Enhanced tolerance to autoimmune uveitis in CD200-deficient mice correlates with a pronounced Th2 switch in response to antigen challenge

A single exposure to inhaled Ag 10 days before immunization leads to long term, Ag-specific tolerance. Respiratory tract myeloid APCs are implicated, but how regulation is invoked, and how tolerance is sustained are unclear. This study examines the in vivo function of the myeloid regulatory molecule CD200 in the process of tolerance induction. Despite earlier onset of experimental autoimmune uveitis in sham-tolerized, CD200-deficient mice, disease incidence and subsequent severity were actually reduced compared with those in wild-type mice. Protection was more effective and long term, lasting at least 28 days. Halting disease progression and tolerance in CD200(-/-) mice correlated with a marked increase in Th2-associated cytokine production by Ag-challenged splenocytes. Reduced overall disease and enhanced tolerance in the CD200-deficient mice in this model system were unexpected and may be related to altered populations of MHC class II(low) APC in the respiratory tract compared with wild-type mice together with associated activation of STAT6 in draining lymph nodes of tolerized mice. These data indicate that in the absence of default inhibitory CD200 receptor signaling, alternative, powerful regulatory mechanisms are invoked. This may represent either permissive dominant Th2 activation or an altered hierarchy of negative signaling by other myeloid cell-expressed regulatory molecules.     

Transmembrane TNF is sufficient to initiate cell migration and granuloma formation and provide acute, but not long-term, control of Mycobacterium tuberculosis infection

TNF is critical for immunity against Mycobacterium tuberculosis infection; however, the relative contributions of the soluble and transmembrane forms of TNF in this immunity are unknown. Using memTNF mice, which express only the transmembrane form of TNF, we have addressed this question. Wild-type (WT), TNF-/-, and transmembrane TNF (memTNF) mice were infected with M. tuberculosis by aerosol. TNF-/- mice developed overwhelming infection with extensive pulmonary necrosis and died after only 33 days. memTNF mice, like WT mice, contained bacterial growth for over 16 wk, developed an Ag-specific T cell response, and initially displayed compact granulomas, comprised of both lymphocytes and macrophages. Expression of mRNA for the chemokines CXCL10, CCL3, CCL5, and CCL7 was comparable in both WT and memTNF mice. As the infection progressed, however, the pulmonary lesions in memTNF mice became larger and more diffuse, with increased neutrophil accumulation and necrosis. This was accompanied by increased influx of activated memory T cells into the lungs of memTNF mice. Eventually, these mice succumbed to infection with a mean time to death of 170 days. The expression of memTNF on T cells is functionally important because the transfer of T cells from memTNF, but not TNF-/- mice, into either RAG-/- or TNF-/- mice conferred the same survival advantage on the M. tuberculosis-infected recipient mice, as the transfer of WT T cells. Therefore, memTNF, in the absence of soluble TNF, is sufficient to control acute, but not chronic, M. tuberculosis infection, in part through its expression on T cells.     

Cysteine-scanning mutagenesis and thiol modification of the Rickettsia prowazekii ATP/ADP translocase: evidence that transmembrane regions I and II, but not III, are structural components of the aqueous translocation channel

