The circadian clock is functional in eosinophils and mast cells

Abstract

Allergic diseases are frequently exacerbated between midnight and early morning, suggesting a role for the biological clock. Mast cells (MC) and eosinophils are the main effector cells of allergic diseases and some MC-specific or eosinophil-specific markers, such as tryptase or eosinophil cationic protein, exhibit circadian variation. Here, we analysed whether the circadian clock is functional in mouse and human eosinophils and MC. Mouse jejunal MC and polymorphonuclear cells from peripheral blood (PMNC) were isolated around the circadian cycle. Human eosinophils were purified from peripheral blood of non-allergic and allergic subjects. Human MC were purified from intestinal tissue. We found a rhythmic expression of the clock genes mPer1, mPer2, mClock and mBmal1 and eosinophil-specific genes mEcp, mEpo and mMbp in murine PMNC. We also found circadian variations for hPer1, hPer2, hBmal1, hClock, hEdn and hEcp mRNA and eosinophil cationic protein (ECP) in human eosinophils of both healthy and allergic people. Clock genes mPer1, mPer2, mClock and mBmal1 and MC-specific genes mMcpt-5, mMcpt-7, mc-kit and mFcεRI α-chain and protein levels of mMCPT5 and mc-Kit showed robust oscillation in mouse jejunum. Human intestinal MC expressed hPer1, hPer2 and hBmal1 as well as hTryptase and hFcεRI α-chain, in a circadian manner. We found that pre-stored histamine and de novo synthesized cysteinyl leukotrienes, were released in a circadian manner by MC following IgE-mediated activation. In summary, the biological clock controls MC and eosinophils leading to circadian expression and release of their mediators and, hence it might be involved in the pathophysiology of allergy.

Keywords: allergy; biological clock; circadian rhythm; eosinophils; mast cells.

Figure 1

Circadian mRNA expression of clock genes (mPer1, mPer2, mBmal1 and mClock) and eosinophil-specific molecules (mMbp, mEpo and mEcp) in murine polymorphonuclear cells (PMNC). PMNC were isolated around the circadian cycle from mouse peripheral blood. Total RNA was extracted, reverse-transcribed and analysed by quantitative real-time PCR. Clock gene expression levels were normalized using mGapdh, n = 4 (measured each in triplicates), CT = circadian time, CT0 is the time when the light came on during the light–dark cycle. PMNC purity was > 95%.

Figure 2

Circadian mRNA expression of clock genes (hPer1, hPer2, hCry1, hBmal1 and hClock) and eosinophil-specific molecules [hEdn and hEcp mRNA and eosinophil cationic protein (ECP)] in human blood eosinophils of non-allergic subjects (a) and allergic subjects (b). Purity of eosinophils was > 99·5%. Eosinophils were cultured and harvested every 3 hr around the circadian cycle. Total RNA was extracted, reverse-transcribed and analysed by quantitative real-time PCR. Clock gene expression levels were normalized using hGapdh, n = 4 (non-allergic donors) and n = 5 (allergic donors), each measured in triplicates. Protein levels of ECP were normalized using GAPDH. Representative Western blots for ECP are shown (top row) alongside the loading control (bottom row).

Figure 3

Circadian mRNA expression of clock genes (mPer1, mPer2, mBmal1 and mClock) and mast cell (MC) -specific molecules (mFcεRI α-chain, mMcpt-7, mMcpt-5 and mc-kit mRNA and mMCPT-5 and mc-Kit protein) in enriched mouse lamina propria mast cells. Mast cell purity was > 90%. Cells were collected around the circadian cycle. Total RNA was extracted, reverse-transcribed and analysed by quantitative real-time PCR. Clock gene expression levels were normalized using mGapdh, n = 4 (measured each in triplicates). Protein levels of mMCPT-5 and mc-Kit were normalized using β-actin, n = 4, CT = circadian time, CT0 is the time when the light came on during the light–dark cycle. Representative Western blots for mMCPT-5 and mc-Kit are shown (top row) alongside the loading control (bottom row).

Figure 4

(a) Circadian expression of clock genes (hPer1, hPer2, hCry1, hBmal1 and hClock mRNA) and mast cell (MC) -specific molecules (hFcεRI α-chain, hc-kit, hCxcl8, hTryptase mRNA) in freshly isolated human intestinal MC (hiMC) harvested every 3 hr around the circadian cycle (one representative out of three experiments measured in triplicates is shown). (b) Circadian expression of clock genes (hPer1, hPer2, hCry1, hBmal1 and hClock mRNA) and mast cell-specific molecules (hFcεRI α-chain, hc-kit, hCxcl8, hTryptase mRNA and tryptase protein) in long-term cultured hiMC. Purity of freshly isolated hiMC was > 90%, purity of long-term cultured hiMC was > 99%. The hiMC were synchronized with 40 μm dexamethasone for 2 hr and subsequently harvested every 3 hr. Total RNA was extracted, reverse-transcribed and analysed by quantitative real-time PCR. Clock gene expression levels were normalized using hGapdh, n = 4 (measured each in triplicates). Protein levels of tryptase were normalized using β-actin. Representative Western blot for tryptase is shown (top row) alongside the loading control (bottom row).

Figure 5

Circadian release of pre-stored histamine (a) and de novo synthesized cysteinyl leukotrienes (cysLT) (b) by human intestinal mast cells (hiMC) in response to FcεRI cross-linking. The hiMCs were synchronized with 40 μm dexamethasone for 2 hr and stimulated by FcεRI cross-linking every 4 hr around the circadian cycle. The release of histamine is shown in per cent of the total cell content for every time-point, n = 2, release of cysLT, n = 4, hiMC purity was > 99%.