Heterodimeric IL-27 (p28/EBV-induced gene 3) is an important member of the IL-6/IL-12 cytokine family. IL-27 is predominantly synthesized by mononuclear phagocytes and exerts immunoregulatory functional activities on lymphocytic and nonlymphocytic cells during infection, autoimmunity or neoplasms. There is a great body of evidence on the bidirectional interplay between the autonomic nervous system and immune responses during inflammatory disorders, but so far IL-27 has not been defined as a part of these multifaceted neuroendocrine networks. In this study, we describe the role of catecholamines (as mediators of the sympathetic nervous system) related to IL-27 production in primary mouse macrophages. Noradrenaline and adrenaline dose-dependently suppressed the release of IL-27p28 in LPS/TLR4-activated macrophages, which was independent of α1 adrenoceptors. Instead, β2 adrenoceptor activation was responsible for mediating gene silencing of IL-27p28 and EBV-induced gene 3. The β2 adrenoceptor agonists formoterol and salbutamol mediated suppression of IL-27p28 production, when triggered by zymosan/TLR2, LPS/TLR4, or R848/TLR7/8 activation, but selectively spared the polyinosinic-polycytidylic acid/TLR3 pathway. Mechanistically, β2 adrenergic signaling reinforced an autocrine feedback loop of macrophage-derived IL-10 and this synergized with inhibition of the JNK pathway for limiting IL-27p28. The JNK inhibitors SP600125 and AEG3482 strongly decreased intracellular IL-27p28 in F4/80+CD11b+ macrophages. In endotoxic shock of C57BL/6J mice, pharmacologic activation of β2 adrenoceptors improved the severity of shock, including hypothermia and decreased circulating IL-27p28. Conversely, IL-27p28 was 2.7-fold increased by removal of the catecholamine-producing adrenal glands prior to endotoxic shock. These data suggest a novel role of the sympathetic neuroendocrine system for the modulation of IL-27–dependent acute inflammation.

Interleukin-27 is a heterodimeric glycosylated protein of the IL-6/IL-12 family of cytokines (13). It consists of the signature subunit p28 (also known as IL-27p28), IL-27A, or IL-30 (2). The second subunit of IL-27, EBV-induced gene 3 (EBI3), is shared with heterodimeric IL-35 (EBI3/p35) (4). The p28 and EBI3 subunits interact with noncovalent bonds for the formation of IL-27 to bind to the specific, high-affinity IL-27RA receptor also known as WSX-1 (5, 6).

The gene expression and release of IL-27 mainly occurs in mononuclear phagocytes such as macrophages and dendritic cells in direct response to microbial patterns or other immune signals (7, 8). The central role of mononuclear phagocytes for IL-27 production is underscored by our previous observation that depletion of these cells resulted in >85% reduction of circulating IL-27 concentrations during acute inflammation (9). IL-27 is released by mononuclear phagocytes during viral, bacterial, protozoan, and fungal infections (1013). For example, living or inactivated Gram-negative bacteria, including Escherichia coli or Salmonella enteritidis, provoke the production of IL-27 (14, 15). On the molecular level, the agonistic activation of pathogen recognition receptors such as TLR2, TLR3, TLR4, TLR7, and TLR9 is sufficient to initiate abundant gene expression of IL-27 (10, 16, 17). Both Toll/IL-1R domain–containing adaptor inducing IFN-β and MyD88 adaptor proteins are required for IL-27p28 production in LPS-activated macrophages (9, 18, 19). Tyrosine kinase 2 specifically promotes the gene expression of IL-27p28 rather than EBI3 following TLR4 activation (20). The downstream signaling pathways involve recruitment of IFN regulatory factors 1, 3, and 9 and NF-κB (c-Rel) transcription factors to the promoter region of the IL-27p28 gene (18, 19, 21). Additionally, IL-27 production is greatly amplified by type I and type II IFNs based on autocrine/paracrine-positive feedback loops (20, 21). Alternatively, PI3K/Akt and STAT3 represent inhibitory signaling pathways for antagonizing IL-27p28 gene expression in LPS/TLR4-activated macrophages (9, 16).

The prominent functions of IL-27 following ligation to the IL-27RA/gp130 receptor complex on various T cell subsets include activation of JAK1/2, asymmetric STAT1/STAT3, and Egr-2 signaling pathways culminating in the release of IL-10 (5, 2225). IL-27 signaling also stimulates dendritic cells for induction of the immunoregulatory molecule CD39 (26). These properties situate IL-27 as a major T cell–suppressive cytokine required for regaining immune homeostasis following clearance of infections and for preventing T cell–mediated lethal inflammation (12). IL-27 antagonizes Th17 cell development and Th17 cell functions via STAT1 (2629). The differentiation of type 1 regulatory T cells is induced by IL-27 (30). Alternatively, IL-27 mediates the survival of both activated CD4+ and CD8+ T cells (31, 32). The clonal expansion of naive CD4+ T cells and Th1-mediated responses including IFN-γ production can be activated by IL-27 (2, 33, 34). Additionally, IL-27 synergizes with type I and type II IFNs for limiting innate lymphoid cell type 2–like innate immune responses (35, 36). Hence, the functional properties of IL-27 can encompass immunosuppression or promotion of inflammation, and its overall effects are most likely dependent on the specific disease context, other inflammatory signals, and the total amounts of IL-27 generated (1).

The autonomic nervous system with its sympathetic and parasympathetic transmitters orchestrates immune responses, inflammation, and immunity (37, 38). Catecholamines are the primary mediators of the sympathetic arm of the autonomic nervous system (39). Noradrenaline is released by nerves innervating the primary and secondary lymphoid organs such as thymus, bone marrow, lymph nodes, and spleen (40, 41). Additionally, noradrenaline is also produced in the medulla of adrenal glands, with the latter representing the predominant source of adrenaline release into the bloodstream (40). Additionally, leukocytes can transform into a relevant source of catecholamines during inflammation (42). Noradrenaline and adrenaline act with varying affinity through nine distinct subtypes of adrenoceptors (α1A, α1B, α1D, α2A, α2B, α2C, β1, β2, β3) (43). Mononuclear phagocytes were shown to express the α1 and the β2 adrenoceptors (44). These receptor subtypes transmit neuroendocrine stress signals for orchestrating the functional participation of mononuclear phagocytes in inflammation. A variety of mononuclear phagocyte functions such as chemotaxis (45, 46), phagocytic efficiency (47), Ag presentation (48), cell proliferation (49), and most notably cytokine release (5052) are affected by adrenoceptor activation.

In the present study, we examined the hypothesis that adrenergic mediators are important for the release of IL-27 by selectively LPS/TLR4-activated macrophages. We show that catecholamines potently suppress the production of IL-27 via activation of β2 adrenoceptors by mechanisms involving IL-10 and the JNK signaling pathway. These data expand our current view on the entanglements of the sympathetic nervous system via catecholamines during acute inflammatory responses.

Mouse studies were performed in accordance with the Animal Protection Act of Germany, the State Investigation Office of Rhineland-Palatinate, the Federation of European Laboratory Animal Science Associations guidelines, and directive 2010/63/EU of the European Parliament and of the Council of the European Union. C57BL/6J mice and IL-10−/− mice (B6.129P2-Il10tm1Cgn/J) were originally obtained from The Jackson Laboratory (Bar Harbor, ME) (53). The generation of α1A-AR−/− and α1B-AR−/− mice has been previously described (5456). These strains were backcrossed for nine generations on a C57BL/6Slc background and used along with the corresponding wild-type (WT) control strain. All mouse strains were bred and genotyped at the animal facilities of the University Medical Center of the Johannes Gutenberg University Mainz in a specific pathogen-free environment under standardized conditions with a 12-h light/dark cycle, temperature of 22 ± 2°C, humidity of 55 ± 10%, and with free access to food and tap water. Adrenalectomized C57BL/6 mice and sham operated C57BL/6 mice were purchased from Taconic Biosciences (Hudson, NY) and were housed as described above. The adrenalectomized C57BL/6 mice received normal saline (NaCl 0.9%) as drinking water to maintain their fluid and electrolyte balances in the absence of adrenal hormones.

