We have reported previously that myristoylated alanine-rich C kinase substrate (MARCKS) is a key regulatory molecule controlling mucin secretion by airway epithelial cells in vitro and in vivo. The results of those studies supported a mechanism whereby MARCKS, upon phosphorylation by protein kinase C (PKC), translocates from plasma membrane to cytoplasm, where its binding to membranes of intracellular mucin granules is a key component of the secretory pathway. It remains unknown how MARCKS is targeted to and/or preferentially attaches to mucin granule membranes. We hypothesized that the chaperone cysteine string protein (CSP) may play an important role in this process. CSP was shown to associate with membranes of intracellular mucin granules in well-differentiated normal human bronchial epithelial (NHBE) cells in vitro, as determined by ultrastructural immunohistochemistry and Western blotting of isolated granule membranes. CSP in these cells complexed with MARCKS, as shown by co-immunoprecipitation. Given reported associations between CSP and a second chaperone, heat shock protein 70 (HSP70), a role for HSP70 in the MARCKS-dependent secretory mechanism also was investigated. HSP70 appeared to form a trimeric complex with MARCKS and CSP associated with mucin granule membranes within airway epithelial cells. Transfection of the HBE1 human bronchial epithelial cell line with siRNAs targeting sequences of MARCKS, CSP, or HSP70 resulted, in each case, in significant knockdown of expression of these proteins and subsequent attenuation of mucin secretion. The results provide the first evidence that CSP and HSP70, and their interactions with MARCKS, are involved in mucin secretion.

Acute lung injury (ALI) is a frequent pulmonary complication in critically ill patients. We characterized a murine model of LPS-induced ALI, focusing on Th cells. Following LPS administration, bronchoalveolar lavage lymphocytes, neutrophils, IL-6, TNF-α, and albumin were increased. Analysis of LPS-induced T cells revealed increased Th cell-associated cytokines (IL-17A, -17F, and -22), as well as increased expression of CD69 (a cell activation marker), Foxp3, and CTLA4 in CD4+ T cells. Administration of anti-CTLA4 Ab decreased LPS-induced bronchoalveolar lavage albumin and IL-17A, while increasing CD4+Foxp3+ cell number and Foxp3 expression in CD4+Foxp3+ cells. These data suggest that pulmonary LPS administration promotes CD4+ T cells and that T cell pathways involving CTLA4 contribute to ALI.

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BackgroundExcess mucus in the airways leads to obstruction in diseases such as chronic bronchitis, asthma, and cystic fibrosis. Mucins, the highly glycosolated protein components of mucus, are stored in membrane-bound granules housed in the cytoplasm of airway epithelial "goblet" cells until they are secreted into the airway lumen via an exocytotic process. Precise mechanism(s) of mucin secretion, including the specific proteins involved in the process, have yet to be elucidated. Previously, we have shown that the Myristoylated Alanine-Rich C Kinase Substrate (MARCKS) protein regulates mucin secretion by orchestrating translocation of mucin granules from the cytosol to the plasma membrane, where the granules dock, fuse and release their contents into the airway lumen. Associated with MARCKS in this process are chaperone (Heat Shock Protein 70 [HSP70], Cysteine string protein [CSP]) and cytoskeletal (actin, myosin) proteins. However, additional granule-associated proteins that may be involved in secretion have not yet been elucidated.MethodsHere, we isolated mucin granules and granule membranes from primary cultures of well differentiated human bronchial epithelial cells utilizing a novel technique of immuno-isolation, based on the presence of the calcium activated chloride channel hCLCA1 (the human ortholog of murine Gob-5) on the granule membranes, and verified via Western blotting and co-immunoprecipitation that MARCKS, HSP70, CSP and hCLCA1 were present on the granule membranes and associated with each other. We then subjected the isolated granules/membranes to liquid chromatography mass spectrometry (LC-MS/MS) to identify other granule associated proteins.ResultsA number of additional cytoskeletal (e.g. Myosin Vc) and regulatory proteins (e.g. Protein phosphatase 4) associated with the granules and could play a role in secretion were discovered. This is the first description of the airway goblet cell "granulome."

Pulmonary surfactant protein D (SP-D), a member of the collectin family, is an innate immune molecule critical for defense that can also modulate adaptive immune responses. We previously showed that SP-D–deficient mice exhibit enhanced allergic responses and that SP-D induction requires lymphocytes. Thus, we postulated that SP-D may decrease adaptive allergic responses through interaction with T cells. In this study, we used two forms of SP-D, a dodecamer and a shorter fragment containing the trimeric neck and carbohydrate recognition domains (SP-D NCRD). Both forms decreased immune responses in vitro and in a murine model of pulmonary inflammation. SP-D NCRD increased transcription of CTLA4, a negative regulator of T cell activation, in T cells. SP-D NCRD no longer decreased lymphoproliferation and IL-2 cytokine production when CTLA4 signals were abrogated. Administration of SP-D NCRD in vivo no longer decreased allergen induced responses when CTLA4 was inhibited. Our results indicate that SP-D decreases allergen responses, an effect that may be mediated by increase of CTLA4 in T cells.