Regulation of the macrophage oxytocin receptor in response to inflammation

Abstract

It has been demonstrated that the neuropeptide oxytocin (OT) attenuates oxidative stress and inflammation in macrophages. In the current study, we examined the role of inflammation on the expression of the oxytocin receptor (OXTR). We hypothesized that OXTR expression is increased during the inflammation through a nuclear factor-κB (NF-κB)-mediated pathway, thus responding as an acute-phase protein. Inflammation was induced by treating macrophages (human primary, THP-1, and murine) with lipopolysaccharide (LPS) and monitored by expression of IL-6. Expression of OXTR and vasopressin receptors was assessed by qPCR, and OXTR expression was confirmed by immunoblotting. Inflammation upregulated OXTR transcription 10- to 250-fold relative to control in THP-1 and human primary macrophages and increased OXTR protein expression. In contrast, vasopressin receptor-2 mRNA expression was reduced following LPS treatment. Blocking NF-κB activation prevented the increase in OXTR transcription. OT treatment of control cells and LPS-treated cells increased ERK1/2 phosphorylation, demonstrating activation of the OXTR/G_αq/11 signaling pathway. OT activation of OXTR reduced secretion of IL-6 in LPS-activated macrophages. Collectively, these findings suggest that OXTR is an acute-phase protein and that its increased expression is regulated by NF-κB and functions to attenuate cellular inflammatory responses in macrophages.

previous studies have shown that the neuropeptide oxytocin (OT) has anti-inflammatory properties and can facilitate wound healing (8), ameliorate carrageenan-induced inflammation (18), reduce sepsis and colonic inflammation (11), and decrease renal injury (1). In our laboratory, we have demonstrated that OT is both anti-inflammatory and antioxidant in human vascular cells, monocytes, and macrophages (25) and that chronic infusion of OT in both mice and rabbit models of atherosclerosis inhibited the progression of this inflammatory disease (15, 26).

The OT receptor (OXTR) has been well characterized (9, 34), and although it is classically understood in the context of reproductive tissues and brain function, OXTR has also been identified throughout the body in endothelial cells, smooth muscle cells, monocytes, macrophages (25, 28), myoblasts, cardiomyocytes, and bone cells (3, 4, 10, 16). OXTR is a G protein-coupled receptor, and in most cases it is coupled via the G_αq/11 protein to its effector phospholipase C-β (PLCβ) (9). PLCβ then catalyzes the cleavage of the membrane-bound phosophatidylinositol 4,5-biphosphate into inositol triphosphate (IP₃) and diacylglycerol (DAG). IP₃ triggers calcium release from intracellular stores and together with DAG stimulates protein kinase C (PKC), which phosphorylates target proteins leading to the cascade activating the ERK1/2 pathway.

OXTR expression can undergo dramatic changes in reproductive tissues (23, 34), which are due largely to activation of estrogen response elements (ERE) located on the OXTR promoter region (17, 29). In addition to EREs, there are a variety of other transcription factor-binding sites located on the OXTR promoter region that may regulate OXTR expression during an inflammatory response. Both nuclear factor-κ light chain enhancer of activated B cells (NF-κB) and nuclear factor interleukin-6 (NF-IL-6) response elements have been identified on the 5′-flanking region of the OXTR gene (20), suggesting that OXTR protein expression may be regulated by activation of either or both of these response elements. NF-κB activation is a critical initial factor in the cellular inflammatory cascade inducing expression of the cytokine IL-6 (14), raising the possibility that OXTR expression can be regulated by NF-κB and/or IL-6 during inflammation.

In the current study, we examined the effects of inflammation on the OXTR expression in cultured human and mouse macrophages. OXTR function was assessed by phosphorylation of ERK1/2 and the ability of OT to inhibit IL-6 secretion. We hypothesized that OXTR expression is increased during the inflammatory response through an NF-κB-mediated pathway, thus responding as an acute-phase protein. This upregulation in the expression of OXTR may contribute to the anti-inflammatory properties of OT.

