Matrix Metalloproteinase Expression by Human Alveolar Macrophages in Relation to Emphysema

Figures & data

Figure 1 Distribution of lung function data. Panel A shows the distribution of forced expiratory volume in 1 second (FEV₁), % predicted (n = 47). Panel B shows the distribution of forced vital capacity (FVC), % predicted (n = 47). Panel C shows the distribution of FEV₁/FVC ratio (n = 49). Panel D shows the distribution of carbon monoxide diffusing capacity (DL_CO), % predicted (n = 40).

Table 1 Characteristics of subjects, smoking history, lung function data, distribution of emphysema score, and cellular content of bal fluid

Variable	Subjects (n = 54)
Age, yr^a	66.6 (1.4)
Sex, n (%)	26 men (48)
Smoking history
Nonsmoker, n (%)	1 (2)
Current smokers, n (%)	24 (44)
Former smokers^b, n (%)	29 (54)
Pack-years^a,^c	46.7 (3.8)
Lung function data^a(range)
FEV1, % predicted	85.7 (2.9) (18–125)
FVC, % predicted	93.6 (2.0) (64–130)
FEV1/FVC	70.2 (1.8) (23–100)
DLCO, % predicted	74.5 (2.6) (27–110)
Distribution of emphysema score, n (%)
Score 0	5 (12)
Score 1	20 (46)
Score 2	14 (33)
Score 3	1 (2)
Score 4	3 (7)
Score 5	0 (0)
Cell content of BAL fluid
Total alveolar macrophages recovered, x 10^6^a	270.7 (42.9)
BAL differential cell counts
Macrophages, %^a	80.3 (2.4)
Neutrophils, %^a	8.5 (1.4)
Lymphocytes, %^a	11.2 (1.5)

^aMean (SE).

^bStudy participants who quit smoking 6 months or more before surgery.

^cNumber of packs of cigarettes smoked per day × number of years of smoking.

BAL, bronchoalveolar lavage; FEV₁, forced expiratory volume in 1 second; FVC, forced vital capacity; DL_CO, carbon monoxide diffusing capacity.

Table 2 Primers and conditions for real-time PCR experiments

Target	Primers	Annealing Temperature	Single Detection	Fragment Length
ACTB	5′-CACCCTGAAGTACCCCATC	56°C	85°C	226 bp
	5′-TAGCACACGCTGGATAGCAAC
CTSL	5′-TCACAGTTTAGAGGCACAGTGG	54°C	80°C	241 bp
	5′-CTTCCTGGGCTTACGGTTTT
CTSS	5′-TTGCCTGATTCTGTGGACTG	56°C	82°C	205 bp
	5′-CACCATTGCAGCCTTTGTTTCC
MMP1	5′-ACAAACCCCAAAAGCGTGT	53°C	79°C	197 bp
	5′-CTCTGTCGGCAAATTCGTA
MMP9	5′-AGACACCTCTGCCCTCACC	56°C	85°C	202 bp
	5′-ACGCATCTCTGCCACCCGA
MMP12	5′-ATGATGAAAGGAGACAGATGATGG	54°C	77°C	203bp
	5′-ACAACCAAACCAGCTATTGC

ACTB, beta-actin; CTSL, cathepsin L; CTSS, cathepsin S; MMP, matrix metalloproteinase.

Figure 2 Comparison of matrix metalloproteinase 1 (MMP1) mRNA expression and emphysema. Panel A shows a plot comparing MMP1 expression with emphysema severity (current smokers, n = 8; former smokers, n = 12). Panel B shows a plot comparing MMP1 expression with emphysema severity, excluding the two outliers (current smokers, n = 7; former smokers, n = 11). The extent of severe emphysema was defined using a density mask cut-off of −950 Hounsfield units. Panel C shows a plot comparing MMP1 expression with emphysema extent (current smokers, n = 8; former smokers, n = 12). The overall extent of emphysema was defined using a density mask cut-off of −855 Hounsfield units.

Figure 3 Matrix metalloproteinase 12 (MMP12) gene expression in current smokers and former smokers. Panel A shows the difference in levels of MMP12 mRNA expression, normalized according to the amount of beta-actin, in study participants who are current smokers (CS) (n = 24) and former smokers (FS) (study participants who had quit smoking for a least 6 months prior to surgery) (n = 29). Panel B shows the difference in levels of MMP12 expression in current smokers and former smokers classified according to the length of smoking cessation: (1) less than 10 years (FS < 10 yrs) (n = 13) or (2) greater than or equal to 10 years (FS > 10 yrs) (n = 16). The ends of the box are the first and third quartiles; the line across the middle of the box identifies the median.

Figure 4 Baseline matrix metalloproteinase 12 (MMP12) mRNA expression and carbon monoxide diffusing capacity (DL_CO). MMP12 expression was significantly inversely correlated with DL_CO (r = −0.43, p = 0.006, n = 40).

Figure 5 Relative gene expression in cultured alveolar macrophages. Alveolar macrophages were cultured for 24 h and 48 h in the presence of medium alone (CTRL), medium + lipopolysaccharide (LPS) (2 μ g/ml), medium + interleukin-1beta (IL-1beta) (20 ng/ml), or medium + tumor necrosis factor (TNF-alpha) (20 ng/ml). Panels A through E show the expression of cathepsin L (CTSL), cathepsin S (CTSS), matrix metalloproteinase 1 (MMP1), MMP9, and MMP12, respectively, with the use of real-time PCR. Samples from 54 subjects were run in triplicate. Data are means ± SE. *Statistically different compared with time control, p < 0.05. **Statistically different compared with time control, p < 0.001. † Statistically different between 24 h and 48 h, p < 0.05. ‡ Statistically different between 24 h and 48 h, p < 0.001. Note the log scale for levels of expression of MMP1.