potentiated by pretreatment with brefeldin A (5 µg/ml; Sigma-Aldrich Corp.; Fig. 5). No staining was observed under basal conditions or when primary Ab was replaced with an isotype-matched control Ab with irrelevant specificity (data not shown). To better characterize the nature of CXCL10-producing cells, double immunofluorescence for CXCL10 (green) and cytokeratin (red) was performed (Fig. 5). The results demonstrated that hZFC were the major source of CXCL10 in our primary cultures (Fig. 5). Negative controls for the double-label immunofluorescence consisted of sections stained with anti-CXCL10 antibody, revealed, and then stained with an isotype control Ab.
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Discussion |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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Increased circulating concentrations of CXCL10 have been reported in several human autoimmune conditions (29, 30, 31). As far as autoimmune endocrine diseases are concerned, high CXCL10 serum levels have been found in GD, HT, and type 1 DM (16, 17, 18, 19). In this respect, a significant increase in CXCL10 found in AD patients may be expected. However, some peculiar aspects of AD should be discussed. Serum CXCL10 levels have been found to be correlated with the time since diagnosis of GD or type 1 DM (16, 18, 19). The lack of correlation we report between CXCL10 and duration of disease should be regarded as a consequence of the particular course of AD. In fact, due to the great physiological reserve of adrenal glands, clinical symptoms of adrenal deficiency become manifest only when massive glandular destruction has occurred (4). It is therefore reasonable to assume that time since diagnosis, as in HT, but not in GD, is not positively representative of the duration of the autoimmune process (16, 17, 18). Similarly, the lack of correlation between CXCL10 and anti-21OHAb and ACA seems in line with the previously reported absence of a correlation between CXCL10 and thyroglobulin Ab or TPO Ab in HT patients (17).
Evaluation of SAD patients has not provided additional information; in this case, also, circulating chemokine levels show great variability among patients. The lack of gender-dependent differences in CXCL10 serum levels in AD is of interest. In fact, in our previous experience, we found a significant increase in serum CXCL10 levels only in type 1 DM females, but comparable levels were observed between type 1 DM males and healthy controls (32). Such results were interpreted as a consequence of the frequently associated presence of other clinical or subclinical autoimmune diseases in females with autoimmune diabetes (33). In this view, the fact that both IAD and APS patients showed increased serum CXCL10 levels with respect to controls regardless of gender together with the finding of comparable levels between NAAD and controls may be regarded as suggesting a specific influence of the adrenal autoimmune process in determining elevated CXCL10 concentrations in such patients.
Intraadrenal cytokine production has been known, and previous reports had identified local immune cells as the main source (21). Furthermore, a recent case report demonstrated overexpression of CXC chemokines (IL-8, ENA-78, Gro-, and Gro-) by an adrenocortical carcinoma (23).
Although there is extensive literature regarding cytokines in the adrenal cortex, there are no reports examining the expression or secretion of the subclass of CXC chemokines and, in particular, of Th1 autoimmunity-associated chemokines. In this study we provide evidence that hZFC when stimulated with proinflammatory cytokines are able to produce chemokines, as assessed by cell supernatant assay and laser confocal immunofluorescence. Such results constitute the first demonstration of CXC chemokine production by stimulated human adrenal cells and suggest a role for the inflamed glandular epithelium in the recruitment of specific subsets of infiltrating lymphocytes. The elevated levels of circulating CXCL10 observed in autoimmune AD patients may result from secretion by both lymphocytes and hZFC stimulated with IFN-, in line with previous evidence from studies of thyroid autoimmune disease (15, 16, 17, 34).
Another finding that should be discussed is the influence of hydrocortisone on IFN--mediated effects. In fact, stimulation of hZFC with IFN- or IFN- plus TNF in the presence of increasing concentrations of hydrocortisone yielded a lower response of CXCL10 secretion. Similar effects were observed after stimulation with proinflammatory cytokines and ACTH rather than hydrocortisone, in line with the prompt response of cortisol secretion by hZFC to treatment with ACTH. GC are known for their potent antiinflammatory and immunosuppressive actions (35). Furthermore, GC are able to suppress the production of several cytokines and chemokines by inhibiting the nuclear factor-
B and activating protein-1 transcription factor families (36). It has been reported previously that GC inhibited IFN- induction of major histocompatibility class II expression, and a recent report has demonstrated that GC may also inhibit IFN--inducible gene expression, indicating that GC can also suppress IFN- activity (37, 38). Taken together, our results indicate that the role of IFN--induced chemokines in the pathogenesis of the glandular autoimmune process is a mechanism at least in part shared by different endocrine organs. A serum CXCL10 assay for the clinical follow-up of such patients is of potential interest; however, only performance of prospective clinical trials will be able to verify the importance of serum determination as a parameter for better management of such patients.
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Footnotes |
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Abbreviations: Ab, Antibody; ACA, adrenal cortex autoantibody; AD, Addison’s disease; APS, autoimmune polyglandular syndrome; CXCL, CXC chemokine ligand; DM, diabetes mellitus; GC, glucocorticoid; GD, Graves’ disease; HT, Hashimoto’s thyroiditis; hZFC, human zona fasciculata cell; IAD, isolated Addison’s disease; IFN-, interferon-; NAAD, Addison’s disease due to nonautoimmune cause; 21OHAb, 21-hydroxylase autoantibody; SAD, subclinical autoimmune Addison’s disease.
Received June 8, 2004.
Accepted January 3, 2005.
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References |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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-inducible chemokine IP-10/CXCL10 are augmented in both newly diagnosed type I diabetes mellitus patients and subjects at risk of developing the disease. Diabetologia 45:1107–1110[CrossRef][Medline]
inducile chemokines in endocrine autoimmunity: an expanding field. J Endocrinol Invest 26:177–180[Medline]
-inducible chemokine IP-10/CXCL10 are augmented in both newly-diagnosed type I diabetes mellitus patients and subjects at risk of developing the disease. Diabetologia 46:1020–1021[CrossRef][Medline]
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