Humans respond to major surgery, trauma, or infection with an orchestrated release of neuroendocrine mediators termed the “stress response.” Researchers who first studied the stress response1,2 determined that it is rapid, proportional to the degree of injury, organized to promote resolution, and finally, enhances resistance to further injury. They also recognized glucocorticoids (GCs) as critical components of an injury response because, in their absence, experimental animals recovered slowly from stress or died.2,3 These initial findings that GCs have a supportive or stimulatory role in the stress response were largely forgotten with the first report of their potent anti-inflammatory properties in 1949.4 This was a largely unexpected finding that directed subsequent GC research toward understanding their therapeutic potential as anti-inflammatory and immunosuppressive drugs.
A strict conception of GCs as anti-inflammatory obscures both preexisting and emerging data that support a more comprehensive role for GCs in stress physiology. Recent research reveals that, depending on dose and timing, GCs induce substantial proinflammatory effects on human immune responses.1,5–8 When GCs were shown to increase expression of proinflammatory cytokine receptors on human immune cells,5 investigators looking for in vivo consequences reported findings suggesting clinical relevance.6–8 For example, cortisol pretreatment markedly augments the in vivo inflammatory response to bacterial endotoxin (lipopolysaccharide [LPS]) in humans,6 an effect reproduced by cortisol concentrations observed during systemic stress.7 Stress-associated concentrations of cortisol (approximately 3 times peak diurnal concentrations) also augment in vivo immune cell infiltration into inflammatory sites in humans.9 These and similar results10–12 are variously termed “adaptive,” “preparatory,” or “priming” because they are usually observed in vivo during a period that follows the early stress response when GCs induce their well-known anti-inflammatory effects. The data, therefore, support a dual role for GCs that includes early anti-inflammatory effects to limit consequences of inflammation followed by delayed, augmenting effects on inflammatory responses that enhance resistance to repeated injury.
Reliable and robust experimental models are needed to study the cellular and molecular events responsible for GC-induced proinflammatory responses. We previously reported that GCs augment interferon-ϒ (IFN-ϒ) mediated activation events in human monocyte/macrophages (MOs).13 MOs, which have a central role in initiation, amplification, and resolution of an in vivo inflammatory response,14 adopt proinflammatory or anti-inflammatory phenotypes when exposed, respectively, to the cell differentiation factors granulocyte-macrophage colony stimulating factor (GM-CSF) or M-CSF.14–17 We hypothesized that cortisol induces a delayed enhancing effect on IFN-ϒ–mediated proinflammatory responses in GM-CSF–differentiated human MOs.
Written consent was obtained by all subjects following a protocol approved by the Dartmouth College Committee for Protection of Human Subjects (institutional review board). Subjects were healthy males and females, 22–50 years of age, taking no medications and with no history of infection or major injury within 90 days. Heparinized whole blood samples were layered over Ficoll-Hypaque-1.077 (Sigma, St Louis, MO, #H8889) and centrifuged at 400g for 30 minutes. Monocytes were isolated from mononuclear cell fractions by CD14+ bead selection (Miltenyi Biotech, Auburn, CA, #130-050-201). Alternatively, mononuclear cells were obtained by leukapheresis and purified using cold aggregation as described.13 Cells obtained by leukapheresis were stored under liquid nitrogen until use, at which time they were thawed at 37°C and washed twice with RPMI/10% fetal bovine serum (Hyclone, Logan, UT, #SH30070) just before culture. Monocytes obtained by both methods were >92% pure, >90% viable, and gave comparable results.
Monocytes were cultured in 96-well flat-bottom plates (Falcon, Durham, NC, #353072) at a final concentration of 1.25 × 106 cells/mL in a final volume of 200 μL RPMI-1640 with 10% fetal bovine serums. Reagents were added concurrently to achieve the final concentrations of cortisol (Steraloids, Newport, RI, #Q3880), IFN-ϒ (20 ng/mL; Actimmune, Palo Alto, CA, #64116-011-12), GM-CSF (100 pg/mL; PeproTech, Rocky Hill, NJ, #300–03), M-CSF (100 pg/mL; PeproTech, #300–25) RU486 (Sigma-Aldrich, St Louis, MO, #M8046), or goat antihuman IFN-ϒ receptor type 1 (IFN-ϒR1) polyclonal antibody (Thermo Fisher, Rockford, IL, #PA-5-47866). All reagents were endotoxin free. After 18-hour overnight incubations, cells were either isolated for immunostaining or incubated with Escherichia coli LPS; E. coli 0111:B4; InvivoGen, San Diego, CA, catalog #tlrl-eblps, Lot 33-505-LPS) at a final concentration of 1 ng/mL for 4 hours, after which culture supernatants were collected.
