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. 2019 Nov 13:10:1313.
doi: 10.3389/fphar.2019.01313. eCollection 2019.

Endogenous Annexin-A1 Negatively Regulates Mast Cell-Mediated Allergic Reactions

Ajantha Sinniah  1 , Samia Yazid  1 , Stefania Bena  1 , Sonia M Oliani  1 , Mauro Perretti  1 , Rod J Flower  1
Affiliations

Endogenous Annexin-A1 Negatively Regulates Mast Cell-Mediated Allergic Reactions

Ajantha Sinniah et al. Front Pharmacol. .

Abstract

Mast cell stabilizers like cromoglycate and nedocromil are mainstream treatments for ocular allergy. Biochemical studies in vitro suggest that these drugs prevent mast cell degranulation through the release of Annexin-A1 (Anx-A1) protein. However, the direct effect of Anx-A1 gene deletion on mast cell function in vitro and in vivo is yet to be fully investigated. Hence, we aim to elucidate the role of Anx-A1 in mast cell function, both in vivo and in vitro, using a transgenic mouse model where the Anx-A1 gene has been deleted. Bone marrow-derived mast cells (BMDMCs) were cultured from wild-type animals and compared throughout their development to BMDMCs obtained from mice lacking the Anx-A1 gene. The mast cell differentiation, maturity, mediator, and cytokine release were explored using multiple biochemical techniques, such as Western blots, ELISA, and flow cytometry analysis. Electron microscopy was used to identify metachromatic granules content of cells. For in vivo studies, Balb/C wild-type and Anx-A1-deficient mice were divided into the following groups: group 1, a control receiving only saline, and group 2, which had been sensitized by prior exposure to short ragweed (SRW) pollen by topical contact with the conjunctival mucosae. Allergic conjunctivitis was evaluated blind after 24 h by trained observers scoring clinical signs. Electron micrographs of BMDMCs from Anx-A1-null mice revealed more vacuoles overall and more fused vacuoles than wild-type cells, suggesting enhanced secretory activity. Congruent with these observations, BMDMCs lacking the Anx-A1 gene released significantly increased amounts of histamine both spontaneously as well as in response to Ig-E-FcεRI cross-linking compared to those from wild-type mice. Interestingly, the spontaneous release of IL-5, IL-6, IL-9, and monocyte chemoattractant protein-1 (MCP-1) were also markedly increased with a greater production observed upon IgE cross-linking. This latter finding is congruent with augmented calcium mobilization in BMDMCs lacking the Anx-A1 gene. In vivo, when compared to wild-type animals, Anx-A1-deficient mice exposed to SRW pollen displayed exacerbated signs and symptoms of allergic conjunctivitis. Taken together, these results suggest Anx-A1 is an important non-redundant regulator of mast cell reactivity and particularly in allergen mediated allergic reactions.

Keywords: Annexin A1; allergic conjunctivitis model; bone marrow-derived mast cells; mast cell; transgenic mouse model.