Bone marrow–derived macrophages (BMDM) were generated by incubating isolated bone marrow for 7 d with L929 cell–conditioned medium in culture dishes and subsequent segregation from nonadherent cells as described by us before (9). Peritoneal elicited macrophages (PEM) were collected 4 d after i.p. injection of thioglycollate (1.5 ml, 2.4% w/v; Becton Dickinson, Franklin Lakes, NJ). Macrophages were cultured in RPMI 1640 (Thermo Fisher Scientific, Waltham, MA) containing 0.1% BSA (Sigma-Aldrich, St. Louis, MO) and 100 U/ml penicillin-streptomycin (Thermo Fisher Scientific) at 37°C, 5% CO2. At the end of experiments, macrophage supernatants were collected, cleared from cellular debris by centrifugation (500 × g, 5 min, 4°C), and cryopreserved at −80°C until further protein analysis. Adherent macrophages were either lysed for later mRNA extraction or carefully detached from plates for flow cytometry analysis.

LPS (10 mg/kg body weight) from E. coli serotype 0111:B4 (Sigma-Aldrich) with and without formoterol (200 nmol/kg body weight; Sigma-Aldrich) was injected into the peritoneum of C57BL/6J mice. At 3–12 h time points, small volumes of EDTA-anticoagulated blood samples were collected by retro-orbital puncture under deep anesthesia with isoflurane. Plasma samples were obtained by centrifugation (2000 × g, 10 min, 4°C) and stored at −80°C until further analysis. At the end of experiments, mice were euthanized by CO2 inhalation and spleens were isolated for mRNA studies.

For survival studies, LPS (10 mg/kg body weight; i.p.) with and without formoterol (200 nmol/kg body weight; i.p.) was administered followed by a second injection of formoterol (200 nmol/kg body weight; i.p.) or an equivalent volume of vehicle (PBS) after 24 h. Body surface temperature was measured with an infrared thermometer over the abdomen (153 IRB; Bioseb, Vitrolles, France). The clinical severity score (CSS; ranging from 1 [no symptoms] to 5 [dead]) was assessed as described elsewhere (57).

To study the role of endogenous glucocorticoid release, C57BL/6J mice were treated with mifepristone (30 mg/kg body weight i.p.) (58), whereas the control group received the same volume of 3.49% DMSO (v/v) in PBS, and both groups of mice received LPS (10 mg/kg body weight; i.p.) 30 min later. EDTA-anticoagulated blood samples were collected after 8 h by retro-orbital puncture as described above.

ELISA kits for mouse IL-27p28, IL-10, MCP1, and KC were purchased from R&D Systems (Minneapolis, MN). The mouse IL-6 ELISA was from BioLegend (San Diego, CA). Cell-free supernatants or plasma were analyzed according to the manufacturer’s protocol. Briefly, the samples were diluted in PBS supplemented with 0.1% BSA to fit into the standard range and the optical densities of oxidized tetramethylbenzidine were measured with an Opsys MR Dynex microplate reader (Dynex Technologies, Chantilly, VA). The IL-27p28 ELISA is specified by the manufacturer to display 4.6% cross-reactivity with recombinant mouse IL-27 (p28/EBI3 fusion).

Macrophages or spleen homogenates (TissueLyser II; Qiagen, Valencia, CA) were processed and total RNA was isolated with help of the RNeasy mini kit (Qiagen). Next, cDNA was generated via reverse transcription (high-capacity cDNA reverse transcription kit; Applied Biosystems, Foster City, CA). For real-time PCR (RT-PCR), 1–2 ng of cDNA was complemented with iQ SYBR Green master mix (Bio-Rad Laboratories, Hercules, CA) as well as specific forward and reverse primers at a concentration of 0.5 μM each. Reactions were performed with a C1000 thermal cycler (Bio-Rad Laboratories) and results were normalized to GAPDH using the 2−ΔΔCt method. Primer sequences were as follows: mouse GAPDH, 5′-TACCCCCAATGTGTCCGTCGTG-3′ (forward), 5′-CCTTCAGTGGGCCCTCAGATGC-3′ (reverse); mouse IL-27p28, 5′-GGCCATGAGGCTGGATCTC-3′ (forward), 5′-AACATTTGAATCCTGCAGCCA-3′ (reverse); mouse EBI3, 5′-GGCTGAGCGAATCATCAA-3′ (forward), 5′-GAGAGAGAAGATGTCCGGGAA-3′ (reverse); mouse IL-10, 5′-AGACACCTTGGTCTTGGAGC-3′ (forward) and 5′-TTTGAATTCCCTGGGTGAGA-3′ (reverse); mouse arginase 1, 5′-CAGAAGAATGGAAGAGTCAG-3′ (forward) and 5′-CAGATATGCAGGGAGTCACC-3′ (reverse).

Intracellular IL-27p28 was trapped in the macrophages by incubation for 12 h with monensin (2 μM; Becton Dickinson). Following gentle detachment of macrophages, Fc receptors were blocked by incubating with anti-CD16/CD32 Ab (TruStain FcX; BioLegend). After surface marker staining for CD11b–Pacific Blue (clone M1/70; BioLegend) and F4/80-allophycocyanin (clone BM8; BioLegend), cells were fixed and permeabilized (Cytofix/Cytoperm Plus kit; Becton Dickinson). Subsequently, the cells were stained with IL-27p28–PE (clone MM27-7B1; eBioscience, San Diego, CA), NO synthase 2 (NOS2)–Alexa Fluor 488 (clone CXNFT; eBioscience), or CD206-PE/Cy7 (clone C068C2; BioLegend). Matched fluorochrome-labeled isotype Abs were used as controls. At least 50,000 cells were acquired with a FACSCanto II (Becton Dickinson), and data analyses were performed using WinList for Win32 3.0 software (Verity Software, Topsham, ME) and FlowJo VX for Windows (FlowJo, Ashland, OR) with pregating on mononuclear phagocytes according to forward and side scatter plots.

The phosphorylation status of JNK was assessed using the phospho-JNK (Thr183/Tyr185) single-plex set from Bio-Rad Laboratories following the manufacturer’s protocol. Briefly, 2 × 106 macrophages were lysed in the presence of inhibitors for proteases and phosphatases (Bio-Plex cell lysis kit; Bio-Rad Laboratories). All magnetic bead washing steps were performed with a Bio-Plex Pro II wash station (Bio-Rad Laboratories). Fluorescence intensities corresponding to phospho-JNK content were measured in a Luminex 200/Bio-Plex 200 system with Bio-Plex Manager software 6.1 (Bio-Rad Laboratories).

LPS (E. coli serotype O111:B4), salbutamol (α-[(tert-butylamino)methyl]-4-hydroxy-m-xylene-α,α′-diol), formoterol ((R*,R*)-N-[2-hydroxy-5-[1-hydroxy-2-[[2-(4-methoxyphenyl)-1-ethylethyl]amino]ethyl]phenyl]formamide fumarate), and mifepristone (RU-486) were purchased from Sigma-Aldrich. Prazosin hydrochloride and salmeterol xinafoate were purchased from Tocris Bioscience (Bristol, U.K.). Orciprenaline (Alupent) was from Boehringer Ingelheim (Ingelheim, Germany). The TLR agonists polyinosinic-polycytidylic acid [poly(I:C)], zymosan (Saccharomyces cerevisiae) and R848 were from InvivoGen (San Diego, CA). Adrenaline was obtained from Jenapharm (Jena, Germany) and noradrenaline was from Sanofi Aventis (Gentilly, France). Recombinant mouse IL-10 and IL-4 were purchased from PeproTech (Rocky Hill, NJ). JNK inhibitors AEG3482 and SP600125 were obtained from Tocris Bioscience. Neutralizing anti-mouse IL-10 receptor Abs (clone 1B1.3a) were affinity purified using protein G–Sepharose (GE Healthcare, Munich, Germany), according to a standard protocol, and used together with corresponding azide-free control rat IgG1κ Abs (BioLegend).

GraphPad Prism v6.00 software was used for statistical analyses. In vitro experiments were repeated three times (biological replicates), and each independent experiment was performed in duplicate wells (technical replicates) unless stated otherwise in the figure legends. In vivo data were generated with the numbers of mice as indicated in the figure legends. All values are depicted as mean with error bars representing SEM. Statistical significance was analyzed using the unpaired two-tailed Student t test, one-way ANOVA with Dunnett multiple comparisons test, two-way ANOVA, or log-rank Mantel–Cox tests for survival studies. We considered differences significant at p < 0.05.