MATERIALS AND METHODS

Cell cultures.

Human THP-1 (cat. no. TIB202; ATCC, Manassas, VA) monocytes were cultured as described (25) in RPMI 1640 medium (cat. no. 30-2001; Life Technologies, Grand Island, NY) containing 10% fetal bovine serum (FBS), activated into macrophages by treatment with phorbol myristate acetate (100 nM) for 24 h, and maintained in culture for an additional 6 days before experimentation. Primary human monocyte-derived macrophages were prepared from whole blood buffy coats (obtained from the local blood bank) from two donors (donor 1, a male; and donor 2, a female), as described previously (2). Briefly, 5 × 10⁶ peripheral blood mononuclear cells (PBMC) were plated in 60-mm culture dishes containing RPMI 1640 with 10% FBS and allowed to adhere for 2 h. Nonadherent cells were removed by rinsing with PBS, and adherent cells were allowed to differentiate into macrophages during 7 days of culture, with media changes every 48 h. Mouse RAW 264.7 (ATCC TIB71) macrophages were maintained in Dulbecco’s modified Eagle’s medium (cat. no. 30-2002; Life Technologies) containing 10% FBS and cultured to near confluence. All cell types were incubated with OT or vasopressin at physiologically relevant levels (100 pM), and inflammation was induced with 100 ng/ml lipopolysaccharide (LPS) for 6 h. The NF-κB inhibitor [caffeic acid phenethylester (CAPE); Millipore, Billerica, MA] was used at a concentration of 25 µg/ml for treatment with cell cultures.

In vivo murine peritoneal macrophages.

Male C57BL/6 mice (12 wk old) were obtained from The Jackson Laboratory (Bar Harbor, ME) and kept in a pathogen-free, temperature-controlled room with a 12-h light-dark cycle and allowed food and water ad libitum. Mice were randomly assigned to vehicle or LPS treatment groups. Those in the experimental group were injected with 50 µg of LPS intraperitoneally and euthanized after 6 h. Resident macrophages were collected by gavage of the peritoneal cavity as previously described (33). Procedures for the care, use, and euthanasia of experimental animals followed the protocols and regulations of and was approved by the Animal Care and Use Committee of the University of Miami and conformed to the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health.

RT-PCR experiments to measure IL-6 and OXTR mRNA expression.

Real-time polymerase chain reaction (RT-PCR) was used to quantify cellular OXTR, IL-6, and vasopressin receptor (AVPR1a, AVPR1b, and AVPR2) mRNA expression levels. Total RNA was isolated using the RNeasy extraction kit (Qiagen, Valencia, CA), and then cDNA was synthesized after DNAse I treatment using reagents from Applied Biosystems (Foster City, CA), following the manufacturer’s instructions.

Quantitative gene expression of human OXTR, AVPRs, and IL-6 by RT-PCR was performed with the TaqMan gene expression assay. The following Applied Biosystems inventoried primers were used: human OXTR (Hs00168573_m1), mouse OXTR (Mm01329577_g1), human IL-6 (Hs00985639_m1), mouse IL-6 (Mm00446190_m1), human AVPR1a (Hs00176122_m1), AVPR1b (Hs00949767_m1), and AVPR2 (Hs04195588_s1). cDNA (50 ng) was amplified with TaqMan Universal PCR Master Mix, and reactions were run using universal cycling conditions on an Applied Biosystems 7500 Real-Time PCR system. Samples were analyzed in triplicates. The ΔΔC_T (threshold cycle) method was used to analyze changes in gene expression. Relative quantification was expressed as the fold change compared with the appropriate control condition (22). 18S RNA was used as the endogenous mRNA control. A nontemplate control was performed to ensure that there was no amplification of genomic DNA.

Western blotting for OXTR.

