Elsevier

Human Pathology

Volume 41, Issue 7, July 2010, Pages 935-943
Human Pathology

Original contribution
High levels of extracellular matrix metalloproteinase inducer are expressed in lymphangioleiomyomatosis

https://doi.org/10.1016/j.humpath.2009.12.009Get rights and content

Summary

Pulmonary lymphangioleiomyomatosis is pathologically characterized by the proliferation of abnormal smooth muscle-like cells (lymphangioleiomyomatosis cells) that synthesize excess matrix metalloproteinases. Extracellular matrix metalloproteinase inducer is minimally expressed in the healthy lung, but is up-regulated in various lung injuries that are apparently associated with matrix metalloproteinases. We therefore immunohistochemically stained extracellular matrix metalloproteinase inducer in lymphangioleiomyomatosis cells and measured extracellular matrix metalloproteinase inducer in bronchoalveolar lavage fluid using an enzyme-linked immunosorbent assay. We also quantified extracellular matrix metalloproteinase inducer messenger RNA expression in the lung using quantitative reverse transcriptase–polymerase chain reaction. Intense staining for extracellular matrix metalloproteinase inducer in lymphangioleiomyomatosis cells indicated high immunoreactivity. Dual immunofluorescence studies revealed diffuse colocalization between extracellular matrix metalloproteinase inducer and α smooth muscle actin, matrix metalloproteinase–2, or matrix metalloproteinase–9. Although levels of extracellular matrix metalloproteinase inducer messenger RNA did not differ in whole lung homogenates from patients with lymphangioleiomyomatosis and healthy controls (1.7 ± 0.1 versus 1.5 ± 0.2 SE, not significant; both n = 4), lymphangioleiomyomatosis nodules retrieved by laser capture microdissection expressed 5.1 (±1.0 SE)-fold higher levels of extracellular matrix metalloproteinase inducer messenger RNA than lung homogenates (P = .0077, n = 4). Levels of extracellular matrix metalloproteinase inducer were significantly elevated in bronchoalveolar lavage fluid from lymphangioleiomyomatosis patients compared with controls (82.7 ± 19.5 versus 38.6 ± 9.0 SE pg/mL, P = .0497; n = 8 and 9, respectively). Increased levels of extracellular matrix metalloproteinase inducer colocalized with increased matrix metalloproteinases in lymphangioleiomyomatosis cells indicate that it potentially functions in pulmonary lymphangioleiomyomatosis.

Introduction

Lymphangioleiomyomatosis (LAM) is a rare multisystem disease characterized by recurrent spontaneous pneumothorax, chylothorax, chylous ascites, and angiomyolipomas that primarily affects women of childbearing age [1]. Abnormally proliferative smooth muscle-like LAM cells underlie the formation of characteristic LAM nodules in the lungs and of angiomyolipomas in the kidney [2]. Although the characteristics of LAM cells are not yet fully understood, immunopathologic studies have found that high levels of matrix metalloproteinase (MMP)–2, MMP-9, and membrane type 1–MMP are expressed in LAM cells, implying their involvement in the destructive cystic formation and/or metastasis associated with pulmonary LAM [3], [4]. We recently reported that levels of MMP-9 are higher in serum from patients with LAM than from healthy controls [5]. However, the regulatory mechanism of MMP induction in LAM remains largely unknown.

Several studies of MMP-inducing factors in tumor cells have led to the identification of the highly glycosylated transmembrane protein extracellular matrix metalloproteinase inducer (EMMPRIN)/CD147/basigin [6], [7]. Levels of EMMPRIN expression are high in fetal lung epithelium [8], suggesting a physiologic role in development. Although essentially minimal in healthy adult lung and other tissues, EMMPRIN is up-regulated in diverse pathologic processes, such as failing myocardium [9], hepatitis C virus–associated cirrhotic liver [10], chronic smoker's lung [11], and interstitial pneumonia [12]. In particular, EMMPRIN has been identified on the surface of cancer cells [13], [14], where it induces several MMPs including MMP-1, -2, -3, and -9 and membrane type 1–MMP in neighboring stromal cells or cancer cells themselves, resulting in invasion or metastasis.

Until recently, LAM cells have been considered histologically benign not only in sporadic LAM, but also in tuberous sclerosis complex (TSC)–related LAM. However, compelling evidence such as LAM recurrence after lung transplantation and the presence of LAM cells with the same mutations in the lungs, lymph nodes, and kidneys suggests that LAM cells can spread among lesions [15], [16]. Thus, metastasis seems to be one mechanism through which LAM cells are disseminated [15], [17].

We therefore postulated that LAM cells produce EMMPRIN that is involved in the induction of MMPs. The present study immunohistochemically localizes EMMPRIN in association with MMP-2 and MMP-9 in LAM nodules; examines the expression of LAM-driven EMMPRIN at the messenger RNA (mRNA) level; and quantifies levels of EMMPRIN, MMP-2, and MMP-9 in bronchoalveolar lavage (BAL) fluids.

Section snippets

Patients

We enrolled 10 female patients with LAM, 6 of whom had been pathologically diagnosed with pulmonary LAM by video-assisted thoracoscopy-guided lung biopsy; the other 4 were diagnosed by clinical and radiographic findings as previously described [18]. Among 10 patients with LAM enrolled in this study, 2 were current smokers. Paraffin-embedded and frozen tissues were obtained from 4 of the 6 pathologically diagnosed patients, and only paraffin-embedded tissues were obtained from 2 of them. All

Characteristics of the participants

Table 1 summarizes the clinical characteristics of each of the enrolled patients with LAM. None of them received hormonal therapy during this study. Airflow limitation was frequent but mild in the patients, most of whom were asymptomatic. Table 2 summarizes the findings of participants who were enrolled in the BAL study. The total number of cells and the total protein in BAL fluid were significantly elevated in the patients compared with healthy controls (19.0 ± 6.2 versus 5.3 ± 0.5 × 104/mL, P

Discussion

Here, we discovered that EMMPRIN is prominently expressed at the mRNA and protein levels in LAM cells. We also found that EMMPRIN colocalized with αSMA, MMP-2, and MMP-9 in LAM cells, suggesting that EMMPRIN in LAM cells could induce MMP-2 and MMP-9 in an autocrine fashion.

EMMPRIN is up-regulated exclusively in epithelial-driven cells, such as aberrant regenerated epithelial cells in fibrotic lung injuries in mice [24] and humans [12], in the bronchiolar epithelium of smokers' lungs [11], and

Acknowledgments

We thank Ms Yoko Suzuki for excellent technical assistance with LCM.

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  • Cited by (0)

    Source of funds: This study was supported by scientific research grants from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (No. 19590877 to T. B.) and from the Ministry of Health, Labour, and Welfare of Japan to the Respiratory Failure Research Group.

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