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. Author manuscript; available in PMC: 2017 Dec 5.
Published in final edited form as: J Neurochem. 2016 Dec 5;139(6):1138–1150. doi: 10.1111/jnc.13870

Interleukin-6 mediated signaling in adrenal medullary chromaffin cells

Danielle E Jenkins a, Dharshini Sreenivasan b, Fiona Carman a, Babru Samal c, Lee E Eiden c, Stephen J Bunn a
PMCID: PMC5177545  NIHMSID: NIHMS825897  PMID: 27770433

Abstract

The pro-inflammatory cytokines, tumor necrosis factor-α and interleukin-1β/α modulate catecholamine secretion, and long-term gene regulation, in chromaffin cells of the adrenal medulla. Since interleukin-6 (IL6) also plays a key integrative role during inflammation, we have examined its ability to affect both tyrosine hydroxylase activity and adrenomedullary gene transcription in cultured bovine chromaffin cells. IL6 caused acute tyrosine/threonine phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2), and serine/tyrosine phosphorylation of signal transducer and activator of transcription 3 (STAT3). Consistent with ERK1/2 activation, IL6 rapidly increased tyrosine hydroxylase phosphorylation (serine-31) and activity, as well as up-regulated genes, encoding secreted proteins including galanin, vasoactive intestinal peptide, gastrin releasing peptide and parathyroid hormone-like hormone. The effects of IL6 on the entire bovine chromaffin cell transcriptome were compared to those generated by G-protein coupled receptor (GPCR) agonists (histamine and pituitary adenylate cyclase-activating polypeptide) and the cytokine receptor agonists (interferon-α and tumor necrosis factor-α). Of 90 genes up-regulated by IL6, only 16 are known targets of IL6 in the immune system. Those remaining likely represent a combination of novel IL6/STAT3 targets, ERK1/2 targets and, potentially, IL6-dependent genes activated by IL6-induced transcription factors, such as hypoxia-inducible factor 1α. Notably, genes induced by IL6 include both neuroendocrine-specific genes activated by GPCR agonists, and transcripts also activated by the cytokines. These results suggest an integrative role for IL6 in the fine-tuning of the chromaffin cell response to a wide range of physiological and paraphysiological stressors, particularly when immune and endocrine stimuli converge.

Keywords: chromaffin cell, cytokine, inflammation, interleukin 6, STAT3, tyrosine hydroxylase

Graphical abstract

The cytokine interleukin-6 (IL6) plays a key integrative role during inflammation. While it was shown that other pro-inflammatory cytokines modulate catecholamine secretion and long-term gene regulation in chromafin cells of the adrenal medulla the effect of IL6 in these cells remains elusive. We provide evidence that IL6 interacts directly with those cells to rapidly increase the phosphorylation and activity of the catecholamine synthesizing enzyme tyrosine hydroxylase. Prolonged exposure to IL6 increased the expression of a wide range of genes, including those for a number of biologically active neuropeptides. IL6 can thus potentially modulate the adrenal medullary stress response.

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Introduction

The chromaffin cells of the adrenal medulla play a major role in physiological adaptation to stress by secreting catecholamines (adrenaline and noradrenaline) and a range of biologically active peptides. Acetylcholine, released from the splanchnic nerve, leads to nicotinic receptor-mediated exocytosis of chromaffin cell secretory granules in the basal state. PACAP, released from the splanchnic nerve at higher firing frequencies, maintains the secretory output of the adrenal medulla during stress, acting via PAC1 receptors expressed on the chromaffin cells (Stroth et al. 2013). Adrenal medullary activity is subject to a number of other physiological inputs including endocrine and paracrine signals. Angiotensin II, for example, promotes both catecholamine secretion and synthesis (Cavadas et al. 2003, Bobrovskaya et al. 2007). Paracrine mediators such as glucocorticoids, prostaglandin E2, and histamine also influence secretory or biosynthetic activity of chromaffin cells (Schinner & Bornstein 2005, Ehrhart-Bornstein & Bornstein 2008, Currie et al. 2000, Jewell et al. 2011, Marley 2003). Thus the physiological secretory activity of the chromaffin cell is influenced by a wide-range of biological factors.

Recent studies using isolated adrenal chromaffin cells have provided evidence that the pro-inflammatory cytokines interleukin-1 (IL1), tumor necrosis factor alpha (TNFα) and interferon-α (IFNα) directly target these cells (Bunn et al. 2012, Samal et al. 2015, Tamura et al. 2014, Ait-Ali et al. 2008, Eskay & Eiden 1992, Rosmaninho-Salgado et al. 2009, Tachikawa et al. 1997, Douglas & Bunn 2009). In each case, stimulation of the appropriate receptor results in the activation of a cytokine-specific intracellular signaling pathway followed by a delayed alteration in gene expression. Recent results suggest an interaction between PACAP transmission at the adrenomedullary synapse during stress-induced catecholamine secretion and cytokine regulation of chromaffin cell plasticity in vivo (Ait-Ali et al. 2010b).

Many of the genes subject to cytokine-mediated regulation code for neuropeptides co-secreted with adrenal medullary catecholamines (Douglas et al. 2010, Bunn et al. 2012). Exposure of isolated bovine chromaffin cells to TNFα, for example, increased mRNA levels for the neuropeptides galanin, vasoactive intestinal peptide (VIP) and secretogranin II (Ait-Ali et al. 2008, Ait-Ali et al. 2004, Eskay & Eiden 1992, Turquier et al. 2002). IL1 had a similar action, increasing mRNA levels for these neuropeptides as well as increasing the secretion of both secretoneurin and enkephalin (Ait-Ali et al. 2004, Eskay & Eiden 1992). This cytokine also increased the release of neuropeptide-Y from both isolated mouse and human adrenal chromaffin cells (Rosmaninho-Salgado et al. 2007, Rosmaninho-Salgado et al. 2009).