OXTR protein expression was examined in total cell homogenates. PBS-washed cells were collected by scraping and solubilized in SDS lysis buffer (50 mM Tris, pH 8.6, 1% SDS), aided by brief sonication. Protein was measured with BCA Protein Assay (Pierce, Rockford, IL). For immunoblotting, 5–20 µg of protein was separated by denaturing and reducing electrophoresis on 10–20% Tris-glycine polyacrylamide gradient gels (Lonza, Walkersville, MD). After separation, proteins were transferred to nitrocellulose. Membranes were blocked in Tris-buffered saline containing 5% nonfat powdered milk and 0.1% Tween-20 for 1 h and then probed with polyclonal anti-rabbit OXTR (cat. no. ab181077; Abcam, Cambridge, MA) diluted 1:1,000 in blocking buffer at 4°C overnight. Immunoreactive bands were detected with appropriate peroxidase-conjugated secondary antibody for 1 h and then visualized with chemiluminescence. To normalize for protein loading, the membranes were stripped and reprobed with chicken anti-actin antibody (cat. no. SAB3500350; Sigma, St. Louis, MO).

To document the specificity of the OXTR antibody, kidney tissue from OXTR-knockout and wild-type mouse controls (generously provided by Dr. Larry Young, Emory University) was probed by Western blot using the OXTR antibody described above. Kidneys, which have been shown to express OXTR (21), were homogenized and plasma membrane fractions prepared as described previously (25). Specificity of the OXTR antibody was demonstrated by the lack of immunoreactivity in tissue from OXTR-knockout mice compared with the wild-type control that shows positive immunoreactivity of protein bands with the molecular size expected for native (~47 kDa) and glycated forms (~70 kDa) of the OXTR (Fig. 1).

Fig. 1.

Specificity of the anti-oxytocin receptor (OXTR) antibody. Plasma membrane fractions were prepared from kidney tissue obtained from wild-type (WT) or OXTR-knockout (KO) mice. Five micrograms of membrane protein was separated by denaturing polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. Left: samples were probed with an anti-OXTR antibody to evaluate specificity of the immunoreactivity. Right: gel lanes stained for total protein to demonstrate comparable protein loading for the samples. Bottom: immunostaining of Na/K ATPase as an additional loading control.

IL-6 assay.

Cell-free culture supernatants were assayed for IL-6 using commercially available reagents (BD Biosciences, San Diego, CA). IL-6 concentrations were expressed as pg/ml IL-6 of cell culture media.

ERK1/2 phosphorylation.

Differentiated macrophages (5 × 10⁶ cells/100 mm culture dish) were treated with LPS in RPMI containing 1% FBS for 6 h at 37°C in a humidified incubator with 5% CO₂.After incubation, cells were washed twice with PBS and then incubated for an additional 1 h in complete RPMI to allow for ERK1/2 dephosphorylation (31). Cells were then treated with OT for 5 or 10 min in a 37°C water bath. At the end of the incubation, cells were immediately chilled on ice, washed three times with ice-cold PBS, and collected by scraping into buffer containing protease and phosphatase inhibitors. Phosphorylated and total ERK1/2 were measured by immunoblotting, as described previously (25), using antibodies from Cell Signaling Technology (Danvers, MA) diluted 1:1,000 in 5% BSA blocking buffer.

Statistical analyses.

Data were obtained from a minimum of three replicates from at least three separate experiments and are presented as means ± SE. Results were compared by paired independent t-tests or ANOVA (1- or 2-way) with post hoc Bonferroni correction. An α-level of 0.05 was required for statistical significance.

RESULTS

Effects of LPS on OXTR expression in human macrophages.