Culture supernatants were collected and frozen at –80oC for batched measurement of interleukin-6 (IL-6) concentrations. IL-6 levels were determined using an IL-6 enzyme-linked immunosorbent assay (ELISA) kit (R&D Systems, Minneapolis, MN, #D6050) according to manufacturer instructions. The assay has a lower detection limit of 3.12 pg/mL. Interassay variability ranged from 1.0% to 9.8% and intraassay variabilities ranged from 3.6% to 12.6%.
Recommended volumes (5–20 ) of fluorescent-labeled antibodies were mixed with 20-μL cell samples plus 20 μL of 12 mg/mL human γ-globulin (from Cohn fraction II, III; Sigma Chemical, St Louis, MO, #G-4386) and incubated on ice for 1 hour in the dark. Cells were washed once in phosphate-buffered saline, pelleted, and fixed with 2% paraformaldehyde. Monocyte cell surface markers were evaluated on CD163-positive cells (Trillium Diagnostics, Brewer, ME, CD163-PE, clone Mac-2–158) that were initially gated by forward and side scatter. Data were acquired for fluorescence intensity using a MACSQuant 8 (Miltenyi Biotec). Files were then analyzed using FlowLogic software (Inivai Technologies, Mentone, Victoria, Australia). UltraComp compensation beads (eBioscience, San Diego, CA, #01-2222-42) were used as directed by the manufacturer for: anti-CD14 (Invitrogen, Frederick, MD, clone TuK4), anti-CD163 (Trillium Diagnostics, clone Mac-2–158), and anti–IFN-ϒR1 (Miltenyi Biotec, clone REA161 and R&D Systems, clone 92101).
Following IL-6 experiments, a separate investigation used the same cell isolation and culture conditions to examine the effect of cortisol on GM-CSF treated MOs using multiplex analysis of soluble mediators. Cell culture supernatants were analyzed by Millipore 41-MultiPlex Human Cytokine/Chemokine Kit (EMD Millipore Corporation, Billerica, MA). The assay was performed according to the manufacturer’s protocol using Luminex fluorescent bead technology. The following cytokines/chemokines were screened: EGF, Eotaxin, G-CSF, GM-CSF, IFN-α2, IFN-γ, IL-10, IL-12p40, IL-12p70, IL-13, IL-15, IL-17A, IL-1Ra, IL-1α, IL-1 β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IP-10, MCP-1, MIP-1α, MIP-1β, RANTES, TNF-α, TNF-β, VEGF, FGF-2, TGF-α, FIT-3L, Fractalkine, GRO, MCP-3, MDC, PDGF-AA, PDGF-AB/BB, and sCD40L. The fluorescence intensity of beads was measured using the Bio-Plex array reader (Bio-Rad, Hercules, CA). Bio-Plex Manager software (Bio-Rad, Hercules, CA) with 5-parametric-curve fitting was used for data analysis. This assay has a lower detection limit of 3.2 pg/mL. Interassay variability ranged from 5% to 19%, and intraassay variabilities ranged from 2% to 13%.
Statistical analyses were performed using GraphPad Prism version 7 for Mac (GraphPad Software, La Jolla, CA). Repeated-measures 1-way analysis of variance followed by the Dunnett multiple comparisons test was performed to assess the relationships between individual group means and the mean of a control group. To assess the interaction of culture conditions, repeated-measures 2-way analysis of variance with the Tukey multiple comparisons test was calculated. For each analysis, n = number of different donors tested. Statistical significance was defined at the 95% level.
Cortisol Pretreatment Enhances IFN-ϒ–Mediated IL-6 Release From Undifferentiated MOs Stimulated With LPS
MOs cultured for 18 hours with IFN-ϒ (20 ng/mL), in the absence of a MO differentiation factor, were subsequently stimulated for 4 hours with LPS. The addition of cortisol during the 18-hour pretreatment induced a highly significant increase in IFN-ϒ–mediated IL-6 release after LPS stimulation (P = .0004). When cortisol at either a 50 nM or 1 μM concentration was added to IFN-ϒ during preincubation, IL-6 release was significantly increased compared to IFN-ϒ alone (P = .0003 and P < .0001, respectively; Figure 1A). In the absence of LPS stimulation, MOs did not release detectable IL-6 (data not shown).