To study the role of adrenergic mediators for the IL-27–dependent immune response, we used cultures of LPS/TLR4-activated macrophages (Fig. 1A). LPS (100 ng/ml) induced a time-dependent appearance of IL-27p28 in supernatants of macrophages derived from C57BL/6J mice. The addition of either adrenaline (10−6 M) or noradrenaline (10−6 M) potently prevented the release of IL-27p28 to supernatants of macrophage cultures at all the time points studied (Fig. 1A). When the mRNA levels for IL-27p28 were assessed by RT-PCR, it was observed that both adrenaline and noradrenaline blunted the gene expression of IL-27p28 (Fig. 1B). In dose-response experiments, the IC50 of adrenaline regarding suppression of IL-27p28 protein release in LPS/TLR4-activated macrophages was ∼10−8 M, whereas noradrenaline appeared to be somewhat less potent at concentrations <10−6 M (Fig. 1C).

FIGURE 1.

Release of IL-27p28 in macrophages is suppressed by catecholamines. (A) Macrophages (BMDM) from C57BL/6J mice were incubated with either LPS (100 ng/ml) alone or in combination with adrenaline (10−6 M) or noradrenaline (10−6 M). At several time points, the supernatants were collected followed by detection of IL-27p28 by ELISA. (B) RT-PCR analysis of mRNA for IL-27p28 in LPS/TLR4-activated macrophages either with or without addition of adrenaline (10−6 M) or noradrenaline (10−6 M), normalized to GAPDH expression. (C) Dose-response curves of the effects of adrenaline (10−9–10−6 M) and noradrenaline (10−9–10−6 M) on the release of IL-27p28 by LPS/TLR4-activated macrophages. IL-27p28 concentrations with LPS alone were used as 100% value (ELISA). (D) IL-27p28 release from macrophages derived from mice with genetic deletion of the α1A-adrenoceptor (α1A-AR−/−), α1B-adrenoceptor (α1B-AR−/−), or macrophages of the corresponding WT control strain (C57BL/6Slc). Macrophages were stimulated with LPS alone or coincubated with either adrenaline (10−6 M) or noradrenaline (10−6 M) for 24 h. For normalization the concentrations of IL-27p28 after LPS alone treatment for each mouse strain were used as 100% values. (E) Inhibitory effects of catecholamines on LPS-induced IL27p28 release after 30 min preincubation with the α1 blocker, prazosin (10−6 M), versus DMSO vehicle (0.004% v/v) (24 h, ELISA). Ctrl denotes resting control macrophages that received either DMSO vehicle or prazosin alone. Statistical significance was tested versus LPS stimulation of DMSO vehicle (*) or LPS stimulation of prazosin-treated cells (#). All data are representative of three independent experiments each performed in duplicate wells. Error bars represent SEM. *p < 0.05, ****p < 0.0001, ####p < 0.0001. ns, not significant.

FIGURE 1.

Release of IL-27p28 in macrophages is suppressed by catecholamines. (A) Macrophages (BMDM) from C57BL/6J mice were incubated with either LPS (100 ng/ml) alone or in combination with adrenaline (10−6 M) or noradrenaline (10−6 M). At several time points, the supernatants were collected followed by detection of IL-27p28 by ELISA. (B) RT-PCR analysis of mRNA for IL-27p28 in LPS/TLR4-activated macrophages either with or without addition of adrenaline (10−6 M) or noradrenaline (10−6 M), normalized to GAPDH expression. (C) Dose-response curves of the effects of adrenaline (10−9–10−6 M) and noradrenaline (10−9–10−6 M) on the release of IL-27p28 by LPS/TLR4-activated macrophages. IL-27p28 concentrations with LPS alone were used as 100% value (ELISA). (D) IL-27p28 release from macrophages derived from mice with genetic deletion of the α1A-adrenoceptor (α1A-AR−/−), α1B-adrenoceptor (α1B-AR−/−), or macrophages of the corresponding WT control strain (C57BL/6Slc). Macrophages were stimulated with LPS alone or coincubated with either adrenaline (10−6 M) or noradrenaline (10−6 M) for 24 h. For normalization the concentrations of IL-27p28 after LPS alone treatment for each mouse strain were used as 100% values. (E) Inhibitory effects of catecholamines on LPS-induced IL27p28 release after 30 min preincubation with the α1 blocker, prazosin (10−6 M), versus DMSO vehicle (0.004% v/v) (24 h, ELISA). Ctrl denotes resting control macrophages that received either DMSO vehicle or prazosin alone. Statistical significance was tested versus LPS stimulation of DMSO vehicle (*) or LPS stimulation of prazosin-treated cells (#). All data are representative of three independent experiments each performed in duplicate wells. Error bars represent SEM. *p < 0.05, ****p < 0.0001, ####p < 0.0001. ns, not significant.

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To approach the question whether the observed effects of catecholamines were mediated through α- or β-adrenoceptors, we compared the suppressive effects of adrenaline and noradrenaline in LPS/TLR4-activated macrophages derived from mice with genetic deficiency of the α1 adrenoceptors (α1A, α1B) to macrophages from WT mice (C57BL/6Slc) after LPS treatment (Fig. 1D). Importantly, catecholamines were equally active to suppress IL-27p28 release by macrophages derived from α1A-AR−/− or α1B-AR−/− mice as compared with macrophages from WT mice (Fig. 1D). For comparison, the concentrations of IL-27p28 are depicted as relative values normalized to LPS alone for each individual mouse strain and pooled from several independent experiments. There was an inconsistent trend of lower absolute concentrations of IL-27p28 in LPS-activated α1B-AR−/− macrophages (data not shown). We also used prazosin as a non–subtype selective α1 adrenoceptor antagonist. Indeed, adrenaline and noradrenaline retained their activities as suppressors of LPS-induced IL-27p28 release during α1 adrenoceptor blockade with prazosin (Fig. 1E). Collectively, the data presented in Fig. 1D and 1E pointed against the involvement of α1 adrenoceptors for modulating the production of IL-27 in macrophages.

To further characterize the role of adrenoceptors for regulating the gene expression of IL-27, we designed experiments using β2 adrenoceptor agonists. Macrophages from C57BL/6J mice were activated with LPS (100 ng/ml) alone or combined with the highly β2 selective adrenoceptor agonist, (R,R)-formoterol (10−6 M). At different time points (0–24 h), the mRNA was extracted and expression levels of IL-27p28 were analyzed by RT-PCR and normalized to GAPDH mRNA (Fig. 2A). The LPS-induced accumulation of mRNA for IL-27p28 peaked around 6 h and was profoundly diminished by the addition of formoterol. The treatment of macrophages with formoterol alone did not result in altered gene expression of IL-27p28 as compared with resting untreated macrophages (Ctrl; Fig. 2A). Next, we used salbutamol (also known as albuterol) as another Food and Drug Administration–approved drug with β2 adrenoceptor agonistic activities. Likewise, salbutamol was a potent inhibitor of IL-27p28 mRNA (Fig. 2B). The gene expression of EBI3 was also studied, because it is the second subunit of heterodimeric IL-27 (2). Indeed, LPS promoted an accumulation of the mRNA for EBI3, but the relative increase (5- to 25-fold) appeared to be less pronounced as compared with IL-27p28 mRNA (∼1000-fold). This observation may be related to the fact that unstimulated resting macrophages already contained higher baseline mRNA for EBI3 as compared with IL-27p28 mRNA (data not shown). Most importantly, the induction of EBI3 was significantly reduced by the selective β2 adrenoceptor agonists formoterol (Fig. 2C) and salbutamol (Fig. 2D) in cultures of LPS/TLR4-activated macrophages.

FIGURE 2.

Gene expression of IL-27p28 and EBI3 is antagonized by β2 adrenoceptor agonists in macrophages. (A) Time course (0–24 h) of mRNA for IL-27p28 from macrophages (C57BL/6J, BMDM) activated by LPS (100 ng/ml) alone or in combination with the selective β2 adrenoceptor agonist formoterol (10−6 M). Formoterol alone did not alter the gene expression levels of IL-27p28 (RT-PCR). (B) Time course of IL-27p28 mRNA in LPS/TLR4-activated macrophages when combined with the selective β2 adrenoceptor agonist salbutamol (10−6 M) (RT-PCR). (C) RT-PCR for mRNA of EBI3 in macrophages at several time points after LPS and formoterol (10−6 M), when used alone or in combination. (D) RT-PCR for EBI3 mRNA in macrophages stimulated with LPS with and without salbutamol or salbutamol (10−6 M) alone at different time points (0–24 h). All data are representative of three independent experiments each performed in duplicate wells. Error bars represent SEM. *p < 0.05, **p < 0.01, ***p < 0.001 for LPS versus LPS plus β2 adrenoceptor agonists.

FIGURE 2.

