Studies of the role of basophils in aspirin-exacerbated respiratory disease pathogenesis

Abstract

Background: Aspirin-exacerbated respiratory disease (AERD) is characterized by the triad of chronic rhinosinusitis with nasal polyps (CRSwNP), asthma, and intolerance to cyclooxygenase-1 enzyme inhibitors. The underlying mechanisms contributing to AERD pathogenesis are not fully understood, but AERD is characterized by an enhanced type 2 inflammatory phenotype. Basophils are potent type 2 effector cells, but their involvement in AERD pathophysiology remains unclear.

Objective: We sought to characterize the systemic and local basophil responses in patients with AERD compared with patients with CRSwNP.

Methods: Sinonasal tissues including inferior turbinate and/or nasal polyps (NPs) and peripheral blood were collected from controls, patients with AERD, and patients with CRSwNP. Expression of cell surface (CD45, FcεRI, CD203c), activation (CD63), and intracellular (2D7) markers associated with basophils was characterized using flow cytometry. Clinical data including Lund-Mackay scores and pulmonary function were obtained.

Results: The mean number of basophils (CD45⁺CD203c⁺FcεRI⁺CD117^-) detected in AERD NPs (147 ± 28 cells/mg tissue) was significantly elevated compared with that detected in CRSwNP NPs (69 ± 20 cells/mg tissue; P = .01). The number of circulating basophils was significantly elevated in patients with AERD (P = .04). Basophils in NPs had significantly higher CD203c and CD63 mean fluorescence intensity compared with blood in both conditions (P < .01). Basophils from AERD NPs had lower expression of the granule content marker 2D7 compared with those from matched blood (P < .01) or NPs of patients with CRSwNP (P = .06), suggesting ongoing degranulation. Basophil 2D7 mean fluorescence intensity significantly correlated with pulmonary function (r = 0.62; P = .02) and inversely correlated with sinonasal inflammation (r = -0.56; P = .004).

Conclusions: Increased basophil numbers and extent of ongoing degranulation in NPs of patients with AERD compared with patients with CRSwNP may contribute to the exaggerated disease pathogenesis and severity unique to AERD.

Keywords: 2D7; AERD; Aspirin-exacerbated respiratory disease; CRSwNP; basophil; chronic sinusitis; nasal polyp.

FIG 1.

Basophils are elevated in NPs of patients with AERD. Flow cytometry was used to quantify the number of CD45⁺ leukocytes (A), CD45⁺ Siglec-8⁺ FcεRI⁻ eosinophils (B), CD45⁺ CD117⁺ FcεRI⁺ mast cells (C), and CD45⁺ CD203c⁺ FcεRI⁺ CD117⁻ basophils (D) in NPs of patients with CRSwNP or AERD. For comparison, the number of leukocytes (Fig 1, A) and basophils (Fig 1, D) was quantified in inferior turbinate tissue of healthy controls. The number of tissue basophils strongly correlated with the number of tissue eosinophils (E) and tissue mast cells (F) in NPs. IT, Inferior turbinate; n.d., not determined. Dot plots illustrate individual data points, with solid lines representing the median and interquartile range. Statistical significance (P < .05) was determined by Mann-Whitney U test comparing AERD and CRSwNP (Fig 1, A–D) or Spearman rank correlation (Fig 1, E and F). *P < .05.

FIG 2.

Basophils are elevated in the peripheral blood of patients with AERD. Flow cytometry was used to quantify the number of CD45⁺ leukocytes (A), CD45⁺ Siglec-8⁺ FcεRI⁻ eosinophils (B), and CD45⁺ CD203c⁺ FcεRI⁺ CD117⁻ basophils (C) in the peripheral blood of patients with CRSwNP or AERD or healthy controls. The number of peripheral blood basophils strongly correlated with the number of circulating eosinophils (D) in patients with CRSwNP and AERD combined. Dot plots illustrate individual data points, with solid lines representing the median and interquartile range. Statistical significance (P < .05) was determined by Mann-Whitney U test comparing AERD and CRSwNP (Fig 2, A–C) or Spearman rank correlation (Fig 2, D). *P < .05. **P < .01.

FIG 3.

Basophil numbers strongly correlated between the peripheral blood and NPs of patients with AERD but not in those with CRSwNP. The number of CD45⁺ CD203c⁺ FcεRI⁺ CD117⁻ basophils in matched peripheral blood and NPs was compared when patients with AERD or CRSwNP were combined (A) or when patients with CRSwNP (B) and patients with AERD (C) were analyzed separately. Dot plots illustrate individual data points, with blue representing a patient with CRSwNP and black representing a patient with AERD. Statistical significance (P < .05) was determined by Spearman rank correlation.

FIG 4.

Basophils expressed higher levels of activation markers CD63 and CD203c in NPs compared with peripheral blood in both AERD and CRSwNP. Flow cytometry was used to quantify the MFI of CD203c (A) and CD63 (B) on basophils isolated from the peripheral blood and from NPs of patients with AERD or CRSwNP. Peripheral blood from healthy controls was used for comparison. The number of CD63^hi basophils was quantified in NPs (C) and peripheral blood (D) from the diseased and healthy conditions. IT, Inferior turbinate. Dot plots illustrate individual data points, with solid lines representing the median and interquartile range. Statistical significance (P < .05) was determined by Kruskal-Wallis test with post hoc analysis using the Dunn’s test for multiple comparison (Fig 4, A and B) and by Mann-Whitney U test comparing AERD and CRSwNP (Fig 4, C and D). **P < .01. ***P < .001.

FIG 5.

Basophils have reduced levels of 2D7 expression in NPs of patients with AERD compared with those with CRSwNP. Flow cytometry was used to quantify the MFI of intracellular 2D7 expression (A) within basophils isolated from the peripheral blood and from NPs of patients with AERD or CRSwNP. Peripheral blood from healthy controls was used for comparison. The percentage of basophils expressing 2D7⁺ in peripheral blood (B) and NPs (C) was also determined. IT, Inferior turbinate. Dot plots illustrate individual data points, with solid lines representing the median and interquartile range. Statistical significance (P < .05) was determined by Kruskal-Wallis test with post hoc analysis using the Dunn’s test for multiple comparison (Fig 5, A) and by Mann-Whitney U test comparing AERD and CRSwNP (Fig 5, B and C). *P < .05. ***P < .001.

FIG 6.

Markers of basophil activation (CD63) and degranulation (2D7 MFI) correlated with sinonasal and pulmonary disease severity. The number of CD63^hi basophils significantly correlated with the LM score (A) and inversely correlated with FEV₁ (B) in patients with either CRSwNP or AERD. In addition, 2D7 MFI inversely correlated with the LM score (C) and positively correlated with FEV₁ (D) in both CRSwNP and AERD. Dot plots illustrate individual data points, with blue representing a patient with CRSwNP and black representing a patient with AERD. Statistical significance (P < .05) was determined by Spearman rank correlation.