High Autofluorescence Research Articles

Separation of human alveolar macrophages by flow cytometry.

Alveolar macrophages (AM), which represent the major resident population of immunocompetent cells in the lower respiratory tract, have been implicated in the pathogenesis of acute lung injury in view of their exceptional capacity to release a large array of inflammatory mediators. The ex vivo analysis of these cells, accessible to bronchoalveolar lavage (BAL) is hampered by the fact that, under conditions of respiratory failure, the AM pool is heavily expanded by polymorphonuclear neutrophils (PMN), which necessitates separation of these cell populations. In the present study, we describe a flow cytometric approach to sort human AM obtained from BAL samples of both healthy volunteers (n = 10) and patients with severe pneumonia demanding mechanical ventilation (n = 10), using forward scatter and high autofluorescence characteristics to discriminate AM from PMN and lymphocytes. This technique yielded highly purified AM populations (>95%) as evidenced by morphological analysis, cytochemistry, and CD71 and CD14 expression of the sorted cells. The flow sorting process, per se, did not induce the expression of the acute-phase cytokine tumor necrosis factor-alpha (TNF-alpha) in control AM as determined by reverse transcriptase-polymerase chain reaction. Unstimulated and lipopolysaccharide-induced TNF-alpha protein secretion were comparable in sorted and unsorted AM as demonstrated by enzyme-linked immunosorbent assay. We suggest flow sorting of viable human AM as an efficient and nonperturbing separation technique to yield highly purified cell populations especially from PMN-rich BAL fluids of critically ill patients.

Surface CD4 Is Critical toin VitroHIV Infection of Human Alveolar Macrophages

The CD4 glycoprotein is the major cellular receptor for HIV. CD4 surface expression of monocytes decreases with time in culture while their susceptibility to HIV-1 increases. Our aim was to investigate whether this phenomenon occurs in macrophages that have differentiated in vivo by investigating CD4 expression and HIV-1 infection of human alveolar macrophages (AMs). Using flow cytometry to detect CD4 expression by Leu-3a labeled indirectly with fluorescein isothiocyanate or allophycocyanin, we found that CD4 was expressed at low but detectable levels, despite the high background autofluorescence well described in AMs. This finding was supported by the detection of CD4 mRNA in AMs using RT-PCR. T cell contamination of mRNA extracts of AMs was excluded by amplifying in parallel with primers to the constant region of the T cell receptor. Despite this low level of surface CD4, recombinant soluble CD4 and anti-CD4 antibody completely inhibited HIV-1 infection of AMs. We conclude that CD4, although expressed at low levels on the surface of AMs, appears to be critical to HIV-1 infection of these cells.

Microautoradiographic detection of CO2 fixation in lenticel chlorenchyma of youngFraxinus excelsior L. stems in early spring

Microautoradiography indicated that 1-year-oldFraxinus excelsior L. stem chlorenchyma assimilated external14CO2 in mid-April, when buds were swollen, but before bud-break. The lenticel regions showed the highest amount of radioactively labeled assimilates. Labeled assimilates declined in the tangential direction with increasing distance from lenticels, suggesting that14CO2 entered the stem through the open intercellular spaces of lenticels. In the radial direction, the amount of radioactively labeled assimilates did not constantly decline with growing distance from the lenticel entrance. It was high in all lenticel phelloderm cells, which had high chlorophyll autofluorescence and very small starch grains, highest in the adjacent 4–6 rows of chlorenchyma, which had larger starch grains that increased in size towards the interior rows, and much lower in the inner cortex chlorenchyma, which had large starch grains. We suggest that the main function of the lenticel chlorenchyma (lenticel phelloderm plus 4–6 rows of adjacent cortex chlorenchyma) is the refixation of respiratory CO2 which could easily leave the stem intercellular spaces, rather than the fixation of external CO2. The lenticel chlorenchyma could reduce the loss of respiratory CO2 by its photosynthetic activity.

Autofluorescence correction for fluorescence in situ hybridization.

Optimal sensitivity of fluorescence in situ hybridization (FISH) requires bright signals and low background fluorescence. Use of locus-specific probes is especially dependent on high sensitivity. Some tissue preparations show high autofluorescence, masking small or dim signals. We have developed a new method for subtracting autofluorescence from digital images on a pixel-by-pixel basis. It is based on the observation that fluorescent labels for FISH have narrower excitation and emission spectra than the chemical components responsible for autofluorescence. Our new approach uses calculation of the ratio of autofluorescence between multiple color images for correction of autofluorescence in each individual image. By subtracting autofluorescence components, we were able to enhance centromeric signals and make previously indistinguishable cosmid signals clearly visible. This image-processing approach to autofluorescence correction may widen the applicability of gene-specific probes in FISH analysis of tumor material.