While TNFα and IL1 are major pro-inflammatory cytokines, they are only part of a complex intercellular cytokine-signaling cascade. In the classical inflammatory response, locally generated TNFα and IL1 act on immunocytes to stimulate the synthesis and release of interleukin-6 (IL6) (Hunter & Jones 2015, Scheller et al. 2011). This latter cytokine has extensive, well-documented, pro- and anti-inflammatory actions on a wide range of target tissues (Scheller et al. 2011, Scheller et al. 2014). While IL6 is an essential stimulator of the adrenal cortex during immune activation and is elevated during stress, its actions on the adrenal medulla are largely unknown (Bethin et al. 2000, Rohleder et al. 2012).

In this study we provide evidence that IL6 interacts directly with isolated bovine adrenal chromaffin cells to stimulate the extracellular signal-regulated kinase 1/2 (ERK1/2) and signal transducer and activator of transcription 3 (STAT3) intracellular signaling pathways with the resultant increase in the site-specific phosphorylation and activation of tyrosine hydroxylase (TH; the rate-limiting enzyme in catecholamine synthesis) and the elevation of mRNA expression for a number of neuropeptides. These observations provide the first evidence that IL6 can regulate adrenal chromaffin cell signaling, protein phosphorylation and gene transcription, with the potential to modulate the secretory output of the adrenal medulla in response to inflammation and stress. IL6, by modifying both catecholamine and neuropeptide synthesis, and thus the secretory cocktail of the chromaffin cell, may have an important role in integrating and limiting the course of the inflammatory response at the adrenal medulla.

Materials and Methods

Isolation and Culture of Bovine Adrenal Medullary Chromaffin Cells

Intact adrenal glands from steers were kindly provided by local licensed abattoirs and placed at 4°C within 20 min post-mortem. Adrenal medullary chromaffin cells were isolated and purified as described previously (Roberts-Thomson et al. 2000, Anouar et al. 1999), and cultured at a density of 1.0 - 1.5 × 106 cells per well in 24- or 12-well collagen-coated plates for qPCR and western blotting experiments. For immunocytochemistry, cells were plated at a density of 100,000 per well in 24-well plates on collagen-coated glass coverslips, while those employed in microarray analysis were cultured in poly-D-lysine-coated 6-well plates at a density of 2 × 106 cells per well. Experiments were performed after 2 - 9 days in culture.

Incubation of chromaffin cells with IL6 and other stimuli

The culture medium was removed and each well washed twice for 5 min with 1 ml HEPES buffer (10 mM HEPES, 144 mM NaCl, 5.2 mM KCl, 1.2 mM MgSO4, 2.2 mM CaCl2, 10 mM glucose, 1% w/v bovine serum albumin, pH 7.4). The second wash was replaced with 500 μl of buffer containing the required concentration of recombinant human IL6 (routinely 1 nM, a concentration indicated in preliminary experiments to be approximately half-maximal for pY-STAT3 response, obtained from PeproTech) and incubated at 37°C for 5 - 60 min. Alternative stimuli (100 nM phorbol 12-myristate 13-acetate (PMA), 100 nM angiotensin II, 10 μM histamine or 3 μM nicotine) were used in the same way with modified incubation times as indicated in Results.

Western blotting

Stimulated cells were lysed in 100 μl of 4% SDS (in 10 mM Tris containing 2 mM EDTA) and processed for western blot by incubating overnight with antibodies to one or more of the following: STAT3 (1/100), tyrosine phosphorylated STAT3 (1/200), ERK1/2 or phosphorylated ERK1/2 (1/1000), p38 or phosphorylated p38 (1/1000), c-Jun N-terminal kinases (JNK) or phosphorylated JNK (1/1000) (Cell Signaling), serine phosphorylated STAT3 (1/1000), TH (1/10,000), TH phosphorylated Ser19 or Ser31 (1/200 and 1/1000 respectively, Chemicon International), TH-ser40 (1/5000, Zymed) or β-actin (1/12,000, Abcam Ltd), followed by appropriate HRP-conjugated secondary antibodies (diluted 1/5000, GE Heathcare) and ECL as per manufacturer’s instructions. Blots, each of which included β-actin loading control, were quantified by density measurement on a BIO-RAD GS-700 Imaging Densitometer.

Immunocytochemistry

Stimulated cells were fixed in cold methanol (−20°C) for 5 min, air dried, blocked with phosphate buffered saline (PBS) containing 4% v/v goat serum and then incubated overnight at 4°C with antibodies to one or more of the following; STAT3 (1/400), tyrosine or serine phosphorylated STAT3 (1/200), TH (mouse monoclonal, 1/10,000) or TH (rabbit polyclonal, 1/20,000), followed by appropriate fluorescently-conjugated secondary antibodies (Alexa Fluor 488 or 568, Molecular Probes) for 1.5 h at room temperature. Coverslips were washed twice in PBS, mounted in Vectashield (Vector Laboratories) and examined under fluorescence microscopy using an Olympus AX70 microscope fitted with a Spot-RT Color digital camera.

Measurement of TH activity

TH activity was determined exactly as described previously using a 3H-water release assay on lysates obtained from IL6-stimulated cells (Bobrovskaya et al. 2004). Data were normalized relative to basal TH activity measured for the same time period in buffer alone and analyzed using a Mann-Whitney U-test.

