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. 2000 Aug;100(4):471-80.
doi: 10.1046/j.1365-2567.2000.00045.x.

Naturally occurring polyphenolic antioxidants modulate IgE-mediated mast cell activation

S Chen  1 , J Gong, F Liu, U Mohammed
Affiliations

Naturally occurring polyphenolic antioxidants modulate IgE-mediated mast cell activation

S Chen et al. Immunology. 2000 Aug.

Abstract

Reactive oxygen species (ROS) are known to modulate activities of a host of kinases, phosphatases and transcription factors. Rutin and chlorogenic acid (CGA) are the major polyphenolic antioxidants present in the small molecular fraction of smokeless tobacco leaf extracts, as ascertained by reverse-phase high-pressure liquid chromatography (HPLC) and mass spectrometry. Levels of intracellular ROS in resting versus antigen-immunoglobulin E (IgE)-challenged murine mast cells were measured at 510 nm by fluorescence-activated cell sorting (FACS) using carboxy-dichlorofluorescein (DCFH-DA). Enhanced ROS production was observed in IgE-sensitized mast cells following antigenic challenge. Rutin and CGA reduced ROS levels in antigen-IgE-activated mast cells. Concomitantly, they also profoundly inhibited histamine release by these activated mast cells. In contrast, rutin and CGA augmented the inducible cytokine messages, i.e. interleukin (IL)-10, IL-13, interferon-gamma (IFN-gamma), IL-6 and tumour necrosis factor-alpha (TNF-alpha) in IgE-sensitized mast cells following antigen challenge. This study indicates that tobacco polyphenolic antioxidants that quench intracellular ROS, differentially affect two effector functions of antigen-IgE-activated mast cells. This model system may be employed to determine the molecular target of polyphenols. The potential role of these polyphenolic antioxidants on IgE-mediated allergy in vivo depends on a balance of their differential effects on mast cell activation.

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Figures

Figure 1
Figure 1
Purification and characterization of non-pyrolysed, naturally occurring polyphenolic antioxidants in tobacco leaf extract. (a) Profile of SM-STE by RP-HPLC. SM-STE with MW of 1000 cut-off were prepared as described in Materials and Methods. One hundred µl of SM-STE were subjected to RP-HPLC (0·1% TFA; ACN 20% to 70% in 45 min) on a C18 column by a Dynamax Rainin's HPLC Method Manager System. (b) Ionogram of SM-STE. M.W. of SM-STE was determined by a SCIEX-API 100 mass spectrometer (Perkin-Elmer) based on ionic spray and subsequent TOF mass determination of the ionized molecular species. (c) Second RP-HPLC of hydrophilic substance obtained from initial purification from (a). (d) Mass determination of CGA in the purified peak of the second round of RP-HPLC from (c).
Figure 2
Figure 2
Inhibition of ROS production in antigen–IgE-activated mast cells by polyphenolic antioxidants. MC/9 mast cells (1 × 106) were incubated with 2 µg/ml IgE along with rutin and CGA at 1–160 µg/ml overnight (a, dose responses). Cells were washed, and challenged with 100 ng/ml DNP-BSA from 1 to 24 hr post-antigenic challenge (b, kinetics). Cells were washed, viability determined by trypan blue and 1 × 106 cells were then incubated with DCFH-DA at 10 µ m final, and emission at 510 nm determined. Histograms of oxidized DCF for the rutin/CGA-treated group versus control at 2 hr post-antigenic challenge, including the medium and baseline controls were depicted (c, FACS diagram).
Figure 3
Figure 3
Effect of polyphenolic antioxidants on histamine release by antigen–IgE-activated mast cells. Mast cells were sensitized with 2 µg/ml IgE in the presence of different concentrations of rutin/CGA for 24 hr in duplicate cultures. Cells were washed, resuspended in Tyrode's buffer (135 mm KCl, 1 mm MgCl2, 1·8 mm CaCl2, 5·6 mm glucose, and 20 mm HEPES, pH 7·4), and incubated with 100 ng/ml DNP-BSA for 1 hr at 37°. Two hundred µl supernatant was then taken from the duplicate cultures. Levels of histamine were determined against the standard curve according to a quantitative histamine RIA kit obtained from Biomerica Inc. (Newport Beach, CA). Approximately 30% histamine was released in supernatants (350 ± 70 ng/106 cells) of antigen-IgE activated control mast cells. Four such experiments were performed for different doses of antioxidants.
Figure 4
Figure 4
Time-course of IFN-γ gene expression in polyphenolic antioxidant-treated, antigen-IgE activated mast cells. Levels of cytokine messages of antioxidant-treated mast cells were determined by RNase protection assay with RiboQuant™, mCK-1 kit from PharMingen (for IL-4, IL-5, IL-10, IL-13, IL-15, IL-9, IL-2, IL-6, IFN-γ). Similar kinetics for various cytokine messages was noted, and IFN-γ was shown. Mast cells were cultured in T-75 flask at 1 × 106/ml with 2 µg/ml IgE overnight in the presence of different concentrations of rutin/CGA overnight. Cells were washed twice with Tyrode's buffer, resuspended in conditioned medium, and challenged at 37° with DNP-BSA at 100 ng/ml at different time-points. Cells were then pelleted, total RNA extracted and cytokine messages calibrated with housekeeping gene as standard by a PhosphoImager (Molecular Dynamics). Units of expression were computed by dividing the intensity of the individual band over that of L32 gene in each respective lane. Mean and SEM of densitometric readings of two similarly performed experiments are presented.
Figure 5
Figure 5
Dosage responses of IL-10, IL-13, and IFN-γ in antigen–IgE-activated mast cells treated with polyphenolic antioxidants. The procedure was similar to that described in the legend to Fig. 4. RiboQuant™, mCK-1 kit from PharMingen was employed and cytokine messages determined at 1 hr post-antigen challenge. (a) A representative autoradiogram. (b) Mean and SEM of densitometric reading of three similarly performed experiments.
Figure 6
Figure 6
Dosage responses of TNF-α, IL-6 in antigen-IgE activated mast cells treated with polyphenolic antioxidants. The procedure was similar to that described in the legend to Fig. 4. RiboQuant™, mCK-3 kit from PharMingen (LTβ, TNF-α, Il-6, IFN-γ) was used and cytokine messages determined at 1 hr post-antigen challenge. (a) A representative autoradiogram. (b) Mean and SEM of densitometric readings of three similarly performed experiments.
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