COPD is associated with a macrophage scavenger receptor-1 gene sequence variation

Abstract

Background: Macrophages play an important role in COPD. We genotyped at-risk smokers to evaluate the role of polymorphisms in the macrophage scavenger receptor-1 gene (MSR1) in COPD susceptibility and related measures of lung function. Then, in macrophages from donors with specific MSR1 genotypes, we determined the effect of MSR1 single nucleotide polymorphisms (SNPs) on macrophage function by examining in vitro adhesion, receptor expression, and cell number in culture as an index of increased survival/reduced apoptosis.

Methods: Smokers (> or = 20 pack-years) who were > 40 years (n = 714) were genotyped for seven SNPs; one nonsense change (ex6R293x_C/T), four missense changes (ex4V113A_T/C, ex4P174Y_G/T, ex11H441R_A/G, and in the ligand binding site ex6P275A_C/G), -176511_A/G in the promoter region, and IVS5-59_C/A in the intron. Nonsmoking healthy volunteers (n = 85) were genotyped, and peripheral blood monocytes were isolated from seven P275A_CG/GG and eight P275A_CC controls and cultured to generate monocyte-derived macrophages (MDM). The effectiveness of trypsin and scraping to dislodge MDM was scored on a four-point subjective scale. MDM were counted on a Z1 particle counter and surface expression of MSR1 was determined by fluorescence-activated cell sorting analysis using secondary staining of antibodies against human macrophage scavenger receptor (MSR1).

Results: The MSR1-coding SNP P275A was associated with susceptibility to COPD in smokers (P < .005) and a lower percent predicted (pp) FEV(1), FEV(1)/FVC, and pp forced expiratory flow (FEF)(25-75) (P = .03). P275A_CG/GG was also associated with increases in maintenance of cell number in culture (increased survival/reduced apoptosis), MSR1 expression, and adhesion of macrophages to plastic in vitro (P < .05).

Conclusions: The MSR1 association with COPD susceptibility, COPD-related measures of lung function, and abnormalities of macrophage function may account for significant COPD morbidity.

Figure 1.

Five of the seven single nucleotide polymorphisms (SNPs) had a minor allele frequency < 0.05 and were not analyzed further. A significant association between SNP P275A (*P < .005) and susceptibility to COPD is demonstrated.

Figure 2.

Cell counts on day 5 and day 10 of the study. Mean ± SEM number of adherent cells at day 5 and at day 10. Asterisk (*) indicates statistical significance at P < .05 compared with corresponding P275A_C controls. Inset: Mean ± SEM total number of cells isolated on first day with lymphocyte and monocyte fractions indicated. Sample size was n = 8 for the P275A_CC group and n = 7 for the P275A_CG/GG group. Lymph = lymphocyte; Mono = monocyte.

Figure 3.

MSR1 expression at day 10. Mean ± SEM percent cells expressing MSR1 surface marker (A). The relative intensity of MSR1 expression, expressed as MFI, is likewise increased in the P275A_CG/GG group (B). Asterisk (*) indicates statistical significance at P < .05 compared with P275A_CC controls. Sample size was n = 8 for the P275A_CC group and n = 7 for the P275A_CG/GG group. MFI = median fluorescence intensity; MSR1 = macrophage scavenger receptor 1.

Figure 4.

Relative strength of MDM cell adherence on day 10; 1 = > 75% of cells removed by trypsin; 2 = 50% to 75% of cells removed by trypsin; 3 = 25% to 50% of cells removed by trypsin; 4 = < 25% of cells removed by trypsin. In all cases, remainder was removed by scraping. Horizontal line indicates median. Asterisk (*) indicates statistical significance at P < .05 compared with MSR1 gene P275A_CC controls by Mann-Whitney test. Sample size was n = 8 for the P275A_CC group and n = 7 for the MSR1 P275A_CG/GG group. MDM = monocyte-derived macrophage. See Figure 3 legend for expansion of other abbreviation.

Figure 5.

Uptake of fluorescein isothiocyanate-labeled AcLDL after 1-h suspension culture. Mean ± SEM MFI. (P = .08 one-tailed t test.) Sample size was n = 8 for the MSR1 P275A_CC group and n = 7 for the P275A_CG/GG group. AcLDL = acetylated low-density lipoprotein. See Figure 3 legend for expansion of other abbreviation.