Double Deficiency of Tetraspanins CD9 and CD81 Alters Cell Motility and Protease Production of Macrophages and Causes Chronic Obstructive Pulmonary Disease-like Phenotype in Mice

Abstract

CD9 and CD81 are closely related tetraspanins that regulate cell motility and signaling by facilitating the organization of multimolecular membrane complexes, including integrins. We show that CD9 and CD81 are down-regulated in smoking-related inflammatory response of a macrophage line, RAW264.7. When functions of CD9 and CD81 were ablated with monoclonal antibody treatment, small interfering RNA transfection, or gene knock-out, macrophages were less motile and produced larger amounts of matrix metalloproteinase (MMP)-2 and MMP-9 than control cells in vitro. In line with this, CD9/CD81 double-knock-out mice spontaneously developed pulmonary emphysema, a major pathological component of chronic obstructive pulmonary disease (COPD). The mutant lung contained an increased number of alveolar macrophages with elevated activities of MMP-2 and MMP-9 and progressively displayed enlarged airspace and disruption of elastic fibers in the alveoli. Secretory cell metaplasia, a finding similar to goblet cell metaplasia in cigarette smokers, was also observed in the epithelium of terminal bronchioles. With aging, the double-knockout mice showed extrapulmonary phenotypes, including weight loss, kyphosis, and osteopenia. These results suggest that the tetraspanins CD9 and CD81 regulate cell motility and protease production of macrophages and that their dysfunction may underlie the progression of COPD.Chronic obstructive pulmonary disease (COPD), 5 a disease defined by incompletely reversible airflow limitation, results from abnormal inflammatory response to chronic cigarette smoking. Pulmonary emphysema is a major component of COPD, and a dominant hypothesis in its pathophysiology is that persistent infiltration of inflammatory cells and production of proteases, including matrix metalloproteinases (MMPs) in the lung, lead to tissue destruction and airspace enlargement (1, 2). In patients with emphysema, there was an increase in bronchoalveolar lavage fluid (BALF) concentrations and macrophage expression of MMP-9 (3). Studies of human samples have shown increases of MMP-2 and MMP-9 in smoking-related emphysema (4). Alveolar macrophages secrete elastolytic enzymes, including MMP-2, MMP-9, and MMP-12, and play a pivotal role in the pathophysiology of COPD. There was a marked increase in the numbers of macrophages in airways, lung parenchyma, BALF, and sputum in patients with emphysema (2). Macrophages are activated by cigarette smoke to release inflammatory mediators such as TNF-␣, chemokines, and reactive oxygen species as well as MMPs, providing a cellular mechanism that links smoking with inflammation in COPD (2). It was recently proposed that lowered activity of histone deacetylases (HDACs), which are suppressors of inflammatory genes, accounts for the persistent activation of macrophages in COPD patients (5).The tetraspanin proteins include at least 33 members, including CD9, CD63, CD81, CD82, and CD151 in mammals. They are characterized by the structure that spans the plasma membrane four times and have a propensity to form co...

Tetraspanin Tspan9 regulates platelet collagen receptor GPVI lateral diffusion and activation

“…Elucidation of tetraspanin function using knockout mice can be complicated by functional redundancy between tetraspanins, which share relatively strong protein sequence identity. For example, CD9 and CD81 share 45% identity, and double-deficient mice have macrophage dysfunction leading to a phenotype that resembles chronic obstructive pulmonary disease; neither single knockout has detectable disease [54]. Tspan9 has relatively strong sequence identity with Tspan4 (55%) and CD53 (43%), for which knockout mice phenotypes have yet to be reported.…”

The TspanC8 Subgroup of Tetraspanins Interacts with A Disintegrin and Metalloprotease 10 (ADAM10) and Regulates Its Maturation and Cell Surface Expression

“…CD81 was shown to partially compensate for the fertilization defect observed in CD9-deficient mice (12). In addition, a role for these tetraspanins in the maintenance of normal lung function was only revealed by the observation of a chronic obstructive pulmonary disease-like phenotype in the double knock-out mouse but not in either of the single knockouts (44). Compensation between TspanC8 tetraspanins is likely to complicate future analyses of knockdown cells and knock-out animals.…”

Deletion of Tetraspanin CD9 Diminishes Lymphangiogenesis in Vivo and in Vitro

“…Mice-CD9-KO and CD81-KO mice were provided by Dr. E. Mekada (Research Institute for Microbial Diseases, Osaka University, Osaka, Japan), and the generation of CD9/CD81-DKO mice was described previously (27). These mice were backcrossed more than eight generations into the C57BL/6J background in a barrier facility, and all of the animal procedures were performed in accordance with the Osaka University guidelines on animal care.…”

“…We have previously shown that CD9 and CD81 coordinately prevent the fusion of macrophages and that DKO mice develop an emphysematous lung phenotype (27,43). Hence, our present data suggest that both CD9 and CD81 additively function not only in macrophages, but also in LEC.…”

“…Likewise, the morphology and abundance of the blood vascular network were also compromised in the DKO mice, although to a much lesser extent. To exclude the possibility that the emphysematous lung phenotype in DKO mice might influence these vascular phenotypes (27), we further examined the diaphragm. Lymphatics in the diaphragm were markedly decreased in DKO mice (Fig.…”