Interaction of phosphatidylcholine liposomes with the human stratum corneum

The interaction of dimyristoylphosphatidylcholine liposomes with the human stratum corneum was investigated by confocal laser scanning microscopy and differential scanning calorimetry. Human skin is characterized by a high autofluorescence. By introducing appropriate optical filters the autofluorescence of the skin was depressed and the penetration profile of fluorescence labelled vesicles was investigated. From optical sectioning it was obvious that neither the vesicles nor the fluorophore N-(lissamine rhodamine B sulfonyl)diacylphosphatidylethanolamine (Rho-PE) penetrates in detectable amounts into the human skin. Differential scanning calorimetry of human stratum corneum revealed, that the peak positions of the human stratum corneum specific endothermic transitions at 10°C, 35°C, 50°C, 62°C, 73°C and 81°C did not change significantly after 18 h of non-occlusive vesicle application. However, the enthalpy of the transitions at 35°C, 50°C, 62°C and 73°C, estimated through peak heights increased, relative to the protein related peak at 81°C. A novel transition at 10°C was observed. From these data we conclude that DMPC liposomes do not penetrate intact into the human skin. We deduce, however, that the vesicles disintegrate at the surface of stratum corneum after non-occlusive application. The individual lipid molecules then interact with the lipid barrier of the stratum corneum and penetrate into the latter, which results in an increase of the enthalpy, related to the lipid components of the SC.

Dendritic cells and their precursors isolated from human bronchoalveolar lavage: immunocytologic and functional properties.

Human bronchoalveolar lavage (BAL) has been described to contain, besides a large number of alveolar macrophages (AM) (approximately 95%), small numbers of monocyte-like cells (approximately 2%) and dendritic cells (DC) (approximately 0.4%). To separate AM (high autofluorescence) from DC, we used a fluorescence activated cell sorter (FACS) to separate BAL cells into a low autofluorescent (LAF) fraction and a high autofluorescent (HAF) fraction. Immunocytologic and functional properties of these fractions were investigated. The LAF fraction was composed of acid phosphatase (APh)- and RFD9-negative cells, which were strongly positive for HLA-DR, L25, RFD1, and CD68. A portion of these cells expressed CD1a (22%) and My4 (60%). The marker pattern of these cells is reminiscent to that of intraepithelial bronchial DC and to that of blood DC. The majority of the LAF cells had a monocyte-like morphology, but after overnight culture the percentage of LAF cells with long cytoplasmic extensions (DC morphology) was strongly augmented (from 18 to 51%). The HAF fraction contained 100% AM, strongly positive for APh, HLA-DR, CD68, RFD7, and RFD9. In culture, the LAF cells formed clusters with T cells and vigorously stimulated the proliferation of allogeneic T cells and naive (CD45RO-negative) T cells. BAL and LAF cells produced higher responses in nonsmokers than in smokers. In contrast, HAF cells did not form clusters with T cells and did not stimulate allogeneic T cell proliferation. HAF cells even suppressed mitogen-driven T cell proliferation.(ABSTRACT TRUNCATED AT 250 WORDS)

Autofluorescence in cataractous human lens and its relationship to light scatter.

The autofluorescence profile of the lens was measured from 84 eyes of 84 patients with cortical, nuclear, posterior subcapsular, or mixed lens opacities. Measurements were performed with a fluorometer in the blue-green autofluorescence range (495 nm/520 nm). The mean maximum autofluorescence value differed in every cataract group statistically significantly from that of the age matched controls (p < or = 0.0058). The highest autofluorescence values were measured in nuclear and mixed cataract groups (p < 0.0001) with high and narrow autofluorescence profile. In cortical cataracts the curve was low and flattened and the mean maximum autofluorescence value was lower than in the control eyes (p < 0.0001). The maximum autofluorescence was related to lens coloration as well as to visual acuity only in nuclear cataract. The regression between maximum autofluorescence and light scatter was statistically significant only in the nuclear cataract group (p = 0.0004). Since the autofluorescence profiles differed not only in height but also in width between the cataract groups, various width/maximum autofluorescence ratios were measured. In nuclear and mixed cataract groups the ratio 75% width/maximum autofluorescence was statistically significantly lower than in other groups (p < 0.0001). In cortical cataracts the ratio (50% width - 75% width)/maximum autofluorescence was statistically significantly higher than in other groups (p < 0.0001).

Flow Cytometric Analysis of I-J Expression on Murine Bone Marrow-Derived Macrophages

