Calcium-dependent, non-apoptotic, large plasma membrane bleb formation in physiologically stimulated mast cells and basophils.

Large membrane derangements in the form of non-detaching blebs or membrane protrusions occur in a variety of cell stress and physiological situations and do not always reflect apoptotic processes. They have been studied in model mast cells under conditions of cell stress, but their potential physiological relevance to mast cell function and formation in primary mast cells or basophils have not been addressed. In the current study, we examine the large, non-detaching, non-apoptotic, membrane structures that form in model and primary mast cells under conditions of stimulation that are relevant to allergy, atopy and Type IV delayed hypersensitivity reactions. We characterized the inflation kinetics, dependency of formation upon external free calcium and striking geometric consistency of formation for large plasma membrane blebs (LPMBs). We describe that immunologically stimulated LPMBs in mast cells are constrained to form in locations where dissociation of the membrane-associated cytoskeleton occurs. Mast cell LPMBs decorate with wheat germ agglutinin, suggesting that they contain plasma membrane (PM) lectins. Electrophysiological capacitance measurements support a model where LPMBs are not being formed from internal membranes newly fused into the PM, but rather arise from stretching of the existing membrane, or inflation and smoothing of a micro-ruffled PM. This study provides new insights into the physiological manifestations of LPMB in response to immunologically relevant stimuli and in the absence of cell stress, death or apoptotic pathways.

Five mouse homologues of the human dendritic cell C-type lectin, DC-SIGN.

DC-SIGN, a human C-type lectin, is expressed on the surface of dendritic cells (DC), while a closely related human gene, DC-SIGNR or L-SIGN, is found on sinusoidal endothelial cells of liver and lymph node. Both DC-SIGN and DC-SIGNR/L-SIGN can bind ICAM-3 and HIV gp120, and transmit HIV to susceptible cells in trans. Here, we report the cloning of five mouse genes homologous to human DC-SIGN and DC-SIGNR/L-SIGN. Only one gene, named mouse DC-SIGN, is highly expressed in DC, and is not found in a panel of mouse macrophage and lymphocyte cell lines. The other four genes, named mouse SIGNR1 (SIGN-Related gene 1), SIGNR2, SIGNR3 and SIGNR4, are expressed at lower levels in various cells according to RT-PCR and Northern blot analyses on RNA. All the genes of mouse DC-SIGN and SIGNRs map to adjacent regions of chromosome 8 A1.2-1.3. However, like human DC-SIGN, only the mouse DC-SIGN gene is closely juxtaposed to the CD23 gene, while the other four SIGNR genes are located close to each other in a neighboring region. mRNAs of mouse DC-SIGN and three SIGNR genes encode type II transmembrane proteins (DC-SIGN, 238 amino acids; SIGNR1, 325 amino acids; SIGNR3, 237 amino acids; SIGNR4, 208 amino acids), but the SIGNR2 gene only encodes a carbohydrate recognition domain (CRD) without a cytosolic domain and a transmembrane domain (SIGNR2, 178 amino acids). Amino acid sequence similarities between the CRD of human DC-SIGN and the mouse homologues are 67% for DC-SIGN, 69% for SIGNR1, 65% for SIGNR2, 68% for SIGNR3 and 70% for SIGNR4 respectively. However, the membrane proximal neck domains in the mouse genes are much shorter than their counterparts in human DC-SIGN and DC-SIGNR/L-SIGN. This family of mouse C-type lectins is therefore complex, but only one of the new genes, DC-SIGN, is juxtaposed to CD23 and is expressed at high levels in DC.

Tissue macrophages associated with angiogenesis in chronic airway inflammation in rats.

