Regulation of cytotoxic T lymphocyte antigen 4 by cyclic AMP.

Recent studies indicate that cyclic AMP (cAMP) induces cytotoxic T lymphocyte antigen (CTLA) 4. CTLA4 is expressed in T cells, and is a negative regulator of T cell activation. CTLA4 expression is regulated by T cell receptor plus CD28 (adaptive immune signaling) at both the transcriptional and post-transcriptional level. Here, we examine the pathways by which cAMP regulates CTLA4 expression, focusing on transcriptional activation. Elevating intracellular cAMP levels by cell-permeable cAMP analogs, the adenylyl cyclase activator, forskolin, or phosphodiesterase inhibitors increases CTLA4 mRNA expression in EL4 murine T cells and primary CD4(+) T cells. Activation of protein kinase A (using the protein kinase A-selective agonist, N6-phenyladenosine-cAMP), but not exchange proteins activated by cAMP (using the exchange proteins activated by cAMP-selective 8-pCPT-2Me-cAMP), increases CTLA4 promoter activity. Mutation constructs of the CTLA4 promoter uncover an enhancer binding site located within the -150 to -130 bp region relative to the transcription start site. Promoter analysis and chromatin immunoprecipitation assays suggest that cAMP response element-binding is a putative transcription factor induced by cAMP. We have previously shown that CTLA4 mediates decreased pulmonary inflammation in an LPS-induced murine model of acute lung injury (ALI). We observed that LPS can induce CTLA4 transcription via the same cAMP-inducible promoter region. The immunosuppressant, rapamycin, decreases cAMP and LPS-induced CTLA4 transcription in vitro. In vivo, LPS induces cAMP accumulation in bronchoalveolar lavage fluid, bronchoalveolar lavage cells, and lung tissues in ALI. We demonstrate that rapamycin decreases cAMP accumulation and CTLA4 expression in ALI. Together, these data suggest that cAMP may negatively regulate pulmonary inflammatory responses in vivo and in vitro by altering CTLA4 expression.

Mucin granule-associated proteins in human bronchial epithelial cells: the airway goblet cell "granulome".

BACKGROUND: Excess 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. METHODS: Here, 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. RESULTS: A 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."

Surfactant protein D-mediated decrease of allergen-induced inflammation is dependent upon CTLA4.

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.

T cell pathways involving CTLA4 contribute to a model of acute lung injury.

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-alpha, 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.

MARCKS and related chaperones bind to unconventional myosin V isoforms in airway epithelial cells.

We have shown previously that myristoylated alanine-rich C kinase substrate (MARCKS) is a key regulatory molecule in the process of mucin secretion by airway epithelial cells, and that part of the secretory mechanism involves intracellular associations of MARCKS with specific chaperones: heat shock protein 70 (Hsp70) and cysteine string protein (CSP). Here, we report that MARCKS also interacts with unconventional myosin isoforms within these cells, and further molecular interactions between MARCKS and these chaperones/cytoskeletal proteins are elucidated. Primary human bronchial epithelial cells and the HBE1 cell line both expressed myosin V and VI proteins, and both MARCKS and CSP were shown to bind to myosin V, specifically Va and Vc. This binding was enhanced by exposing the cells to phorbol-12-myristate-13-acetate, an activator of protein kinase C and stimulator of mucin secretion. Binding of MARCKS, Hsp70, and CSP was further investigated by His-tagged pull down assays of purified recombinant proteins and multiple transfections of HBE1 cells with fusion proteins (MARCKS-HA; Flag-Hsp70; c-Myc-CSP) and immunoprecipitation. The results showed that MARCKS binds directly to Hsp70, and that Hsp70 binds directly to CSP, but that MARCKS binding to CSP appears to require the presence of Hsp70. Interrelated binding(s) of MARCKS, chaperones, and unconventional myosin isoforms may be integral to the mucin secretion process.

MARCKS regulation of mucin secretion by airway epithelium in vitro: interaction with chaperones.

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.

Protease-activated receptor-2 (PAR-2) is a weak enhancer of mucin secretion by human bronchial epithelial cells in vitro.

PAR-2, a member of a family of G-protein-coupled receptors, can be activated by serine proteases via proteolytic cleavage. PAR-2 expression is known to be upregulated in respiratory epithelium subsequent to inflammation in asthma and chronic obstructive pulmonary disease (COPD). Since these diseases also are characterized by excessive mucus production and secretion, we investigated whether PAR-2 could be linked to mucin hypersecretion by airway epithelium. Normal human bronchial epithelial (NHBE) cells in primary culture or the human bronchial epithelial cell lines, NCI-H292 and HBE-1, were used. NHBE, NCI-H292, and HBE-1 cells expressed prominent levels of PAR-2 protein. Short-term (30min) exposure of cells to the synthetic PAR-2 agonist peptide (SLIGKV-NH2) elicited a small but statistically significant increase in mucin secretion at high concentrations (100microM and 1000microM), compared to a control peptide with reversed amino acid sequence (VKGILS-NH2). Neither human lung tryptase nor bovine pancreatic trypsin, both PAR-2 agonists, affected NHBE cell mucin secretion when added over a range of concentrations. Knockdown of PAR-2 expression by siRNA blocked the stimulatory effect of the AP. The results suggest that, since PAR-2 activation only weakly increases mucin secretion by human airway epithelial cells in vitro, PAR-2 probably is not a significant contributor to mucin hypersecretion in inflamed airways.