Mast Cell Stabilizer&Rsquo; in Mice
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Laboratory Investigation (2012) 92, 1472–1482 & 2012 USCAP, Inc All rights reserved 0023-6837/12 $32.00 Evidence questioning cromolyn’s effectiveness and selectivity as a ‘mast cell stabilizer’ in mice Tatsuya Oka, Janet Kalesnikoff, Philipp Starkl, Mindy Tsai and Stephen J Galli Cromolyn, widely characterized as a ‘mast cell stabilizer’, has been used in mice to investigate the biological roles of mast cells in vivo. However, it is not clear to what extent cromolyn can either limit the function of mouse mast cells or influence biological processes in mice independently of effects on mast cells. We confirmed that cromolyn (at 10 mg/kg in vivo or 10–100 mM in vitro) can inhibit IgE-dependent mast cell activation in rats in vivo (measuring Evans blue extravasation in passive cutaneous anaphylaxis (PCA) and increases in plasma histamine in passive systemic anaphylaxis (PSA)) and in vitro (measuring peritoneal mast cell (PMC) b-hexosaminidase release and prostaglandin D2 synthesis). However, under the conditions tested, cromolyn did not inhibit those mast cell-dependent responses in mice. In mice, cromolyn also failed to inhibit the ear swelling or leukocyte infiltration at sites of PCA. Nor did cromolyn inhibit IgE-independent degranulation of mouse PMCs induced by various stimulators in vitro. At 100 mg/kg, a concentration 10 times higher than that which inhibited PSA in rats, cromolyn significantly inhibited the increases in plasma concentrations of mouse mast cell protease-1 (but not of histamine) during PSA, but had no effect on the reduction in body temperature in this setting. Moreover, this concentration of cromolyn (100 mg/kg) also inhibited LPS-induced TNF production in genetically mast cell-deficient C57BL/6-KitW-sh/W-sh mice in vivo. These results question cromolyn’s effectiveness and selectivity as an inhibitor of mast cell activation and mediator release in the mouse. Laboratory Investigation (2012) 92, 1472–1482; doi:10.1038/labinvest.2012.116; published online 20 August 2012 KEYWORDS: anaphylaxis; cromolyn; disodium cromoglycate; DSCG; mast cell; mouse; rat Mast cells have long been regarded as exceptionally efficient been clarified, cromolyn has been used clinically as an anti- initiators and amplifiers of certain innate and acquired im- asthma drug for more than 30 years.10 Clinical studies mune responses, especially IgE-dependent acute responses to showed that inhalation of cromolyn by subjects with allergic challenge with specific antigen.1–5 In addition to their well- asthma blocked allergen-induced bronchospasm, presumably known functions in IgE-dependent responses, mast cells also by inhibiting the release of mediators from mast cells,11 an can secrete mediators in response to a variety of other signals, idea that was supported by the drug’s ability to inhibit the including products of pathogens, components of animal degranulation of rat peritoneal mast cells (PMCs) in response venoms,6,7 products of complement activation, neurotrans- to challenge with IgE and specific antigen in vitro.12–15 For mitters,8 vascular factors,9 and stem cell factor (SCF), this reason, cromolyn is often characterized clinically as a suggesting that mast cells have a much larger spectrum of ‘mast cell stabilizer’,16 and this drug is now used for the treat- potential roles in health and disease than was originally ment of other diseases thought to involve mast cell activa- thought.1–5 tion, including allergic rhinitis, allergic conjunctivitis, and One approach for understanding the roles of mast cells mastocytosis.17 in health and disease would be to employ drugs that can Although the early studies of cromolyn were performed effectively and selectively inhibit the function of this cell predominantly in rats and humans, many groups are now in vivo. Cromolyn (cromolyn sodium, sodium cromoglycate, employing this agent to ‘stabilize’ mast cells or to suppress SCG, disodium cromoglycate, and DSCG) is often used as mast cell functions in mice. Indeed, treatment of mice with such an agent. Although its mechanism of action has not cromolyn in vivo has become commonplace as part of efforts Department of Pathology, Stanford University School of Medicine, Stanford, CA, USA Correspondence: SJ Galli, MD, Department of Pathology, Stanford University School of Medicine, Lane Building, L-235, 300 Pasteur Drive, Stanford, CA 94305-5324, USA. E-mail: sgalli@stanford.edu Received 6 April 2012; revised 12 June 2012; accepted 19 June 2012 1472 Laboratory Investigation | Volume 92 October 2012 | www.laboratoryinvestigation.org Effects of cromolyn in mice versus rats T Oka et al to investigate roles of mast cells in mouse models of host PMCs with cromolyn before stimulating the cells to defense or disease that are thought to be independent of degranulate substantially reduced the