Mast cells regulate CD4+ T-cell differentiation in the absence of antigen presentation.

BACKGROUND: Given their unique capacity for antigen uptake, processing, and presentation, antigen-presenting cells (APCs) are critical for initiating and regulating innate and adaptive immune responses. We have previously shown the role of nicotinamide adenine dinucleotide (NAD+) in T-cell differentiation independently of the cytokine milieu, whereas the precise mechanisms remained unknown. OBJECTIVE: The objective of this study is to further dissect the mechanism of actions of NAD+ and determine the effect of APCs on NAD+-mediated T-cell activation. METHODS: Isolated dendritic cells and bone marrow-derived mast cells (MCs) were used to characterize the mechanisms of action of NAD+ on CD4+ T-cell fate in vitro. Furthermore, NAD+-mediated CD4+ T-cell differentiation was investigated in vivo by using wild-type C57BL/6, MC-/-, MHC class II-/-, Wiskott-Aldrich syndrome protein (WASP)-/-, 5C.C7 recombination-activating gene 2 (Rag2)-/-, and CD11b-DTR transgenic mice. Finally, we tested the physiologic effect of NAD+ on the systemic immune response in the context of Listeria monocytogenes infection. RESULTS: Our in vivo and in vitro findings indicate that after NAD+ administration, MCs exclusively promote CD4+ T-cell differentiation, both in the absence of antigen and independently of major APCs. Moreover, we found that MCs mediated CD4+ T-cell differentiation independently of MHC II and T-cell receptor signaling machinery. More importantly, although treatment with NAD+ resulted in decreased MHC II expression on CD11c+ cells, MC-mediated CD4+ T-cell differentiation rendered mice resistant to administration of lethal doses of L monocytogenes. CONCLUSIONS: Collectively, our study unravels a novel cellular and molecular pathway that regulates innate and adaptive immunity through MCs exclusively and underscores the therapeutic potential of NAD+ in the context of primary immunodeficiencies and antimicrobial resistance.

Binding of WIP to actin is essential for T cell actin cytoskeleton integrity and tissue homing.

The Wiskott-Aldrich syndrome protein (WASp) is important for actin polymerization in T cells and for their migration. WASp-interacting protein (WIP) binds to and stabilizes WASp and also interacts with actin. Cytoskeletal and functional defects are more severe in WIP(-/-) T cells, which lack WASp, than in WASp(-/-) T cells, suggesting that WIP interaction with actin may be important for T cell cytoskeletal integrity and function. We constructed mice that lack the actin-binding domain of WIP (WIPΔABD mice). WIPΔABD associated normally with WASp but not F-actin. T cells from WIPΔABD mice had normal WASp levels but decreased cellular F-actin content, a disorganized actin cytoskeleton, impaired chemotaxis, and defective homing to lymph nodes. WIPΔABD mice exhibited a T cell intrinsic defect in contact hypersensitivity and impaired responses to cutaneous challenge with protein antigen. Adoptively transferred antigen-specific CD4(+) T cells from WIPΔABD mice had decreased homing to antigen-challenged skin of wild-type recipients. These findings show that WIP binding to actin, independently of its binding to WASp, is critical for the integrity of the actin cytoskeleton in T cells and for their migration into tissues. Disruption of WIP binding to actin could be of therapeutic value in T cell-driven inflammatory diseases.

Binding of the WASP/N-WASP-interacting protein WIP to actin regulates focal adhesion assembly and adhesion.

The actin cytoskeleton is essential for cell adhesion and migration, functions important for tumor invasion. In addition to binding N-WASP/WASP, WIP binds and stabilizes F-actin. WIP(-/-) fibroblasts were used to test the role of WIP in F-actin function. WIP(-/-) cells had defective focal adhesion (FA), stress fiber assembly, and adherence to substrates, functions that were restored by transduction of wild-type WIP. Protein and mRNA levels of several FA constituents regulated by the myocardin-related transcription factor (MRTF)­serum response factor (SRF) transcription factor complex were reduced in WIP(-/-) fibroblasts. The level of G-actin, which sequesters MRTF in the cytoplasm, was increased, and nuclear localization of MRTF-A and SRF was reduced, in WIP(-/-) fibroblasts. Transfection of an MRTF-A mutant that constitutively translocates to the nucleus or transfection of constitutively active SRF restored FA and stress fiber assembly. Fibroblasts from knock-in mice expressing a WIP mutant that fails to bind actin phenocopied WIP(-/-) fibroblasts. Thus, WIP is a novel regulator of FA assembly and cell adhesion.

