Epidermal growth factor receptor signaling mediates vesicant-induced airway epithelial secretion of interleukin-6 and production of mucin.

The chemical warfare agent sulfur mustard (HD) and its analogue nitrogen mustard (HN2) are highly reactive vesicants that can cause airway epithelial injury. However, little is known about the mechanisms governing vesicant-related airway damage. This study assessed the role of epidermal growth factor receptor (EGFR) signaling in mediating the effects of exposure to vesicants on the secretion of cytokines and production of mucin in human airway epithelial cells. Normal human bronchial epithelial cells (NHBECs) at an air-liquid interface were challenged apically with either 200 µM HN2 or medium alone (mock treatment, MT), and cultures were evaluated for receptor fate, the secretion of IL-6, and the production of both total mucin and Mucin 5AC (MUC5AC). Exposure to HN2 induced the activation of both EGFR and (44/42)mitogen-activated protein kinase ((44/42)MAPK), as well as the ubiquitination and colocalization of EGFR within lysosomal structures. Moreover, challenge with HN2 induced the up-regulation of IL-6 and MUC5AC at the mRNA and protein levels, and stimulated the secretion of total mucin in NHBECs. HN2-related effects on the secretion of IL-6 and the production of total mucin and MUC5AC were reversed by the selective EGFR inhibitor AG1478 and by an EGFR-blocking antibody. The HN2-induced activation of (44/42)MAPK and the up-regulation of IL-6 secretion in NHBECs were also largely reversed by a transforming growth factor-α (TGF-α)-blocking antibody and by the metalloprotease inhibitor GM 6001, suggesting that the HN2-related effects on EGFR signaling were TGF-α-dependent. Collectively, these findings suggest that EGFR signaling may play a significant role in mediating vesicant-induced airway epithelial injury.

ß-Lactones Inhibit N-acylethanolamine Acid Amidase by S-Acylation of the Catalytic N-Terminal Cysteine.

The cysteine amidase N-acylethanolamine acid amidase (NAAA) is a member of the N-terminal nucleophile class of enzymes and a potential target for anti-inflammatory drugs. We investigated the mechanism of inhibition of human NAAA by substituted ß-lactones. We characterized pharmacologically a representative member of this class, ARN077, and showed, using high-resolution liquid chromatography-tandem mass spectrometry, that this compound forms a thioester bond with the N-terminal catalytic cysteine in human NAAA.

Endocytosis of MHC molecules by distinct membrane rafts.

In B-lymphocytes, endocytosis of MHC I and MHC II molecules is important for the cross-priming and presentation of labile antigens, respectively. Here, we report that MHC I and MHC II were internalized by separate endocytic carriers that lacked transferrin receptor. Cholera toxin B was co-internalized with MHC II, but not with MHC I, suggesting that the CLIC/GEEC pathway is involved in the uptake of MHC II. Endocytosis of MHC I and MHC II was inhibited by filipin, but only MHC II showed a strong preference for a membrane raft environment in a co-clustering analysis with G(M)1. By using a novel method for the extraction of detergent-resistant membranes (DRMs), we observed that MHC I and MHC II associate with two distinct types of DRMs. These differ in density, protein content, lipid composition, and ultrastructure. The results of cell surface biotinylation and subsequent DRM isolation show that precursors for both DRMs coexist in the plasma membrane. Moreover, clustering of MHC proteins at the cell surface resulted in shifts of the respective DRMs, revealing proximity-induced changes in the membrane environment. Our results suggest that the preference of MHC I and MHC II for distinct membrane rafts directs them to different cellular entry points.

A clinically relevant in vitro model for evaluating the effects of aerosolized vesicants.

The chemical warfare vesicant sulfur mustard (HD) is a known toxic agent to the human respiratory tract and the major airways are considered to be a primary target of HD-induced injury. However, there is no consensus regarding which model systems are most appropriate for studying the effects of aerosolized vesicants on human airway epithelium. In this study, we evaluated the consequences of exposure of differentiated human respiratory epithelial cells in air-liquid interface to mechlorethamine (HN2), an HD functional analog. HN2 challenge was administered via the apical (air) interface over a wide dose range (20-400 microM) to differentiated HBE1 cells. Cultures were observed over 1-48 h for evidence of HN2-induced morphologic abnormalities as well as for possible cellular cytotoxicity, apoptotic changes, and induction of cytokine secretion. HN2 at concentrations of > or =200 microM caused disruption and denudation of the airway epithelial architecture within 24h of exposure. Moreover, HN2-induced cytotoxic and apoptotic changes in HBE1 cells in a dose- and time-dependent fashion. HN2 challenge also induced secretion of chemokines and proinflammatory cytokines including TNF-alpha, IL-1 alpha, IL-1 beta, IL-6, IL-8, RANTES, MCP-1, IP-10, G-CSF, GM-CSF and IL-15. These observations parallel those described in the lungs of HD-exposed victims and underscore the utility and potential applicability of this model to future mechanistic studies of vesicant-induced pulmonary injury.

