A circadian clock in murine bone marrow-derived mast cells modulates IgE-dependent activation in vitro

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Abstract

Circadian rhythm is expressed in most organisms, and many functions and parameters in the immune system are associated with time-of-day. However, it is largely unknown if local circadian clocks in immune cells directly control physiological outcomes. We hypothesized that a circadian clock in murine bone marrow derived mast cells (BMMCs) modulates IgE-dependent activation in vitro. Mature BMMCs, grown from bone marrow of C57BL/6 mice, were synchronized with serum rich media (50% horse serum). Total RNA was harvested from BMMCs at 4 h intervals for up to 72 h following synchronization and expression of circadian genes (mPer1, mPer2, Bmal1, Rev-erbα, and Dbp) was measured by quantitative PCR. Serum shock synchronized expression of circadian genes (mPer2, Bmal1, Rev-erbα, and Dbp) in BMMCs. Synchronized BMMCs stimulated via the high affinity IgE receptor (FcεRI) at different time intervals display circadian rhythms in IL-13 and IL-6 mRNA expression. The expression of fcer1a gene and FcεRIα protein displayed a circadian pattern following serum shock, with mean periods of 18.9 and 28.6 h, respectively. These results demonstrate that synchronized BMMCs provide an in vitro model to study circadian mechanism(s) associated with allergic disease and that circadian oscillation of cytokine production following IgE-dependent activation is at least in part due to circadian oscillation of FcεRIα.

Introduction

Circadian rhythm is a conserved feature of organisms ranging from cyanobacteria to humans (Dunlap, 1999) that is manifested as oscillations of biological processes with a periodicity of approximately 24 h. The circadian system consists of three components: sensors, pacemakers, and effectors. Sensors include both photic and non-photic receptors that entrain the central clock to environmental cues. Afferent pathways convey information from the sensors to pacemakers that establish the rhythm, and efferent pathways alter effector organ function according to the established rhythm. In mammals, a central circadian pacemaker located in the suprachiasmatic nucleus (SCN) of the ventral hypothalamus (Gekakis et al., 1998) coordinates oscillators in peripheral organs (Reppert and Weaver, 2002).

The core circadian molecular clock in mammals consists of a series of transcriptional and translational loops involving Period1–3, Clock, Bmal1, and Cry1–2 genes (Takahashi et al., 2008). CLOCK and BMAL1 heterodimers translocate into the nucleus, bind to E-box motifs (CACGTG), and activate transcription of Per and Cry genes (Gekakis et al., 1998). PER and CRY proteins synthesized in the cytoplasm accumulate to a critical level and bind to CKIε/δ kinase. The phosphorylated heterotrimer translocates into the nucleus and inhibits CLOCK–BMAL1 heterodimer, thereby inhibiting transcription of their own genes and other clock-controlled genes (CCGs), such as Dbp (Kume et al., 1999). Another feedback loop directs alternating activation and repression of BMAL1 expression by the nuclear receptors RORα and REV-ERBα (Etchegaray et al., 2003), respectively (Sato et al., 2004, Emery and Reppert, 2004).

In vivo, peripheral tissues and cells are synchronized by the SCN. However, in vitro, cycling of circadian oscillations in individual cells that are not entrained to an external signal is uncoordinated, and therefore, self-sustained oscillation in populations of cells is unsynchronized. Detuned circadian oscillators observed in cell lines (Nagoshi et al., 2004) and some primary cells (Keller et al., 2009) can be synchronized by a serum shock (Durgan et al., 2005, Nagoshi et al., 2004, Balsalobre et al., 1998) or pharmacological substances (Yagita et al., 2010, Huang et al., 2009, Wu et al., 2008, Wu et al., 2007, Balsalobre et al., 2000a).

Many diseases, including immune mediated diseases such as allergic asthma, exhibit circadian variation of symptoms. Nocturnal symptoms of asthma are common and include bronchoconstriction, airway inflammation and hyperreactivity, dyspnea, cough, and apnea during the night (Smolensky et al., 2007, Kelly et al., 2004, Irvin et al., 2000, Kraft et al., 1999, Ballard et al., 1989). Mast cells play a central role in allergic diseases (Brown et al., 2008); and while circadian rhythmicity is an established feature of many immune cells, including peripheral blood mononuclear cells (PBMCs) (Born et al., 1997; Murphy et al., 2007), natural killer (NK) cells (Arjona and Sarkar, 2006), and peritoneal macrophages (Keller et al., 2009), it is unknown if or how circadian rhythms may influence mast cell function. While it remains unknown how circadian variation may directly influence mast cell function, it has been reported that plasma histamine levels in patients with mastocytosis exhibit a circadian variation (Friedman et al., 1989), and circadian variation in mast cell number has been observed in thyroid gland (Catini et al., 1994), ovaries, (Gaytan et al., 1991), tongue, pinna, and dorsal skin (Chen and He, 1989). Despite this evidence, it remains unknown whether circadian clock genes are expressed in mast cells and whether these genes influence mast cell function.

Crosslinking of FcεRI (high-affinity IgE receptor) on the surface of mast cells by allergen bound IgE results in immediate release of preformed inflammatory mediators including histamine and proteases, that initiate an immediate hypersensitivity reaction (Metcalfe et al., 2009). In addition, activation of mast cells stimulates production of cytokines and chemokines, including IL-6, IL-13, CXCL8, and CCL3, which promote the late-phase inflammatory reactions.

The present study was conducted to test the hypothesis that a circadian clock expressed in murine bone marrow derived mast cells (BMMCs) modulates IgE-dependent activation in vitro. BMMCs were serum shocked with a high concentration of horse serum, and expression of circadian clock genes (mPer1, mPer2, Bmal1, Rev-erbα, and Dbp) were monitored for up to 72 h. Inflammatory cytokines were measured following FcεRI stimulation to examine the influence of circadian rhythm on mast cell activation. Lastly, rhythmic expression of FcεRI was evaluated by both quantitative real-time PCR and flow cytometry.

Section snippets

Animals

Four week-old C57BL/6 mice were obtained from The Jackson Laboratories (Bar Harbor, ME). Mice were euthanized by CO2 asphyxiation followed by collection of femoral bone marrow for generation of BMMCs. All animal protocols were approved by the Institutional Animal Care and Use Committee of East Carolina University.

Cell culture

Mouse BMMCs were cultured from femoral bone marrow of C57BL/6 mice. Bone marrow was collected from 3 to 4 mice per BMMC culture and all experiments were repeated 6 times with 6

Expression of circadian clock genes in serum synchronized BMMCs

Expression of mPer2, Bmal1, Rev-erbα, and Dbp exhibited a robust oscillation over a 72 h period following 2 h synchronization with serum rich media (50% horse serum) (Fig. 2). mPer2 and Rev-erbα, which are negative regulatory arms of the circadian transcriptional complex, displayed peak expression levels at 19.8 and 18.5 h following synchronization. In contrast, Bmal1, a positive regulatory arm of the circadian transcriptional complex, exhibited peak phase at 5.9 h after synchronization (Table 1).

Discussion

To our knowledge, this is the first report on expression and function of circadian clock genes in mast cells. The most important finding in this study is that circadian clock genes are rhythmically expressed in serum synchronized mouse bone marrow-derived mast cells. Cytokine mRNA expression following IgE-dependent activation of BMMCs and serum synchronization exhibited a trend towards a circadian pattern. However, IgE/Ag stimulation of BMMCs did not shift existing circadian expression of a key

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