HS3ST2 modulates breast cancer cell invasiveness via MAP kinase- and Tcf4 (Tcf7l2)-dependent regulation of protease and cadherin expression.

Heparan sulfate 3-O-sulfotransferase 2 (HS3ST2), an enzyme mediating 3-O-sulfation of heparan sulfate (HS), is silenced by hypermethylation in breast cancer. As HS has an important co-receptor function for numerous signal transduction pathways, the phenotypical changes due to HS3ST2 reexpression were investigated in vitro using high and low invasive breast cancer cell lines. Compared to controls, highly invasive HS3ST2-expressing MDA-MB-231 cells showed enhanced Matrigel invasiveness, transendothelial migration and motility. Affymetrix screening and confirmatory real-time PCR and Western blotting analysis revealed increased expression of several matrix metalloproteinases, cadherin-11, E-cadherin and CEACAM-1, while protease inhibitor and annexin A10 expression were decreased. Low invasive HS3ST2 -expressing MCF-7 cells became even less invasive, with no change in gelatinolytic MMP activity. HS3ST2 expression increased HS-dependent basal and FGF2-specific signaling through the constitutively active p44/42 MAPK pathway in MDA-MB-231 cells. Increased MAPK activation was accompanied by upregulation of ß-catenin in MDA-MB-231, and of the transcription factor Tcf4 in both cell lines. Dysregulation of Tcf4-regulated ion transporters and increased cytosolic acidification were observed in HS3ST2-expressing MDA-MB-231 cells, which is a possible underlying cause of increased chemosensitivity towards doxorubicine and paclitaxel in these cells. This study provides the first in vitro evidence of the involvement of HS3ST2 in breast cancer cell invasion and chemosensitivity.

Cdc42 interacting protein 4 (CIP4) is essential for integrin-dependent T-cell trafficking.

The F-BAR domain containing protein CIP4 (Cdc42 interacting protein 4) interacts with Cdc42 and WASP/N-WASP and is thought to participate in the assembly of filamentous actin. CIP4(-/-) mice had normal T- and B-lymphocyte development but impaired T cell-dependent antibody production, IgG antibody affinity maturation, and germinal center (GC) formation, despite an intact CD40L-CD40 axis. CIP4(-/-) mice also had impaired contact hypersensitivity (CHS) to haptens, and their T cells failed to adoptively transfer CHS. Ovalbumin-activated CD4(+) effector T cells from CIP4(-/-)/OT-II mice migrated poorly to antigen-challenged skin. Activated CIP4(-/-) T cells exhibited impaired adhesion and polarization on immobilized VCAM-1 and ICAM-1 and defective arrest and transmigration across murine endothelial cell monolayers under shear flow conditions. These results demonstrate an important role for CIP4 in integrin-dependent T cell-dependent antibody responses and GC formation and in integrin-mediated recruitment of effector T cells to cutaneous sites of antigen-driven immune reactions.

Vaccinia virus inoculation in sites of allergic skin inflammation elicits a vigorous cutaneous IL-17 response.

Eczema vaccinatum (EV) is a complication of smallpox vaccination occurring in patients with atopic dermatitis. In affected individuals, vaccinia virus (VV) spreads through the skin, resulting in large primary lesions and satellite lesions, and infects internal organs. BALB/c mice inoculated with VV at sites of Th2-biased allergic skin inflammation elicited by epicutaneous ovalbumin (OVA) sensitization exhibited larger primary lesions that were erosive, more satellite lesions, and higher viral loads in skin and internal organs than mice inoculated in saline-exposed skin, unsensitized skin, or skin sites with Th1-dominant inflammation. VV inoculation in OVA-sensitized skin induced marked local expression of IL-17 transcripts and massive neutrophil infiltration compared to VV inoculation in saline-exposed skin. Treatment with anti-IL-17 decreased the size of primary lesions, numbers of satellite lesions, and viral loads. Addition of IL-17 promoted VV replication in skin explants. These results suggest that IL-17 may be a potential therapeutic target in EV.

WIP is critical for T cell responsiveness to IL-2.