inhibitory effect of IgE.18–29 However, surprisingly, there is little published cromolyn on PMC degranulation (an effect of the drug called information supporting the conclusion that cromolyn is ‘tachyphylaxis’12,13), we focused primarily on the effects of either an effective or selective ‘stabilizer’ (ie, inhibitor) of the cromolyn treatment observed when the agent is administered activation of mast cell populations in the mouse. at the time of mast cell activation. We also used genetically On the contrary, one group reported that cromolyn (at mast cell-deficient mice to assess whether the actions of this 200 mg/kg) did not inhibit Evans blue extravasation asso- agent are selective for mast cells in vivo. ciated with IgE-dependent passive cutaneous anaphylaxis (PCA) reactions in mice, one of the most common assays MATERIALS AND METHODS used to measure mast cell function in vivo.30 Moreover, Animals other groups reported that treatment with cromolyn (at KitW-sh/ þ mice generously provided by Peter Besmer 1–100 mM31 or 1–100 mg/ml,15 respectively) at the time of (Memorial Sloan-Kettering Cancer Center) were backcrossed antigen challenge did not inhibit IgE plus antigen-induced with C57BL/6 J mice (Jackson Laboratories) for more than degranulation31 or cysteinyl leukotriene release15 in mouse 11 generations to produce mast cell-deficient C57BL/ bone-marrow-derived cultured mast cells (BMCMCs) 6-KitW-sh/W-sh (herein: KitW-sh/W-sh) mice.34 Cpa3-Cre; in vitro. However, Marquardt et al. reported that long-term Mcl-1 þ / þ and mast cell- and basophil-deficient Cpa3-Cre; culture of mouse BMCMCs with cromolyn, as opposed to Mcl-1fl/fl mice were bred in our laboratory.35 C57BL/6J short-term treatment with the drug, resulted in a significant mice or Wistar rats were purchased from Jackson inhibition of IgE- and antigen-induced mediator release by Laboratories or Charles River, respectively. All animal care BMCMCs, but the basis for the difference in the response and experimentation was conducted in accordance with the of these cells to short-term vs long-term treatment with ‘Guide for the Care and Use of Laboratory Animals’ prepared cromolyn was not defined.31 In addition, Forbes et al.32 by the Institute of Laboratory Animal Resources, National reported that repeated treatment of mice with cromolyn Research Council, and published by the National Academy inhibited the increases in blood concentrations of the mast Press (revised, 1996) and with the approval of the Stanford cell-associated mediator, mouse mast cell protease-1 University Committee on Animal Welfare. (mMCP-1), a product of mucosal mast cells, in mice that genetically overexpressed IL-9. Taken together, such studies PCA Reaction raise the possibility that cromolyn might have inhibitory Rats (female, 8 weeks) and mice (male or female, 8 weeks) effects on some mast cell functions (such as the release of were sensitized by intradermal injection of anti-DNP IgE MCP-1 by mucosal mast cells32) but not others (eg, IgE- (aDNP clone e26, generously provided by Dr Fu-Tong Liu, dependent activation of skin mast cells30), and that the effects UC Davis; 2.5 ng for rat dorsal skin; 10 ng for mouse ear of the drug on even the same type of mast cells may depend pinnae) in saline (0.9% sodium chloride; 50 ml for rats; on the conditions of cromolyn exposure.15,31 10 ml for mice). Twenty-four hours later, DNP-HSA (Sigma; However, there are very few reports analyzing the effects 1 mg/kg for rats; 10 mg/kg for mice) was injected i.v. with or of cromolyn on mouse mast cells, and we are aware of no without cromolyn sodium salt (MP Biomedicals). Mouse ear reports on the effects of cromolyn on mouse PMCs— thickness was measured with a dial thickness gauge (G-1 A, the mast cell type most thoroughly investigated in studies of Ozaki, Tokyo, Japan). Mouse ear pinnae collected from mice 12–15 the effects of cromolyn on mast cell activation in the rat. killed by exposure to CO2 6 h after challenge were fixed in Accordingly, we think that it is difficult to draw any conclu- 10% formalin for preparation of paraffin sections stained sion about the effectiveness or selectivity of cromolyn as with hematoxylin and eosin, and leukocyte numbers were a mast cell stabilizer in mice other than more studies of quantified by light microscopy as per mm of horizontal field this topic are needed. In contrast, there have been many length of the ear (by an observer not aware of the identity of reports showing that cromolyn can inhibit rat PMC degranu- the individual sections). For measuring Evans blue extra- 12–15 33 lation or prostaglandin D2 (PGD2) synthesis in vitro, vasation, Evans blue (Sigma; 10 mg/kg for rats; 100 mg/kg for as well as reduce the extent of IgE-dependent degranulation mice) was injected i.v. with DNP-HSA.36 Thirty minutes of rat skin mast cells in PCA reactions in vivo.13,15,30 later, skin areas were photographed and cut out.