To evaluate the effect of an inflammatory stimulus (confirmed by IL-6 expression and secretion) on OXTR gene expression, human THP-1 macrophages were incubated with LPS for ≤18 h (Fig. 2). OXTR and IL-6 mRNA expression were increased significantly by LPS treatment, with maximal expression occurring by 3 and 6 h of incubation. For both OXTR and IL-6 expression, there was a significant treatment (LPS vs. control) by time interaction: F(5, 12) = 15.58, P < 0.001; F(5, 12) = 9.93, P < 0.001, respectively (Fig. 2, A and D). There was a gradual decrease back to baseline levels for both OXTR and IL-6 mRNA by 18 h of LPS treatment. Under control conditions, THP-1 macrophages exhibited two bands of OXTR immunoreactivity that were consistent with a 46-kDa immature form and a 67-kDa mature glycosylated form of the receptor (Fig. 2B) (25). After incubation of cells with LPS for 6 h, OXTR protein expression was increased significantly in both immunoreactive bands (Fig. 2, B and C). For IL-6 secretion, there was also a significant treatment by time interaction, F(5, 20) = 61.79, P < .001, such that secretion increased by 6 h and reached peak levels by 9–12 h, remaining elevated over the course of the experiment (Fig. 2E). When LPS-treated cells were also incubated with OT, there was a 34% decrease in IL-6 secretion, P < 0.01 (Fig. 2F). This suggests that the LPS-stimulated increase in OXTR expression yielded functional OXTR receptors capable of attenuating inflammation [as demonstrated previously by Szeto et al. (25)].

Fig. 2.

Treatment of THP-1 macrophages with LPS-induced expression of OXTR (A–C) and IL-6 (D–F). Cells were treated with LPS (100 ng/ml; ■) or vehicle (●), and then OXTR mRNA (A), IL-6 mRNA (D), and IL-6 protein secretion (E) were measured over time. OXTR protein expression in control and LPS-treated cells was determined by Western blotting (B). There was a significant increase in OXTR mRNA expression over time that paralleled IL-6 mRNA expression (both P < 0.001), and IL-6 protein secretion was also increased over time. OXTR protein expression normalized for actin was significantly increased (P < 0.05) after LPS treatment (C). Pretreatment of cells with OT (100 pM) for 30 min prior to LPS stimulation for 6 h (F) caused significant reduction in IL-6 secretion, whereas pretreatment with100 pM vasopressin (VP) had no effect. Data are expressed as means ± SE of n = 3 or 4 replicates and are representative of at least 3 experiments. *P < 0.05; **P < 0.01; ***P < 0.001. RQ, relative quantification.

Given the potential functional overlap between the OXTR and the AVPRs (9), we also evaluated the effects of LPS on AVPR1a, AVPR1b, and AVPR2 in THP-1 macrophages. AVPR1a and AVPR1b gene expression was not detectable by PCR in control cells and remained undetectable following treatment with LPS. AVPR2 gene expression was detectable in control cells, and in contrast to the increased expression seen in OXTR, AVPR2 expression was decreased by 65% after LPS treatment (P < 0.001; data not shown). Given that vasopressin has affinity for the OXTR (9), we also evaluated whether vasopressin would produce a reduction in IL-6 secretion in LPS-treated THP-1 macrophages, similar to OT. At a dose comparable with that used for OT (100 pM), vasopressin elicited no significant change in IL-6 secretion from LPS-treated cells (Fig. 1F).

To demonstrate that the LPS-mediated change in OXTR gene expression is not limited to the THP-1 macrophage, primary human macrophage cultures from two individual donors were treated with LPS (Fig. 3). LPS induced a significant increase in OXTR expression in the cells from both donors compared with control incubations (P < 0.01), and this change was paralleled by a significant increase in IL-6 expression (P < 0.001). Vasopressin receptor expression was also evaluated and similar to THP-1 macrophages, AVPR1a and AVPR1b gene expression was not detectable in control or LPS-treated primary macrophages. AVPR2 gene expression was detectable in control cells, and unlike OXTR and IL-6 expression, AVPR2 expression was decreased after LPS treatment (P < 0.01; Fig. 3). OXTR protein expression before and after LPS treatment was also evaluated in primary human macrophage cultures from donor 1 (Fig. 4). Similar to the LPS-induced change in OXTR expression in THP-1 cells, PBMCs exhibited a towfold increase in the 67-kDa OXTR-immunoreactive band, representing the mature form of the receptor, whereas levels of the 46-KDa immunoreactive band were near the level of detection and could not be quantified.

