Gene expression profiling identifies MMP-12 and ADAMDEC1 as potential pathogenic mediators of pulmonary sarcoidosis

Abstract

Rationale: Little is known about the genetic regulation of granulomatous inflammation in sarcoidosis.

Objectives: To determine if tissue gene array analysis would identify novel genes engaged in inflammation and lung remodeling in patients with sarcoidosis.

Methods: Gene expression analysis was performed on tissues obtained from patients with sarcoidosis at the time of diagnosis (untreated) (n = 6) compared with normal lung tissue (n = 6). Expression of select genes was further confirmed in lung tissue from a second series of patients with sarcoidosis and disease-free control subjects (n = 11 per group) by semi-quantitative RT-PCR. Interactive gene networks were identified in patients with sarcoidosis using Ingenuity Pathway Analysis (Ingenuity Systems, Inc., Redwood, CA) software. The expression of proteins corresponding to selected overexpressed genes was determined using fluorokine multiplex analysis, and immunohistochemistry. Selected genes and proteins were then analyzed in bronchoalveolar lavage fluid in an independent series of patients with sarcoidosis (n = 36) and control subjects (n = 12).

Measurements and main results: A gene network engaged in Th1-type responses was most significantly overexpressed in the sarcoidosis lung tissues, including genes not previously reported in the context of sarcoidosis (e.g., IL-7). MMP-12 and ADAMDEC1 transcripts were most highly expressed (> 25-fold) in sarcoidosis lung tissues, corresponding with increased protein expression by immunohistochemistry. MMP-12 and ADAMDEC1 gene and protein expression were increased in bronchoalveolar lavage samples from patients with sarcoidosis, correlating with disease severity.

Conclusions: Tissue gene expression analyses provide novel insights into the pathogenesis of pulmonary sarcoidosis. MMP-12 and ADAMDEC1 emerge as likely mediators of lung damage and/or remodeling and may serve as markers of disease activity.

Figure 1.

Interactive gene networks in lung tissues derived from patients with sarcoidosis. (A) The gene network most highly overrepresented in the sarcoidosis group relative to control subjects based on statistical analysis (see Methods) includes STAT1, IL7, and IL15. (B) The gene network in which the two most highly overexpressed genes (MMP12 and ADAMDEC1) in the sarcoidosis lung samples were found. Genes and gene products are represented by their common abbreviated names according to established direct (solid lines) or indirect (dashed lines) interactions and the specific nature of the interaction. These include binding (straight line), activation (arrow), inhibition (truncated line), or either activation or inhibition (truncated line with arrow). Genes that are significantly overexpressed in the sarcoidosis group relative to control subjects are highlighted in red; green denotes reduced expression, and white indicates no difference in expression level. The shape of each gene “node” denotes the function of the gene product.

Figure 2.

Semi-quantitative reverse transcriptase–polymerase chain reaction (RT-PCR) analysis demonstrates enhanced select gene expression in lung tissues of patients with sarcoidosis. Semi-quantitative RT-PCR analysis was performed on lung samples obtained from in independent series of patients with sarcoidosis (n = 11) and control subjects (n = 11), the clinical characteristics of which are shown in Table 1 (mean ± SEM; *P < 0.05; Wilcoxon Rank-Sum test with a multiple comparison adjustment using the Bonferonni-Holm's procedure). See online supplement for actual RT-PCR images.

Figure 3.

Cytokine analysis in lung tissues of patients with sarcoidosis. Bio-Plex Human Cytokine Bead Array analysis of lung tissue samples identified a number of proinflammatory cytokine proteins that were significantly elevated in the lungs of patients with sarcoidosis relative to control subjects (mean ± SEM; *P < 0.05; Wilcoxon Rank-Sum test with a multiple comparison adjustment using the Bonferonni-Holm's procedure).

Figure 4.

Immunohistochemical analyses of MMP-12 and ADAMDEC1 protein expression in pulmonary sarcoidosis. Immunohistochemical staining of frozen lung samples for CD68, MMP-12, elastin, and ADAMDEC1 from representative control subjects and patients with sarcoidosis. Granulomas labeled “G.” Comparison of anti-CD68 immunostaining with anti–MMP-12 showed colocalization within the granulomas of sarcoidosis tissues. Elastin staining was depleted in granulomas but was distributed uniformly throughout the lung tissue otherwise. Anti-ADAMDEC1 immunostaining was uniformly distributed at lower levels in control lungs but was expressed at high levels in tissue adjacent to granulomas in the lungs of patients with sarcoidosis.

Figure 5.

MMP12 and ADAMDEC1 are overexpressed in bronchoalveolar cells from patients with sarcoidosis. MMP12 and ADAMDEC1 (gene) expression were determined by real-time PCR in bronchoalveolar lavage cells obtained from an independent series of normal subjects and patients with sarcoidosis. RNA expression was normalized to housekeeping genes as described in Methods. Sarcoidosis was associated with (A) increases in MMP12 and (B) ADAMDEC1 expression in bronchoalveolar lavage cells, which was highest in those with severe disease (mean ± SD; Tukey's test). HC = healthy control subjects; NS = nonsevere; SEV = severe.

Figure 6.

Soluble MMP-12 and ADAMDEC1 are elevated in acellular bronchoalveolar lavage (BAL) in sarcoidosis. Western blot analysis of concentrated BAL fluid from patients with sarcoidosis (n = 12) and healthy control subjects (n = 8). Gel loading was normalized to 10 μg total protein per lane using the bicinchoninic acid protein assay. Images are representative of two independent sample sets. (A) For MMP-12, recombinant pro–MMP-12 was used as a control with the expected band seen at 54 kD (arrow). (B) For ADAMDEC1, two bands were visualized: a species around the expected size (53 kD, arrow) and a dimer of approximately 28 kD that represents a cleavage product (arrowhead). The unlabeled bands likely represent nonspecific binding. (C and D) Densitometry results. (C) The MMP-12 level was higher overall in patients with sarcoidosis compared with healthy control subjects (1.65-fold; P = 0.003; Student's t test); (D) the increase was nearly significant for ADAMDEC1 (1.28-fold; P = 0.056). Stratification of the subjects by clinical phenotype revealed that MMP12 and ADAMDEC1 levels were elevated in severe versus nonsevere subjects (1.45-fold and 1.39-fold, respectively; P < 0.001 for both; Tukey's test). HC = healthy controls; NS = nonsevere; SEV = severe.