Angiogenesis is a feature of chronic inflammation produced by Mycoplasma pulmonis infection of the respiratory tract. The mechanism of this angiogenesis is unknown, but cellular growth factors and matrix remodeling proteases produced by inflammatory cells are likely to be involved. The goal of this study was to determine the relationship between changes in the number, shape, and distribution of ED2-immunoreactive macrophages and the development of angiogenesis in the tracheal mucosa of Wistar rats after M. pulmonis infection. In pathogen-free rats, ED2-positive cells were scattered in the airway mucosa (261 +/- 42 cells/mm2 of surface, mean +/- SE). Most cells were irregularly shaped and had moderate ED2 immunoreactivity. No lymphoid tissue was present. The number of ED2-positive cells increased rapidly after infection, was 120% above baseline at 1 wk, and remained significantly increased throughout the 4-wk study (P < 0.05). Angiogenesis was first detected at 2 wk, and at 3 wk the vessel length density was nearly 8-fold the pathogen-free value. At 3 and 4 wk, focal sites of angiogenesis coincided with discrete clusters of round, strongly immunoreactive ED2-positive cells (1,340 +/- 124 cells/mm2) in polyp-like collections of mucosal lymphoid tissue. The close association of distinctive ED2-positive cells with angiogenic blood vessels suggests a relationship between a subset of tissue macrophages and the angiogenesis associated with M. pulmonis infection. The time course of the changes indicates that the initial influx of ED2-positive macrophages precedes the angiogenesis, and the rounding of the cells parallels the growth of new vessels.

Glucocorticoid-induced apoptosis of dendritic cells in the rat tracheal mucosa.

Dendritic cells are antigen-presenting cells that constitutively express high levels of major histocompatibility complex class II (Ia) antigen on their plasma membrane. Previous studies have shown that the number of dendritic cells in the rat airway mucosa decreases rapidly after glucocorticoid treatment. We sought to determine whether apoptosis contributes to this steroid-induced cell decrease. Dendritic cells in tracheal whole mounts were revealed by immunoperoxidase staining using the OX-6 (anti-Ia) monoclonal antibody. In untreated rats, a dense network of Ia-immunoreactive (Ia+) cells with highly branched cytoplasmic processes was observed just beneath the tracheal epithelium (1,405 +/- 140 cells/mm2 mucosa; mean +/- SEM, n = 6). In rats treated with dexamethasone (10 mg/kg, intraperitoneally), four distinct changes in dendritic cell morphology were evident 4 to 8 h after injection: (1) appearance of large Ia+ granules in cytoplasmic processes, (2) narrowing of cytoplasmic processes, (3) loss of Ia immunoreactivity from the cell surface, and (4) fragmentation of cells into small Ia+ bodies. These changes accompanied a 56% decrease in the number of Ia+ cells over 8 h. The contribution of apoptosis to this decrease in Ia+ cells was determined by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling (TUNEL) of nucleosomal DNA fragments in histologic sections. The number of TUNEL+ bodies increased from a control value of 174 +/- 47 bodies/mm2 mucosa to 2,108 +/- 294 bodies/mm2 mucosa at 4 h and 936 +/- 343 bodies/ mm2 mucosa at 8 h (n = 4 rats per time point). The location of TUNEL+ bodies closely corresponded to that of Ia+ cells stained in adjacent histologic sections. We conclude that apoptosis contributes to the rapid decrease in airway dendritic cells after glucocorticoid treatment.

Organ-specific endothelial cell uptake of cationic liposome-DNA complexes in mice.

This study identified the organ and cellular distribution of cationic liposome-DNA complexes injected intravenously into CD-1 mice for gene delivery. DOTIM-cholesterol liposomes were labeled with the fluorescent dye CM-Dil and complexed with plasmid DNA encoding the chloramphenicol acetyltransferase reporter gene. The distribution of the complexes was examined in 29 organs and tissues by fluorescence, confocal, and electron microscopy from 5 min to 24 h after injection. The complexes formed clusters in blood, which were cleared within 20 min. Complexes visible by fluorescence microscopy were taken up by endothelial cells, leukocytes, and macrophages and did not leave the vasculature except in the spleen. At 5 min, the complexes formed a patchy coating on the endothelial surface, but by 4 h, they were internalized into endosomes and lysosomes in organ- and vessel-specific patterns. Uptake by capillary endothelial cells was greatest in the lung, ovary, and anterior pituitary, less in muscle and the heart, and nearly absent in the brain and pancreatic islets. In lymph nodes and intestinal Peyer's patches, the uptake was sparse in capillaries but abundant in high endothelial venules. In the liver and spleen, most of the uptake was in Kupffer cells and macrophages. Measurements of chloramphenicol acetyltransferase reporter gene expression were generally consistent with the pattern of uptake by endothelial cells. The uptake and gene expression were accompanied by a decrease in circulating leukocytes and platelets. Overall, our results showed that the complexes were internalized by endothelial cells in organ- and vessel-specific patterns that did not match any previously identified properties of the microvasculature. The unusual distribution of endothelial cell uptake may be explained by a heterogeneously distributed membrane receptor for which the complexes are ligands.