Inflammatory stimuli up-regulate transient receptor potential vanilloid-1 expression in human bronchial fibroblasts

Lung fibroblasts are involved in interstitial lung disease, chronic asthma, and chronic obstructive pulmonary disease (COPD). The expanded fibroblast population in airway disease leads to airway remodeling and contributes to the inflammatory process seen in these diseases. The cation channel transient receptor potential vanilloid-1 (TRPV1) is activated by noxious stimuli, including capsaicin, protons, and high temperatures and is thought to have a role in inflammation. Although TRPV1 expression is primarily reported to be neuronal, some extraneuronal expression has been reported. The authors therefore sought to determine whether human primary bronchial fibroblasts (HPBFs) express TRPV1 and whether inflammatory mediators can induce TRPV1 expression. The authors show that fibroblasts are predominantly TRPV1 negative; however, following stimulation with 3 common inflammatory mediators, tumor necrosis factor α (TNF-α), lipopolysaccharide (LPS), and interleukin-1α (IL-1α), TRPV1 mRNA was observed at 24 and 48 hours post treatment with all 3 mediators. Using Western blotting an increase in TRPV1 expression with all 3 inflammatory mediators was detected with significant increases seen at 72 hours post LPS and IL-1α treatment. In stark contrast to the untreated fibroblasts, significant calcium signaling in response to capsaicin and resiniferatoxin in HPBFs treated for 24 and 48 hours with TNF-α, LPS, or IL-1α was also observed. These results indicate that TRPV1 can be expressed on bronchial fibroblasts in situations where an underlying inflammatory stimulus exists, as is the case in airway diseases such as asthma and COPD.     

TRPA1 is activated by direct addition of cysteine residues to the N-hydroxysuccinyl esters of acrylic and cinnamic acids

The nociceptor TRPA1 is thought to be activated through covalent modification of specific cysteine residues on the N terminal of the channel. The precise mechanism of covalent modification with unsaturated carbonyl-containing compounds is unclear, therefore by examining a range of compounds which can undergo both conjugate and/or direct addition reactions we sought to further elucidate the mechanism(s) whereby TRPA1 can be activated by covalent modification. Calcium signalling was used to determine the mechanism of activation of TRPA1 expressed in HEK293 cells with a series of related compounds which were capable of either direct and/or conjugate addition processes. These results were confirmed using physiological recordings with isolated vagus nerve preparations. We found negligible channel activation with chemicals which could only react with cysteine residues via conjugate addition such as acrylamide, acrylic acid, and cinnamic acid. Compounds able to react via either conjugate or direct addition, such as acrolein, methyl vinyl ketone, mesityl oxide, acrylic acid NHS ester, cinnamaldehyde and cinnamic acid NHS ester, activated TRPA1 in a concentration dependent manner as did compounds only capable of direct addition, namely propionic acid NHS ester and hydrocinnamic acid NHS ester. These compounds failed to activate TRPV1 expressed in HEK293 cells or mock transfected HEK293 cells. For molecules capable of direct or conjugate additions, the results suggest for the first time that TRPA1 may be activated preferentially by direct addition of the thiol group of TRPA1 cysteines to the agonist carbonyl carbon of α,β-unsaturated carbonyl-containing compounds.     

Effect of High Glucose on Human Alveolar Macrophage Phenotype and Phagocytosis of Mycobacteria

Purpose

Diabetes mellitus (DBM) reduces immunological activity and increases susceptibility to various infections, including tuberculosis (TB). Human alveolar macrophage (hAM) functions are altered in DBM.

Methods

To mimic hyperglycemic conditions in the lung alveolus, we co-cultured a hAM cell line (Daisy cell line) with human umbilical vein endothelial cells for 48 h in the presence of culture media alone, normal glucose (5 mM), and high glucose (22 mM). Using flow cytometry, immunophenotype characterization included cell surface markers CD 11c, CD14, CD16, CD86, CD163, CD169, CD206, CX3CR-1, CSF-1R, and matrix metalloproteinase-9 (MMP9). Phagocytic function was measured by immunofluorescence microscopy at 24 h after inoculation of cells with GFP-expressing Mycobacterium smegmatis.

Results

Direct exposure of AMs to high glucose and exposure in the co-culture system yield different results for the same phenotypic markers. MMP9 expression was increased under both conditions. CD169 and CX3CR1 expressions were decreased when AMs were exposed directly to high glucose but increased under co-culture. Immunofluorescence assay revealed that phagocytosis decreased in AMs when directly exposed to increased glucose levels from 2.5 mM to normal glucose (5 mM), yet AMs under co-culture did not show decreased phagocytosis until concentrations were raised to 25 mM.