Gene expression of IL-27p28 and EBI3 is antagonized by β2 adrenoceptor agonists in macrophages. (A) Time course (0–24 h) of mRNA for IL-27p28 from macrophages (C57BL/6J, BMDM) activated by LPS (100 ng/ml) alone or in combination with the selective β2 adrenoceptor agonist formoterol (10−6 M). Formoterol alone did not alter the gene expression levels of IL-27p28 (RT-PCR). (B) Time course of IL-27p28 mRNA in LPS/TLR4-activated macrophages when combined with the selective β2 adrenoceptor agonist salbutamol (10−6 M) (RT-PCR). (C) RT-PCR for mRNA of EBI3 in macrophages at several time points after LPS and formoterol (10−6 M), when used alone or in combination. (D) RT-PCR for EBI3 mRNA in macrophages stimulated with LPS with and without salbutamol or salbutamol (10−6 M) alone at different time points (0–24 h). All data are representative of three independent experiments each performed in duplicate wells. Error bars represent SEM. *p < 0.05, **p < 0.01, ***p < 0.001 for LPS versus LPS plus β2 adrenoceptor agonists.

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Time course studies revealed that the release of IL-27p28 (as detected by ELISA) was constantly suppressed by the presence of formoterol in cultures of LPS/TLR4-activated macrophages derived from C57BL/6J mice (Fig. 3A). Similar effects were noted when salbutamol was used as a selective β2 adrenoceptor agonist (Fig. 3B). Comparable results were obtained in peritoneal-elicited and bone marrow–derived C57BL/6J macrophage preparations (BMDM; data not shown). In dose-response studies, formoterol used in concentrations as low as 10−10 M displayed a high potency for inhibition of IL-27p28 (Fig. 3C). Alternatively, the inhibitory effects of the short-acting salbutamol were less pronounced with concentrations <10−7 M (Fig. 3D). We confirmed that the β2 adrenoceptor agonist formoterol was equally effective to suppress IL-27p28 in macrophages with genetic deficiency of the α1 adrenoceptors (α1A, α1B) as compared with WT macrophages (Fig. 3E). This was in accordance with the subsequent observation that incubation of macrophages with the α1 antagonist, prazosin, did not alter the inhibitory effects of formoterol (Fig. 3F). The nonselective β12 adrenoceptor agonist orciprenaline and the long-acting β2 adrenoceptor agonist salmeterol resulted in a similar blockade of IL-27p28 release by LPS/TLR4-activated macrophages (Fig. 1G). Finally, it was confirmed that formoterol and salbutamol also downregulated other proinflammatory chemokines (KC, MCP1) and cytokines (IL-6) in LPS-activated macrophage cultures (Supplemental Fig. 1A–C) (59).

FIGURE 3.

Dose-dependent and time-dependent modulation of IL-27 release by β2 adrenoceptor agonists in macrophages. (A) Time course of IL-27p28 release in supernatants of macrophage cultures (C57BL/6J, PEM) when incubated with LPS alone, LPS in combination with formoterol (10−6 M), or formoterol (10−6 M) alone (ELISA). (B) Time course of macrophage-derived IL-27p28 release in cultures incubated with LPS alone, LPS in combination with salbutamol (10−6 M), or salbutamol (10−6 M) alone (ELISA). (C) Dose-response studies of the suppressive effects of formoterol (10−12–10−6 M) in LPS/TLR4-activated macrophages (24 h, ELISA). (D) Dose-response studies of the inhibitory effects of salbutamol (10−9–10−6 M) in LPS/TLR4-activated macrophages (24 h, ELISA). (E) Comparison of the suppressive effects of formoterol in LPS/TLR4-activated macrophages derived from either WT mice (C57BL/6Slc), α1A adrenoceptor–deficient mice (α1A-AR−/−), or α1B adrenoceptor–deficient mice (α1B-AR−/−). For each genotype the concentrations of IL-27p28 in supernatants were normalized to LPS alone (=100% value). Formoterol was equally effective to suppress IL-27p28 in all strains. (F) Inhibitory effect of formoterol on LPS-induced IL-27p28 release by BMDM following 30 min preincubation with the α1 blocker prazosin (10−6 M) or DMSO vehicle (0.004% v/v) (24 h, ELISA). (G) Comparison of the inhibitory effects of the β2 adrenoceptor agonists formoterol, orciprenalin, and salmeterol (10−6 M each) on IL-27p28 release in LPS/TLR4-activated macrophages (24 h, ELISA). Ctrl denotes resting control macrophages. All data are representative of three independent experiments each performed in duplicate wells. Error bars represent SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, LPS versus LPS plus β2 adrenoceptor agonist. ns, not significant.

FIGURE 3.

Dose-dependent and time-dependent modulation of IL-27 release by β2 adrenoceptor agonists in macrophages. (A) Time course of IL-27p28 release in supernatants of macrophage cultures (C57BL/6J, PEM) when incubated with LPS alone, LPS in combination with formoterol (10−6 M), or formoterol (10−6 M) alone (ELISA). (B) Time course of macrophage-derived IL-27p28 release in cultures incubated with LPS alone, LPS in combination with salbutamol (10−6 M), or salbutamol (10−6 M) alone (ELISA). (C) Dose-response studies of the suppressive effects of formoterol (10−12–10−6 M) in LPS/TLR4-activated macrophages (24 h, ELISA). (D) Dose-response studies of the inhibitory effects of salbutamol (10−9–10−6 M) in LPS/TLR4-activated macrophages (24 h, ELISA). (E) Comparison of the suppressive effects of formoterol in LPS/TLR4-activated macrophages derived from either WT mice (C57BL/6Slc), α1A adrenoceptor–deficient mice (α1A-AR−/−), or α1B adrenoceptor–deficient mice (α1B-AR−/−). For each genotype the concentrations of IL-27p28 in supernatants were normalized to LPS alone (=100% value). Formoterol was equally effective to suppress IL-27p28 in all strains. (F) Inhibitory effect of formoterol on LPS-induced IL-27p28 release by BMDM following 30 min preincubation with the α1 blocker prazosin (10−6 M) or DMSO vehicle (0.004% v/v) (24 h, ELISA). (G) Comparison of the inhibitory effects of the β2 adrenoceptor agonists formoterol, orciprenalin, and salmeterol (10−6 M each) on IL-27p28 release in LPS/TLR4-activated macrophages (24 h, ELISA). Ctrl denotes resting control macrophages. All data are representative of three independent experiments each performed in duplicate wells. Error bars represent SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, LPS versus LPS plus β2 adrenoceptor agonist. ns, not significant.

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We and others have reported before that several different TLR/pathogen recognition receptor pathways can induce the production of IL-27p28 in mononuclear phagocytes (9, 16, 18, 21). To investigate whether the suppressive effects of β2 adrenoceptor agonists were specific for the TLR4 pathway or a rather global phenomenon, we incubated macrophages with receptor agonists for TLR2, TLR3, TLR4, and TLR7/8 (Fig. 4A). The receptor agonists for TLR2 (zymosan), TLR3 [poly(I:C)], and TLR7/8 (R848) all promoted the appearance of IL-27p28, as detected by ELISA after 24 h, in a similar fashion as observed for the TLR4 (LPS) (Fig. 4A). Whereas the activation of the pathways for TLR2, TLR4, and TLR7/8 were significantly inhibited by formoterol (β2), no such effects were noted when poly(I:C) was used to stimulate TLR3-dependent IL-27p28 release (Fig. 4A). Next, we compared the inhibitory effects of adrenaline, noradrenaline, formoterol, and salbutamol, when macrophages were coincubated with either LPS or poly(I:C) (Fig. 4B). In fact, neither of these adrenoceptor agonists displayed any significant activity regarding the reduction of poly(I:C)-induced IL-27p28 in macrophages, whereas the release of LPS-induced IL-27p28 was strongly suppressed (Fig. 4B).

FIGURE 4.

Selective suppression of IL-27 by β2 adrenoceptor agonists in dependency of activated pattern recognition receptor pathways. (A) Macrophages (C57BL/6J, PEM) were activated with LPS (TLR4, 100 ng/ml), zymosan (TLR2, 10 μg/ml), poly(I:C) (TLR3, 1 μg/ml), or R848 (TLR7/8, 5 μg/ml) alone or in combination with the β2 adrenoceptor agonist formoterol (10−6 M). IL-27p28 was detected in supernatants by ELISA after 24 h. (B) IL-27p28 release from LPS/TLR4-activated macrophages was compared with poly(I:C)/TLR3-activated macrophages, when coincubated with adrenaline (10−6 M), noradrenaline (10−6 M), formoterol (10−6 M), or salbutamol (10−6 M). All data are representative of three (A) or two (B) independent experiments each performed in duplicate wells. Error bars represent SEM. *p < 0.05, **p < 0.01, ***p < 0.001, LPS versus LPS plus adrenoceptor agonists. ns, not significant.

