Mast cell inhibitory activity

Mast cells derive from distinct bone marrow precursors5 and enter the tissues as undifferentiated cells where they mature under the influence of "microenvironmen-tal" conditions; these include stem cell factor (CSF or c-kit ligand) and interleukins

3, 4, and 6.6 Rat connective tissue mast cells (CTMC), found primarily in skin and lungs, contain rat mast cell protease I (RMCP I), whereas mucosal mast cells (MMC) contain RMCP I.7 All human mast cells (HMC) contain the proteolytic enzyme tryptase, but human CTMC contain yet another protease, chymase.7 Mast cells are the main source of tissue histamine, which is released from secretory granules when mast cells are triggered with IgE and specific antigen.6 Such granules also store numerous other vasoactive, pro-inflammatory, and nociceptive molecules,8,9 some of which may be released differentially without exocytosis.10-13 In addition to the well-known immunologic stimuli, CTMC also secrete in response to a number of neuropeptides.14

Mast cells play a central role in the pathogenesis of diseases such as allergic asthma, rhinoconjunctivitis, asthma, anaphylaxis, urticaria, and systemic mastocytosis; they are now considered to be important players in other chronic inflammatory disorders, such as arthritis and inflammatory bowel disease.61415 Mast cells may also participate in sterile inflammatory conditions exacerbated by stress such as atopic dermatitis, interstitial cystitis, irritable bowel syndrome, migraines, and multiple sclerosis.14 Basophils, the circulating "equivalent" of the tissue mast cells, are considered as important cells in the pathogenesis of late phase allergic reactions.16-19

Both mast cells and basophils possess high affinity receptors for IgE in their plasma membranes. Cross-linking of these receptors is essential to trigger the secretion of histamine and other preformed, granule-associated mediators and to initiate the generation of newly formed phospholipid-derived mediators. Various flavonoids have been shown in a number of systems to inhibit this secretory process.20,21 Definitive evidence of flavonoid regulation of secretion was first provided in studies of the secretion of histamine from rat mast cells and basophils stimulated with antigen. Quercetin, kaempferol, and myricetin were found to inhibit the release of rat mast cell histamine.22,23 Quercetin also induced histamine accumulation, expression of mRNA for RMCP II,24 and development of secretory granules in rat basophil leukemia (RBL) cells.24 Flavonoids have also been shown to have antiproliferative effects on a number of transformed cells.1

Inhibitory activity of some flavon-3-ols was associated with the following structural features: a C4 keto group, an unsaturated double bond at position C2-C3 in the Y-pyrone ring, and an appropriate pattern of hydroxylation in the B ring. These characteristics were near identical to those identified for other inhibitory activities. The flavonoid glycosides, rutin and naringin, were inactive in vitro as were the flavanones (reduced C2-C3 bond) taxifolin and hesperitin. Morin, catechin, and cyanidin were also inactive.1 It is important to note that, while quercetin, kaempferol, and myricetin were potent inhibitors of histamine release from rat peritoneal mast cells, morin was not. The addition of a single hydroxyl group at position 2' appears to be sufficient to reduce inhibition of mast cell secretion. This hydroxyl group may be interacting with the oxygen at position 1 forming a cyclic structure that possibly interferes with some key biological event.

The basophil histamine-releasing effect of different secretagogues could be inhibited by some, but not all, of 11 flavonoids representing several chemical classes: (a) anti-IgE or concanavalin A (IgE-dependent histamine releasing agents); (b) the che-moattractant peptide, f-MetLeuPhe or the tumor promoter phorbol ester, TPA (both

IL-6

Histamine Tryptase

IL-6

IL-8

TNF-alpha

FIGURE 15.2 Inhibition of release of inflammatory molecules from human umbilical cord blood-derived mast cells (hCBMCs) by pretreatment with quercetin (0.5 mM) for 5 min before stimulation with anti-IgE for either 30 min for histamine and tryptase, and 24 hours for IL-6, IL-8, and TNF-a (n = 6, *p<0.05). Note that inhibition was 80% or higher for all mediators except for histamine.

f-MetLeuPhe and TPA are receptor-dependent, IgE-independent, histamine-releasing agents); and (c) the divalent cation ionophore A23187 (bypasses receptor-dependent processes and carries Ca2+ directly into the cytoplasm).1 The effect of quercetin to uniformly inhibit basophil histamine secretion stimulated by a variety of agonists strongly suggests that there is a final common pathway utilized by each of these agonists which is sensitive to quercetin and other structurally similar flavonoids.