Fig. 3.

mRNA expression of OXTR, IL-6, and arginine vasopressin receptor 2 (AVPR2) in primary monocyte-derived macrophages isolated from 2 donors (donor 1 was male, and donor 2 was female).. Cells were treated with LPS (100 ng/ml) or control media, and then OXTR mRNA (A and D), IL-6 mRNA (B and E), and AVPR2 mRNA (C and F) were measured after a 6-h incubation. There was a significant increase in OXTR and IL-6 mRNA expression to LPS treatment. In contrast, expression of AVPR2 mRNA was significantly decreased with LPS treatment. **P < 0.01 and ***P < 0.001 by t-test (n = 4/condition).

Fig. 4.

Treatment of human peripheral blood mononuclear cell-derived macrophages with LPS induced expression of OXTR protein. A: cells from donor 1 in Fig. 2 were treated with LPS (100 ng/ml) or vehicle, and OXTR protein expression in control and LPS-treated cells was determined by Western blotting. B: OXTR 67-kDa protein expression normalized for actin was significantly increased (*P < 0.05) after LPS treatment.

Effects of LPS on OXTR expression in murine macrophages.

The effects of LPS on OXTR mRNA expression and IL-6 mRNA were also evaluated in a cultured murine macrophage cell line (RAW 264.7) and in peritoneal macrophages harvested 6 h after an in vivo intraperitoneal injection of LPS. Unlike the human macrophage OXTR response, the cultured cell line and the tissue resident mouse macrophages did not exhibit any significant change in OXTR mRNA expression relative to controls following LPS stimulation (P > 0.05; Fig. 5, A and C). In both cultured cells and peritoneal resident macrophages, LPS treatment significantly increased IL-6 mRNA expression (P < .0.01 and P < 0.001, respectively; Fig. 5, C and D) and demonstrating the increased inflammatory response by LPS treatment of cells.

Fig. 5.

Effect of LPS treatment on OXTR and IL-6 mRNA expression in murine macrophages. A and C: treatment of cultured murine macrophages (RAW 264.7) with LPS (100 ng/ml) or control media for 6 h and expression of OXTR and IL-6 mRNA. B and D: effect of in vivo LPS (50 µg injected intraperitoneally) treatment on resident murine peritoneal macrophages OXTR and IL-6 mRNA compared with vehicle controls. LPS did not significantly affect OXTR mRNA expression in either cultured or tissue resident murine cells but did significantly increase IL-6 mRNA expression. Data are expressed as relative quantification (RQ) normalized to 18S and are the mean ± SE of triplicate dishes for the RAW 264.7 cells representative of 3 experiments, and peritoneal macrophages were obtained from 4 vehicle-treated and 5 LPS-treated mice. **P < 0.01; ***P < 0.001.

The increased expression of IL-6 was paralleled by a >100-fold increase in secretion of IL-6 into the media of cultured cells and the serum of treated mice (data not shown). These data show that mouse macrophages, unlike the human macrophages described above, do not increase expression of OXTR in response to an inflammatory stimuli. Since the effect occurred only in human cells, the remaining experiments focused on cultured THP-1 macrophages.

The role of NF-κB and IL-6 on OXTR expression during LPS stimulation.

NF-κB is a key regulator of the cellular inflammatory response. To identify the role of NF-κB transcriptional activity on OXTR expression, THP-1 macrophages were incubated with an NF-κB inhibitor, CAPE, in the presence or absence of LPS. It is also possible that LPS leads to OXTR upregulation through increased IL-6 secretion and subsequent activation of NF-IL-6 via the IL-6 receptor. Therefore, we also assessed the effects of exogenous IL-6 (50 ng/ml) on OXTR expression. Cells stimulated with LPS significantly increased expression of OXTR and IL-6 by 88- and 35,000-fold, respectively (Table 1). The LPS-stimulated increase in cell expression of OXTR and IL-6 was abolished by treatment with CAPE. These data suggest that NF-κB activity is essential for OXTR transcription during inflammation. Incubation of cells with exogenous IL-6 had no significant effect on the mRNA expression of either OXTR or IL-6, suggesting that IL-6 per se is not involved in the upregulation of OXTR expression.