FIGURE 4.

Selective suppression of IL-27 by β2 adrenoceptor agonists in dependency of activated pattern recognition receptor pathways. (A) Macrophages (C57BL/6J, PEM) were activated with LPS (TLR4, 100 ng/ml), zymosan (TLR2, 10 μg/ml), poly(I:C) (TLR3, 1 μg/ml), or R848 (TLR7/8, 5 μg/ml) alone or in combination with the β2 adrenoceptor agonist formoterol (10−6 M). IL-27p28 was detected in supernatants by ELISA after 24 h. (B) IL-27p28 release from LPS/TLR4-activated macrophages was compared with poly(I:C)/TLR3-activated macrophages, when coincubated with adrenaline (10−6 M), noradrenaline (10−6 M), formoterol (10−6 M), or salbutamol (10−6 M). All data are representative of three (A) or two (B) independent experiments each performed in duplicate wells. Error bars represent SEM. *p < 0.05, **p < 0.01, ***p < 0.001, LPS versus LPS plus adrenoceptor agonists. ns, not significant.

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To shed light on the molecular mechanisms mediating the suppression of IL-27 by β2 adrenoceptor agonists, we first turned our attention to IL-10 (59). The presence of formoterol in cultures of LPS/TLR4-activated macrophages (C57BL/6J) significantly increased the amounts of mRNA for IL-10 at all the time points studied (3–24 h; Fig. 5A). Likewise, formoterol promoted the release of IL-10 protein as detected by ELISA in supernatants of activated macrophages, when formoterol was used in a concentration range of 10−10–10−6 M (Fig. 5B). Salbutamol significantly augmented the release of IL-10 by LPS/TLR4-activated macrophages in concentrations of 10−8–10−6 M (Fig. 5C). When β2 adrenoceptor agonists were used alone (i.e., in the absence of LPS), no induction of IL-10 release was detectable (ELISA; data not shown). To further characterize the relationship between IL-10 and IL-27p28 in LPS/TLR4-activated macrophages, we used monoclonal neutralizing anti–IL-10R Abs. When anti–IL-10R Abs were added together with LPS and the β2 adrenoceptor agonist formoterol (β2), the suppressive effects of formoterol regarding the release of IL-27p28 were almost completely intercepted (Fig. 5D). Matched isotype control Abs (IgG1) were used as a negative control and did not interfere with the inhibition of LPS-induced IL-27p28 formation by formoterol (Fig. 5D). Next, the role of IL-10 was studied by using macrophages derived from IL-10–deficient mice (IL-10−/−). The IL-10−/− mice were sacrificed ≤6 wk of age before the spontaneous development of chronic enterocolitis and colon carcinoma in situ, which is frequently observed in this mouse strain (53). We compared the suppressive effects of formoterol in LPS/TLR4-activated macrophages from either C57BL/6J (WT) mice to macrophages from IL-10−/− mice (Fig. 5E). Relative values for IL-27p28 were calculated for each strain and each individual time point by setting LPS alone to 100%. Although coincubation with formoterol nullified the release of IL-27p28 in LPS/TLR4-activated WT macrophages, the release of IL-27p28 by IL-10−/− macrophages partly remained intact (Fig. 5E). In detail, in IL-10−/− macrophages undergoing the LPS plus formoterol treatment the remaining release of IL-27p28 was 41% at the 6 h time point, 58% at the 9 h time point, and 78% at the 24 h time point as compared with LPS alone in IL-10−/− macrophages (Fig. 5E). Hence, IL-10 appeared to be especially important to mediate the β2 adrenergic effects at later time points. This idea is supported by the fact that several hours are required for the generation and build-up of substantial amounts of IL-10 in response to LPS and formoterol. The peak of mRNA for IL-10 after LPS and formoterol was observed after 6 h (Fig. 5A). Fig. 5F shows the distinct inhibitory effects of formoterol (β2) in LPS-activated macrophages from either C57BL/6J (WT) mice or IL-10−/− mice. The absolute concentrations of IL-27p28 after LPS alone were higher in IL-10−/− macrophages as compared with WT macrophages (Fig. 5F). Finally, recombinant mouse IL-10 greatly suppressed the release of IL-27p28 from LPS-activated macrophages (Fig. 5G). No such activities of recombinant mouse IL-10 were observed, when IL-27p28 was induced by poly(I:C) activation (Fig. 5G). This observation may provide an explanation for the data presented in Fig. 4A and 4B, where a selective sparing of IL-27p28 inhibition by neuroendocrine sympathomimetic agonists was observed in poly(I:C)-activated macrophages.

FIGURE 5.

β2 adrenoceptor agonists activate an autocrine/paracrine IL-10 loop to limit IL-27p28. (A) RT-PCR of mRNA for IL-10 in macrophages (C57BL/6J, PEM) incubated with LPS (100 ng/ml) with and without formoterol (10−6 M) at different time points (0–24 h). Ctrl denotes untreated resting macrophages. (B) IL-10 release by macrophages treated with LPS with and without formoterol in different concentrations (10−12–10−6 M) (24 h, ELISA). (C) IL-10 release by macrophages treated with LPS with and without salbutamol in different concentrations (10−9–10−6 M) (24 h, ELISA). (D) IL-27p28 release from LPS/TLR4-activated macrophages with addition of a monoclonal blocking anti–IL-10R Ab (αIL-10R; 10 μg/ml) using isotype-matched IgG1 Ab (10 μg/ml) as control. Interception of IL-10R signaling nullified the suppressive effects of formoterol (β2, 10−6 M) (24 h, ELISA). (E) Relative release of IL-27p28 at different time points (6–24 h) from macrophages derived from C57BL/6J (WT) mice as compared with macrophages from IL-10−/− mice, when incubated with the combination of LPS plus formoterol (10−6 M) (ELISA). For each time point and mouse strain the IL-27p28 concentration with LPS alone was calculated as 100% value. (F) IL-27p28 in supernatants from WT macrophages and IL-10−/− macrophages incubated with LPS or LPS plus β2 adrenoceptor agonist (formoterol, 10−6 M) (24 h, ELISA). (G) Comparison of the effects of recombinant mouse IL-10 (10 ng/ml) on the release of IL-27p28 from macrophages (WT) either induced by a TLR4 agonist (LPS 100 ng/ml) or a TLR3 agonist [poly(I:C) 1 μg/ml]. All data are representative of three independent experiments each performed in duplicate wells. Error bars represent SEM. *p < 0.05, **p < 0.01, ***p < 0.001. For (A)–(C), statistical significance was tested for LPS versus LPS plus β2 adrenoceptor agonist. n.s., not significant.

FIGURE 5.

β2 adrenoceptor agonists activate an autocrine/paracrine IL-10 loop to limit IL-27p28. (A) RT-PCR of mRNA for IL-10 in macrophages (C57BL/6J, PEM) incubated with LPS (100 ng/ml) with and without formoterol (10−6 M) at different time points (0–24 h). Ctrl denotes untreated resting macrophages. (B) IL-10 release by macrophages treated with LPS with and without formoterol in different concentrations (10−12–10−6 M) (24 h, ELISA). (C) IL-10 release by macrophages treated with LPS with and without salbutamol in different concentrations (10−9–10−6 M) (24 h, ELISA). (D) IL-27p28 release from LPS/TLR4-activated macrophages with addition of a monoclonal blocking anti–IL-10R Ab (αIL-10R; 10 μg/ml) using isotype-matched IgG1 Ab (10 μg/ml) as control. Interception of IL-10R signaling nullified the suppressive effects of formoterol (β2, 10−6 M) (24 h, ELISA). (E) Relative release of IL-27p28 at different time points (6–24 h) from macrophages derived from C57BL/6J (WT) mice as compared with macrophages from IL-10−/− mice, when incubated with the combination of LPS plus formoterol (10−6 M) (ELISA). For each time point and mouse strain the IL-27p28 concentration with LPS alone was calculated as 100% value. (F) IL-27p28 in supernatants from WT macrophages and IL-10−/− macrophages incubated with LPS or LPS plus β2 adrenoceptor agonist (formoterol, 10−6 M) (24 h, ELISA). (G) Comparison of the effects of recombinant mouse IL-10 (10 ng/ml) on the release of IL-27p28 from macrophages (WT) either induced by a TLR4 agonist (LPS 100 ng/ml) or a TLR3 agonist [poly(I:C) 1 μg/ml]. All data are representative of three independent experiments each performed in duplicate wells. Error bars represent SEM. *p < 0.05, **p < 0.01, ***p < 0.001. For (A)–(C), statistical significance was tested for LPS versus LPS plus β2 adrenoceptor agonist. n.s., not significant.