Microarray Procedure and Data Analysis

These procedures were conducted as described previously (Samal et al. 2013). Briefly, total RNA was isolated using Trisol and RNAqueous kit (Ambion Life Technologies) and RNA quality and quantity were evaluated by Bioanalyzer (Agilent, Inc) in the NHGRI-NINDS-NIMH Microarray Core Facility. Only RNA with an RNA Integrity Number greater than 7 was used for microarray analysis. 5 µg of RNA was employed for One-Cycle Target Labeling, according to the Affymetrix recommended protocol (Affymetrix P/N 900493). Hybridizations were performed in triplicate using linear-amplified RNA from cells incubated with or without IL6 (10 nM, a maximally effective concentration, for 24 h), human IFNα (PeproTech, 1 nM for 24 h), or histamine (10 μM for 6 h). The hybridization cocktail containing biotin-labeled cDNAs was added to the Affymetrix GeneChip Bovine Genome Array (P/N 900562). Using the Affymetrix Fluidics Station and following standard Affymetrix protocol, chips were hybridized and washed and then stained with streptavidin phycoerythrin solution (Molecular Probes), and enhanced with a 0.5 mg/mL biotinylated anti-streptavidin antibody solution (Vector Laboratories). The probe arrays were scanned using an Affymetrix Gene Chip Scanner 3000. Scanned arrays were analyzed to identify transcripts significantly up- or down-regulated by various treatments applying robust multichip analysis (RMA) normalization and ANOVA in the Partek Genomic Suite platform (Partek, Inc.) as previously described (Samal et al. 2015). Gene lists were subsequently parsed to include only those transcripts significantly up- or down-regulated ≥2.0 fold in the treated groups compared to the untreated controls. TranscriptIDs were used to obtain transcript sequences from the Affymetrix website (http://www.affymetrix.com/) which were BLASTed at NCBI (http://www.ncbi.nlm.nih.gov/) to obtain latest gene symbols. Bioinformatic analyses were performed on up-regulated gene cohorts from IL6-, IFNα- and histamine-treated chromaffin cell samples, as well as parallel gene lists obtained as described previously for regulation by PACAP and TNFα (Samal et al. 2013, Samal et al. 2007, Ait-Ali et al. 2010a).

Vennture, a Venn diagram investigation tool (http://www.grc.nia.nih.gov/), was used to identify the regulation of activated genes and their upstream transcription factors associated with the five different first messenger stimuli (Martin et al. 2012). Transcripts or transcription factors which were found to be significant for the activation by the above stimuli (2-fold or greater) were presented as input files to generate the graphical outputs presented in Figure 9.

Quantitative Polymerase Chain Reaction (qPCR)

The effect of IL6 on the mRNA levels for a number of selected transcripts were determined exactly as described previously (Ait-Ali et al. 2010a). Freshly isolated chromaffin cells were cultured for 24 h in 12-well plates at a density of 1.2 × 106 cells per well and then IL6 (10 nM) was added for a further 24 h. Total RNA was isolated using RNeasy Mini Spin Columns (QIAGEN) and quantified by spectrophotometry. Approximately 2 μg was then reverse transcribed using random hexamers pdN6 (Invitrogen) and SuperScript II RNase H reverse transcriptase (Invitrogen). The forward and reverse primer pairs, spanning exon-exon boundaries, were designed using Primer 3 as follows:

Gene Forward Reverse
SOCS 5′-CCT CAA GAC CTT CAG CTC CA-3′ 5′-ACG CTG AGG GTG AAA AAG TG-3′
galanin 5′-GAC AGC CAC AGG TCA TTT CAA-3′ 5′-GCC GGG CTT CGT CTT CA-3′
VIP 5′-GGC AGC GTT ATC CTG CTA GG-3′ 5′-CTG TTC AGG GTC CAA CCT CT-3′
PTHLH 5′- AGC AAT GGA GCG TCG CAG TGT-3′ 5′-ACA GCC CTT TTG AGT CGG CG-3′
GRP 5′-ACC ACT GGG CTG TCG GAC ACT TAA-3′ 5′-GGC ATA CTC CCT CAG CTG CTC CT-3′
STC1 5′-GCA ACC CAT GAG GCG GAG CAG-3′ 5′-GAG GCA GCG AAT CAC TTC AGC TGA G-3′
ANGPT2 5′-CGC GCC CCT TGA CTA CGA CG-3′ 5′-TCT CAA GCT TCA TGA GCC ACT GTG-3′
HPRT1 5′-CTT CAG GGA CTT GAA TCA CGT GTG TGT-3′ 5′-CGC CAG GTA TTT CCA AAC TCA ACT CGG-3′
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Statistical analysis

For qPCR, fold changes in mRNA levels were determined by normalizing against hypoxanthine-guanine phosphoribosyltransferase-1 (HPRT1) mRNA, a non-variable control transcript using the ΔΔCt method, and statistical analysis was performed using a Mann-Whitney U-test, comparing IL6 treated cells with those incubated for the same time period with buffer alone. For western blotting semi-quantitative analysis was always made within the same blot with reference to a control value (see Figure legends for specific details) and statistical analysis was performed using a Mann-Whitney U-test, comparing IL6 treated cells with those incubated for the same time period with buffer alone.

