BACKGROUND The mutations that have been implicated in pulmonary fibrosis account for only a small proportion of the population risk. METHODS Using a genomewide linkage scan, we detected linkage between idiopathic interstitial pneumonia and a 3.4-Mb region of chromosome 11p15 in 82 families. We then evaluated genetic variation in this region in gel-forming mucin genes expressed in the lung among 83 subjects with familial interstitial pneumonia, 492 subjects with idiopathic pulmonary fibrosis, and 322 controls. MUC5B expression was assessed in lung tissue. RESULTS Linkage and fine mapping were used to identify a region of interest on the p-terminus of chromosome 11 that included gel-forming mucin genes. The minor-allele of the single-nucleotide polymorphism (SNP) rs35705950, located 3 kb upstream of the MUC5B transcription start site, was present at a frequency of 34% among subjects with familial interstitial pneumonia, 38% among subjects with idiopathic pulmonary fibrosis, and 9% among controls (allelic association with familial interstitial pneumonia, P = 1.2×10−15; allelic association with idiopathic pulmonary fibrosis, P = 2.5×10−37). The odds ratios for disease among subjects who were heterozygous and those who were homozygous for the minor allele of this SNP were 6.8 (95% confidence interval [CI], 3.9 to 12.0) and 20.8 (95% CI, 3.8 to 113.7), respectively, for familial interstitial pneumonia and 9.0 (95% CI, 6.2 to 13.1) and 21.8 (95% CI, 5.1 to 93.5), respectively, for idiopathic pulmonary fibrosis. MUC5B expression in the lung was 14.1 times as high in subjects who had idiopathic pulmonary fibrosis as in those who did not (P<0.001). The variant allele of rs35705950 was associated with up-regulation in MUC5B expression in the lung in unaffected subjects (expression was 37.4 times as high as in unaffected subjects homozygous for the wild-type allele, P<0.001). MUC5B protein was expressed in lesions of idiopathic pulmonary fibrosis. CONCLUSIONS A common polymorphism in the promoter of MUC5B is associated with familial interstitial pneumonia and idiopathic pulmonary fibrosis. Our findings suggest that dys-regulated MUC5B expression in the lung may be involved in the pathogenesis of pulmonary fibrosis. (Funded by the National Heart, Lung, and Blood Institute and others.)

Mucus hypersecretion is a crucial feature of pulmonary diseases such as asthma, chronic bronchitis and cystic fibrosis. Despite much research, there is still no effective therapy for this condition. Recently, we showed that the myristoylated, alanine-rich C-kinase substrate (MARCKS) protein is required for mucus secretion by human bronchial epithelial cells in culture. Having synthesized a peptide corresponding to the N-terminal domain of MARCKS, we now show that the intratracheal instillation of this peptide blocks mucus hypersecretion in a mouse model of asthma. A missense peptide with the same amino acid composition has no effect. Based on quantitative histochemical analysis of the mouse airways, the peptide seems to act by blocking mucus release from goblet cells, possibly by inhibiting the attachment of MARCKS to membranes of intracellular mucin granules. These results support a pivotal role for MARCKS protein, specifically its N-terminal region, in modulating this secretory process in mammalian airways. Intratracheal administration of this MARCKS-related peptide could therapeutically reduce mucus secretion in the airways of human patients with asthma, chronic bronchitis and cystic fibrosis.

Neutrophil migration into infected tissues is essential for host defense, but products of activated neutrophils can be quite damaging to host cells. Neutrophil influx into the lung and airways and resultant inflammation characterizes diseases such as chronic obstructive pulmonary disease, bronchiectasis, and cystic fibrosis. To migrate, neutrophils must reorganize the actin cytoskeleton to establish a leading edge pseudopod and a trailing edge uropod. The actin-binding protein myristoylated alanine-rich C-kinase substrate (MARCKS) has been shown to bind and cross-link actin in a variety of cell types and to co-localize with F-actin in the leading edge lamellipodium of migrating fibroblasts. The hypothesis that MARCKS has a role in the regulation of neutrophil migration was tested using a cell-permeant peptide derived from the MARCKS myristoylated aminoterminus (MANS peptide). Treatment of isolated human neutrophils with MANS significantly inhibited both their migration and b2 integrin-dependent adhesion in response to N-formyl-methionyl-leucyl-phenylalanine (fMLF), IL-8, or leukotriene (LT)B 4 . The IC 50 for fMLF-induced migration and adhesion was 17.1 mM and 12.5 mM, respectively. MANS significantly reduced the F-actin content in neutrophils 30 seconds after fMLF stimulation, although the peptide did not alter the ability of cells to polarize or spread. MANS did not alter fMLF-induced increases in surface b2 integrin expression. These results suggest that MARCKS, via its myristoylated aminoterminus, is a key regulator of neutrophil migration and adhesion.

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Leukocytes synthesize a variety of inflammatory mediators that are packaged and stored in the cytoplasm within membrane-bound granules. Upon stimulation, the cells secrete the granule contents via an exocytotic process whereby the granules translocate to the cell periphery, the granule membranes fuse with the plasma membrane, and the granule contents are released extracellularly. We have reported previously that another exocytotic process, release of mucin by secretory cells of the airway epithelium, is regulated by the myristoylated alanine-rich C kinase substrate ( Keywords: MARCKS protein; leukocytes; degranulationLeukocytes synthesize a number of inflammatory mediators that are packaged and stored in cytoplasmic membrane-bound granules. These mediators include myeloperoxidase (MPO) in neutrophils (1), eosinophil peroxidase (EPO) and major basic pro- (2), lysozyme in monocytes/macrophages (3, 4), and granzyme in natural killer (NK) cells and cytotoxic lymphocytes (5-8). These mediators are released at sites of injury and contribute to inflammation and repair in the lung and elsewhere. Leukocytes release these granules via an exocytotic mechanism (9, 10), but the regulatory molecules and specific pathways involved in the exocytotic process have not been fully described.Several exogenous stimuli can provoke degranulation of leukocytes via a pathway that involves activation of protein kinase C (PKC) and subsequent phosphorylation events (9-13). MARCKS (myristoylated alanine-rich C kinase substrate), a ubiquitous phosphorylation target of PKC, is highly expressed in leukocytes (14-16). We have previously demonstrated that MARCKS protein is involved in exocytotic secretion of mucin by goblet cells that line the respiratory airways (17, 18). In airway epithelial cells, the N-terminus of MARCKS seems to be integral to the secretory process. The mechanism seems to involve the binding of MARCKS to membranes of intracellular mucin granules because a peptide against the N-terminus of MARCKS blocked mucin secretion and binding of MARCKS to mucin granule membranes in these cells (18). Because MARCKS is a prominent protein in leukocytes, we investigated whether or not MARCKS, and specifically its N-terminus, could play a role in leukocyte degranulation.In these studies, we used four different leukocyte types or models that secrete specific granule contents in response to phorbol ester-induced activation of PKC. First, neutrophils were isolated from human blood, and the in vitro release of MPO by these cells was assessed. Due to difficulties in isolating sufficient amounts of other leukocyte types from blood, we investigated the release of membrane-bound inflammatory mediators from commercially available human leukocyte cell lines. The human promyelocytic cell line HL-60 clone 15 was used to assess secretion of , the monocytic leukemia cell line U937 was used to assess secretion of lysozyme (3,4,23), and the lymphocyte NK cell line NK-92 was used to assess the release of granzyme (6-8). In all cases, the cells were preinc...