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Because IL-10 is a determinant factor for the polarization of macrophages into an M2 phenotype (60, 61), we studied the role of IL-10–inducing β2 adrenoceptor agonists related to M1/M2 differentiation. Formoterol and salbutamol increased the amounts of mRNA for arginase 1, an established M2 marker, when combined with LPS (Supplemental Fig. 1D). Recombinant mouse IL-4 induced M2 polarization as studied by CD206 expression on F4/80+CD11b+ macrophages by flow cytometry, and the presence of CD206 was further augmented by β2 adrenoceptor agonists (Supplemental Fig. 1E, 1F). The intracellular content of LPS/TLR4-induced NOS2, a known M1 marker, was profoundly reduced by formoterol or salbutamol (74.7% versus 38.5% or 42.3% F4/80+CD11b+NOS2+ macrophages; Supplemental Fig. 1G, 1H). These findings are in accordance with a recent report describing the M2 macrophage phenotype promoting activities of β2 adrenoceptor agonists (62).

To investigate the intracellular mechanisms responsible for the suppression of IL-27 by catecholamines, we turned our attention to the JNK signaling pathway. We have reported before that β2 adrenoceptor agonists selectively affect phosphorylation of JNK (59). When macrophages derived from C57BL/6J mice were incubated with LPS (100 ng/ml) alone or in combination with formoterol (10−6 M), a significant reduction in intracellular phospho-JNK levels (Thr183/Tyr185) was observed with formoterol (Fig. 6A). Similarly, treatment with recombinant mouse IL-10 (10 ng/ml) resulted in a confinement of JNK activation patterns in LPS/TLR4-activated macrophage cultures (Fig. 6B).

FIGURE 6.

Role of the JNK signaling pathway in the regulation of IL-27. (A) Phospho-JNK (Thr183/Tyr185) in untreated resting macrophages (Ctrl, C57BL/6J, PEM) as compared with 1 h incubation with LPS (100 ng/ml) or LPS plus formoterol (10−6 M); bead-based assay (Bio-Plex 200/Luminex 200). (B) Phospho-JNK in macrophages after 1 h incubation with LPS or LPS plus recombinant mouse IL-10 (10 ng/ml; preincubated overnight). (C) IL-27p28 release from LPS-activated macrophages together with the JNK inhibitor, AEG3482, in different concentrations (5–20 μM; 90 min preincubation) (12 h, ELISA). (D) Dose-response curve of the effects of the JNK inhibitor, SP600125, on the release of IL-27p28 by macrophages (12 h, ELISA). (E) Flow cytometry of intracellular IL-27p28 in F4/80+CD11b+ macrophages incubated with LPS alone or in combination with either AEG3482 or SP600125 (12 h). Regions were defined according to isotype Ab controls (data not shown). All data are representative of three independent experiments each performed in duplicate wells. Error bars represent SEM. *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 6.

Role of the JNK signaling pathway in the regulation of IL-27. (A) Phospho-JNK (Thr183/Tyr185) in untreated resting macrophages (Ctrl, C57BL/6J, PEM) as compared with 1 h incubation with LPS (100 ng/ml) or LPS plus formoterol (10−6 M); bead-based assay (Bio-Plex 200/Luminex 200). (B) Phospho-JNK in macrophages after 1 h incubation with LPS or LPS plus recombinant mouse IL-10 (10 ng/ml; preincubated overnight). (C) IL-27p28 release from LPS-activated macrophages together with the JNK inhibitor, AEG3482, in different concentrations (5–20 μM; 90 min preincubation) (12 h, ELISA). (D) Dose-response curve of the effects of the JNK inhibitor, SP600125, on the release of IL-27p28 by macrophages (12 h, ELISA). (E) Flow cytometry of intracellular IL-27p28 in F4/80+CD11b+ macrophages incubated with LPS alone or in combination with either AEG3482 or SP600125 (12 h). Regions were defined according to isotype Ab controls (data not shown). All data are representative of three independent experiments each performed in duplicate wells. Error bars represent SEM. *p < 0.05, **p < 0.01, ***p < 0.001.

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To define the role of JNK activation for the release of IL-27 by macrophages, we used specific pharmacologic JNK inhibitors. The small molecule inhibitor, AEG3482, dose-dependently suppressed IL-27p28 release from LPS/TLR4-activated macrophages (Fig. 6C). Likewise, the competitive and reversible JNK inhibitor, SP600125, antagonized the appearance of IL-27p28 in cell culture supernatants of macrophages (Fig. 6D). In flow cytometry experiments, F4/80+CD11b+ double-positive macrophages were costained for intracellular IL-27p28 in the presence of a Golgi transport inhibitor. These experiments confirmed that the blockade of JNK signaling with either AEG3482 or SP600125 resulted in profoundly reduced numbers of IL-27p28–producing macrophages as indicated by the lower frequencies of IL-27p28+F4/80+ and IL-27p28+CD11b+ cells, with the latter induced by the presence of LPS (Fig. 6E). In summary, these studies suggested that β2 adrenoceptor activation antagonized JNK signaling to blunt the production of IL-27p28 in macrophages.

To investigate whether the neuroendocrine regulatory circuits defined in macrophage cultures are also present in vivo, we employed a model of endotoxic shock in C57BL/6J mice during which splenic macrophages are the major cellular source of IL-27p28 (9, 20).

Because adrenaline and noradrenaline have a short plasmatic half-live in vivo (t1/2 of ∼2 min), we used formoterol, as a long-lasting β2 adrenoceptor agonist in endotoxic shock. One group of mice received injections of LPS alone, whereas the other group received formoterol i.p. together with LPS and again formoterol i.p. after 24 h. Formoterol treatment significantly reduced the severity of LPS-induced hypothermia at early time points (Fig. 7A). Hypothermia is a predictor of septic shock mortality (63). Additionally, all mice were monitored and assessed using a CSS. Formoterol reduced the CSS values, indicating an improvement of the adverse clinical signs during the course of endotoxic shock (Fig. 7B). There was a nearly significant trend (p = 0.067) for a better survival of the formoterol-treated mice (n = 10 per group; Fig. 7C).

FIGURE 7.

Influence of adrenergic activation on the severity and cytokine release of endotoxic shock. (A) Body surface temperature during the early phase of endotoxic shock (LPS 10 mg/kg body weight i.p.; n = 10) with and without formoterol (200 nmol/kg body weight i.p.; n = 10). (B) CSS during endotoxic shock with and without formoterol (n = 10 per group). (C) Survival of endotoxic shock in the presence or absence of formoterol (200 nmol/kg body weight i.p.; two injections at 0 and 24 h; the control group received PBS vehicle in addition to LPS; n = 10 per group). (D) Serial measurements of IL-27p28 in plasma at 3–12 h after administration of LPS with and without formoterol in two groups of C57BL/6J mice (ELISA). (E) Plasma concentrations of IL-10 from the same experiment as shown in (D) (ELISA). (F) RT-PCR of mRNA for IL-27p28 in spleen homogenates of endotoxemic mice (n = 7) or endotoxemic mice with cotreatment of formoterol (n = 4) (12 h). (G) IL-27p28 in plasma 8 h following endotoxemia of C57BL/6 mice with prior sham surgery (Sham-OP) or adrenalectomy surgery (ADR-X) several weeks before LPS injection. (H) IL-27p28 in plasma 8 h following endotoxemia of C57BL/6J mice with the selective glucocorticoid receptor antagonist mifepristone (30 mg/kg body weight i.p., 30 min before LPS challenge) or DMSO vehicle injection (n = 5 per group). Experiments were performed with cohoused n = 4–10 male mice per group, and each symbol (circles, squares, triangles) represents one animal. Error bars represent SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. ns, not significant.

FIGURE 7.