Results

Incubation of chromaffin cells with IL6 (1 nM) resulted in a time-dependent increase in tyrosine-705 (Y-pSTAT3) and serine-727 (S-pSTAT3) phosphorylation of STAT3 (Figure 1). The Y-pSTAT3 response was relatively slow, peaking between 15 - 30 min at approximately 250% basal levels (Figure 1a). In contrast, the increase in S-pSTAT3 was more rapid and transient, being maximally elevated at 5 min (Figure 1b). Immunocytochemistry revealed an IL6-stimulated (15 min) increase in Y-pSTAT3 in the nuclei of some, but not all, the chromaffin cells (Figure 2). The number of Y-pSTAT3 immunoreactive chromaffin cells increased with time, reaching a maximum of approximately 70% after 30 min stimulation (data not shown). Staining for non-phosphorylated STAT3 revealed a punctate nuclear localization in all cells, which was independent of IL6 (Figure 2). S-pSTAT3 immunoreactivity was low, but increased with IL6 incubation (15 min) to appear as a perinuclear ring in some cells (Figure 2). Incubation with IL6 rapidly (5 min) elevated the phosphorylation, and thus activation, of ERK1/2 (Figure 3a). Immunocytochemistry revealed that the IL6 stimulated increase was predominantly cytoplasmic (Figure 2). In contrast to its activation of ERK1/2, IL6 failed to elevate the phosphorylation of either p38 or JNK over a 5 - 30 min time period (Figure 3b and 3c). To investigate the possible interaction between STAT3 and ERK1/2 signaling, cells were incubated in the presence of the mitogen-activated protein kinase kinase (MEK) inhibitor PD98059 which abolished the IL6-stimulated increase in pERK1/2 and S-pSTAT3 but not Y-pSTAT3 (Figure 4). The association between pERK1/2 and S-pSTAT3 was further investigated by examining the effect of a range of agonists in addition to IL6. PMA (100 nM), angiotensin II (100 nM), histamine (10 μM) and nicotine (3 μM) all significantly increased pERK1/2 and S-pSTAT3. IL6 was the only stimulus that also elevated Y-pSTAT3 levels (Figure 5). Given that the serine-31 residue of TH is a known substrate for pERK1/2 in chromaffin cells (Dunkley et al. 2004), the ability of IL6 to increase the phosphorylation of this site was examined. Incubation with IL6 resulted in a rapid increase in TH phosphorylation on serine-31 (approximately 180% basal after 15 min) with no significant increase in either serine-19 or serine-40 phosphorylation (Figure 6a). Coincident with increasing serine-31 phosphorylation IL6 elevated TH activity to approximately 150% basal (Figure 6b).

Figure 1. IL6 stimulated a time-dependent increase in the tyrosine and serine phosphorylation of STAT3.

Figure 1

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Adrenal chromaffin cells were incubated for increasing periods of time with (shaded columns) or without (open columns) IL6 (1 nM). Panel a) shows the level of tyrosine-705 phosphorylated STAT3 (Y-pSTAT3) and panel b) the level of serine-727 phosphorylated STAT3 (S-pSTAT3). The insert above panel a. presents a representative Western blot of both the pY-STAT3 and pS-STAT3 responses together with the level of non-phosphorylated STAT-3. The bands presenting the time course for pY-STAT3 and STAT3 levels are from a single Western blot, while those for pS-STAT3 are from the same protein samples run on a separate blot. Data are presented as a percentage of the appropriate signal in cells incubated for 5 min with buffer alone and represent the mean ± SEM of 3-6 independent determinations. * p< 0.05, ** p< 0.01 compared to the corresponding basal response at the same time point (Mann-Whitney U test).

Figure 2. Immunohistochemical detection of IL6 mediated signaling.

Figure 2

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Adrenal chromaffin cells, cultured on glass coverslips, were incubated for 15 min with or without IL-6 (1 nM). Images in the left-hand column present cells incubated with buffer alone while those on the right show cells incubated with IL6. The scale bar indicates 20 μm and the results are representative of at least 3 independent repeats on separate culture preparations. Arrows indicate the presence of a perinuclear ring of S-pSTAT3 immunoreactivity in IL6 treated cells.

Figure 3. IL6 selectively activated the ERK1/2 but not the p38 or JNK signaling pathways.

Figure 3

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Adrenal chromaffin cells were incubated for increasing periods of time with (shaded columns) or without (open columns) IL6 (1 nM). Panel a) shows the level of phosphorylated ERK1 (pERK1) (pERK2 gave essentially identical results), panel b) the level of phosphorylated p38 (p-p38) and panel c) the levels of phosphorylated JNK (p-JNK). The insert on each panel presents a representative Western blot. In panels b and c “–” indicate buffer only and “+” the presence of a stimulus. In the absence of an IL6 response panels b) and c) present the effect of a 10 min incubation with histamine (10 μM) as a positive control. Responses are expressed as a percentage of that measured after 5 min incubation with buffer alone and represent the mean ± SEM of 3 independent determinations. Note while this Figure presents the data for pERK1 the pERK2 response was essentially identical. * p< 0.05, ** p< 0.01 compared to the corresponding basal response at the same time point (Mann-Whitney U test)

Figure 4. IL6 stimulated serine-727 STAT3 phosphorylation is mediated by ERK1/2.

Figure 4

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Adrenal chromaffin cells were incubated for 15 min with (shaded columns) or without (open columns) IL6 (1 nM) in the presence or absence of the MEK inhibitor PD98059 (50 μM). Panel a) shows the level of phosphorylated ERK1 (pERK1) (pERK2 gave essentially identical results), panel b) the level of serine-727 phosphorylated STAT3 (S-pSTAT3) and panel c) the levels of tyrosine-705 phosphorylated STAT3 (Y-pSTAT3). Responses are expressed as a percentage of that measured after 5 min incubation with buffer alone and represent the mean ± SEM of 3 independent determinations. * p< 0.05, ** p< 0.01 compared to the matched basal response (Mann-Whitney U test)

Figure 5. A number of different stimuli increase ERK1/2 phosphorylation and serine but not tyrosine phosphorylation of STAT3.

Figure 5

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Adrenal chromaffin cells were incubated for 15 min with buffer alone (B), IL6 (1 nM), PMA (100 nM), angiotensin II (AII, 100 nM), histamine (His, 10 μM) or nicotine (Nic, 3 μM). Panel a) shows the level of phosphorylated ERK1 (pERK1) (pERK2 gave essentially identical results), panel b) the level of serine-727 phosphorylated STAT3 (S-pSTAT3) and panel c) the levels of tyrosine-705 phosphorylated STAT3 (Y-pSTAT3). Responses are expressed as a percentage of that measured after incubation with buffer alone and represent the mean ± SEM of 3 independent determinations. * p< 0.05, ** p< 0.01 compared to the basal response (Kruskal-Wallis test followed by a Dunn’s multiple comparison).

