S1P₄ Regulates Passive Systemic Anaphylaxis in Mice but Is Dispensable for Canonical IgE-Mediated Responses in Mast Cells

Abstract

Mast cells are key players in the development of inflammatory allergic reactions. Cross-linking of the high-affinity receptor for IgE (FcεRI) on mast cells leads to the generation and secretion of the sphingolipid mediator, sphingosine-1-phosphate (S1P) which is able, in turn, to transactivate its receptors on mast cells. Previous reports have identified the expression of two of the five receptors for S1P on mast cells, S1P₁ and S1P₂, with functions in FcεRI-mediated chemotaxis and degranulation, respectively. Here, we show that cultured mouse mast cells also express abundant message for S1P₄. Genetic deletion of S1pr4 did not affect the differentiation of bone marrow progenitors into mast cells or the proliferation of mast cells in culture. A comprehensive characterization of IgE-mediated responses in S1P₄-deficient bone marrow-derived and peritoneal mouse mast cells indicated that this receptor is dispensable for mast cell degranulation, cytokine/chemokine production and FcεRI-mediated chemotaxis in vitro. However, interleukin-33 (IL-33)-mediated enhancement of IgE-induced degranulation was reduced in S1P₄-deficient peritoneal mast cells, revealing a potential negative regulatory role for S1P₄ in an IL-33-rich environment. Surprisingly, genetic deletion of S1pr4 resulted in exacerbation of passive systemic anaphylaxis to IgE/anti-IgE in mice, a phenotype likely related to mast cell-extrinsic influences, such as the high circulating levels of IgE in these mice which increases FcεRI expression and consequently the extent of the response to FcεRI engagement. Thus, we provide evidence that S1P₄ modulates anaphylaxis in an unexpected manner that does not involve regulation of mast cell responsiveness to IgE stimulation.

Keywords: IL-33; S1P4; S1pr4; anaphylaxis; chemotaxis; degranulation; mast cell; mediator release; sphingosine-1-phosphate.

Figure 1

S1P₄ is expressed in mast cells. Quantitative PCR data showing the relative expression of S1P receptor message normalized to glyceraldehyde 3-dehydrogenase (GAPDH) in bone marrow-derived mast cells (BMMC, left) and peritoneal-derived mast cells (PDMC, right). Plots represent the mean ± SE (or SD for PDMC) of data pooled from 7 independent BMMC or 2 PDMC cultures. ND: Not detected.

Figure 2

Analysis of degranulation, cytokine and chemokine responses in primary S1P₄-deficient mast cells. (A–C) Degranulation response to antigen stimulation (A,B) or to pharmacological stimulation with thapsigargin (B). Mast cells from S1pr4^+/+ (solid) and S1pr4^−/− mice (open) were grown in the presence of stem cell factor (SCF) and recombinant mIL-3 for 6–7 weeks (BMMC) or 14 days (PDMC) and sensitized with 100 ng/mL anti-dinitrophenyl (DNP)-IgE in cytokine-free media for 14 h. Degranulation was assessed by measuring the release of β-hexosaminidase into the media after 30 min of stimulation with the indicated concentrations of DNP (antigen; Ag) (A), 1 µM thapsigargin (B), or antigen in addition to 1 ng/mL recombinant IL-33 (C). Data represent the mean ± SE of results pooled from 4–8 independent cultures. (D,E) BMMC from S1pr4^+/+ (solid bars) and S1pr4^−/− mice (open bars) were sensitized overnight with 100 ng/mL anti-DNP IgE in cytokine-free media. Cells were washed, stimulated with the indicated concentrations of Ag and the amounts of IL-6 (D) and TNF-α (E) secreted into the media measured by ELISA at 4 h post-stimulation. The limit of detection for IL-6 and TNF-α quantitation by ELISA are shown by a dotted line in panels C and D at 0.0018 ng/mL and 0.00721 ng/mL, respectively. Data is pooled from 4 independent cultures. (F,G) Validation by ddPCR of the normalized relative expression of select chemokines (F) and cytokines (G) identified as being variably upregulated in S1pr4^+/+ and S1pr4^−/− BMMC cultures following Ag stimulation. Relative expression of Il6 is included for comparison. Data show mean ± SE of values obtained from at least seven independent cultures of BMMC for each genotype. All comparisons between S1pr4^+/+ and S1pr4^−/− cells were found to be not statistically significant unless otherwise indicated. * p < 0.05.

