In vitro desensitization of human skin mast cells

Abstract

Desensitization is a clinical procedure whereby incremental doses of a drug are administered over several hours to a sensitive patient until a therapeutic dose and clinical tolerance are achieved. Clinical tolerance may occur in part by attenuating the mast cell response. In the present study, primary human skin mast cells were used to establish and characterize an in vitro model of desensitization. Mast cells in culture were armed with allergen-specific (4-hydroxy-3-nitrophenylacety and Der p2) and non-specific IgE antibodies, and then desensitized by incremental exposures to 4-hydroxy-3-nitrophenylacety-BSA. This desensitization procedure abrogated the subsequent degranulation response to the desensitizing allergen, to an unrelated allergen, and to IgG anti-FcεRI, but not to C5a, substance P, compound 48/80, and calcium ionophore. Desensitized cells regained their FcεRI-dependent degranulation capability by 24-48 h after free allergen had been removed. Therefore, sensitized human skin mast cells are reversibly desensitized in vitro by exposure to incremental doses of that allergen, which also cross-desensitizes them to an unrelated allergen.

Fig. 1

NP-BSA-induced degranulation of human skin mast cells. a % NP-specific IgE. Human skin mast cells, sensitized with different percentages of NP-specific IgE (total IgE=1 μg/ml), were washed and activated with NP-BSA (10 ng/ml) or 22E7 (1 μg/ml; n=3). b NP-BSA dose–response. Mast cells, sensitized with 10% NP-specific IgE (1 μg/ml total IgE), were stimulated with different concentrations of NP-BSA (n=3). Mean±SD, dagger, p<0.001 compared to non-stimulated cells

Fig. 2

Desensitization of mast cells with sequential NP-BSA concentrations. Bars: black, degranulation during desensitization or exposure to buffer alone; grey, degranulation during exposure to activating concentrations of stimulants. a Sequential allergen desensitization. Mast cells sensitized with 10% NP-specific IgE (1 μg/ml total IgE) were desensitized with increasing NP-BSA concentrations (“Methods” section; n=4). Degranulation of desensitized mast cells was compared to that of corresponding non-desensitized mast cells. b Degranulation during each step of sequential allergen desensitization (n=3) as performed in a with 10% NP-specific IgE-sensitized (1 μg/ml total IgE) mast cells. Degranulation at each dose of NP-BSA was compared to that at 1 pg/ml. c Degranulation of mast cells sensitized with 100% NP-specific IgE (1 μg/ml) after sequential allergen desensitization (as in a; n=3). 22E7 and NP-BSA-challenged desensitized cells were compared to NP-BSA-challenged non-desensitized cells, and to desensitized and non-desensitized cells challenged with buffer. a–c Asterisk, p<0.05; dagger, p<0.001. d Calculations of %Des and %DegDD using simulated bar values. A, net degranulation during desensitization; C, net degranulation of desensitized cells upon stimulation; B, net degranulation of non-desensitized cells upon stimulation after subtracting spontaneous degranulation; D=B-A, net degranulation of non-desensitized cells after subtracting degranulation during desensitization

Fig. 3

Attempted desensitization of mast cells with fixed concentrations of NP-BSA. Mast cells sensitized with 100% NP-specific IgE (1 μg/ml) were incubated overnight with various fixed concentrations of NP-BSA (“Methods” section), and then stimulated with 10 ng/ml of NP-BSA (n=3). Asterisk, p=0.01 comparing each value to the buffer control. Bars: black, degranulation during desensitization; grey, degranulation during activation

Fig. 4

Effect of sequential desensitization on surface CD63 and on Syk, Fyn, and Lyn. Mast cells sensitized with 10% NP-specific IgE (100 μg/ml total IgE) were either not desensitized or sequentially desensitized with NP-BSA. a Surface CD63 (n=3) after stimulation with NP-BSA (10 ng/ml). Filled, isotype control; unfilled, anti-CD63. b Syk, Fyn, and Lyn Western blot 5 min after stimulation with 22E7. c Analysis of Western blot band densities (n=4)

Fig. 5

Sequential desensitization and calcium flux. Mast cells sensitized with 10% NP-specific IgE (1 μg/ml total IgE) were either not desensitized (a, c) or desensitized (as in Fig. 2; b, d) and were then stimulated with 22E7 (100 ng/ml, a, b) or C5a (100 ng/ml, c, d) as indicated by arrows. Representative real-time OD 340/380 ratios from one of three independent experiments are shown

Fig. 6

Allergen cross-desensitization. Skin mast cells sensitized with 10% NP-specific and 10% Der p2-specific IgE (1 μg/ml total IgE) were sequentially desensitized with NP-BSA (%Degranulation=17± 5%), and then challenged with NP-BSA, conjugated Der p2, or 22E7, and net% degranulation was calculated (n=3). Asterisk, p<0.05

Fig. 7

Sequential desensitization after FcεRI upregulation. Mast cells were incubated with 10% NP-specific IgE (1 μg/ml) for 1 week and analyzed. a Surface expression of FcεRI by flow cytometry with 22E7 (n=3). Grey fill-in, isotype control; black line, baseline; grey line, upregulated. b Sequential desensitization of mast cells with NP-BSA as in Fig. 2. Mast cells were then stimulated with NP-BSA (10 ng/ml) or 22E7 (100 ng/ml) and assessed for degranulation (n=3). Dagger, p< 0.001 comparing desensitized to non-desensitized cells

Fig. 8

Recovery of mast cells after sequential desensitization. a Mast cells, sensitized with 10% IgE anti-NP (100 μg/ml total IgE), were desensitized as in Fig. 2a, washed, placed in fresh medium for 1–72 h, and then stimulated with 22E7 (100 ng/ml) or NP-BSA (10 ng/ml; n=3). Asterisk, p<0.01; dagger, p<0.001 comparing each time point and the IgE⁻ (only stimulated with 22E7) to the corresponding non-desensitized IgE⁺ cells. b Mast cells, sensitized and desensitized as above, were washed, incubated in fresh medium with NP-IgE for 24–72 h, and stimulated with NP-BSA (10 ng/ml). Asterisk, p<0.05; dagger, p<0.001 comparing each time point to the IgE⁺ cells (n=3)