We were the first to show that while cromolyn was a weak inhibitor of histamine release from human umbilical cord-derived cultured mast cells, quercetin was a much better inhibitor with morin having intermediate activity. Quercetin, moreover, was a potent inhibitor of release of both the unique mast cell proteolytic enzyme tryptase, as well as of the cytokines interleukin-6, interleukin-8, and tumor necrosis factor (Figure 15.2). Cromolyn could not inhibit the release of these molecules, whereas morin was considerably weaker than quercetin.25 These results indicate that findings from rodent mast cells are not necessarily applicable to human cells, and that quercetin is a better mast cell inhibitor than cromolyn. Quercetin could also inhibit selective release of IL-6 in response to IL-1 (Figure 15.3).

Fewtrell and Gomperts22 and Middleton et al.1 demonstrated that only activated mast cells or activated basophils were affected by quercetin and other inhibitory fla-vonoids.1 In other words, the unstimulated cells could be exposed to the flavonoids, be washed, and subsequently be shown to react normally to a secretagogue with his-tamine release. They also observed that pretreatment of rat mast cells with disodium cromoglycate (cromolyn), a "mast cell stabilizing" anti-allergic drug, for 30 min completely abolished the inhibition normally observed upon subsequent exposure to quercetin, added together with antigen. This finding suggested that cromolyn and

Concanavalin Mitogen

FIGURE 15.3 Structure of quercetin with possible targets, inhibition of which may explain its ability to inhibit mast cell secretion. The insert shows a dose-response of the inhibitory activity of quercetin (5 min pretreatment) on release of IL-6 from hCBMCs stimulated by IL-1 (100 mM) for 24 hours. (n = 8, *p < 0.05.) MAPK, mitogen activation protein kinase; MC, mast cells; PI3K, phosphotidyl inositol-3 kinase.

FIGURE 15.3 Structure of quercetin with possible targets, inhibition of which may explain its ability to inhibit mast cell secretion. The insert shows a dose-response of the inhibitory activity of quercetin (5 min pretreatment) on release of IL-6 from hCBMCs stimulated by IL-1 (100 mM) for 24 hours. (n = 8, *p < 0.05.) MAPK, mitogen activation protein kinase; MC, mast cells; PI3K, phosphotidyl inositol-3 kinase.

quercetin acted at the same or a closely associated molecular site. It was not known, however, just what cellular component in activated mast cells or basophils first interacts with cromolyn or active flavonoids to inhibit the secretory process.

One possible explanation was considered by the similarity in the structure of quercetin with that of cromolyn. It had been shown that secretagogue-induced exo-cytosis in rat mast cells was associated with stimulation of Ca2+-dependent protein phosphorylation.26 Purified rat peritoneal mast cells which had been labeled with 32P and then stimulated by addition of compound 48/80 resulted in the phosphorylation of four proteins of apparent molecular weights 78, 68, 59, and 42 kD. Phosphorylation of the proteins with apparent molecular weights of 68, 59, and 42 kD was evident within 10 s after addition of 48/80; phosphorylation of the 78 kD molecular weight protein, however, was not evident until 30-60 s after addition of the secretagogue.26 These experiments clearly indicated that the exocytosis of the mast cell was associated with phosphorylation of certain proteins, while recovery from secretion was related to phosphorylation of a unique protein.