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Figures

Figure 1
Figure 1
Differences in ultrastructural morphology between wild-type and Anx-A1 null BMDMCs. (A–F) Electron micrographs of BMDMCs from the wild-type control strain (A–C) and from the Anx-A1 null line (D–F). The morphological features of the vacuoles from the Anx-A1 null BMDMs were shown in (E and F). The white arrow shown in (E indicates fused vacuoles and the dotted arrow, mitochondria and extracellular granule remnants are seen in (F) (white arrow). Whereas “empty” (B) (white arrows), “partly filled,” and dense vacuoles in (C) (white arrow) are observed in the BMDMCs from the WT strain. (G–L) Results of a planimetric analysis of the median number of different vacuoles cells from the two genotypes. There was an overall trend towards a greater number of dense and partly filled vacuoles in the Anx-A1 null strain, although this was not statistically significant. We did, however, observe a significant (p 0.05) increase in the number of “fused vacuoles” (J) as well as the total number of all vacuoles (K) in the Anx-A1 null strain. Scale bars, 10 µm * p < 0.05 vs WT. Data are expressed as the median ± semi-quartile ranges and were analyzed using the Mann–Whitney test.
Figure 2
Figure 2
Anx-A1 regulates mast cell maturity and spontaneous degranulation. BMDMCs were prepared from WT and Anx-A1 KO mice as described, sensitized with anti-DNP-IgE (100 ng/ml; Sigma), and challenged with DNP-BSA (1 µg/ml; Sigma Aldrich, Dorset, UK). (A) At week 3 culture, the Anx-A1 KO BMDMCs expressed approximately 30% more c-Kit-positive cells as compared to their WT counterparts. At week 4 culture, both WT and Anx-A1 KO BMDMCs reached 90% c-Kit-positive cells. (B) BMDMCs from the Anx-A1 KO mice are significantly (p 0.05) more sensitive to ionomycin (1 µM) when compared to WT. (C) The total intracellular histamine level in Anx-A1-/- BMDMCs is significantly (p 0.05) lower than the WT cells; however, (D) demonstrates that these cells undergo higher net histamine release in comparison to the WT control (p 0.05). (E and F) Unstimulated Anx-A1 KO BMDMCs spontaneously release more histamine and tryptase when compared to WT BMDMCs. * p < 0.05; ** p < 0.01 and *** p < 0.001 vs WT. Data are expressed as mean ± SEM and analyzed using the two-tailed t test.
Figure 3
Figure 3
Spontaneous, and Ig-E challenged, release of pro-inflammatory cytokines by Anx-A1-/- BMDMCs. BMDMCs were prepared from WT and Anx-A1 KO mice as described, sensitized with anti-DNP-IgE (100 ng/ml; Sigma), and challenged with DNP-BSA (1 µg/ml; Sigma Aldrich, Dorset, UK). (A and B) Unstimulated WT and Anx-A1 KO BMDMCs did not express IL-4 and IL-5. But upon sensitization, the expression of IL-4 was significantly (p 0.05) decreased while IL-5 expression was significantly (p 0.05) increased in Anx-A1 KO BMDMCs in comparison to their WT counterpart. (C) BMDMCs lacking the Anx-A1 gene release TNF-α in both unstimulated and stimulated conditions. (D–F) Unstimulated Anx-A1 KO BMDMCs spontaneously release IL-9, IL-6, and MCP-1 when compared to WT cells. The level of IL-9 was increased in both WT and KO BMDMCs upon stimulation, but the expression of IL-6 (p 0.001) and MCP-1 (p 0.05) was significantly increased in the Anx-A1 KO BMDMCs compared to the WT cells. * p < 0.05 and ** p < 0.01 vs WT. Data are expressed as the mean ± SEM and analyzed using the one tailed t test.
Figure 4
Figure 4
Anx-A1 regulates phospho-Stat 5. BMDMCs were prepared from WT and Anx-A1-/- mice as previously described, sensitized with anti-DNP-IgE (100 ng/ml; Sigma), and challenged with DNP-BSA (1 µg/ml; Sigma Aldrich, Dorset, UK). Analysis of Stat-5 phosphorylation in WT and Anx-A1 KO BMDMCs in the presence of human recombinant Anx-A1 (10 ng/ml) at various time points was accomplished by Western blot analysis (A). Following challenge, KO BMDMCs expressed more than fourfold increase of Stat-5 phosphorylation (p 0.001) in comparison with WT cells. However, hr-Anx-A1 (10 ng/ml) reversed this effect in a time-dependent manner. (B) Densitometry values of three independent experiments were expressed as the mean percentage of WT control values ± SEM and analyzed by one-way ANOVA. Phospho Stat-5: **p 0.01 and ***p 0.001 vs. WT.
Figure 5
Figure 5
Anx-A1-/- mice develop enhanced allergic conjunctivitis upon SRW pollen exposure. (A–C) Anx-A1-/- and WT mice were topically challenged for 8 days with SRW pollen or sterile PBS (control group) and the clinical scores were evaluated on the final day 20 min post-challenge. Anx-A1-/- (D) mice exposed to SRW pollen exhibited pronounced clinical signs of conjunctivitis when compared to WT (B), including a significant increase of conjunctival redness (E), lid edema (F), tearing (G), and mucus (H). Data were expressed as the mean ± SEM and analyzed by Student’s t test. *p 0.05, **p 0.01, ***p 0.001.
Figure 6
Figure 6
Influx of mast cells into the bulbar conjunctival tissue of Anx-A1-null mice. Histological analysis of conjunctival tissue 24 h post-SRW pollen-challenge indicates an increased influx of mast cells in the Anx-A1 KO mice (C and D) as compared to WT (A and B). There were a higher proportion of degranulated mast cells observed in the conjunctival tissue of Anx-A1-/- mice, whereas intact metachromatic granules were observed in the WT mice. Quantitative analysis of the number of mast cells in both WT and Anx-A1-/- was shown in (E). Data represent the mean ± SEM of the number of cells per 0.1 mm2 for each group (each group = 4 mice). Stain: Toluidine blue. Scale bar, 10 µm. Data expressed as the mean ± SEM and analyzed by Student’s t test. *p 0.05 vs WT.
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