Table 1.

Effects of NF-κB inhibition on OXTR and IL-6 mRNA gene expression

	IL-6 (RQ)	P Value	OXTR (RQ)	P Value
Control	1.00 ± 0.22		1.00 ± 0.26
LPS	34743 ± 7139	0.029	88 ± 28	0.018
IL-6	0.55 ± 0.13	0.515	0.23 ± 0.11	0.129
CAPE	0.42 ± 0.20	0.013	0.01 ± 0.00	0.019
LPS + CAPE	0.52 ± 0.25	0.104	0.14 ± 0.07	0.003

Data are the means ± SE of triplicate culture dishes from combined 3 independent experiments. OXTR, oxytocin receptor; RQ, relative quantification; CAPE, caffeic acid phenethylester. OXTR and IL-6 expression after NF-κB inhibition (25 µg/ml CAPE) in LPS-treated THP-1 macrophages. A 30-min preincubation with CAPE followed by a 6-h LPS treatment completely inhibited the increase in OXTR and IL-6 mRNA. Treatment with exogenous IL-6 (50 ng/ml) had no significant effect on gene expression. P values comparing treatments with control condition for each gene.

OT-stimulated ERK1/2 phosphorylation.

To demonstrate that the inflammation-related increase in expression of OXTR is functional, we evaluated downstream signaling of the PKC pathway by measuring increased ERK1/2 phosphorylation after adding OT to the cells. For this experiment (Fig. 6), there was a significant interaction of treatment (LPS vs. control) by time, F(2, 36) = 16.64, P < .001, and a main effect of treatment, F(1, 36) = 85.73, P < .001, Mean comparisons revealed that OT increased ERK1/2 phosphorylation at 10 min in control cells relative to baseline (P < 0.05). Incubation of LPS-treated cells with OT induced a greater increase in ERK1/2 phosphorylation relative to the noninflammatory control cells at both 5 and 10 min (P < 0.001). These findings suggest that in the presence of inflammation, OT induced a more rapid and greater increase in ERK1/2 phosphorylation than in the noninflammatory condition, which is consistent with an increase in OXTR density due to inflammation.

Fig. 6.

Time course of ERK1/2 phosphorylation in control and LPS-treated THP-1 macrophages after incubation with OT. OT increased ERK1/2 phosphorylation in control and LPS-treated cells. Data are means ± SE of triplicate culture dishes. Results are from 4 (ERK1/2) separate experiments. ***P < 0.001 compared with control; §P < 0.05 compared with 0 min for control.

DISCUSSION

There is increasing evidence that administration of exogenous OT can attenuate inflammation in a variety of experimental models (1, 8, 12, 13, 15, 18, 25, 26). In the current study, we confirmed that OT reduced LPS-stimulated IL-6 secretion in THP-1 macrophages, a result reported previously in macrophages and intestinal cells (13, 25). It is possible that endogenous increases in plasma OT levels may be responsible for its anti-inflammatory actions in vivo. Alternatively, rapid induction of OXTR expression could also mediate enhanced anti-inflammation, even with relatively constant, low levels of OT. In both human THP-1 and primary monocyte-derived macrophages, the current study demonstrated that OXTR mRNA and protein expression were significantly upregulated in the face of a potent inflammatory stimulus. The increase in OXTR expression paralleled that of IL-6, suggesting that this receptor functions as an acute-phase protein. Interestingly, the same inflammatory stimulus caused a decreased expression of AVPR2, which suggests that OT and AVP receptors are differentially regulated by inflammation. It is likely that OXTR protein is produced in response to inflammation to facilitate OT’s anti-inflammatory effects (11, 13, 18, 25, 26, 30, 32). This suggests that OT’s ability to decrease cellular inflammation is due not only to the amount of circulating OT but also to the regulation of its receptor.