Mucolipin-2 localizes to the Arf6-associated pathway and regulates recycling of GPI-APs.

In mammals, the mucolipin family includes three members mucolipin-1, mucolipin-2, and mucolipin-3 (MCOLN1-3). While mutations in MCOLN1 and MCOLN3 have been associated with mucolipidosis type IV and the varitint-waddler mouse phenotype, respectively, little is known about the function and cellular distribution of MCOLN2. Here we show that MCOLN2 traffics via the Arf6-associated pathway and colocalizes with major histocompatibility protein class I (MHCI) and glycosylphosphatidylinositol-anchored proteins (GPI-APs), such as CD59 in both vesicles and long tubular structures. Expression of a constitutive active Arf6 mutant, or activation of endogenous Arf6 by transfection with EFA6 or treatment with aluminum fluoride, caused accumulation of MCOLN2 in enlarged vacuoles that also contain MHCI and CD59. In addition, overexpression of MCOLN2 promoted efficient activation of Arf6 in vivo, thus suggesting that MCOLN2 may have a role in the traffic of cargo through the Arf6-associated pathway. In support of this we found that overexpression of a MCOLN2 inactive mutant decreases recycling of CD59 to the plasma membrane. Therefore, our results indicate that MCOLN2 localizes to the Arf6-regulated pathway and regulates sorting of GPI-APs.

MHC II molecules and invariant chain reside in membranes distinct from conventional lipid rafts.

Major histocompatibility complex class II (MHC II) peptide complexes can associate with lipid rafts, and this is a prerequisite for their recruitment to the immunological synapse and for efficient T cell stimulation. One of the most often used criterion for raft association is the resistance to extraction by the detergent Triton X-100 (TX-100) at low temperature. For MHC II, a variety of detergents have been used under different conditions, leading to variable and often conflicting conclusions about the association of MHC II with detergent-resistant membranes (DRMs). To clarify whether these inconsistencies were caused by variations in the isolation protocols or reflect different biochemical properties of MHC II lipid complexes, we used two standardized procedures for the isolation of membranes resistant to TX-100, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), or Brij 98. Our results suggest that some of the reported variations in the association of MHC II with DRMs are caused by differences in the methods. We also show that in our hands, specific and efficient flotation of MHC II and the MHC II-associated invariant chain from mouse B-lymphoma cells was only achieved with Brij 98, but not with TX-100 and CHAPS. We furthermore used DRMs prepared from hen egg lysozyme-fed B-lymphoma cells to activate the T cell hybridoma 3A9. In agreement with our biochemical data, T cell activation could only be achieved with Brij 98- but not with TX-100-resistant membranes. Thus, MHC II and also the invariant chain belong to a set of proteins comprising the T cell receptor, prominin, and the prion protein, which reside in membrane environments distinct from conventional lipid rafts.

Association of major histocompatibility complex II with cholesterol- and sphingolipid-rich membranes precedes peptide loading.

Major histocompatibility complex class II protein (MHC II) molecules present antigenic peptides to CD4-positive T-cells. Efficient T cell stimulation requires association of MHC II with membrane microdomains organized by cholesterol and glycosphingolipids or by tetraspanins. Using detergent extraction at 37 degrees C combined with a modified flotation assay, we investigated the sequence of events leading to the association of MHC II with cholesterol- and glycosphingolipid-rich membranes (DRMs) that are distinct from tetraspanins. We find two stages of association of MHC II with DRMs. In stage one, complexes of MHC II and invariant chain, a chaperone involved in MHC II transport, enter DRMs in the Golgi stack. In early endosomes, these complexes are almost quantitatively associated with DRMs. Upon transport to late endocytic compartments, MHC II-bound invariant chain is stepwise proteolyzed to the MHC class II-associated invariant chain peptide (CLIP) that remains MHC II-bound and retains a preference for DRMs. At the transition between the two stages, CLIP is exchanged against processed antigens, and the resulting MHC II-peptide complexes are transported to the cell surface. In the second stage, MHC II shows a lower overall association with DRMs. However, surface MHC II molecules occupied with peptides that induce resistance to denaturation by SDS are enriched in DRMs relative to SDS-sensitive MHC II-peptide complexes. Likewise, MHC II molecules loaded with long-lived processing products of hen-egg lysozyme containing the immunodominant epitope 48-61 show a very high preference for DRMs. Thus after an initial mainly intracellular stage of high DRM association, MHC II moves to a second stage in which its preference for DRMs is modulated by bound peptides.