Influence of adrenergic activation on the severity and cytokine release of endotoxic shock. (A) Body surface temperature during the early phase of endotoxic shock (LPS 10 mg/kg body weight i.p.; n = 10) with and without formoterol (200 nmol/kg body weight i.p.; n = 10). (B) CSS during endotoxic shock with and without formoterol (n = 10 per group). (C) Survival of endotoxic shock in the presence or absence of formoterol (200 nmol/kg body weight i.p.; two injections at 0 and 24 h; the control group received PBS vehicle in addition to LPS; n = 10 per group). (D) Serial measurements of IL-27p28 in plasma at 3–12 h after administration of LPS with and without formoterol in two groups of C57BL/6J mice (ELISA). (E) Plasma concentrations of IL-10 from the same experiment as shown in (D) (ELISA). (F) RT-PCR of mRNA for IL-27p28 in spleen homogenates of endotoxemic mice (n = 7) or endotoxemic mice with cotreatment of formoterol (n = 4) (12 h). (G) IL-27p28 in plasma 8 h following endotoxemia of C57BL/6 mice with prior sham surgery (Sham-OP) or adrenalectomy surgery (ADR-X) several weeks before LPS injection. (H) IL-27p28 in plasma 8 h following endotoxemia of C57BL/6J mice with the selective glucocorticoid receptor antagonist mifepristone (30 mg/kg body weight i.p., 30 min before LPS challenge) or DMSO vehicle injection (n = 5 per group). Experiments were performed with cohoused n = 4–10 male mice per group, and each symbol (circles, squares, triangles) represents one animal. Error bars represent SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. ns, not significant.

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Subsequently, plasma was collected by repetitive bleeding from the retro-orbital sinus at 3, 6, and 12 h time points (Fig. 7D). At all the time points studied, formoterol significantly reduced the plasma concentrations of IL-27p28 in mice with endotoxic shock. IL-10 was quantified in the plasma samples of the same experiment (Fig. 7E). Formoterol promoted the appearance of IL-10 in plasma during endotoxemia at 3 and 6 h (Fig. 7E). Additionally, the amounts of mRNA for IL-27p28 were quantified in spleen homogenates of C57BL/6J mice with endotoxemia with and without formoterol (Fig. 7F). In fact, formoterol significantly diminished the gene expression of IL-27p28 during endotoxemia after 12 h (Fig. 7F). No reciprocal effect was observed for mRNA of IL-10 after 12 h (data not shown), which may be explained by the concept that IL-10 gene expression already occurs earlier during endotoxemia (Fig. 7E).

To study the influence of endogenous catecholamines for the LPS/TLR4-induced release of IL-27p28 during endotoxic shock, we used C57BL/6J mice after surgical removal of the adrenal glands. Mice were allowed to recover from the surgery for several weeks before they were subjected to endotoxemia. As a control group, mice after sham surgery were treated in the same fashion. Adrenalectomized mice displayed 2.7-fold higher concentrations of IL-27p28 in plasma 8 h after injection of LPS (Fig. 7G). Of note, in one outlier mouse within the group of adrenalectomy the concentrations of IL-27p28 were even substantially lower as in the sham operated control group. We speculate that in this mouse the intra-abdominal injection of LPS may not have been i.p. but rather accidental intraintestinal or retro-abdominal. Alternatively, in this outlier animal the surgical removal of the adrenal glands may have been unintentionally incomplete. In any case, the concentrations of IL-27p28 were significantly higher (p < 0.01) in adrenalectomized mice after endotoxemia. To evaluate a potential contribution of endogenous glucocorticoids in adrenalectomized mice, we used the glucocorticoid receptor antagonist, mifepristone, according to a published dosing regimen (58). The appearance of IL-27p28 in plasma was not significantly influenced by mifepristone treatment during endotoxic shock (Fig. 7H), suggesting that endogenous catecholamines rather than endogenous glucocorticoids are major regulators of IL-27p28 release in endotoxic shock (Figs. 7G, 8).

FIGURE 8.

Schematic of the proposed mechanisms of IL-27 regulation by neuroendocrine mediators in macrophages. Activation of the TLR4 pathway by LPS promotes phospho-JNK (and other signaling cascades; data not shown) for facilitating the gene expression and release of IL-27 by macrophages. Catecholamines via β2 adrenoceptor activation antagonize phospho-JNK and suppress the release of IL-27. β2 adrenoceptor agonists (e.g., formoterol, salbutamol) promote the release of IL-10 in an autocrine/paracrine feedback loop that via IL-10R limits phospho-JNK and reduces the release of IL-27. Activation of IL-27 production by the TLR3 pathway is unresponsive to the effects of β2 adrenoceptors and/or IL-10.

FIGURE 8.

Schematic of the proposed mechanisms of IL-27 regulation by neuroendocrine mediators in macrophages. Activation of the TLR4 pathway by LPS promotes phospho-JNK (and other signaling cascades; data not shown) for facilitating the gene expression and release of IL-27 by macrophages. Catecholamines via β2 adrenoceptor activation antagonize phospho-JNK and suppress the release of IL-27. β2 adrenoceptor agonists (e.g., formoterol, salbutamol) promote the release of IL-10 in an autocrine/paracrine feedback loop that via IL-10R limits phospho-JNK and reduces the release of IL-27. Activation of IL-27 production by the TLR3 pathway is unresponsive to the effects of β2 adrenoceptors and/or IL-10.

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In this study, we have investigated the influence of neuroendocrine sympathetic mediators on the gene expression and production of IL-27 by macrophages. Our findings suggest that adrenaline and noradrenaline interact with β2 adrenoceptors to mediate a context-dependent suppression of IL-27 release by LPS/TLR4-activated macrophages rather than poly(I:C)/TLR3-activated macrophages. The underlying mechanisms of these phenomena appear to rely on a feedback loop with IL-10, wherein poly(I:C)/TLR3-induced IL-27 is insensitive to the counterbalancing effects of IL-10. Additionally, β2 adrenoceptor agonists synergistically with IL-10 downmodulate the IL-27–stimulating intracellular JNK signaling pathway. The proposed molecular mechanisms are summarized in Fig. 8.

The data of our current study extend the knowledge in the neuroimmune field by to our knowledge, first describing that IL-27 is regulated by catecholamines, the principal hormones of multifaceted acute stress responses (64). Adrenaline and noradrenaline are well known to affect a multitude of immune functions such as Ag presentation, leukocyte proliferation and traffic, secretion of cytokines and Abs, and balancing Th1/Th2 lymphocyte responses (64). The cellular effects of catecholamines are mediated by several classes of adrenoceptors, which all belong to the 7-transmembrane domain spanning G protein–coupled receptor family (41). These adrenoceptors activate cAMP and several kinase signaling pathways (e.g., PKA, CREB, p38 MAPK), but mediate inhibition of NF-κB (41, 65). In general, adrenaline exerts a higher affinity for β2 adrenoceptors than does noradrenaline. This is reflected by our initial observation that equimolar concentrations of adrenaline were more potent for the inhibition of IL-27p28 as compared with noradrenaline (Fig. 1C). Indeed, subsequent experiments revealed that the inhibition of IL-27 in macrophages appeared as mainly mediated by β2 adrenoceptor agonists (Figs. 1D, 1E, 2, 3). The single doses of catecholamines added to our macrophage cultures were typically higher as compared with continuous physiologic concentrations (1–20 × 10−9 M), although plasma catecholamine levels can increase up to 20-fold (2–8 × 10−8 M) during severe stress and sepsis (66, 67).

We have focused our studies on macrophages because mononuclear phagocytes are considered an exclusive cellular source of IL-27 (7, 9), although this view was recently challenged by uncovering a unique population of IL-27–producing Foxp3CD11a+CD49d+ Ag-specific CD4+ T cells during malaria infection (68).

Our data suggest that catecholamines suppress IL-27p28 release by inhibition of LPS/TLR4-induced phosphorylation of the JNK signaling pathway in primary mouse macrophages (Figs. 6, 8). JNK proteins belong to the MAPK family of stress-induced intracellular signals. In fact, we have demonstrated before that catecholamines via their β2 adrenergic activities negatively regulate the phosphorylation of JNK (59, 69). The IFN-γ–mediated IL-27 protein content was positively regulated by JNK in human monocytic cells (70). Additionally, using Theiler’s murine encephalomyelitis virus–infected RAW264.7 cells the pharmacologic blockage of JNK was associated with a reduction of mRNA for IL-27p28 (10). Theiler’s murine encephalomyelitis virus infection of macrophages is thought to activate the TLR3 and TLR7 pattern recognition pathways (10). Interestingly, there was a negligible potency of catecholamines regarding the selective inhibition of poly(I:C)/TLR3-induced IL-27p28 in our experiments. This is partly explained by the insensitivity of poly(I:C)/TLR3-induced IL-27p28 for autocrine/paracrine IL-10 (Fig. 5G). In conclusion, catecholamines appear to modulate IL-27p28 release in a context-dependent manner rather during bacterial than viral infections.