Cortisol Pretreatment Enhances IFN-ϒ–Mediated IL-6 Release From GM-CSF–Treated MOs Stimulated With LPS
MOs cultured for 18 hours with IFN-ϒ, in the presence of the MO differentiation factor GM-CSF, were subsequently stimulated for 4 hours with LPS. The addition of cortisol during the 18-hour pretreatment induced a highly significant increase in IFN-ϒ–mediated IL-6 release after LPS stimulation (P = .0002). When cortisol at either a 50 nM or 1 μM concentration was added with IFN-ϒ during preincubation, IL-6 release increased significantly compared to IFN-ϒ alone (P = .003 and P < .0001, respectively; Figure 1B).
Cortisol Pretreatment Enhances IFN-ϒ–Mediated IL-6 Release From M-CSF–Treated MOs Stimulated With LPS
MOs cultured for 18 hours with IFN-ϒ, in the presence of the MO differentiation factor M-CSF, were stimulated for 4 hours with LPS. The addition of cortisol during the 18-hour pretreatment induced a significant increase in IFN-ϒ–mediated IL-6 release after LPS stimulation (P = .007). When cortisol at either a 50 nM or 1 μM concentration was added with IFN-ϒ during preincubation, IL-6 release significantly increased compared to IFN-ϒ alone (P = .0183 and P = .0011, respectively; Figure 1C).
Cortisol Increases IFN-α2 Release and Inhibits IL-1 Receptor Antagonist Release From GM-CSF–Treated MOs Cultured With IFN-ϒ and Stimulated With LPS
Multiplex analysis of IL-6 release by GM-CSF–treated MOs stimulated with LPS showed concordance with the ELISA IL-6 analysis (Supplemental Digital Content, Document, http://links.lww.com/AA/C417). Cortisol at a 1 μM concentration induced a significant (>4-fold) increase in the release of IFN-α2 by GM-CSF–treated MOs cultured with IFN-ϒ compared to cells cultured with GM-CSF and IFN-ϒ but without cortisol (P = .003; Figure 2A). Cortisol at 50 nM and 1 μM concentrations also induced a significant decrease in the release of IL-1 receptor antagonist by GM-CSF–treated MOs cultured with IFN-ϒ compared to cells cultured with GM-CSF and IFN-ϒ without cortisol (P = .002 and P = .009, respectively; Figure 2B).
Blocking the GC Receptor or the IFN-ϒR1 Inhibits Cortisol Enhancement of IFN-ϒ–Mediated IL-6 Release by LPS-Stimulated MOs
Because GM-CSF MOs showed the most vigorous interaction between IFN-ϒ and cortisol, GM-CSF–treated MOs were cultured with IFN-ϒ plus cortisol and without or with the GC receptor (GR) antagonist, RU486 at a 500 nM concentration (Figure 3A) or a polyclonal antibody to IFN-ϒR1 at a 2 μg/mL concentration (Figure 3B), both followed by LPS stimulation. Both RU486 and the polyclonal antibody significantly decreased IL-6 release by LPS-stimulated MOs exposed to cortisol plus IFN-ϒ (P < .0001 for both).
Cortisol Increases Expression of IFN-ϒR1 on Undifferentiated and GM-CSF–Treated MOs
Untreated and GM-CSF–treated MOs were cultured for 18 hours with cortisol alone to assess its effect on expression of the IFN-ϒ receptor, IFN-ϒR1. There was a significant increase in expression of IFN-ϒR1 on undifferentiated MOs treated with 1 μM cortisol concentrations (P < .0001; Figure 4). Culture of GM-CSF–exposed MOs with cortisol also induced an increased expression of IFN-ϒR1 that was significant at both the 50 nM concentration and at the 1 μM concentration (P = .001 and P < .00001, respectively). IFN-ϒ alone decreased expression of IFN-ϒR1 as previously reported18 and there was no observed effect of cortisol on expression of IFN-ϒ receptor type 2 (data not shown).