The contribution of transmembrane regions I, II, and III of the Rickettsia prowazekii ATP/ADP translocase to the structure of the putative water-filled ATP translocation channel was evaluated from the accessibility of hydrophilic, thiol-reactive, methanethiosulfonate reagents to a library of 68 independent cysteine-substitution mutants heterologously expressed in Escherichia coli. The MTS reagents used were MTSES (negatively charged) and MTSET and MTSEA (both positively charged). Mutants F036C, Y042C, and R046C (TM I), K066C and P072C (TM II), and F101C, F105C, F108C, Y113C, and P114C (TM III) had no assayable transport activity, indicating that cysteine substitution at these positions may not be tolerated. All three MTS reagents inhibit the transport of ATP in mutants of TM I (L039C, S043C, S047C, I048C) and TM II (S061C, S063C, T067C, I069C, V070C, A074C). Further, these residues appear to cluster along a single face of the transmembrane domain. Preexposure of MTS-reactive mutants S047C (TM I) and T067C (TM II) to high levels of ATP resulted in protection from MTS-mediated inhibition. This indicated that both TM I and TM II make major contributions to the structure of an aqueous ATP translocation pathway. Finally, on the basis of the lack of accessibility of charged MTS reagents to the thiol groups in mutants of TM III, it appears that TM III is not exposed to the ATP translocation channel. Cysteine substitution of residues constituting a highly conserved "phenylalanine face" in TM III resulted in ablation of ATP transport activity. Further, substituting these phenylalanine residues for either isoleucine or tyrosine also resulted in much lower transport activity, indicating that some property of phenylalanine at these positions that is not shared by cysteine, isoleucine, or tyrosine is critical to translocase activity.     

MT1-MMP-dependent neovessel formation within the confines of the three-dimensional extracellular matrix

During angiogenesis, endothelial cells initiate a tissue-invasive program within an interstitial matrix comprised largely of type I collagen. Extracellular matrix-degradative enzymes, including the matrix metalloproteinases (MMPs) MMP-2 and MMP-9, are thought to play key roles in angiogenesis by binding to docking sites on the cell surface after activation by plasmin- and/or membrane-type (MT) 1-MMP-dependent processes. To identify proteinases critical to neovessel formation, an ex vivo model of angiogenesis has been established wherein tissue explants from gene-targeted mice are embedded within a three-dimensional, type I collagen matrix. Unexpectedly, neither MMP-2, MMP-9, their cognate cell-surface receptors (i.e., beta3 integrin and CD44), nor plasminogen are essential for collagenolytic activity, endothelial cell invasion, or neovessel formation. Instead, the membrane-anchored MMP, MT1-MMP, confers endothelial cells with the ability to express invasive and tubulogenic activity in a collagen-rich milieu, in vitro or in vivo, where it plays an indispensable role in driving neovessel formation.     

A standardized kinesin nomenclature

In recent years the kinesin superfamily has become so large that several different naming schemes have emerged, leading to confusion and miscommunication. Here, we set forth a standardized kinesin nomenclature based on 14 family designations. The scheme unifies all previous phylogenies and nomenclature proposals, while allowing individual sequence names to remain the same, and for expansion to occur as new sequences are discovered.     

Regulation of myeloid cell function through the CD200 receptor

Myeloid cells play pivotal roles in chronic inflammatory diseases through their broad proinflammatory, destructive, and remodeling capacities. CD200 is widely expressed on a variety of cell types, while the recently identified CD200R is expressed on myeloid cells and T cells. CD200 deletion in vivo results in myeloid cell dysregulation and enhanced susceptibility to autoimmune inflammation, suggesting that the CD200-CD200R interaction is involved in immune suppression. We demonstrate in this study that CD200R agonists suppress mouse and human myeloid cell function in vitro, and also define a dose relationship between receptor expression and cellular inhibition. IFN-gamma- and IL-17-stimulated cytokine secretion from mouse peritoneal macrophages was inhibited by CD200R engagement. Inhibitory effects were not universal, as LPS-stimulated responses were unaffected. Inhibition of U937 cell cytokine production correlated with CD200R expression levels, and inhibition was only observed in low CD200R expressing cells, if the CD200R agonists were further cross-linked. Tetanus toxoid-induced human PBMC IL-5 and IL-13 secretion was inhibited by CD200R agonists. This inhibition was dependent upon cross-linking the CD200R on monocytes, but not on cross-linking the CD200R on CD4+ T cells. In all, we provide direct evidence that the CD200-CD200R interaction controls monocyte/macrophage function in both murine and human systems, further supporting the potential clinical application of CD200R agonists for the treatment of chronic inflammatory diseases.     

