ovalbumin and tetramethylrhodamine-isothiocyanate

Perivascular cells, including pericytes, macrophages, smooth muscle cells, and other specialized cell types, like podocytes, participate in various aspects of vascular function. However, aside from the well-established roles of smooth muscle cells and pericytes, the contributions of other vascular-associated cells are poorly understood. Our goal was to ascertain the function of perivascular macrophages in adult tissues under nonpathological conditions.. We combined confocal microscopy, in vivo cell depletion, and in vitro assays to investigate the contribution of perivascular macrophages to vascular function. We found that resident perivascular macrophages are associated with capillaries at a frequency similar to that of pericytes. Macrophage depletion using either clodronate liposomes or antibodies unexpectedly resulted in hyperpermeability. This effect could be rescued when M2-like macrophages, but not M1-like macrophages or dendritic cells, were reconstituted in vivo, suggesting subtype-specific roles for macrophages in the regulation of vascular permeability. Furthermore, we found that permeability-promoting agents elicit motility and eventual dissociation of macrophages from the vasculature. Finally, in vitro assays showed that M2-like macrophages attenuate the phosphorylation of VE-cadherin upon exposure to permeability-promoting agents.. This study points to a direct contribution of macrophages to vessel barrier integrity and provides evidence that heterotypic cell interactions with the endothelium, in addition to those of pericytes, control vascular permeability.

Topics: Animals; Antigens, CD; Cadherins; Capillaries; Capillary Permeability; Cell Communication; Cell Movement; Cells, Cultured; Coculture Techniques; Dextrans; Endothelial Cells; Fluorescein-5-isothiocyanate; Humans; Macrophages, Peritoneal; Mesentery; Mice, Inbred C57BL; Mice, Nude; Mice, Transgenic; Ovalbumin; Peritoneum; Phenotype; Phosphorylation; Rhodamines; Skin; Time Factors; Transfection

The glycoprotein, avidin, conjugated either to the enzyme horseradish peroxidase, or to the fluorochrome dyes, fluorescein or rhodamine, identifies the granules of mast cells in both tissues and cell suspensions. In the absence of prior fixation, mast cells were not identified with conjugated avidin; however, granules released from these cells were stained with this labeled glycoprotein. The specificity of avidin for mast cells was confirmed by the absence of conjugated avidin-positive cells in the skin of mice (S1/S1d) deficient in mature dermal mast cells. Electron microscopic studies confirmed that avidin binds specifically to individual mast cell granules rather than to other cellular structures. Rodent and human mast cells were readily stained with avidin conjugated to horseradish peroxidase or to either of the fluorochrome dyes. The conjugated avidin staining technique is a reliable and simple method for identifying rodent and human mast cells, one that is useful as both an investigative and a clinical tool.

Topics: Animals; Ascitic Fluid; Avidin; Cytoplasmic Granules; Fixatives; Fluorescein-5-isothiocyanate; Fluoresceins; Fluorescent Dyes; Histocytochemistry; Horseradish Peroxidase; Humans; Male; Mast Cells; Mice; Mice, Inbred BALB C; Microscopy, Electron; Ovalbumin; Rats; Rats, Inbred Strains; Rhodamines; Skin; Staining and Labeling

Fluorescein and tetramethylrhodamine conjugates to protein or dextran were used to determine subcellular pH. The pH dependence of fluorescence of fluorescein isothiocyanate (FITC) conjugates could be described by a single proton dissociation (pK'a approximately 6.8). This allowed pH to be derived accurately from spectra using the simple Henderson-Hasslebach equation. FITC and TRITC conjugates were delivered to mouse macrophage lysosomes by pinocytosis. Lysosomal pH was then determined in several different ways. First, by direct matching of the subcellular fluorescence spectrum with calibration spectra obtained in free solution. Secondly, monensin was used to equilibrate internal and extracellular pH. Subcellular pH could then be determined by the relative increase in fluorescence of the FITC conjugate without loss of probe from the lysosomes. This allowed the calibration of pH dependence with the probe in situ. Finally, macrophages were permitted to pinocytose FITC and TRITC dextran conjugates simultaneously. pH could be determined from the ratio of emissions from the two dyes within the lysosomes. Each of these different methods gave a similar value for lysosomal pH (4.8 +/- 0.1).

Topics: Animals; Cells, Cultured; Coloring Agents; Dextrans; Evaluation Studies as Topic; Fluorescein-5-isothiocyanate; Fluoresceins; Hydrogen-Ion Concentration; Lysosomes; Macrophages; Mice; Monensin; Ovalbumin; Pinocytosis; Rhodamines; Spectrometry, Fluorescence; Thiocyanates; Xanthenes

The transport of proteins across continuous capillary endothelium is believed to be mediated by micropinocytic vesicles which shuttle molecules between the lumenal and abluminal plasma membrane. We have studied the ability of capillary endothelial cells isolated from rat epididymal fat to endocytose fluorescently labelled ovalbumin within micropinocytic vesicles. Net association of fluorescent ovalbumin with endothelial cells reaches an equilibrium after 40 minutes of incubation. This equilibrium is presumably due to a balance between endocytosis and subsequent exocytosis of this protein. Capillaries equilibrated with fluorescent ovalbumin exhibited rapid exocytosis of this protein when it was removed from the external medium. The rate of endocytosis was concentration dependent and obeyed the kinetics expected for adsorptive phase endocytosis. High concentrations of ovalbumin stimulated the ingestion of 14C-sucrose, a marker of fluid endocytosis, suggesting that protein can affect the movement of vesicles within the endothelial cytoplasm. These results imply that capillary endothelium isolated from rat epididymal fat exhibits the ability to endocytose and subsequently exocytose protein. This demonstrates that the two components of endothelial vesicular transport or transcytosis can be observed and studied in a system of isolated capillary endothelium.

Topics: Animals; Biological Transport; Capillaries; Endocytosis; Endothelium; Exocytosis; Fluorescein-5-isothiocyanate; Fluoresceins; Fluorescent Dyes; Furans; Male; Ovalbumin; Rats; Rats, Inbred Strains; Rhodamines; Spectrometry, Fluorescence; Thiocyanates

Isolated capillary endothelial cells exhibit an enhanced vesicular ingestion of nonenzymatically glucosylated myoglobin and ovalbumin. Proteins were incubated in the presence of glucose and the extent of nonenzymatic glucosylation was assessed using NaB3H4 reduction. Unmodified and glucosylated proteins were fluorescently labeled, and vesicular ingestion was quantified using a system of isolated capillary endothelium. Glucosylated myoglobin exhibited a 3.5-fold greater rate of vesicular injection as compared to unmodified myoglobin during the initial 30 min of ingestion. Glucosylated ovalbumin was vesicularly ingested at a rate 15-fold greater than the rate observed for unmodified ovalbumin during the initial 30 min of ingestion. These results indicate that glucosylation of small proteins may alter their subsequent recognition by the endothelial cell plasma membrane and result in increased sequestration within micropinocytic vesicles.

Topics: Animals; Biological Transport; Capillaries; Dogs; Endothelium; Fluorescein-5-isothiocyanate; Fluoresceins; Glucose; Lung; Myoglobin; Ovalbumin; Rhodamines; Spectrometry, Fluorescence; Thiocyanates