We then showed that cromolyn promoted the incorporation of radioactive phosphate into a single rat mast cell protein of apparent molecular weight 78 kD.27 The time course and dose-dependence of phosphorylation of this protein closely paralleled inhibition of mast cell secretion.27 This finding provided an insight into the mechanism of inhibition by cromolyn of mast cell secretion triggered by an immu-nologic stimulus, anti-rat IgE. In additional experiments, we showed that other inhibitors of rat mast cell histamine secretion, also increased the incorporation of radioactive phosphate into a single protein band with an apparent molecular weight of 78 kD.28

We subsequently showed that the 78 kD mast cell phosphoprotein had high homology to moesin,29 a member of the ezrin-radixin-moesin family of proteins,30 which have been shown to regulate signal-transduction by coupling the cell surface to the cytoskeleton.31 Phosphorylation of this protein was shown to take place by a calcium and phorbol ester-independent PKC isozyme.32 More recently, this 78 kD phosphoprotein was cloned and was shown to be identical to moesin;33 it was further shown that its phosphorylation by cromolyn induced some conformational change that permitted covalent binding to actin and resulted in preferential clustering around the mast cell secretory granules, thus possibly preventing them from undergoing exocytosis.33 Because of its apparent involvement in mast cell inhibition, this protein was also called MAst CEll DegranulatiON Inhibitory Agent (MACEDONIA).14 This protein was associated with the plasma membrane, but there were no intramembranous domains. During secretion, the protein could not be recognized by immunocytochemistry and gave the impression it had disappeared; however, Western blot analysis showed that it had not been secreted. Pretreatment with quercetin prevented this apparent "disappearance," suggesting some conformational rearrangements that changed the antigenicity of this protein.

DISCUSSION

The present findings extend previous reports showing that quercetin inhibits stimulated histamine release from rat CTMC22,34 and MMC,35,36 from human lung and intestinal mast cells,37 as well as from activated basophils.23 Various flavonoids, but not morin, had previously also been shown to inhibit polymorphonuclear leukocyte function38 and lymphocyte secretion of interleukin-239 in concentrations similar to the ones used here. Our results indicate that the structural requirements of flavonoids with respect to the inhibition of both proliferation and accumulation of mediators was the same as it had previously been reported for inhibition of rat mast cell secre-tion.125 Similarly, morin could not inhibit mitogen-induced lymphocyte proliferation40 or proliferation of a human lymphoblastoid cell line.41 The fact that morin was inactive supports the notion that the effect of flavonoids is fairly specific. Morin has one additional hydroxyl group in the 2' position of the B ring,4 not shared by the other flavonoids tested, suggesting that there must exist some steric interference that leads to loss of inhibitory activity. The structural requirements for the inhibitory effect reported here may be related to the ability of quercetin and other flavonoids to inhibit various enzymatic systems in vitro,1,4 such as protein kinase C,42, 43 which regulates secretion.44

PKC is the ubiquitous, largely Ca2+- and phospholipid-dependent, multifunctional serine- and threonine-phosphorylating enzyme. It is involved in a wide range of cellular activities including tumor-promotion, mitogenesis, secretory processes and inflammatory cell function. Accumulated evidence indicates that quercetin may exert its mast cell inhibitory effect by inducing the phosphorylation of MACEDONIA through activation of the protein kinase C (PKC) calcium and phorbol ester independent isozyme zeta.45 However, it may also be blocking mast cell activation by inhibiting other PKC isozymes critical in stimulus-response coupling. PKC could be inhibited in vitro by certain flavonoids that appeared to competitively block the

ATP binding site on the catalytic unit of PKC.1 Quercetin was also shown to inhibit tyrosine kinases (PTK) which are implicated in the regulation of cell transformation and cell growth, gene expression, cell-cell adhesion interactions, cell motility, and shape. Again, it acted as a competitive inhibitor of ATP binding and the pattern of B-ring hydroxylation, C2-C3 unsaturation, and C4 keto groups were recognized as strongly affecting inhibitory activity.1

