Valsamma Abraham

Delayed Apoptotic Cell Clearance and Lupus-like Autoimmunity in Mice Lacking the c-mer Membrane Tyrosine Kinase

Mice lacking the membrane tyrosine kinase c-mer have been shown to have altered macro-phage cytokine production and defective phagocytosis of apoptotic cells despite normal phagocytosis of other particles. We show here that c-mer–deficient mice have impaired clearance of infused apoptotic cells and that they develop progressive lupus-like autoimmunity, with antibodies to chromatin, DNA, and IgG. The autoimmunity appears to be driven by endogenous antigens, with little polyclonal B cell activation. These mice should be an excellent model for studying the role of apoptotic debris as an immunogenic stimulus for systemic autoimmunity.

Type XV collagen exhibits a widespread distribution in human tissues but a distinct localization in basement membrane zones.

Abstract.The collagen family of proteins consists of 19 types encoded by 33 genes. One of the more recently discovered collagens is the α1 chain of type XV. Type XV collagen is comprised of a 577-amino-acid, highly interrupted, triple-helical region that is flanked by amino and carboxy noncollagenous domains of 555 and 256 residues, respectively. To address questions of where this collagen is localized and what its function may entail, we produced a bacteria-expressed recombinant protein representing the first half of the type XV collagen carboxy-terminal domain in order to generate highly specific polyclonal antisera. Immunoscreening of an expression library with the affinity-purified antibody revealed three clones coding for part of the type XV triple-helical region and the entire noncollagenous carboxy-terminus. Western blot analysis of human tissue homogenates identified a 116-kDa collagenase-sensitive protein and a 27-kDa collagenase-resistant fragment, whose electrophoretic mobilities were unchanged in the presence and absence of reductant. Northern blot hybridization to human tissue RNAs indicated that type XV has a prevalent and widespread distribution. To determine the precise localization of type XV collagen, immunohistochemical analyses at the light- and electron-microscopic levels were performed. Type XV exhibited a surprisingly restricted and uniform presence in many human tissues as evidenced by a strong association with vascular, neuronal, mesenchymal, and some epithelial basement membrane zones. These data suggest that type XV collagen may function in some manner to adhere basement membrane to the underlying connective tissue stroma.

Phenotypic control of gap junctional communication by cultured alveolar epithelial cells

We examined phenotype-specific changes in gap junction protein [connexin (Cx)] expression and function by cultured rat alveolar type II cells. Type II cells cultured on extracellular matrix in medium containing keratinocyte growth factor (KGF) and 2% fetal bovine serum (FBS; KGF/2) retained expression of surfactant protein C and the 180-kDa lamellar body membrane protein (lbm180). These markers were lost when cells were cultured in medium containing 10% FBS (MEM/10). With RT-PCR, cells cultured in MEM/10 showed transient increases in Cx43 and Cx46 mRNA expression, whereas Cx32 and Cx26 decreased and Cx30.3 and Cx37 were unchanged. Transient changes in Cx32, Cx43, and Cx46 protein expression were confirmed by immunoblot. In contrast, cells cultured in KGF/2 retained expression of Cx32 and showed increased expression of Cx30.3 and Cx46 mRNAs, compared with that in day 0 cells. With immunofluorescence microscopy, Cx32 and Cx43 were at the plasma membrane of cells grown in KGF/2, whereas Cx46 was exclusively intracellular. Type II cells cultured in MEM/10 showed approximately 3- to 4-fold more intercellular transfer of microinjected lucifer yellow through gap junctions than cells grown in 2% FBS. Thus type II cells dynamically alter gap junctional communication, and distinct alveolar epithelial cell phenotypes express different connexins.We examined phenotype-specific changes in gap junction protein [connexin (Cx)] expression and function by cultured rat alveolar type II cells. Type II cells cultured on extracellular matrix in medium containing keratinocyte growth factor (KGF) and 2% fetal bovine serum (FBS; KGF/2) retained expression of surfactant protein C and the 180-kDa lamellar body membrane protein (lbm180). These markers were lost when cells were cultured in medium containing 10% FBS (MEM/10). With RT-PCR, cells cultured in MEM/10 showed transient increases in Cx43 and Cx46 mRNA expression, whereas Cx32 and Cx26 decreased and Cx30.3 and Cx37 were unchanged. Transient changes in Cx32, Cx43, and Cx46 protein expression were confirmed by immunoblot. In contrast, cells cultured in KGF/2 retained expression of Cx32 and showed increased expression of Cx30.3 and Cx46 mRNAs, compared with that in day 0 cells. With immunofluorescence microscopy, Cx32 and Cx43 were at the plasma membrane of cells grown in KGF/2, whereas Cx46 was exclusively intracellular. Type II cells cultured in MEM/10 showed ∼3- to 4-fold more intercellular transfer of microinjected lucifer yellow through gap junctions than cells grown in 2% FBS. Thus type II cells dynamically alter gap junctional communication, and distinct alveolar epithelial cell phenotypes express different connexins.