Figure 6. Interleukin-6 increased the activity and serine-31 phosphorylation of tyrosine hydroxylase.

Figure 6

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a) Adrenal chromaffin cells were incubated for 5 - 60 min with IL6 (1 nM) and the processed for Western blotting. Blots were probed for TH phosphorylated at serine-19 (□), serine-31 (●), or serine-40 (○). Data are expressed as a percentage of the relevant response measured after 5 min incubation with buffer alone and represent the mean ± SEM of 4 independent experiments. * p< 0.05, ** p<0.01 compared to the 5 min basal response. Insert shows a representative Western blot for the TH phosphorylation sites together with a β-actin loading control. b) Adrenal medullary chromaffin cells were incubated with buffer alone (B), IL-6 (IL6) or forskolin (F) for 20 min (used as a positive control) and the TH activity measured as described in Methods. Data are expressed as a percentage of TH activity measured in cells incubated with buffer alone and presented as the mean ± SEM from 4 independent experiments on three separate cell culture preparations. * p< 0.05 compared to basal (Kruskal-Wallis test followed by a Dunn’s multiple comparison).

Affymetrix GeneChip Bovine arrays were used to analyze IL6-mediated changes in mRNA expression profiles in the chromaffin cell transcriptome. Gene symbols were obtained for transcript IDs by BLAST analysis of transcript sequences (at least two-fold up- or down-regulated by the treatment with a P value of equal or less than 0.05), after averaging the expression values of transcripts with multiple presences in the array. A final list of up- and down-regulated genes was obtained by filtering out any transcript for which a gene symbol could not be linked to the transcript ID. IL6 treatment induced the up-regulation of 90 genes and down-regulation of 26 genes (Fig 7a and Supplementary Tables 1a and 1b). For comparison, we also examined changes in gene cohort with two cytokines (IFNα and TNFα), and two GPCR agonists (histamine and PACAP) whose receptors are present on the chromaffin cell membrane (Ait-Ali et al. 2008, Mustafa et al. 2007, Marley 2003). The IFNα and histamine microarrays were performed in tandem with the present IL6 data; data for PACAP and TNFα have been published previously (Samal et al. 2013), with transcript annotation brought up-to-date. All array results are deposited in GEO (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE85901)

Figure 7. Summary of the up- or down-regulation of gene expression in bovine chromaffin cells following incubation with IL6 compared to four other stimuli.

Figure 7

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Adrenal medullary chromaffin cells were incubated with or without IL6 (10 nM for 24 h) or one of four other stimuli; PACAP (100 nM for 6h), Histamine (Hist, 10 μM for 6 h), TNFα (10 nM for 6 h) or IFNα (1 nM for 24 h) after which time total mRNA was extracted and analyzed by microarray. Panel A presents the total number of genes up- or down-regulated 2-fold or greater, compared to cells maintained in culture medium alone (for full details see Supplementary Tables 1a and 1b). Panel B presents the data from Panel A subdivided by IPA gene-function analysis.

An initial examination of the functional classification of transcripts was conducted using IPA (Figure 7b). At this level of analysis the profile of IL6 stimulated transcripts more closely resembles that of PACAP and histamine than the cytokines, with a relatively high representation of intracellular signaling molecules including kinases, phosphatases, GPCRs and channels. It is also noteworthy that of the 90 transcripts up-regulated only 16 are recognized as IL6 targets by the IPA database (https://www.ingenuity.com). Given that this database is drawn primarily from immune system data the findings reported here suggest that the majority of IL6 targets in chromaffin cells may be neuroendocrine specific. Secreted neuropeptides were among the transcripts most strongly increased by IL6 (approximately 40% of the 12 most responsive targets), they were therefore validated using qPCR (Figure 8). The IL6-dependent increase in galanin, PTHLH, GRP and STC1 (stanniocalcin-1) mRNA levels was confirmed by qPCR. VIP was included in this analysis because, while falling below the 2-fold cut-off at 1.6-fold basal on the microarray this was statistically significant and has been shown to be elevated in chromaffin cells in response to other cytokines (Turquier et al. 2002). ANGPT2, while increased 4-fold on the microarray, was only marginally elevated (P < 0.06) in the qPCR analysis. SOCS3 was included as a positive control since it is known to be a direct downstream target of pSTAT3 and thus of IL6.

Figure 8. IL6 induced increases in the expression of specific genes confirmed by qPCR.

Figure 8

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Adrenal medullary chromaffin cells were incubated for 24 h with or without IL6 (10 nM) after which time total mRNA was extracted and subjected to qPCR. Data is expressed as a percentage of the relevant mRNA levels in cells incubated in media alone (indicated by the dotted line) and represent the mean ± SEM of three independent experiments * p<0.05, ** p< 0.01 compared to levels in cells cultured in media alone (Kruskal-Wallis test followed by a Dunn’s multiple comparison). Abbreviations as defined in text.

In order to facilitate comparison of the IL6 stimulated gene expression profile with those of the other stimuli the data were analyzed and presented using the Vennture software. Approximately 65% of the transcripts up-regulated by IL6 were unique to this stimulus (Figure 9a and Supplementary Table 2a). The greatest overlap between IL6-regulated transcripts and those elevated by the other stimuli occurred between IL6 and PACAP (16 transcripts) and IL6 and histamine (14 transcripts). Eight members of this cohort, including the neuropeptide galanin, were common to all three stimuli. IL6-regulated transcripts also showed some overlap with those regulated by IFNα (9 transcripts) and TNFα (6 transcripts). It should also be noted that STC1 was increased (2-6 fold) by all stimuli except TNFα, and that heme oxygenase 1 (HMOX1) was the only transcript significantly increased (2-3 fold) by all five stimuli.

