Cysteinyl leukotriene overproduction in aspirin-exacerbated respiratory disease is driven by platelet-adherent leukocytes

Abstract

Cysteinyl leukotriene (cysLT) overproduction is a hallmark of aspirin-exacerbated respiratory disease (AERD), but its mechanism is poorly understood. Because adherent platelets can convert the leukocyte-derived precursor leukotriene (LT)A(4) to LTC(4), the parent cysLT, through the terminal enzyme LTC(4) synthase, we investigated the contribution of platelet-dependent transcellular cysLT production in AERD. Nasal polyps from subjects with AERD contained many extravascular platelets that colocalized with leukocytes, and the percentages of circulating neutrophils, eosinophils, and monocytes with adherent platelets were markedly higher in the blood of subjects with AERD than in aspirin-tolerant controls. Platelet-adherent subsets of leukocytes had higher expression of several adhesion markers than did platelet nonadherent subsets. Adherent platelets contributed more than half of the total LTC(4) synthase activity of peripheral blood granulocytes, and they accounted for the higher level of LTC(4) generation by activated granulocytes from subjects with AERD compared with aspirin-tolerant controls. Urinary LTE(4) levels, a measure of systemic cysLT production, correlated strongly with percentages of circulating platelet-adherent granulocytes. Because platelet adherence to leukocytes allows for both firm adhesion to endothelial cells and augmented transcellular conversion of leukotrienes, a disturbance in platelet-leukocyte interactions may be partly responsible for the respiratory tissue inflammation and the overproduction of cysLTs that characterize AERD.

Figure 1

Detection of platelet-leukocyte aggregates in nasal polyp tissue. (A) H&E staining of nasal polyp tissue from a subject with AERD shows many eosinophils (black arrowheads). (B) Immunofluorescent staining of the same tissue shows leukocytes (green, CD45⁺) with adherent platelets (red, CD61⁺; white arrowheads). Photographs are shown at 400× magnification. (C) Total numbers of CD45⁺ cells that colocalized with CD61. (D) Percentages of CD45⁺ cells that colocalized with CD61 in the nasal polyp tissue from aspirin-tolerant controls with sinusitis (n = 4) and subjects with AERD (n = 6). Data are expressed as mean + SD.

Figure 2

Platelet-adherent leukocytes are identifiable in peripheral blood. (A) Representative histograms of platelet-adherent eosinophils (identified as CCR3⁺CD45⁺ cells in the granulocyte side scatter [SSC] gate), neutrophils (CD16⁺CD45⁺ cells in the granulocyte SSC gate), monocytes (CD45⁺ in the monocyte SSC gate), and lymphocytes (CD45⁺ cells in lymphocyte SSC gate) in blood from an ATA control (top) and a subject with AERD (bottom). The percentages of each cell type with adherent platelets are shown. (B) Percentages of leukocytes with adherent platelets (as determined by staining with CD61) in blood of nonasthmatic controls (n = 9), ATA controls (n = 13), and subjects with AERD (n = 15). Data are expressed as mean + SD (★P < .001).

Figure 3

Expression of integrins by platelet-adherent and -nonadherent leukocyte subsets. (A) Representative histograms of relative CD18 expression by CD61⁻ platelet-nonadherent (solid gray) and CD61⁺ platelet-adherent (black line) peripheral blood eosinophils (top), neutrophils (middle), and monocytes (bottom) are shown for a subject with AERD. (B-D) Relative expression of CD18 and CD49d on eosinophils (B), CD18 on neutrophils (C), and CD18 and CD11b on monocytes (D), comparing the platelet-adherent and platelet-nonadherent leukocyte subsets in nonasthmatic controls (n = 7), ATA controls (n = 10), and subjects with AERD (n = 9). MFI indicates mean fluorescence intensity. Platelet-free CD61⁻ leukocyte subsets are shown in white columns, and CD61⁺ leukocyte subsets are shown in hatched columns. Data are expressed as mean + SEM (★P < .05, ★★P < .01, ★★★P < .001).

Figure 4

Contribution of platelet LTC₄S to cysLT production by peripheral blood granulocytes. (A) Western blot analysis of platelets for LTC₄S protein in 3 ATA controls and 4 subjects with AERD. (B) Removal of adherent platelets by trypsinization is shown cytofluorographically for a subject with AERD. Isolated granulocytes were stained for CD45 and analyzed for their expression of CD61 before trypsinization (black line) and again after trypsinization (solid gray). (C) Conversion of LTA₄-ME to LTC₄-ME by washed platelets (left section of panel) and by granulocytes (right section of panel) without and with trypsinization to remove adherent platelets from subjects with ATA (gray columns; n = 6) and AERD (black columns; n = 7). (D) A23187-induced production of LTC₄ (top) and the sum of all 5-LO pathway products [LTB₄, LTC₄, LTD₄, (5,6)-dihydroxy-7,9-trans-11,14-cis-eicosatetraenoic acid [5,6-DiHETE], and 6-trans-LTB₄; bottom] by granulocytes without and without trypsinization to remove adherent platelets from subjects with ATA (n = 8) and AERD (n = 10). Data in panels C and D are expressed as mean +SEM (★P < .05, ★★P < .01).

Figure 5

Platelet-adherent leukocytes correlate with systemic cysLT production. (A) Baseline urinary LTE₄ (top) and TXB₂ (bottom) levels analyzed by gas chromatography-mass spectrometry from nonasthmatic controls (n = 8), ATA controls (n = 9) and subjects with AERD (n = 10). Data are expressed as mean + SD (★★★P < .001). (B) Baseline urinary LTE₄ levels plotted against the corresponding percentages of platelet-adherent eosinophils (top), neutrophils (middle), and monocytes (bottom) in the peripheral blood of each subject. Effect size, determined with Pearson correlation coefficient, is denoted as an r value displayed for each cell type. White circles, nonasthmatic controls; gray squares, ATA controls; black triangles, AERD subjects.