The Wiskott-Aldrich syndrome (WAS) interacting protein (WIP) stabilizes the WAS protein (WASP), the product of the gene mutated in WAS. WIP-deficient T cells have low WASP levels, limiting the usefulness of WIP KO mice in defining the role of WIP in T cell function. To define this role, we compared WIP/WASP double KO (DKO) mice to WASP KO mice on DO11.10 background. T cell development was normal in both strains, but peripheral T cell numbers were significantly decreased in DKO mice. WASP KO T cells proliferated and secreted IL-2 normally in response to OVA peptide (OVAp). In contrast, T cells from DKO mice proliferated poorly in response to OVAp in vitro, and cutaneous hapten hypersensitivity was deficient in these mice. DKO T cells up-regulated CD25 expression and secreted normal amounts of IL-2 after antigen stimulation, but had defective response to IL-2, evidenced by failure to further up-regulate CD25 expression, phosphorylate STAT5, and induce expression of STAT5-dependent genes. DKO, but not WASP KO, T cells had a disrupted subcortical actin cytoskeleton and impaired actin polymerization after T cell antigen receptor (TCR) ligation. These results indicate that WIP is essential for IL-2 signaling and responsiveness in T cells, possibly because of its critical role in TCR-triggered actin cytoskeletal reorganization.

IL-21R is essential for epicutaneous sensitization and allergic skin inflammation in humans and mice.

Atopic dermatitis (AD) is a common allergic inflammatory skin disease caused by a combination of intense pruritus, scratching, and epicutaneous (e.c.) sensitization with allergens. To explore the roles of IL-21 and IL-21 receptor (IL-21R) in AD, we examined skin lesions from patients with AD and used a mouse model of allergic skin inflammation. IL-21 and IL-21R expression was upregulated in acute skin lesions of AD patients and in mouse skin subjected to tape stripping, a surrogate for scratching. The importance of this finding was highlighted by the fact that both Il21r-/- mice and WT mice treated with soluble IL-21R-IgG2aFc fusion protein failed to develop skin inflammation after e.c. sensitization of tape-stripped skin. Adoptively transferred OVA-specific WT CD4+ T cells accumulated poorly in draining LNs (DLNs) of e.c. sensitized Il21r-/- mice. This was likely caused by both DC-intrinsic and nonintrinsic effects, because trafficking of skin DCs to DLNs was defective in Il21r-/- mice and, to a lesser extent, in WT mice reconstituted with Il21r-/- BM. More insight into this defect was provided by the observation that skin DCs from tape-stripped WT mice, but not Il21r-/- mice, upregulated CCR7 and migrated toward CCR7 ligands. Treatment of epidermal and dermal cells with IL-21 activated MMP2, which has been implicated in trafficking of skin DCs. These results suggest an important role for IL-21R in the mobilization of skin DCs to DLNs and the subsequent allergic response to e.c. introduced antigen.

Biochemical and functional characterization of cation dependent (Mr 46,000) goat mannose 6-phosphate receptor.

The Mannose 6-phosphate receptor (MPR's) proteins are important for transporting lysosomal enzymes from trans-golgi to the pre-lysosomal compartment. These are conserved in the vertebrates from fish to mammals. We have cloned the full length cDNA for the goat MPR 46 protein and compared its sequences to the other known vertebrate MPR 46 proteins. In the present study the full-length cDNA for the goat MPR 46 protein was expressed in MPR deficient cells. The expressed protein was purified on the multivalent phosphomannan gel in the presence of divalent metal ions. The apparent molecular mass of the expressed protein was found to be approximately 46 kDa and also exhibits oligomeric nature as observed in the other species, by using an MSC1 antibody (that recognizes the MPR 46 from molluscs to mammals) as well as with a peptide specific antibody corresponding to amino acid residues (218-237) of the cytoplasmic tail of human MPR 46 protein. Furthermore the distribution of the expressed protein was visualized by immunofluorescence using MSC1 and LAMP1 antibody. Additionally in the goat MPR 46 expressing cells, the sorting function of the expressed protein to sort cathepsin D to lysosomes was studied by confocal microscopy using cathepsin D antiserum and LAMP1 antibody. The binding of goat MPR 46 to cathepsin D was shown in far Western blotting and the mannose 6-phosphate dependent binding was shown by co-immunoprecipitation.

A novel anti-WIP monoclonal antibody detects an isoform of WIP that lacks the WASP binding domain.

The majority of Wiskott-Aldrich syndrome protein (WASP) in T cells is in a complex with WASP interacting protein (WIP), a 503 a.a. long proline rich protein. Here we demonstrate that a novel anti-WIP mAb, 3D10, recognizes an epitope in the N-terminal domain of the WIP protein, within the sequence 13PTFALA18. mAb 3D10 competes with actin, but not with WASP or Nck, for WIP binding. Analysis of 3D10 immunoprecipitates failed to demonstrate dissociation of the WASP-WIP complex after TCR ligation that we previously reported using a polyclonal anti-WIP anti-serum raised against a C-terminal peptide (a.a. 459-503) that spanned the WASP binding site. 3D10 mAb allowed the detection of a novel isoform of WIP consisting of a truncated 403 a.a. long protein that includes the 377 a.a. encoded by the first 4 exons of WIP followed by a 26 a.a. sequence encoded by intron 4.