Figure 9. Vennture diagram of up-regulated transcripts and associated transcription factors in bovine adrenal medullary chromaffin cells.

Figure 9

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Cells were incubated and processed as described in Figure 7. Panel A presents a Vennture diagram describing the overlap of transcripts up-regulated 2-fold or greater by the five different stimuli. See Supplementary Table 2a for the names of the up-regulated transcripts within each of these cohorts. Panel B uses the same format to present the transcription factors associated with mRNA transcript up-regulation by IL6 and the four other stimuli. The transcript cohorts associated with stimulus were analyzed using IPA in order to determine transcription factors (significant at p<0.000001) previously associated with increased activity at the promoters for these genes. Vennture was used to construct the Venn diagram associated with these, individually and in combination. See Supplementary Table 2b for the names of the 56 transcription factors associated solely with TNFα regulation, and the 14 transcription factors associated solely with TNFα and IFNα regulation. All others are shown within the relevant Vennture domains. Interleukin-6 (IL6), pituitary adenylate cyclase-activating polypeptide (PACAP), histamine (HIS), tumor necrosis factor-α (TNF) and interferon-α (IFN).

Vennture analysis of the transcription factors with significant association (P < 0.000001; IPA analysis) with the regulation of genes increased by the five different stimuli revealed the strongest overlap between the cytokines TNFα and IFNα, substantial overlap between histamine and PACAP, and IL6 transcription factors overlapping with both groups (Figure 9b and Supplementary Table 2b).

Discussion

These data provide the first direct evidence for IL6-mediated responses in the adrenal medullary chromaffin cell, supporting an interaction between the immune and neuroendocrine systems at the level of the adrenal medulla (Nussdorfer & Mazzocchi 1998, Bunn et al. 2012, Kanczkowski et al. 2015). It should be noted however that previous studies have reported that IL6 receptor mRNA is expressed in both rodent and human adrenal medulla (Path et al. 1997, Gadient et al. 1995). IL6 is secreted by many cell types including lymphocytes, fibroblasts, adipocytes, endothelial cells and skeletal muscle in response to various physiological circumstances including exercise, obesity and aging as well as conditions of acute or chronic inflammation (Hunter & Jones 2015, Ershler & Keller 2000, Scheller et al. 2014, Maggio et al. 2006, Path et al. 2000). The IL6-mediated responses described here have been generated with high (nM) concentrations of the cytokine. While circulating levels of this order occur during sepsis (Maier et al. 2005, Damas et al. 1992, Hunter & Jones 2015) it is also appropriate to consider local elevation of IL6 within the adrenal tissue as the potential physiological correlate of these cell culture findings (Kanczkowski et al. 2015). This latter possibility is supported by our observation that TNFα increased the expression of IL6 mRNA (approximately 8-fold) in cultured bovine chromaffin cells (Supplementary Table 1a and (Samal et al. 2013)). Finally, in what has been described as the “top-down” regulation of IL6, it has been proposed that the stress axis can drive IL6 elevation in a non-inflammatory state (Straub 2006).

While IL6 signaling classically occurs via the ligand-binding IL6 receptor (IL6R) in conjunction with the signal-transducing gp130 subunit, cells may also respond via trans-signaling, whereby the cytokine, bound to a soluble IL6 receptor, interacts with membrane located gp130 (Hunter & Jones 2015). It is unknown which mechanism is responsible for IL6 signaling to chromaffin cells although it is arguable that responses in isolated cells in culture may be more likely to involve a direct action. Such a direct action is supported by immunohistochemical studies indicating the presence of the IL6R within the adrenal medulla of some species (Gadient et al. 1995, Path et al. 1997). In addition, expression values (EV, expressed in log2 scale) for IL6R obtained by microarray analysis were always ≥5, in all conditions including the basal (untreated) state, indicating that IL6R mRNA is in fact expressed in the chromaffin cells studied here (data not shown).

Western blotting and immunohistochemistry demonstrated that, as with other cell-types, IL6 treatment of chromaffin cells resulted in the elevation of Y-STAT3 (Heinrich et al. 2003, Garbers et al. 2012). There is only very limited information about STAT-mediated signaling in these cells. We have previously shown that IFNα rapidly increases nuclear Y-pSTAT1 and Y-pSTAT2 in isolated bovine chromaffin cells (Douglas & Bunn 2009). Similarly, interferon-γ slowly (60 min) increased nuclear Y-pSTAT3 staining in these cells (Perez-Rodriguez et al. 2009). Cultured porcine chromaffin cells also show a relatively rapid (5 min) increase in Y-pSTAT3 and Y-pSTAT5 in response to leptin or angiotensin II (Takekoshi et al. 2001, Ishii et al. 2001). An unexpected observation in the current study was the presence of intense IL6-independent non-phosphorylated STAT3 staining within the nuclei of all chromaffin cells. While nuclear-located non-phosphorylated STAT3 has been described in other cells its function is poorly understood (Yu et al. 2014, Meyer et al. 2002, Timofeeva et al. 2012).

As with many cell types, IL6 rapidly activated the ERK1/2 but not p38 or JNK pathways in chromaffin cells. ERK1/2 activation in chromaffin cells occurs in response to diverse stimuli, including depolarization, and stimulation with GPCR and cytokine receptor agonists (Figure 5). pERK1/2 is known to phosphorylate TH at serine-31 in chromaffin cells (Dunkley et al. 2004), a proposal supported by the current data (Figure 6a). TH phosphorylation is important because it increases the rate of catecholamine synthesis (Dunkley et al. 2004, Daubner et al. 2011). While phosphorylation of TH at ser-40 has the greatest effect, phosphorylation at ser-31 also enhances enzyme activity as demonstrated by these current studies (Figure 6b). The IL6-mediated increase in catecholamine synthesis is potentially physiologically important because while the cytokine does not directly stimulate catecholamine secretion (unpublished observations) its ability to elevate catecholamine content could enhance the secretory response to other stimuli such as acetylcholine or PACAP.