Given that the OXTR gene promoter region contains NF-κB and NF-IL-6 binding sites, we examined whether either or both transcription factors drive OXTR expression during inflammation. In the presence of an NF-κB inhibitor, the LPS-stimulated increase in OXTR expression was abolished. In contrast, incubation of cells with exogenous IL-6 had no effect on transcription of OXTR or IL-6. This latter observation implies that the NF-IL-6 promoter in the OXTR gene is nonfunctional or dormant in THP-1 macrophages. The data demonstrate that NF-κB plays a critical role in OXTR expression during inflammation, possibly by activation of the NF-κB promoter region of the OXTR gene.

In similar experiments conducted in mouse macrophage cell line (RAW 264.7) and murine peritoneal resident tissue macrophages, LPS effectively elevated IL-6 but failed to increase OXTR expression. This observation contrasts with previous reports showing that NF-κB activation with LPS increases murine myometrial cell OXTR expression in vitro (27) and that injection of LPS of pregnant mice in vivo increased OXTR in uterine tissues. This may be due to epigenetic differences between mouse macrophages and reproductive tissues. It is also possible that sex difference or prior exposure to sex hormones could contribute to these differences. Additional studies are needed to address tissue and sex-specific differences in transcription factor activation of OXTR promoter regions.

The current study highlights the importance of examining not only regulation of the ligand but also the dynamics of the receptor. OT plasma concentrations can vary but are typically reported to be in the low picomolar range (24). Furthermore, plasma OT has a short half-life (i.e., 3–5 min) (19), and therefore, sustained OT elevations in the face of inflammation are unlikely. Alternatively, induction of OXTR in response to inflammatory stimuli could provide a mechanism by which prolonged anti-inflammation could occur, even with low OT titers.

Further studies are necessary to elucidate the signaling pathways by which OXTR induces anti-inflammation. Data from the current study showed that OT stimulation of OXTR induced ERK1/2 phosphorylation, suggesting activation of the Gα_q/11/PKC signaling pathway, which is the conventional OXTR signaling pathway (9). Interestingly, an alternative, nonconventional OT signaling pathway (the G_sα/PKA pathway) is recruited under some circumstances and may act as a protective mechanism for cells under distress. This pathway has been demonstrated to have an antiproliferative effect in breast cancer, endometrial, bone, and nervous system tumors (5–7). Perhaps in a similar manner, this pathway can be recruited in inflamed cells to decrease further damage and/or cell death.

In conclusion, the current study demonstrates that in human macrophages the OXTR is significantly upregulated in response to an inflammatory stimulus. The increased receptor expression occurred in parallel with changes in the inflammatory cytokine IL-6, and therefore, it should be considered an acute-phase protein. The inflammation-induced OXTR upregulation is mediated via NF-κB activation, but not by elevations in IL-6. This increased expression of OXTR likely contributes to the anti-inflammatory actions of OT.

GRANTS

This work was supported by the National Heart, Lung, and Blood Institute (HL-116387 to P. M. McCabe; N. Sun-Suslow was supported by training grant HL-04726]. K. V. Wagner was supported by the Miami Exchange Program of the Max Planck Institute of Neurobiology, Munich, Germany.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

A.S., N.S.-S., A.J.M., R.I.H., and K.V.W. performed experiments; A.S., N.S.-S., A.J.M., R.I.H., and P.M.M. analyzed data; A.S., N.S.-S., A.J.M., and P.M.M. interpreted results of experiments; A.S., N.S.-S., and A.J.M. prepared figures; A.S., N.S.-S., A.J.M., and P.M.M. approved final version of manuscript; A.J.M. and P.M.M. drafted manuscript; A.J.M. and P.M.M. edited and revised manuscript.