The inhibitory effect of flavonoids on secretory processes is not limited to baso-phils and mast cells, but they are also capable of inhibiting stimulated rabbit neutro-phil lysosomal enzyme release, and mitogen-activated adherent human neutrophils. Anti-IgE-induced H2O2 generation and human basophil histamine release was also inhibited by quercetin. Oxygen free radicals and nonradical reactive oxygen intermediates released by neutrophils and other phagocytes have been increasingly implicated in inflammatory/immune disorders. Flavonoids could profoundly impair the production of reactive oxygen intermediates by neutrophils and other phagocytic cells. This may be accomplished by interference with NADPH oxidase, a powerful oxidant-producing enzyme localized on the surface membrane of neutrophils. Flavonoids could also inhibit neutrophil myeloperoxidase (MPO), a source of reactive chlorinated intermediates. Impairment by flavonoids of the production of active oxygen intermediates by neutrophils and other phagocytes might contribute to the anti-inflammatory activity of these compounds.1 Quercetin inhibited the activation of rabbit peritoneal neutrophils stimulated by f-MetLeuPhe, as determined by measurement of degranulation and superoxide formation; quercetin also inhibited tyrosine phosphorylation, mitogen-activated protein kinase (MAPkinase), and phos-pholipase D. Neutrophil protein tyrosine phosphorylation stimulated by chemotactic factors was diminished by genistein. Ionophore A23187-induced eosinophil secretion of Charcot-Leyden crystal protein and eosinophil cationic protein was inhibited by quercetin, but not by taxifolin (dihydroquercetin), in a concentration-dependent manner. Thus, the activated eosinophil appears to respond to these flavonoids in the same fashion as basophils and mast cells. Eosinophil degranulation stimulated by IgA- or IgG-coated beads was inhibited by genistein; at the same time, several phos-phorylated proteins were decreased in quantity, and PLC activation was inhibited.1

We have reported that flavone, quercetin, and kaempferol, but not morin, led to accumulation of secretory granules in RBL cells,25 which do not normally accumulate secretory granules even at stages expressing increased membrane receptors for IgE. RBL cells are considered similar to MMC 46 and have been used as a model for studying IgE-mediated processes leading to secretion of histamine.47 Quercetin had previously been reported to induce the accumulation of rat mast cell protease II in RBL cells.24 Moreover, quercetin was reported to "endow" RBL cells with secretory responsiveness to cationic mast cell secretagogues, such as compound 48/80.48 In an analogous manner, bone marrow-derived mouse mast cells were shown to acquire responsiveness to SP when cultured in the presence of both SCF and IL-4.49 Fla-vonoids had previously been shown to inhibit two human lymphoid tissue-derived cell lines.39 Moreover, quercetin inhibited proliferation of human acute myeloid and lymphoid leukemic cells without affecting normal hematopoiesies.50 These findings are in accordance with previous studies showing that quercetin blocks proliferation of HL-60 leukemia cells by inducing an accumulation of the cells in the G2/M phase of the cell cycle 51 and progression of human gastric cancer cells from Gj to the S phase of growth.52 Quercetin has been reported to inhibit the growth of estrogen sensitive cells, such as human breast cancer cells in culture.53 It is interesting that quercetin binds to estrogen type II receptors 41 and human mast cells were shown to express cytoplasmic estrogen receptors.54 In fact, the growth inhibitory effect of quercetin on a human lymphoblastoid cell line was shown to be through action on a type-II estrogen-binding site, an effect not shared by hesperidin, which does not bind to such sites.41

Taken together, such findings indicate that certain flavonoids can induce differentiation of cancer cells, possibly by mimicking the action of certain cytokines. Select flavonoids may, therefore, be useful for the treatment of mast cell proliferative disorders, such as systemic mastocytosis55 or interstitial cystitis of the bladder,56 conditions that have been associated with constitutive release of mediators.57 Flavonoids have the potential to be used as therapeutic agents,1 especially because their inhibitory effect on mast cell secretion is additive to that recently discovered for chondroi-tin sulfate,58 one of the major proteoglycans of rat and human mast cells.5960

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