Activated ERK1/2 is believed to phosphorylate STAT3 on ser-727 (Gough et al. 2013, Decker & Kovarik 2000, Huang et al. 2014), a proposal supported here by the observation that PD98059 inhibited IL6-mediated increase in S-pSTAT3 and that known ERK1/2 activators also elevated levels of S-pSTAT3 (Figures 4 and 5). The role of S-pSTAT3 is controversial, with evidence suggesting that while it has limited transcriptional activity of its own, it modulates (generally enhances) the transcriptional activity of Y-pSTAT3 dimers (Decker & Kovarik 2000). It may also have a non-transcriptional action at mitochondria enhancing oxidative phosphorylation and aerobic glycolysis during cellular stress (Demaria et al. 2014). This latter proposal is consistent with our observation of a perinuclear distribution of S-pSTAT3 following IL6 stimulation, which is similar to that of a mitochondrial subpopulation in these cells (Villanueva et al. 2014).

IL6 treatment for 24 h increased the expression of a wide range of genes by 2-fold or greater (Figure 7 and Supplementary Table 1a). We have previously reported that TNFα and PACAP, which are known to act on chromaffin cells during immune-related stress, showed remarkably little overlap in their transcriptional targets, with more than 90% of up-regulated transcripts being stimulus specific (Samal et al. 2013). This concept of relative exclusivity is supported here with the inclusion of three additional first messengers; IL6, histamine and IFNα, with between 38% (histamine) and 80% (IFNα) of transcripts being unique to a specific stimulus (Figure 9a and Supplementary Table 2). While there was transcription overlap between the GPCR agonists (histamine and PACAP) and between the cytokines (TNFα and IFNα) there was no overlap between these two cohorts. In contrast, IL6 shared transcripts with both the GPCR (18 transcripts) and cytokine receptor agonists (11 transcripts). This ability of IL6 to “cross signal” was reflected in the profile of its identified transcription factors: STAT1 and STAT3 being implicated in gene regulation by TNFα, IFNα and IL6, but not histamine or PACAP, and CREB1 implicated in gene regulation by histamine, PACAP and IL6, but not IFNα or TNFα. This overlap suggests that the importance of IL6 in integrating inflammatory and stress transduction at the level of the adrenal medulla is to link autocoid/transmitter (GPCR) signaling to cytokine-related control of adrenomedullary gene expression (Figure 9b).

Known IL6 targets among the elevated transcripts constitute a relatively low percentage of nominally IL6-regulated genes in chromaffin cells. Notably, among the most robustly up-regulated transcripts (3-fold or greater), over 70% are not recognized as IL6 targets by IPA analysis. This may reflect the identification of novel IL6-regulated transcripts unique to neuroendocrine cells, compared to immunocyte gene regulation from which most information about IL6 gene regulation has been previously drawn. Some IL6 gene targets might have been initially elevated, but returned to basal levels by 24 h. Furthermore, prolonged exposure to IL6 may have indirectly regulated some transcripts as a consequence of early-regulated IL6-dependent trans-acting factors acting upon genes which are themselves insensitive to short-term exposure to IL6.

Among the known IL6 targets, the action of the B-cell chemotactic CXCL13 contrasts with that of TNFα, increasing the expression of a number of neutrophil attracting chemokines. This supports the proposal that IL6 assists in the resolution of acute tissue inflammation (Scheller et al. 2014). Hypoxia-inducible factor 1-α (HIF1a) is known to mediate IL6 actions in many cells, where it drives expression of multiple downstream factors supportive of cell survival in hypoxic conditions (Demaria et al. 2014, Semenza 2014). It should be noted, however, that increased HIF1a expression in these experiments was specific to IL6 and thus not a generic response to hypoxia. The largest response seen to IL6 was a 15-fold rise in the expression of Abi3bp, a poorly understood multifunctional autocrine/paracrine factor. While previous reports have implicated it in limiting the proliferation of mesenchymal cells (Hodgkinson et al. 2013) Abi3bp has yet to be attributed a role in neuronal or neuroendocrine tissue. Further investigation is required to determine if Abi3bp regulation by IL6 is a unique feature of the chromaffin cell and whether it is a directly, or an indirectly regulated IL6-responsive transcripts. Some IL6-regulated transcripts in the adrenal medulla are likely to be neuroendocrine-specific, and, given the neurosecretory role of the chromaffin cells, of particular importance in integrating the inflammatory and hormonal roles of the adrenal medulla during inflammation, the stress response, and physiological conditions in which both occur simultaneously.

The effect of IL6 on secretion from chromaffin cells is likely to be broader than its impact on catecholamine output discussed earlier, in that among the most responsive of IL6 targets were a number of neuropeptides. Of these GRP and PTHLH were unique to IL6, while galanin, STC1 and VIP were also elevated by PACAP and histamine. The modest increase in VIP expression determined by microarray analysis (approx. 1.6-fold) compared to qPCR (approx. 6-fold) may reflect in part the compressed dynamic range of transcript fold increase by the former technique (Allanach et al. 2008, Etienne et al. 2004). The physiological roles of VIP, galanin and other neuropeptides produced by the adrenal medulla in response to stress and inflammation are not resolved and may be somewhat species-dependent. In general, neuropeptides released by chromaffin cells show rather dramatic up-regulation in response to paraphysiological stimuli e.g. prolonged hypoglycemia or septicemia as modeled by lipopolysaccharide (LPS) injection (Stroth et al. 2013, Ait-Ali et al. 2010b, Anouar & Eiden 1995). These neuropeptides may have paracrine effects on neighboring tissues such as the adrenal cortex to enhance steroidogenesis, on sensory neurons innervating the adrenal gland that bear neuropeptide receptors, and even on the chromaffin cell itself. In vivo, neuropeptide gene induction in the adrenal medulla is often dependent on PACAP-mediated signaling; however, data obtained in cultured chromaffin cells suggests that this PACAP action displays a strong synergy with TNFα, which may account, in part, for inflammatory responses to LPS in vivo (Ait-Ali et al. 2010b). It has yet to be determined if IL6 induction of neuropeptides in the adrenal medulla has a similar synergistic interaction with either PACAP or TNFα.

