Flow cytometric analysis of colonic and small intestinal mucosal lymphocytes obtained by endoscopic biopsy in the healthy dog.

Flow cytometric analysis of the lymphocyte population of the gut could provide useful information on the immune cells present in the gut that would not be easily obtained in tissue sections. However, little is known of the normal lymphocyte population in the canine gut as determined by flow cytometry, which allows for simultaneous staining of multiple cell surface antigens and identification of specific lymphocytic subsets. Therefore, intraepithelial lymphocytes were obtained from biopsies of the healthy canine proximal small intestine and colon taken with an endoscope, and flow cytometric analysis was used to characterize the lymphocyte subsets present. Endoscopic biopsy of the intestine is a minimally invasive technique commonly used for diagnostic purposes. Although CD3+ lymphocytes were the most abundant subset in both colon and small intestine, CD3+/CD8- lymphocytes predominated in the proximal small intestine, whereas CD3+/CD8+ lymphocytes did in the colon. Canine CD8+ intraepithelial lymphocytes were predominantly CD8alphabeta+ in both small intestine and colon. CD4+ intraepithelial lymphocytes were always much less numerous than CD8+ intraepithelial lymphocytes. As in man, a majority of intraepithelial lymphocytes expressed the T-cell receptor, TCRalphabeta, but TCRgammadelta was expressed by a third of intraepithelial T-cells in the proximal small intestine, and approximately 15% of those in the colon. Very few CD21+ lymphocytes were detected in samples of healthy canine colon and small intestinal intraepithelial cells. We have showed that canine intraepithelial lymphocytes are regionally specialized, and that those from the small intestine are unique in comparison to those of other species such as man and rodents due to the large numbers of CD3+/CD8- intraepithelial lymphocytes. This study provides a baseline for comparison with intraepithelial lymphocytes obtained from canine patients with intestinal disease.

Flow cytometric analysis of canine colonic mucosal lymphocytes from endoscopically obtained biopsy specimens.

OBJECTIVE: To validate use of canine colonic biopsy specimens obtained via endoscopy as a source of mucosal lymphocytes (ML) for flow cytometric analysis. SAMPLE POPULATION: Mucosal biopsy specimens from 10 adult dogs. PROCEDURE: Mucosal lymphocyte subsets obtained from excised colon were compared with ML subsets obtained from biopsy specimens obtained by use of an endoscopic forceps (6 dogs). Endoscopic colonic biopsy specimens from 4 other dogs were used to define whether obtained ML were predominantly of intraepithelial or lamina propria origin. Mucosal lymphocytes were isolated and labeled, using commercially available monoclonal antibodies directed against canine cell surface antigens. Lymphocyte subsets (cytotoxic or helper T cells; B cells) were determined by use of flow cytometric analysis. RESULTS: A large number of viable ML was obtained after dissociation of the colonic epithelium from excised colon (45.5 + 21.5 X 10(6)) and endoscopic (7.2+/-3.4 X 10(6)) biopsy specimens. Lymphocyte subsets obtained with both methods were identical for each dog and consisted predominantly of intraepithelial lymphocytes, with some lymphocytes from the lamina propria. Collagenase digestion of excised colon also yielded a large number of viable lymphocytes from the lamina propria (56.7+/-20.4 X 10(6)), but collagenase digestion of endoscopic biopsy specimens was less rewarding. CONCLUSION AND CLINICAL RELEVANCE: A representative sample of viable intraepithelial ML is obtainable from endoscopic biopsy specimens. Flow cytometric analysis, a minimally invasive technique, can be used to study ML of client-owned animals.

Evidence for redistribution-associated intracellular pK shifts of the pH-sensitive fluoroprobe carboxy-SNARF-1.