Conclusion

Alteration in the expression of certain receptors may contribute to defective sentinel function of AMs, promoting susceptibility to TB in a diabetic host. Variability in cell surface marker expression under direct glucose exposure compared to exposure via co-culture reveals that cell signaling between endothelial cells and AMs may play a crucial role in the phenotypic expression of AMs.

     

Expression and characterization of the intracellular vanilloid receptor (TRPV1) in bronchi from patients with chronic cough

TRPV1 is a modulator of noxious stimuli known to be important in the cough reflex. We have compared the expression of TRPV1 in normal human airways and those from patients with chronic cough and found that there is up regulation in airways smooth muscle in disease. This increased expression appears to be intracellular and we have therefore examined the role of intracellular rat and human TRPV1 activity was found using intracellular calcium signalling with human intracellular TRPV1 being located in a thapsigargin insensitive compartment. Increase in TRPV1 activity may have a role in the airway hypersensitivity seen in chronic cough.     

The RAG1 N-terminal domain is an E3 ubiquitin ligase

RAG1 and RAG2 initiate V(D)J recombination, which is the assembly of immunoglobulin and T cell receptor genes. The N-terminal region of RAG1 can be deleted, leaving an enzymatic "core" able to catalyze the complete reaction. Here we report that the N-terminal portion of RAG1 has a distinct enzymatic role separate from the rest of the protein. It acts as an E3 ligase in the ubiquitylation of a test substrate and formation of polyubiquitin chains in vitro. This finding suggests a new way in which V(D)J recombination can be regulated and coupled to other aspects of cell physiology.     

Impaired V(D)J recombination and lymphocyte development in core RAG1-expressing mice

RAG1 and RAG2 are the lymphocyte-specific components of the V(D)J recombinase. In vitro analyses of RAG function have relied on soluble, highly truncated "core" RAG proteins. To identify potential functions for noncore regions and assess functionality of core RAG1 in vivo, we generated core RAG1 knockin (RAG1(c/c)) mice. Significant B and T cell numbers are generated in RAG1(c/c) mice, showing that core RAG1, despite missing approximately 40% of the RAG1 sequence, retains significant in vivo function. However, lymphocyte development and the overall level of V(D)J recombination are impaired at the progenitor stage in RAG1(c/c) mice. Correspondingly, there are reduced numbers of peripheral RAG1(c/c) B and T lymphocytes. Whereas normal B lymphocytes undergo rearrangement of both JH loci, substantial levels of germline JH loci persist in mature B cells of RAG1(c/c) mice, demonstrating that DJH rearrangement on both IgH alleles is not required for developmental progression to the stage of VH to DJH recombination. Whereas VH to DJH rearrangements occur, albeit at reduced levels, on the nonselected alleles of RAG1(c/c) B cells that have undergone D to JH rearrangements, we do not detect VH to DH rearrangements in RAG1(c/c) B cells that retain germline JH alleles. We discuss the potential implications of these findings for noncore RAG1 functions and for the ordered assembly of VH, DH, and JH segments.     

Characterisation of a New Human Alveolar Macrophage-Like Cell Line (Daisy)

Lung - There is currently no true macrophage cell line and in vitro experiments requiring these cells currently require mitogenic stimulation of a macrophage precursor cell line (THP-1) or ex vivo...     

Recombination-activating gene proteins: more regulation, please

Summary: Developing B and T cells assemble gene segments in order to create the variable regions of immunoglobulin and T‐cell receptors required by our adaptive immune response. The chemistry of this recombination pathway requires a specific nuclease and a more general repair pathway for double‐strand breaks. A complex of the recombination‐activating gene 1 (RAG1) and RAG2 proteins provides the nuclease activity. In fact, RAG1 and RAG2 probably coordinate many steps involving the coding and signaling DNA sequences. Studies using deletion and truncation mutants of the RAG proteins demonstrate that each of these contain a functional core region, representing about two‐thirds of the polypeptides. While the core regions are sufficient to catalyze recombination in test systems, the full‐length proteins seem to show more complicated behaviors in vivo. A plausible explanation is that regions outside the core help in the proper regulation of recombination. The non‐core region of RAG1 has been found to contain a ubiquitin ligase. Regulatory functions may contribute to autoregulation of the proteins involved, fidelity of the reaction, protection of the cell from translocations, coordination of recombination with the cell cycle, and possibly modification of the chromatin structure of target DNA.