Mast cell-associated TNF promotes dendritic cell migration

Mast cells represent a potential source of TNF, a mediator which can enhance dendritic cell (DC) migration. Although the importance of mast cell-associated TNF in regulating DC migration in vivo is not clear, mast cells and mast cell-derived TNF can contribute to the expression of certain models of contact hypersensitivity (CHS). We found that CHS to FITC was significantly impaired in mast cell-deficient Kit(W-sh/W-sh) or TNF(-/)(-) mice. The reduced expression of CHS in Kit(W-sh/W-sh) mice was fully repaired by local transfer of wild-type bone marrow-derived cultured mast cells (BMCMCs), but was only partially repaired by transfer of TNF(-/)(-) BMCMCs. Thus, mast cells, and mast cell-derived TNF, were required for optimal expression of CHS to FITC. We found that the migration of FITC-bearing skin DCs into draining lymph nodes (LNs) 24 h after epicutaneous administration of FITC in naive mice was significantly reduced in mast cell-deficient or TNF(-/)(-) mice, but levels of DC migration in these mutant mice increased to greater than wild-type levels by 48 h after FITC sensitization. Mast cell-deficient or TNF(-/)(-) mice also exhibited significantly reduced migration of airway DCs to local LNs at 24 h after intranasal challenge with FITC-OVA. Migration of FITC-bearing DCs to LNs draining the skin or airways 24 h after sensitization was repaired in Kit(W-sh/W-sh) mice which had been engrafted with wild-type but not TNF(-/)(-) BMCMCs. Our findings indicate that mast cell-associated TNF can contribute significantly to the initial stages of FITC-induced migration of cutaneous or airway DCs.     

Genetic divergence of Connecticut Melanoplus femurrubrum populations

We surveyed Melanoplus femurrubrum populations within the state of Connecticut for genetic diversity at multiple genetic markers, including three mitochondrial [cytochrome oxidase subunit 1 (COI), reduced form of nicotinamide adenine dinucleotide dehydrogenase subunit 2 (ND2), and AT rich] and one nuclear [internal transcribed spacers of the ribosomal DNA cluster (ITS1)] gene regions. All markers were variable, and the AT-rich gene showed the highest sequence divergence. Analysis of molecular variance (AMOVA), fixation index (Fst) analysis, and phylogeographic patterns showed little divergence between northern and southern regions. Estimates of genetic diversity (pi) showed higher mitochondrial diversity in the northern region but nearly equal diversity for the ITS1 gene. This study shows for the first time in Melanoplus genetic variation for the ND2, AT rich, and ITS genes within a small geographic area. Our methods and results should be useful for other researchers interested in conducting population-level studies on closely related species.     

Mast cells enhance T cell activation: importance of mast cell costimulatory molecules and secreted TNF

We recently reported that mast cells stimulated via FcepsilonRI aggregation can enhance T cell activation by a TNF-dependent mechanism. However, the molecular mechanisms responsible for such IgE-, Ag- (Ag-), and mast cell-dependent enhancement of T cell activation remain unknown. In this study we showed that mouse bone marrow-derived cultured mast cells express various costimulatory molecules, including members of the B7 family (ICOS ligand (ICOSL), PD-L1, and PD-L2) and the TNF/TNFR families (OX40 ligand (OX40L), CD153, Fas, 4-1BB, and glucocorticoid-induced TNFR). ICOSL, PD-L1, PD-L2, and OX40L also are expressed on APCs such as dendritic cells and can modulate T cell function. We found that IgE- and Ag-dependent mast cell enhancement of T cell activation required secreted TNF; that TNF can increase the surface expression of OX40, ICOS, PD-1, and other costimulatory molecules on CD3(+) T cells; and that a neutralizing Ab to OX40L, but not neutralizing Abs to ICOSL or PD-L1, significantly reduced IgE/Ag-dependent mast cell-mediated enhancement of T cell activation. These results indicate that the secretion of soluble TNF and direct cell-cell interactions between mast cell OX40L and T cell OX40 contribute to the ability of IgE- and Ag-stimulated mouse mast cells to enhance T cell activation.