Figure 2

Analysis of degranulation, cytokine and chemokine responses in primary S1P₄-deficient mast cells. (A–C) Degranulation response to antigen stimulation (A,B) or to pharmacological stimulation with thapsigargin (B). Mast cells from S1pr4^+/+ (solid) and S1pr4^−/− mice (open) were grown in the presence of stem cell factor (SCF) and recombinant mIL-3 for 6–7 weeks (BMMC) or 14 days (PDMC) and sensitized with 100 ng/mL anti-dinitrophenyl (DNP)-IgE in cytokine-free media for 14 h. Degranulation was assessed by measuring the release of β-hexosaminidase into the media after 30 min of stimulation with the indicated concentrations of DNP (antigen; Ag) (A), 1 µM thapsigargin (B), or antigen in addition to 1 ng/mL recombinant IL-33 (C). Data represent the mean ± SE of results pooled from 4–8 independent cultures. (D,E) BMMC from S1pr4^+/+ (solid bars) and S1pr4^−/− mice (open bars) were sensitized overnight with 100 ng/mL anti-DNP IgE in cytokine-free media. Cells were washed, stimulated with the indicated concentrations of Ag and the amounts of IL-6 (D) and TNF-α (E) secreted into the media measured by ELISA at 4 h post-stimulation. The limit of detection for IL-6 and TNF-α quantitation by ELISA are shown by a dotted line in panels C and D at 0.0018 ng/mL and 0.00721 ng/mL, respectively. Data is pooled from 4 independent cultures. (F,G) Validation by ddPCR of the normalized relative expression of select chemokines (F) and cytokines (G) identified as being variably upregulated in S1pr4^+/+ and S1pr4^−/− BMMC cultures following Ag stimulation. Relative expression of Il6 is included for comparison. Data show mean ± SE of values obtained from at least seven independent cultures of BMMC for each genotype. All comparisons between S1pr4^+/+ and S1pr4^−/− cells were found to be not statistically significant unless otherwise indicated. * p < 0.05.

Figure 3

BMMC chemotaxis in the absence of S1pr4 expression. (A) BMMC from S1pr4^+/+ (solid bars) and S1pr4^−/− mice (open bars) grown in the presence of SCF and IL-3 for 6 weeks were sensitized with anti-DNP IgE in serum-free, cytokine-free media supplemented with 0.04% fatty acid-free bovine serum albumin (BSA) for 14 h. Sensitized cells were subject to transwell migration analysis using antigen (DNP; Ag) with or without 100 nM S1P as a chemoattractant at the indicated concentrations in the bottom chamber. (B) Chemotaxis of BMMC towards 10 ng/mL SCF in the bottom chamber. After 4 h incubation at 37 °C in 5% CO₂, cells that migrated into the lower chamber were collected and counted. In each experiment, three to six technical replicates were performed for the S1pr4^+/+ and S1pr4^−/− cultures. Percent specific migration was calculated by taking the average number of cells in the bottom chamber/total input cells × 100. Data represents the mean ± SE normalized migration for three independent BMMC cultures for each genotype. In each experiment, migration of S1pr4^+/+ and S1pr4^−/− cultures were normalized to the average migration to Ag (10 ng/mL) across the 3 S1pr4^+/+ cultures. * p < 0.05.

Figure 4

S1pr4 deletion exacerbates PSA. (A) S1pr4^+/+ and S1pr4^−/− mice were injected i.v. with 3 µg of mouse IgE. 24 h later, systemic anaphylaxis was induced by i.v. injection of 9 µg of anti-mouse IgE. Body temperature was monitored at the indicated times (S1pr4^+/+ n = 4, S1pr4^−/− n = 7). The asterisks between the curves indicate significant differences (p < 0.001) between genotypes using a two way-ANOVA test. (B,C) Dorsal skin biopsies (B) and inguinal lymph nodes (LN) (C) harvested from S1pr4^+/+ and S1pr4^−/− mice were fixed in 10% neutral buffer formalin, embedded in paraffin and sectioned. Three sections per skin biopsy and two sections per lymph node were stained with toluidine blue and eosin. Each dot represents the average number of metachromatic staining cells/10× field (B) or inguinal LN section (C) in one mouse and was calculated from five fields for each section examined, averaging values from 3 (B) or 2 (C) different sections for each tissue/animal. Floating bars represent the mean ± SE for each group of mice.