Invariant natural killer T cells in children with eosinophilic esophagitis.

Eosinophilic esophagitis (EoE) is an atopic disease characterized by eosinophilic inflammation in which dietary antigens (in particular, milk) play a major role. EoE is most likely a mixed IgE and non‐IgE food‐mediated reaction in which overexpression of Th2 cytokines, particularly IL‐13, play a major role; however, the cells responsible for IL‐13 overexpression remain elusive. Th2‐cytokines are secreted following the ligation of invariant natural killer T cell receptors to sphingolipids (SLs). Sphingolipids (SLs) are presented via the CD1d molecule on the INKTs surface. Cows milk‐derived SL has been shown to activate iNKTs from children with IgE‐mediated food allergies to milk (FA‐MA) to produce Th2 cytokines. The role of iNKTs and milk‐SL in EoE pathogenesis is currently unknown.

Disrupted Mer receptor tyrosine kinase expression leads to enhanced MZ B-cell responses

Control of lymphocyte homeostasis is essential to ensure efficient immune responses and to prevent autoimmunity. Splenic marginal zone B cells are important producers of autoantibodies, and are subject to stringent tolerance mechanisms to prevent autoimmunity. In this paper, we explore the role of the Mer tyrosine kinase (Mertk) in regulating autoreactive B cells. This receptor tyrosine kinase serves to bind apoptotic cells, to mediate their phagocytosis, and to regulate subsequent cytokine production. Mice lacking Mertk suffer from impaired apoptotic cell clearance and develop a lupus-like autoimmune syndrome. Here we show that such Mertk-KO mice have expanded numbers of splenic marginal zone B cells. Mertk-KO mice bearing a DNA-specific immunoglobulin heavy-chain transgene (3H9) produced anti-DNA antibodies that appeared to be secreted largely by marginal zone B cells. Finally, Mertk-KO mice developed greater antibody responses after NP-Ficoll immunization than their B6 counterparts. Taken together, our data show that Mertk has a major effect on the development of the marginal zone B-cell compartment. Mertk is also important in establishing DNA-specific B-cell tolerance in 3H9 anti-DNA transgenic mice.

Mechanism of Collaborative Enhancement of Binding of Paired Antibodies to Distinct Epitopes of Platelet Endothelial Cell Adhesion Molecule-1

Monoclonal antibodies (mAbs) directed to extracellular epitopes of human and mouse Platelet Endothelial Cell Adhesion Molecule-1 (CD31 or PECAM-1) stimulate binding of other mAbs to distinct adjacent PECAM-1 epitopes. This effect, dubbed Collaborative Enhancement of Paired Affinity Ligands, or CEPAL, has been shown to enhance delivery of mAb-targeted drugs and nanoparticles to the vascular endothelium. Here we report new insights into the mechanism underlying this effect, which demonstrates equivalent amplitude in the following models: i) cells expressing a full length PECAM-1 and mutant form of PECAM-1 unable to form homodimers; ii) isolated fractions of cellular membranes; and, iii) immobilized recombinant PECAM-1. These results indicate that CEPAL is mediated not by interference in cellular functions or homophilic PECAM-1 interactions, but rather by conformational changes within the cell adhesion molecule induced by ligand binding. This mechanism, mediated by exposure of partially occult epitopes, is likely to occur in molecules other than PECAM-1 and may represent a generalizable phenomenon with valuable practical applications.