Mannose 6-phosphate receptor (MPR 300) proteins from goat and chicken bind human IGF-II.

Mannose 6-phosphate receptor proteins (MPR 300 and 46) in mammals have been shown to mediate transport of lysosomal enzymes to lysosomes intracellularly. Both receptors are also expressed on the plasma membrane. Only MPR 300 protein on the plasma membrane has been shown to be a multifunctional protein which in addition to binding mannose 6-phosphate containing proteins also binds human insulin-like growth factor-II (IGF-II) causing its internalization [Hille-Rehfeld, A. (1995) Mannose 6-phosphate receptors in sorting and transport of lysosomal enzymes. Biochim. Biophys. Acta. 1241: 177-194]. This property has been shown to be exhibited by other mammalian receptors but not by the chicken and frog receptors. In a recent study however it was shown that the fish embryo MPR 300 binds human IGF-II. [Mendez, E., Planas, J.V., Castillo, J., Navarro, I. and Gutierrez, J. (2001) Identification of a type II insulin-like growth factor receptor in fish embryos. Endocrinology, 142: 1090-1097]. In the present study, we demonstrate that the purified goat and chicken liver receptors bind human IGF-II by employing cross-linking experiments (purified receptors and radiolabeled IGF-II) and by ligand blotting (using purified receptors and biotinylated IGF-II). Further CEF cells (chicken embryonic fibroblasts) that are known to contain the putative MPR 300 protein were employed to demonstrate that the CEF cell receptor binds human IGF-II.

The early vertebrate Danio rerio Mr 46000 mannose-6-phosphate receptor: biochemical and functional characterisation.

Mannose-6-phosphate receptors (MPRs) have been identified in a wide range of species from humans to invertebrates such as molluscs. A characteristic of all MPRs is their common property to recognize mannose-6-phosphate residues that are labelling lysosomal enzymes and to mediate their targeting to lysosomes in mammalian cells by the corresponding receptor proteins. We present here the analysis of full-length sequences for MPR 46 from zebrafish (Danio rerio) and its functional analysis. This is the first non-mammalian MPR 46 to be characterised. The amino acid sequences of the zebrafish MPR 46 displays 70% similarity to the human MPR 46 protein. In particular, all essential cysteine residues, the transmembrane domain as well as the cytoplasmic tail residues harbouring the signals for endocytosis and Golgi-localizing, gamma-ear-containing, ARF-binding protein (GGA)-mediated sorting at the trans-Golgi network, are highly conserved. The zebrafish MPR 46 has the arginine residue known to be essential for mannose-6-phosphate binding and other additional characteristic residues of the mannose-6-phosphate ligand-binding pocket. Like the mammalian MPR 46, zebrafish MPR 46 binds to the multimeric mannose-6-phosphate ligand phosphomannan and can rescue the missorting of lysosomal enzymes in mammalian MPR-deficient cells. The conserved C-terminal acidic dileucine motif (DxxLL) in the cytoplasmic domain of zebrafish MPR 46 essential for the interaction of the GGAs with the receptor domains interacts with the human GGA1-VHS domain. Interestingly, the serine residue suggested to regulate the interaction between the tail and the GGAs in a phosphorylation-dependent manner is substituted by a proline residue in fish.

Biochemical and immunological characterization of a glycosylated alpha-fucosidase from the invertebrate Unio: interaction of the enzyme with its in vivo binding partners.

Mammalian alpha-fucosidase (EC 3.2.1.51) is a lysosomal enzyme that catalyzes the removal of fucose residues from glycosphingolipids and its absence in humans results in a rare metabolic disorder called fucosidosis. Among the invertebrates in the molluscs (Unio) two forms of the enzyme have been reported, a 68 kDa non-glycosylated form and a 56 kDa glycosylated form. The glycosylated form has been purified from the seminal fluid of Unio [Biochem. Biophys. Res. Commun. 234 (1997) 54]. In the present study, the 56 kDa glycosylated form has been purified to homogeneity from the whole body tissue of Unio using a series of chromatographic steps. The purified enzyme migrated as a single protein species in 10% SDS-PAGE. Antibodies to the purified enzyme were raised in a rabbit in order to study its biochemical and immunological properties. The purified enzyme is a glycoprotein that exhibits strong binding to Con A-Sepharose gel and can be deglycosylated by PNGase F enzyme suggesting it to be N-glycosylated. The enzyme has been shown to specifically interact with the mannose 6-phosphate receptor protein (MPR 300) purified from goat and Unio. This specific interaction is discussed in view of its possible in vivo binding partners.