It has been postulated that induction of IL6 expression in TNFα-stimulated cells could provide a potential autocrine control mechanism for cytokine effector function during inflammation (Bunn et al. 2012). To determine if such a relationship might exist in chromaffin cells, we examined the TNFα-regulated transcript cohort in bovine chromaffin cells after 6 h of treatment, and found that the IL6 gene was prominently activated (~8-fold, Supplementary Table 1a). Comparison of transcripts up-regulated in chromaffin cells by IL6 with those we previously identified to be TNFα responsive supported this potential cytokine cascade (Samal et al. 2013). Most notably the neuropeptides VIP and galanin are among those regulated by IL6 in bovine chromaffin cells at 24 h (this report) and by TNFα (Ait-Ali et al. 2008) or LPS in vivo (Ait-Ali et al. 2010b). The physiological relevance of up-regulating neuropeptides by a TNF-to-IL6 cascade may be related to their involvement in steroidogenesis in the adjacent adrenal cortex, with initiation by TNFα, and then IL6, allowing cytokine inflammatory effects occurring systemically to be temporally limited by a linked but delayed anti-inflammatory response. Such a mechanism might serve to optimize the antiseptic actions of TNFα, while minimizing damage to the host during sepsis (Mazzocchi et al. 1992, Andreis et al. 2007, Belloni et al. 2007, Mazzocchi et al. 1998, Pinter et al. 2014, Kormos & Gaszner 2013).

As noted above IL6 uniquely increased the gene expression of both GRP and PTHLH. While neither of these peptides have identified roles within the adrenal gland, early immunochemical studies suggested the presence of both GRP and neuromedin B within chromaffin cell secretory granules (Lemaire et al. 1986) and we have previously found that activation of BB1 (neuromedin-B-preferring) bombesin receptors on these cells induces a concentration-dependent increase in inositol phospholipid metabolism (Bunn et al. 1990). PTHLH has diverse physiological effects in addition to its established role in regulating bone metabolism (Wysolmerski 2012). It has, for example, been reported to enhance secretion from PC12 cells via L-type voltage-sensitive Ca2+ channels, although this has not been confirmed on chromaffin cells (Brines & Broadus 1999). Interestingly, a number of reports have indicated that, as with VIP and galanin, PTHLH enhances steroid production by adrenal cortical cells, thus potentially providing an additional mechanism by which IL6 could support the immunosuppressive actions of the glucocorticoids (Mazzocchi et al. 2001, Kawashima et al. 2005). Interestingly, IL6 administration has been shown to increase ACTH-independent cortisol output from the human adrenal (Mastorakos et al. 1993). Most importantly in the current context, such extrapituitary regulation of the adrenal cortex appears to be of greatest physiological significant during period of chronic stress (Bornstein & Chrousos 1999).

In summary, these data provide the first evidence that IL6 directly interacts with the chromaffin cells of the adrenal medulla. This interaction is complex involving both the STAT3 and ERK1/2 signaling pathways. As a result of this interaction IL6 has the potential to modulate both catecholamine and neuropeptide output of the chromaffin cells and thus to influence the stress response of the adrenal. While the functions of the adrenal medullary peptides are poorly understood, a paracrine action may allow them to enhance glucocorticoid production from the adrenal cortex, an action compatible with the well-recognized anti-inflammatory influence of IL6. While it remains to be determined if these actions arise solely in response to extreme IL6 elevation during sepsis, or from locally produced cytokine within the medulla, they reinforce the concept of a dynamic interaction between the immune and neuroendocrine stress systems.

Supplementary Material

Supp DataS1
Supp Table S1-2

Acknowledgements

We thank Chang-Mei Hsu and Shirley Douglas for their expert technical assistance in preparation adrenal medullary cell cultures, RNA for microarray and qRT-PCR analysis. Djida Ait-Ali is thanked for substantive intellectual and technical contributions to this project, which cannot be acknowledged by authorship solely because she could not be contacted and therefore her assent to authorship could not be obtained. This work was supported by NIMH Intramural Research Program Project Z01 MH002386-21 and a University of Otago Research Grant.

Abbreviations

ERK1/2

extracellular signal-regulated kinase 1/2

GPCR

G-protein couple receptor

GRP

gastrin releasing peptide

HIF1a

hypoxia-inducible factor 1-α

HMOX1

heme oxygenase 1

HPRT1

hypoxanthine-guanine phosphoribosyltransferase-1

IFNα

interferon-α

IL1β

interleukin-1β

IL6

interleukin 6

IL6R

IL6 receptor

IPA

ingenuity pathway analysis

JNK

c-Jun N-terminal kinases

LPS

lipopolysaccharide

PACAP

pituitary adenylate cyclase-activating polypeptide

PBS

phosphate buffered saline

PMA

phorbol 12-myristate 13-acetate

PTHLH

parathyroid hormone-like hormone

STC1

stanniocalcin-1

STAT3

signal transducer and activator of transcription 3

TH

tyrosine hydroxylase

TNFα

tumor necrosis factor-α

VIP

vasoactive intestinal peptide

Footnotes

The authors declare no conflicts of interest.

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Supp Table S1-2
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