Influence of PECAM-1 ligand interactions on PECAM-1-dependent cell motility and filopodia extension

Platelet endothelial cell adhesion molecule (PECAM‐1) has been implicated in angiogenesis through processes that involve stimulation of endothelial cell motility. Previous studies suggest that PECAM‐1 tyrosine phosphorylation mediates the recruitment and then activation of the tyrosine phosphatase SHP‐2, which in turn promotes the turnover of focal adhesions and the extension of filopodia, processes critical to cell motility. While these studies have implicated PECAM‐1‐dependent signaling in PECAM‐1‐mediated cell motility, the involvement of PECAM‐1 ligand binding in cell migration is undefined. Therefore to investigate the role of PECAM‐1 binding interactions in cell motility, mutants of PECAM‐1 were generated in which either homophilic or heparin/glycosaminoglycan (GAG)‐mediated heterophilic binding had been disabled and then expressed in an endothelial cell surrogate. We found that the ability of PECAM‐1 to stimulate cell migration, promote filopodia formation and trigger Cdc42 activation were lost if PECAM‐1‐dependent homophilic or heparin/GAG‐dependent heterophilic ligand binding was disabled. We further observed that PECAM‐1 concentrated at the tips of extended filopodia, an activity that was diminished if homophilic, but not heparin/GAG‐mediated heterophilic binding had been disrupted. Similar patterns of activities were seen in mouse endothelial cells treated with antibodies that specifically block PECAM‐1‐dependent homophilic or heterophilic adhesion. Together these data provide evidence for the differential involvement of PECAM‐1‐ligand interactions in PECAM‐1‐dependent motility and the extension of filopodia.

Isolation of Endothelial Cells from Mouse Lung

The isolation of endothelial cells (ECs) from knockout and transgenic mouse lines provides the opportunity to study the endothelial‐specific activities of a targeted molecule. As a means of pursuing these types of investigations, the protocols described in this unit provide a reliable method for isolating lung microvascular ECs from mouse neonatal pups that can be serially passaged. These protocols are useful in settings where mouse age is irrelevant and a pure population of pulmonary vascular ECs, uncontaminated by other cells, is needed. When a specific source of ECs is not required, these procedures also represent a reliable means of obtaining murine ECs in general. Curr. Protoc. Toxicol. 61:24.2.1‐24.2.9.

Involvement of TIMP-1 in PECAM-1-mediated tumor dissemination

Platelet endothelial cell adhesion molecule-1 (PECAM-1) is expressed on the vascular endothelium and has been implicated in the late progression of metastatic tumors. The activity of PECAM-1 appears to be mediated by modulation of the tumor microenvironment (TME) and promotion of tumor cell proliferation, rather than through the stimulation of tumor angiogenesis. The present study aimed to extend those initial findings by indicating that the presence of functional PECAM-1 on the endothelium promotes a proliferative tumor cell phenotype in vivo, as well as in tumor cell (B16-F10 melanoma and 4T1 breast cancer cell lines) co-culture assays with mouse endothelial cells (ECs) or a surrogate EC line (REN-MP). The pro-proliferative effects were mediated by soluble endothelial-derived factors that were dependent on PECAM-1 homophilic ligand interactions, but which were independent of PECAM-1-dependent signaling. Further analysis of the conditioned media obtained from tumor/EC and tumor/REN-MP co-cultures identified TIMP metallopeptidase inhibitor-1 (TIMP-1) as a PECAM-1-regulated factor, the targeting of which in the tumor cell/REN-MP system inhibited tumor cell proliferation. In addition, TIMP-1 expression was decreased in metastatic tumors from the lungs of PECAM-1-null mice, thus providing evidence of the in vivo significance of co-culture studies. Taken together, these studies indicated that endothelial PECAM-1, through PECAM-1-dependent homophilic binding interactions, may induce release of TIMP-1 from the endothelium into the TME, thus leading to increased tumor cell proliferation.

Mechanisms of autoimmunity in mice lacking the mer membrane tyrosine kinase

There has been increased recent interest in the role of macrophages (Mp) and dendritic cells in systemic lupus erythematosus pathogenesis. The mer receptor tyrosine kinase, expressed on Mp and dendritic cells, mediates the binding of the former but not the latter to apoptotic cells. mer also is important in determining the cytokine profile that ensues after phagocytosis. Mice lacking mer develop antinuclear antibodies and rheumatoid factor. These autoantibodies appear to arise mostly from the splenic marginal zone. Stimulated spleen cells and peritoneal Mp from mer-deficient mice have increased expression of tumor necrosis factor alpha and IκBα, along with increased spontaneous expression of CD30L. The proinflammatory cytokine profile of mer-deficient mice may contribute to the immunogenicity of apoptotic debris. In vitro ligation of mer from normal Mp leads to diminished tumor necrosis factor alpha expression and increased IL-10 and IL-4. mer, which controls both phagocytosis and cytokine synthesis after exposure to apoptotic cells, may be an attractive target for therapeutic intervention in inflammatory and autoimmune disorders.