Allergens induce activation of blood CCR3+ granulocyte subsets in patients with perennial allergic rhinitis

doi:10.12122/j.issn.1673-4254.2026.02.16

Abstract

Abstract:

Objective To explore the effects of allergens on activation of blood CCR3⁺ granulocyte subtypes in patients with allergic rhinitis (AR). Methods Flow cytometry was used to examine the effects of allergens on CD63 and CD203c expressions on CCR3⁺CD203c^- eosinophils and CCR3⁺CD203c⁺ basophils of AR patients. Plasma levels of IL-25 and TSLP of the patients were detected with multiplex immunoassay and ELISA, respectively, and their correlations with eosinophils and basophils were analyzed. RNA-sequencing of blood CD16^- granulocytes from AR patients was performed, and enrichment analysis was used to explore the functions and potential mechanisms of the differentially expressed genes. Results The percentages of eosinophils and basophils in CCR3⁺ granulocytes increased significantly in AR patients (P<0.005), and exposure to allergens further upregulated the percentage of basophils (P<0.005). CD63 expression was significantly increased in patients with seasonal AR (sAR) and perennial AR (pAR) (P<0.005) but not in AR patients negative for skin prick test (nAR). Allergen exposures enhanced the expressions of CD63 and CD203c on eosinophils and basophils in pAR patients (P<0.005). Elevated plasma IL-25 and TSLP levels in AR patients were associated with increased eosinophil and basophil counts (P<0.005). The 3999 differentially expressed genes in CD16^- granulocytes from AR patients positive for skin prick test (SPT⁺) were involved in protein synthesis, energy metabolism and immune function and in endocytosis, phagosome and carbon metabolism pathways. Conclusion The findings in this study enrich the understanding of the roles of CCR3⁺ eosinophils and basophils in the pathogenesis of SPT⁺AR and reveal potential therapeutic targets for AR.

Key words: allergen, allergic rhinitis, basophils, eosinophil, activation

Qiuli WANG, Weihua XU, Fangqiu GU, Siqin WANG, Shaoheng HE, Junling WANG. Allergens induce activation of blood CCR3⁺ granulocyte subsets in patients with perennial allergic rhinitis[J]. Journal of Southern Medical University, 2026, 46(2): 385-393.

Figures/Tables 8

Tab.1 Basic information of volunteers

Population	Positive allergen	Sample size (n)	Age (year)	Medical history (year)	Onset age (year)
HC	——	30	34.5 (23.8-42.0)	——	——
nAR	——	14	35.5 (18.0-42.3)	3.0 (2.0-6.3)	30.5 (15.3-34.5)
sAR	ASE	16	32.0 (24.5-39.8)	4.0 (3.0-10.0)	26.5 (13.3-30.8)
pAR	ASE+DPE+Others	24	26.0 (19-44.5)	3.5 (2.0-10.0)	19.0 (14.0-38.8)
	DPE+Cockroach+Others	1	9.0 (9.0-9.0)	6.0 (6.0-6.0)	3.0 (3.0-3.0)
	Cockroach+Others	4	38.5 (14.0-48.0)	4.0 (1.3-12.8)	34.0 (12.8-35.8)
In total		29	28.0 (18.5-44.0)	4.0 (2.0-10.0)	20.0 (11.5-35.5)
——: not applicable; Age, medical history and onset age were expressed as Median (P₂₅-P₇₅). HC: Healthy control; nAR: AR with negative skin prick test; sAR: Seasonal AR with positive skin prick test; pAR: Perennial AR with positive skin prick test.

Fig.1 Ratios of eosinophils and basophils in blood granulocytes of AR patients. A: Flow cytometry gating strategy for human blood granulocytes. B, C: Representative flow cytometry graphs of CCR3+ cells (B), CCR3+CD203c- eosinophils and CCR3+CD203c+ basophils (C) in granulocytes. D-F: Statistical graphs of the expression ratios of CCR3+ cells (D), eosinophils (E), basophils (F) in granulocytes. *P<0.05 vs HC, #P<0.05 vs nAR Medium, △P<0.05 vs sAR Medium, ¶P<0.05 vs pAR Medium.

Fig.2 Effects of allergens on MFI expression levels of CD63 on eosinophils and CD203c on basophils in blood granulocytes of AR patients. A, B: Flow cytometry for analyzing MFI expression levels of CD63 on eosinophils (A) and CD203c on basophils (B) in granulocytes. C, D: Statistical graph of the MFI expression levels of CD63on eosinophils (C) and CD203c on basophils (D). *P<0.05 vs HC, #P<0.05 vs nAR Medium, △P<0.05 vs sAR Medium, ¶P<0.05 vs pAR Medium.

Fig.3 Plasma levels of IL-25 (A) and TSLP (B) in AR patients. *P<0.05 vs HC.

Fig.4 Eosinophil (A) and basophil (B) counts in blood granulocytes of AR patients. *P<0.05 vs HC.

Tab.2 Spearman's rank correlation coefficient (R) between cytokine levels and eosinophil and basophil counts in granulocytes

Group	Compound	IL-25	TSLP	Eosinophil	Basophil
HC	IL-25	——	-0.343	0.165	-0.056
HC	TSLP	-0.343	——	-0.401	-0.231
nAR	IL-25	——	0.741^⁋	0.587^⁋	0.630^⁋
nAR	TSLP	0.741^⁋	——	0.582^⁋	0.669^⁋
sAR	IL-25	——	0.577^⁋	0.802^⁋	0.895^⁋
sAR	TSLP	0.577^⁋	——	0.590^⁋	0.537^⁋
pAR	IL-25	——	0.772^⁋	0.880^⁋	0.601^⁋
pAR	TSLP	0.772^⁋	——	0.666^⁋	0.429^⁋
——: not applicable;^⁋P<0.05.

Fig.5 RNA-seq analysis of blood CD16- granulocytes of AR patients. A, B: Volcano plot (A) and cluster heatmap (B) of differential expression genes. C, D: GO (C) and KEGG (D) enrichment diagramsof differential expression genes.

Tab.3 RNA-seq analysis of the expression levels of CCR3, ENPP3 and CD63 mRNA in CD16- granulocytes

Protein name	Gene number	Gene name	log₂ fold change (AR vs HC)
CCR3	ENSG00000183625	CCR3	2.55**
CD203c	ENSG00000154269	ENPP3	-0.44
CD63	ENSG00000135404	CD63	2.34**
**P<0.01vs HC.

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