Evidence questioning cromolyn's effectiveness and selectivity as a 'mast cell stabilizer' in mice

Abstract

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 μM 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) β-hexosaminidase release and prostaglandin D(2) 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-Kit(W-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.

Figure 1

Effects of cromolyn on PCA reactions in vivo. Female rats (a) or male mice (b – d) injected i.d. with vehicle (saline) or anti-DNP IgE (IgE) were challenged i.v. with DNP-HSA with or without cromolyn (injected simultaneously); we then measured Evans blue extravasation (a and b), ear swelling (c) and leukocyte migration (d). N = 4 rats (a), 5 mice (b) and 7 – 8 mice (c and d) per group from at least 2 independent experiments. ** p < 0.01, * p < 0.05; n.s.: p > 0.05.

Figure 2

Effect of cromolyn on PSA reactions in vivo. Female rats (a) or male mice (b – d) sensitized i.v. with anti-DNP IgE were challenged i.v. with DNP-HSA with or without cromolyn (injected simultaneously); we then measured plasma histamine (a and b), body temperature (c) and plasma mMCP-1 (d). N = 4 rats (a), and 5 – 10 mice (b – d) per group from at least 2 independent experiments. ** p < 0.01, * p < 0.05; n.s.: p > 0.05.

Figure 3

Effects of cromolyn on IgE-dependent PMC degranulation (a – f) or PGD₂ synthesis (g – i) in vitro. IgE-sensitized PMCs from female rats (a, d and g) or mice (b, c, e, f, h and i) were stimulated with DNP-HSA with or without cromolyn (added simultaneously). N = 3 – 10 per group from 1 – 2 independent experiments. ## p < 0.01, # p < 0.05, †† p < 0.01, † p < 0.05; N.S.: not significantly different versus corresponding values (vehicle or 100 μM cromolyn) for groups not treated with stimuli. ** p < 0.01, * p < 0.05; n.s.: p > 0.05.

Figure 4

Effect of cromolyn on IgE-independent degranulation of PMCs in vitro. PMCs from female rats (a – d) or mice (e – l) were stimulated with thapsigargin (a and e), ionomycin (b and f), substance P (c and g), compound 48/80 (d and h), PMA with ionomycin (i), endothelin-1 (j), adenosine (k), or mouse SCF (l), with or without cromolyn (added simultaneously). N = 3 – 6 per group from 1 – 2 independent experiments. ## p < 0.01, # p < 0.05, †† p < 0.01, † p < 0.05; N.S. not significantly different versus corresponding values (vehicle or 100 μM cromolyn) for groups not treated with stimuli. ** p < 0.01, * p < 0.05; n.s.: p > 0.05.

Figure 5

Effects of cromolyn on LPS-induced TNF production in C57BL/6J wild type or mast cell-deficient Kit^W-sh/W-sh mice in vivo (a and b) or in vitro (c – f). Male wild type (a) or Kit^W-sh/W-sh (b) mice were injected i.p. with vehicle or 100 mg/kg cromolyn 30 min before i.p. challenge with LPS. Spleen cells (c and d) or peritoneal cells (e and f) from wild type (c and e) or Kit^W-sh/W-sh (d and f) mice incubated for 15 min with or without cromolyn were stimulated with LPS. N = 4 – 13 mice per group from 3 (a) or 2 (b) independent experiments or 4 per group from one of 2 – 3 independent experiments, each of which gave similar results (c – f). ** p < 0.01, * p < 0.05.