Cortisol Suppresses LPS-Stimulated Monocyte IL-6 Release After 4-Hour Incubation With GM-CSF and IFN-ϒ But Enhances IL-6 Release When Cells Are Incubated for 18 Hours Before LPS Stimulation
Because cortisol is widely understood to acutely suppress inflammation, we cultured both untreated and GM-CSF–treated monocytes with IFN-ϒ and cortisol for 4 hours. These cells were simultaneously stimulated with LPS during the 4-hour incubation. Culture supernatants were then collected to determine IL-6 concentrations. In the same experiment, to parallel the findings shown in Figure 1B, GM-CSF plus IFN-ϒ–exposed cells were cultured with cortisol for 18 hours, after which they were stimulated with LPS for 4 hours, followed by analysis of culture supernatants for IL-6. Results showed that cortisol did not demonstrate enhancing properties at 4 hours and instead suppressed the IL-6 response in both untreated and GM-CSF–treated monocytes (P = .03 for both at the 1 μM cortisol concentration; Figure 5A, B). In the same experiment, but after the 18-hour incubation of monocytes with GM-CSF plus IFN-ϒ, cortisol had a significant enhancing effect on the IFN-ϒ–mediated IL-6 response to LPS (P = .0035 at the 1 μM cortisol concentration; Figure 5C).
A 6-Hour Exposure to Cortisol Is Sufficient to Prime an Enhanced IL-6 Response to LPS Stimulation in GM-CSF Plus IFN-ϒ–Pretreated MOs
Clinical studies show that cortisol enhancement of the human in vivo response to LPS is achieved after a 6-hour experimental increase in plasma cortisol concentration.6 We, therefore, exposed MOs to GM-CSF and IFN-ϒ plus cortisol at 50 nM and 1 μM concentrations for 6 hours, after which cortisol concentration was decreased to 10 nM (an in vivo concentration observed in nonstressed humans). Incubation was continued for an additional 12 hours with GM-CSF plus IFN-ϒ, followed by LPS stimulation for 4 hours. Exposure to cortisol for 6 hours was sufficient to significantly enhance IFN-ϒ–mediated IL-6 release after LPS stimulation (P = .0034 at the 1 μM cortisol concentration; Figure 5D).
There are compelling reasons to expect that GCs have a comprehensive regulatory role in the human stress response to injury or infection. First, GCs are regulated by the diencephalon via hypothalamic endocrine signals. From a phylogenetic standpoint, this suggests a fundamental, vital, and wide-ranging role in homeostasis. Second, GRs and other GC responsive molecules are found in virtually all nucleated human cells. Third, GCs have been shown to regulate up to 20% of the human genome in nonstressed individuals19 further indicating a broad role in human physiology. Finally, the well-known diurnal variation in GC concentrations that parallel activity/rest cycles suggests that preactivity GC peaks stimulate or support important survival mechanisms. According to most physiological models, the acute surge of in vivo GC concentrations that follows systemic stress limits destructive effects of an early proinflammatory innate immune response. This occurs via well-known anti-inflammatory responses mediated by GR genomic regulation.1,5 The current report adds to a growing literature demonstrating that stress levels of GCs, while suppressing the early proinflammatory response to an external stimulus, also prime the immune system for an augmented response to a subsequent stimulus. The latter effect has been interpreted as an adaptive response to repeated stress such as a postinjury infection.17 Notably, it appears that GC priming of innate immunity occurs simultaneously with GC anti-inflammatory effects.20
Although GCs may prime inflammatory responses, primed cells do not constitutively induce inflammation; they require a second stimulus. Whether an augmented response to a second stimulus manifests as beneficial or detrimental depends on the outcome of interest. An augmented response could be beneficial by increasing resistance to infection after injury. Animals exposed to a sterile systemic stress show increased resistance to experimental infection days later.21 Stressed animals also demonstrate enhanced responses to antigenic stimulation via GC-mediated pathways.8 In humans, a single dose of dexamethasone during cardiac surgery is associated with reduced infection rates after surgery.22 However, adverse effects have also been reported in animals due to an increased inflammatory response.23 Such results may parallel the adverse consequences of the “second-hit” phenomenon observed in critically ill patients. In this clinical scenario, a second systemic stimulus, usually following sterile trauma,24 augments systemic inflammation and worsens clinical outcomes.