A traditional concept of stress hormones such as catecholamines (and glucocorticoids) has been that these mediators act as unilateral suppressors of immune responses and inflammation. The rationale for this view is that reducing the severity of inflammation (e.g., joint swelling, pain) is vitally important to preserve organ function during a “fight or flight” type stress response. Indeed, the idea of catecholamines acting as profound antagonists of the acute inflammatory response is supported by our findings that β2 adrenoceptor agonists increase the release of anti-inflammatory IL-10 by macrophages (Fig. 5). These data are in accordance with the clinical observations that catecholamines induce IL-10 release in patients suffering from acute myocardial infarction or accidental brain injury (71, 72).

In LPS-activated human whole blood, both noradrenaline and adrenaline increase the release of IL-10 but decrease IL-12 concentrations (51). These effects are reversible by administration of the nonselective β12 adrenoceptor blocker, propranolol (51). In human monocytes, adrenaline downregulates TNF-α but increases TNF receptor expression (52). The production of the chemokine CCL3 (MIP1α) is antagonized by exogenous and endogenous catecholamines in murine macrophages and mice after endotoxic shock (73). Catecholamines suppress the intracellular concentrations of IL-6 but do not affect IL-8 in LPS-activated human monocytes (74). All these findings would point toward a uniform immunosuppression by catecholamines.

Alternatively, the conceptual data interpretation is more complex for the suppression of IL-27 by catecholamines via β2 adrenoceptors. It is well established that IL-27 exerts both proinflammatory and anti-inflammatory properties (1). IL-27 can drive a rapid clonal expansion of IFN-γ–producing CD4+ T cells (2). The chemotaxis of activated T cells to the site of local inflammation may be stimulated by IL-27 via the induction of CXCL10 release in lung fibroblasts (75). Additionally, IL-27 is required for the IL-21–dependent production of high-affinity Abs in the germinal centers of lymph nodes (76). In contrast, the anti-inflammatory actions of IL-27 include the promotion of T cell–derived release of IL-10 and antagonizing Th17 cells (22, 27, 28). In skin fibroblasts, IL-27 stimulates the expression of IL-18 binding protein, which is a soluble receptor analog and natural antagonist of the T cell activating cytokine, IL-18 (77). IL-27 induces the expression of immune-regulatory CD39 on dendritic cells to limit T cell responses (26). In consequence of these findings, it is not surprising that genetic disruption of IL-27RA signaling results in lethal inflammation because of the inability to normalize T cell reactivity after clearance of an infectious pathogen (12). Thus, fine-tuning of IL-27–dependent immune responses by catecholamines presumably relays multilayered context-dependent influences on the prevailing inflammatory states and not by any means transmits an obstinate adrenergic immunosuppression. In fact, emerging studies have recently substantiated the idea about proinflammatory properties of catecholamines. For instance, a β2 adrenoceptor agonist promoted neutrophil infiltration and damage of colon tissue in a DSS-induced colitis model, whereas opposite effects were noted by administration of a nonselective β12 adrenergic blocker (78). In another study, noradrenaline induced elevated microarray gene expression profiles of inflammatory cytokines and chemokines in resting and activated memory CD8+ T cells from humans (79). Lymphocyte trafficking is controlled by β2 adrenoceptor activation (80). Finally, blockade of α2 adrenoceptors or catecholamine-synthesizing enzymes greatly suppressed inflammation in two models of lung injury (42). Some of the apparently discrepant findings on the inflammatory properties of catecholamines are apparently reconciled by data suggesting differential roles of β2 adrenoceptors (anti-inflammatory) as compared with α adrenoceptors (proinflammatory) (81, 82). Nonetheless, for IL-27p28 release we did not observe any roles of α1 adrenoceptor signaling relaying the effects of catecholamines in macrophages (Fig. 1D, 1E), although radioligand binding studies have shown such cells to express α1 adrenoceptors (44).

In the past, the adrenal glands and sympathetic nerves were considered as the major sources of catecholamines. However, accumulating data suggest that catecholamines are also produced by immune cells. For example, human neutrophils contain catecholamines and their metabolites, suggesting autoregulatory adrenergic mechanisms (83). Activation of macrophages with LPS has been reported to induce tyrosine hydroxylase, the rate-limiting enzyme responsible for catecholamine generation, and consequently the synthesis of noradrenaline and dopamine (49). Catecholamines derived from mononuclear phagocytes enhance acute inflammatory injury (42). Alternatively activated macrophages may produce catecholamines to sustain adaptive thermogenesis (84), but this view has been recently challenged by negative findings in a mouse strain with inducible hematopoietic cell–specific deletion of tyrosine hydroxylase (85). In any case, there remains evidence for the existence of endogenous catecholamine production by lymphocytes for regulating lymphocyte activities via an autocrine feedback loop (86). Notably, the production of IFN-γ (an IL-27–inducing cytokine) in activated Th1 cells is enhanced by exposure to noradrenaline (87).

In experimental mouse models of endotoxic shock or polymicrobial sepsis, therapeutic interception of IL-27 by neutralizing Abs, a soluble IL-27RA–Fc receptor fusion protein, as well as genetic absence of IL-27RA or EBI3 are all protective against lethality and the adverse peritoneal immune responses (9, 11, 20). This correlates with our findings that formoterol reduced plasma IL-27p28 together with the severity of endotoxic shock (Fig. 7).

In human patients with sepsis and septic shock, continuous infusions of noradrenaline and (in severe cases) adrenaline are routinely used as powerful vasopressors to treat hypotension and concomitant organ hypoperfusion. We speculate that the extent of immune-modulatory IL-27 production is most likely affected by catecholamine therapy and this may determine the overall functional effects of this immune-modulatory cytokine in human sepsis. Moreover, IL-27 has been suggested as a biomarker for the diagnosis of pediatric and adult sepsis patients (88, 89), but we now caution that catecholamine therapy as a confounding factor may distort IL-27 concentrations. A similar situation is imagined for chronic pulmonary obstructive disease, which is associated with elevated serum concentrations of IL-27 during disease exacerbations that are frequently treated with inhalations of β2 adrenoceptor agonists and glucocorticoids (90). Low doses of glucocorticoids are also included in supportive pharmacotherapy regimens of sepsis. Despite our results with the glucocorticoid antagonist mifepristone (Fig. 7H), we cannot exclude the possibility that persistent and abnormal concentrations of endogenous or exogenous glucocorticoids may modulate IL-27. In light of the importance of an adequate stress-hormone response for critically ill patients, it is not surprising that experimental blockade of β12 adrenergic signaling is a disadvantage for sepsis survival (67, 91).

In summary, our present study adds new information to the diverse interactions of neuroendocrine pathways with the immune system. To our knowledge, we provide the first evidence that IL-27–dependent immune functions are under the control of β2 adrenoceptor signaling in mononuclear phagocytes. In the future, it will be interesting to continue the research on the functional consequences of these observations, especially related to IL-27RA–expressing target cells of immune-modulatory IL-27 during inflammatory milieus.

We cordially thank Fabien Meta and Foruzandeh Samangan for technical assistance and Lisa Kubacki for secretarial assistance. M.B. thanks Peter A. Ward, Ulrich Walter, and Heiko Mühl for constant support and mentorship.

This work was supported by the B. Braun Foundation, Federal Ministry of Education and Research Grant 01EO1503 (to M.B.), Deutsche Forschungsgemeinschaft Grants BO3482/3-1, BO3482/3-3, and BO3482/4-1 (to M.B.), European Union Marie Curie Career Integration Grant 334486 (to M.B.), and by a Clinical Research Fellowship of the European Hematology Association (to M.B.). The authors are responsible for the contents of this publication.

Parts of the data in this study appeared in the thesis of M.H.

The online version of this article contains supplemental material.

Abbreviations used in this article:

     
  • BMDM

    bone marrow–derived macrophage

  •  
  • CSS

    clinical severity score

  •  
  • EBI3

    EBV-induced gene 3

  •  
  • NOS2

    NO synthase 2

  •  
  • PEM

    peritoneal elicited macrophage

  •  
  • poly(I:C)

    polyinosinic-polycytidylic acid

  •  
  • RT-PCR

    real-time PCR

  •  
  • WT

    wild-type.

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The authors have no financial conflicts of interest.

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