Properties and peculiarities of the pH-sensitive fluoroprobe carboxy-seminaphthorhodafluor-1 (carboxy-SNARF-1), in view of pHi measurements in single cells, were evaluated using confocal laser scanning microscopy. It was found that in human malignant glioma cells (U 118 MG) grown in multicellular spheroid culture, intracellular calibration curves (nigericin method) varied from one cell to another despite emission ratioing of the fluorescence signals. In addition, considerable deviations between indicator calibration in cell-free solution and intracellular calibration were observed. Microspectrofluorometric measurements revealed that these deviations are attributable to intracellular pK shifts of the indicator rather than to spectral changes of the fluorescence emission. The observed pK shifts are probably due to intracellular redistribution of the indicator between cytosol and lipophilic cell compartemants, e.g. plasma membrane, since the indicator can even be loaded efficiently into the cells via its active acid form (instead of the acetoxymethyl ester form). An approximate theoretical derivation of a cellular calibration curve confirms that a reversible, pH-dependent intracellular redistribution of the protonated indicator component results in an apparent pK shift of delta pK = log(1 + epsilon.P), with P the partition coefficient and epsilon a factor that depends on the different mean layer thicknesses of the cytosol and plasma membrane. Since the apparent pK shift amounts to about 1 pH unit in tumour cells of spheroids, the intracellular pH measuring range of carboxy-SNARF-1 is almost restricted to alkaline pH values. Further consequences of the redistribution phenomenon are discussed with special respect to intracellular ion imaging.

Three-dimensional imaging of rhodamine 123 fluorescence distribution in human melanoma cells by means of confocal laser scanning microscopy.

Three-dimensional (3D) imaging of intracellular rhodamine 123 fluorescence distribution was performed by means of confocal laser scanning microscopy (CLSM). Human IGR melanoma cells grown in monolayer or multicellular spheroid culture were studied for elucidating mitochondrial membrane potential characteristics, and cell and nucleus volume dimensions. Microspheres 6 microns in diameter loaded with rhodamine B were used to calibrate our instruments for performing 3D imaging of optical sections as obtained by CLSM. Accurate optical slicing is only possible taking into consideration the physical characteristics of the objectives used like chromatic and spherical aberrations, depth discrimination or cover slip correction and the temperature dependence of the immersion medium. While 3D imaging of optical slices can be carried out showing the original shape of the object being tested without physical distortion, 3D images of microspheres show well-reproducible structures of rhodamine B fluorescence. These can be explained by a superposition of two effects, namely scattering of the fluorescence light and a gradient of the electromagnetic field strength of the laser beam due to the shape of the object. 3D imaging of optical slices of IGR cells in monolayer or multicellular spheroid culture, which have been loaded with rhodamine 123, show the location of the dye predominantly within the cytoplasm of the cells with a remarkable heterogeneity of fluorescence intensity within and between single cells, indicating differences in the mitochondrial membrane potential and thus in the metabolic activity. Due to the heterogeneity of the cell shape the cell nucleus occupies between 4 and 14% of the total cell volume. These data reveal calibrated 3D imaging as a valuable noninvasive tool to visualize the heterogeneity of cell parameters under different cell culture conditions.

Rac1, and not Rac2, is involved in the regulation of the intracellular hydrogen peroxide level in HepG2 cells.

In order to elucidate the components of the oxygen sensory complex in HepG2 cells which regulates the production of erythropoietin, we have microinjected recombinant variants of the human small GTP-binding protein hRac1 and measured their effects on the production of reactive oxygen species (ROS) by the dihydrorhodamine-123 technique. The dominant-negative mutant hRac1(T17N) inhibits the NADH-stimulated production of ROS in HepG2 cells, whereas the constitutively activated hRac1(G12V) leads to an increase in intracellular ROS concentration. Reverse transcriptase PCR analysis showed that the hRac1, but not the hRac2, gene is expressed in HepG2 cells. These results demonstrate that hRac1, and not hRac2, is involved in the regulation of ROS production in HepG2 cells and suggest that hRac1 specifically functions in the non-phagocytic NAD(P)H oxidase complex.

Cytochromes and oxygen radicals as putative members of the oxygen sensing pathway.

This study applies biophysical methods like light absorption spectrophotometry of cytochromes, determination of NAD(P)H-dependent superoxide anion (O2-) formation and localisation of hydroxyl radicals (*OH) by 3-dimensional (3D) confocal laser scanning microscopy to reveal in human cells putative members of the oxygen sensing signal pathway leading to enhanced gene expression under hypoxia. A cell membrane localised non-mitochondrial cytochrome b558 seems to be involved as an oxygen sensor in the hepatoma cell line HepG2 in cooperation with the mitochondrial cytochrome b563 probably probing additionally metabolic changes. *OH the putative second messenger of the oxygen sensing pathway generated by a Fenton reaction could be visualized in the perinuclear space of the three human cell lines used. Substances like cobalt or the iron chelator desferrioxamine, which have been applied in HepG2 cells to mimic hypoxia induced gene expression, interact on various sides of the oxygen sensing pathway confirming the importance of b-type cytochromes and the Fenton reaction.