Neutral sphingomyelinase-2, acid sphingomyelinase, and ceramide levels in COPD patients compared to controls

Figures & data

Table 1 Subject demographics

	Immunohistochemistry			PCR			Ceramide determination
	NS	S	COPD	NS	S	COPD	NS	S	COPD
Number	12	14	14	4	12	16	2	5	10
Age (years)	69 (8.4)	62 (11.7)	66 (7.2)	72 (10.7)	67 (7.8)	65 (5.7)	74.5 (13.4)	66 (14.7)	65 (9.2)
Male/female	3/9	5/9	6/8	1/3	5/7	12/4	0/2	2/3	7/3
Smoking status (current/ex)	0	14/0	14/0	0	6/6	9/7	0	2/3	5/5
Smoking history (pack-years)	0	39 (19.3)	51 (26.7)	0	33 (24.8)	51 (13.6)	0	25.9 (22.8)	49.5 (15.4)
FEV1 % predicted	117 (19.6)	106 (11.8)	57 (8)	111 (3.5)	101 (18.3)	65 (14.5)	109 (4.2)	93.2 (26)	60.1 (10.9)
FEV1/FVC ratio	75 (7.8)	77 (6.3)	54 (10.7)	81 (9.3)	74 (4.7)	58 (8.6)	72.6 (6.2)	70.7 (5.2)	57.4 (8.2)
ICS usage %	0	0	43	0	0	38	0	0	40

Note: Data are presented as mean (SD).

Abbreviations: COPD, chronic obstructive pulmonary disease; FEV₁, forced expiratory volume in 1 second; FVC, forced vital capacity; ICS, inhaled corticosteroids; NS, nonsmoker; PCR, polymerase chain reaction; S, smoker; SD, standard deviation.

Figure 1 nSMase-2 labeling in human lung tissue.

Notes: Representative images for nSMase-2 expression (brown) within the peripheral lung tissue from (A, D, G) NS, (B, E, H) S, and (C, F, I) COPD patients. nSMase-2 was detected within the small airway epithelium (A–F) with a more pronounced expression toward the apical surface (inset arrow). nSMase-2 was also detected within the alveolar macrophages and alveolar walls (G–I). nSMase-2-positive alveolar macrophages (black arrows) observed near alveolar macrophages lacking nSMase-2 expression (red arrows). Magnification ×200 (A–C) ×600 (D–I).

Abbreviations: NS, nonsmoker; S, smoker.

Figure 2 Quantification of nSMase-2 in lung tissue.

Notes: All data are presented in graphs as means and standard errors of the mean. (A) The percentages of intact small airways with nSMase-2 labeling, (B) the number of cells with nSMase-2 labeling per mm² of the subepithelium, (C) the number of cells with nSMase-2 labeling per mm of the alveolar wall, and (D) the percentage of alveolar macrophages with nSMase-2 labeling. *P<0.05 according to Dunnett’s multiple comparison test.

Figure 3 aSMase labeling in human lung tissue.

Notes: Representative images for aSMase expression (brown) within the peripheral lung tissue from (A, D, G) NS, (B, E, H) S, and (C, F, I) COPD patients. aSMase was detected within the small airway epithelium (A–F) with a pronounced nuclear expression (inset arrow). aSMase was also detected within the alveolar macrophages and alveolar walls (G–I). aSMase-positive alveolar macrophages (black arrows) observed near alveolar macrophages lacking nSMase-2 expression (red arrows). Magnification ×200 (A–C) ×600 (D–I).

Abbreviations: NS, nonsmoker; S, smoker.

Figure 4 Quantification of aSMase labeling in lung.

Notes: All data are presented in graphs as means and standard errors of the mean. (A) The percentages of intact small airways with aSMase labeling, (B) the number of cells with aSMase labeling per mm² of the subepithelium, (C) the number of cells with aSMase labeling per mm of the alveolar wall, and (D) the percentage of alveolar macrophages with aSMase labelling. *P<0.05 according to Dunnett’s multiple comparison test.

Figure 5 nSMase-2 and aSMase gene expression in alveolar macrophages.

Notes: RNA was extracted from NS (n=4), S (n=13), and COPD (n=14) alveolar macrophages. Complementary DNA was synthesized by RT-PCR and analyzed for (A) nSMase-2 and (B) aSMase expression using Taqman gene expression primers in a QPCR. Relative expression levels were determined using the ΔCt method normalizing to the endogenous control (GAPDH). Both box and whisker plots illustrate data as medians with the maximum/minimum values out with the upper/lower quartile.
Abbreviations: GAPDH, glyceraldehyde-3-phosphate dehydrogenase; RT-PCR, reverse transcription polymerase chain reaction; QPCR, quantitative polymerase chain reaction; NS, nonsmoker; S, smoker.

Figure 6 Quantification of ceramide species (C16, C18, and C20) in alveolar macrophages.

Notes: Ceramide species (C20, C16, and C18) were quantified by mass spectrometry. A significant increase in C20 but not in C16 or C18 was observed in alveolar macrophages from COPD patients (n=10) compared with non-COPD patients (n=7), according to Dunnett’s multiple comparison test.