The results reported here from in vitro experiments were designed to represent, as far as possible, relevant inflammatory consequences of hormone–cytokine interactions in vivo. Ongoing investigations of how monocytes differentiate into macrophages with varying phenotypes, for example, are related. GM-CSF induces a proinflammatory (M1, classical) phenotype in MOs while M-CSF leads to an anti-inflammatory, wound healing (M2, alternative) phenotype.16 A protective versus pathological outcome of MO activation is further dependent on exposure to other proinflammatory mediators such as IFN-ϒ or anti-inflammatory mediators such as IL-10.25 In addition, to mimic in vivo events, we exposed MOs to both a “physiological” cortisol concentration (concentrations achieved in vivo during systemic stress7) and a pharmacological concentration (achievable only by exogenous administration of cortisol). While GCs acutely suppress in vivo and in vitro responses to LPS, we found that when MOs were exposed to IFN-ϒ plus physiological and/or pharmacological cortisol concentrations before LPS, the MO inflammatory response was significantly greater for cortisol-treated cells. The implications of this result are potentially broad because IFN-ϒ can decrease human infections26 and regulates an array of MO responses, including antigen presentation, reactive oxygen species production, and monocyte localization.18 As a biomarker of immune cell activation, IL-6 has the advantage of prolonged release compared to tumor necrosis factor-α and IL-1ß.27 However, because IL-6 has pleiotropic effects, including some anti-inflammatory effects,28 we used multiplex analysis to examine cellular release of more uniformly proinflammatory and anti-inflammatory mediators. IFN-α2 has immune-stimulating effects that are the basis for its therapeutic use in the treatment of human disease,29 while IL-1 receptor antagonist is a potent anti-inflammatory mediator used clinically to treat inflammatory conditions.30 Both of these mediators were regulated by the cortisol/IFN-ϒ interaction in a way that was distinctly proinflammatory further supporting a dual-effect model for GC regulation of inflammation. This model includes an adaptive, proinflammatory response of M1 type MOs after their exposure to cortisol and IFN-ϒ.
Because the reported results were observed in all subjects, this report presents a vigorous and reproducible experimental system by which to interrogate potential mechanisms of GC-enhanced IFN-ϒ signaling. The “genomic storm” that accompanies major injury or infection31 directs a complex in vivo environment32 that complicates interpretation of our results. Nonetheless, some relevant signaling modifications are known. For example, GCs increase expression of toll-like receptors (TLRs) TLR2 and TLR4 on multiple cell types leading to augmented inflammatory responses.10,33 Exposure of MO to GCs increases expression of inflammasomes that sense the sepsis mediator High Mobility Group Box 1, leading to augmented inflammatory responses to LPS in vivo.34,35 GCs also enhance in vivo concentrations of High Mobility Group Box 1 in human plasma at the same time that concentrations of tumor necrosis factor-α are decreased.36 Further investigations could examine, for example, if GC enhancement of IFN-ϒ–stimulated responses is affected by GC-induced chromatin decompaction,37 increased cellular expression of IFN-ϒR1,38 or modulation of histone acetylation. Because IFN-ϒ signals mainly through STAT1, which is regulated by histone acetyltransferases, GC regulation of IFN-ϒ responsiveness may be dependent on occupancy of the GR. Finally, intracellular suppressors of cytokine synthesis (SOCS) such as SOCS3 (a feedback inhibitor39) are regulated by GCs which may also induce biphasic regulation of SOCS3 mRNA in human MOs.40
There is a surprisingly robust, proinflammatory interaction between the endogenous stress hormone cortisol and the endogenous immune stimulant, IFN-ϒ. These results are consistent with in vivo studies showing early suppression of LPS-stimulated inflammation by GCs followed by augmented responses to a second stimulus.6,7 The reproducibility and robustness of this model identify a previously unreported experimental platform for studies designed to examine control pathways and molecular mechanisms. Data from such studies could inform development of new interventions in which GCs may be therapeutically manipulated to enhance immune responses before or after clinical events such as elective surgery or vaccine inoculation.
The authors gratefully acknowledge the assistance of D. David Glass, MD, who reviewed the manuscript and provided helpful comments and suggestions.
Name: Mark P. Yeager, MD.
Contribution: This author helped design and conduct the study, analyze the data, and write the manuscript.
Name: Cheryl A. Guyre, PhD.
Contribution: This author helped analyze the data and write the manuscript.
Name: Brian D. Sites, MD, MS.
Contribution: This author helped analyze the data and write the manuscript.
Name: Jane E. Collins, MLT (ASCP).
Contribution: This author helped design and conduct the study, analyze the data, and write the manuscript.
Name: Patricia A. Pioli, PhD.
Contribution: This author helped design the study, analyze the data, and write the manuscript.
Name: Paul M. Guyre, PhD.
Contribution: This author helped design and conduct the study, analyze the data, and write the manuscript.
This manuscript was handled by: Alexander Zarbock, MD.
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