Attempts to analyze bone marrow-derived macrophages (BMDM) by flow cytometry have been prohibited because of their high autofluorescence. Using an autofluorescence reduction method of Steinkamp and Stewart to reduce the autofluorescence of BMDM, we were able to examine several macrophage populations for their expression of I-A, I-J, and Mac-1 cell surface determinants. Bone marrow cells examined immediately after removal from the femur contain 50-60% Mac 1-positive cells (mainly granulocytes). During the next few days granulocytes and nonmacrophage precursor cells die, and the number of Mac 1-positive cells decrease. Once the bone marrow cells have been maintained in L cell conditioned medium (LCM) for 2 to 3 days, the number of cells expressing Mac 1 increases rapidly from 20% to 98% during the next 3 to 4 days. Bone marrow cells grown in LCM do not express I-J until these cells have been in culture 3 to 4 days, and the number of cells expressing I-J (up to 90% positive) parallels the increase in macrophages. Bone marrow cells maintained in LCM did not express detectable I-A during the 14 days these cells were examined. Two other macrophage populations often used in a variety of immunological studies were analyzed by flow cytometry. We found that the majority (up to 80%) of peritoneal cells expressed I-A, and only 20% of peritoneal cells had I-J cell surface determinants. On the other hand, peritoneal exudate cells collected 4 days after thioglycolate medium treatment were predominantly I-J positive (up to 70%), and only about 30% of these cells expressed I-A cell surface antigens. The binding of anti-I-J IgM antibody to BMDM was not to Fc receptors because pretreating these cells with up to 25 micrograms of an IgG2a myeloma protein did not block anti-I-J antibody binding. The addition of 25-200 micrograms of monoclonal anti-Fc receptor antibody was also ineffective in blocking the binding of a monoclonal anti-I-Jk antibody to BMDM. Pretreatment of BMDM with the IgM fraction of several control IgM antibody preparations did not block the specific binding of fluoresceinated anti-I-J IgM antibody. BMDM provide a pure population of macrophages that express a significant level of cell surface I-J antigens. Bone marrow cells grown in LCM are essentially devoid of other contaminating cells, and the increase in the number of I-J-positive cells parallels the increase in macrophages in these cultures.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of subsets of bone marrow-derived macrophages by flow cytometry analysis.

Normal C3H bone marrow cells were grown 7 days in medium containing L cell-derived colony stimulating factor-1 (CSF-1). During the first 4 days of culture, erythroid and granulocytic cells decreased while macrophages increased exponentially with a doubling time of about 31 hr. Only 0.3% of all cells in the initial bone marrow suspension formed discrete colonies of mononuclear phagocytes, but by day 6 60% of the nonadherent cells were capable of forming macrophage colonies, representing a 200-fold enrichment of the original progenitor population. Using flow cytometry, mononuclear phagocytes obtained after 4 days of culture were separated into two distinct phenotypes based on their autofluorescence. Nonadherent cells were a discrete population of small cells exhibiting low autofluorescence, and the adherent cells were a broad heterogeneous population of large cells exhibiting high autofluorescence. A panel of currently available rat monoclonal antibodies (MABs) against murine hematopoietic cells were used to determine whether unique subsets of macrophages could be resolved. The MABs RA 31B6 and H-11 stained virtually all the nonadherent cells but not adherent cells. The MABs E-2 and 11-4.1 (anti-H-2Kk) stained almost all the adherent cells and demonstrated no significant staining of nonadherent cells. Nearly all the nonadherent and adherent cells were stained by the MABs DNL 4.4 and MAC-1. Additionally, the data suggest that the epitopes for MAC-2 and MAC-3 and gamma 2a Fc receptors develop late in nonadherent progenitor cells as they mature into adherent macrophages.

Rapid acidification of endocytic vesicles containing asialoglycoprotein in cells of a human hepatoma line.

Acidification of endocytic vesicles has been implicated as a necessary step in various processes including receptor recycling, virus penetration, and the entry of diphtheria toxin into cells. However, there have been few accurate pH measurements in morphologically and biochemically defined endocytic compartments. In this paper, we show that prelysosomal endocytic vesicles in HepG2 human hepatoma cells have an internal pH of approximately 5.4. (We previously reported that similar vesicles in mouse fibroblasts have a pH of 5.0.) The pH values were obtained from the fluorescence excitation profile after internalization of fluorescein labeled asialo-orosomucoid (ASOR). To make fluorescence measurements against the high autofluorescence background, we developed digital image analysis methods for estimating the pH within individual endocytic vesicles or lysosomes. Ultrastructural localization with colloidal gold ASOR demonstrated that the pH measurements were made when ligand was in tubulovesicular structures lacking acid phosphatase activity. Biochemical studies with 125I-ASOR demonstrated that acidification precedes degradation by more than 30 min at 37 degrees C. At 23 degrees C ligand degradation ceases almost entirely, but endocytic vesicle acidification and receptor recycling continue. These results demonstrate that acidification of endocytic vesicles, which causes ligand dissociation, occurs without fusion of endocytic vesicles with lysosomes. Methylamine and monensin raise the pH of endocytic vesicles and cause a ligand-independent loss of receptors. The effects on endocytic vesicle pH are rapidly reversible upon removal of the perturbant, but the effects on cell surface receptors are slowly reversible with methylamine and essentially irreversible with monensin. This suggests that monensin can block receptor recycling at a highly sensitive step beyond the acidification of endocytic vesicles. Taken together with other direct and indirect estimates of endocytic vesicle pH, these studies indicate that endocytic vesicles in many cell types rapidly acidify below pH 5.5, a pH sufficiently acidic to allow receptor-ligand dissociation and the penetration of some toxin chains and enveloped virus nucleocapsids into the cytoplasm.

Flow cytometry analysis of lung cells from normal and acid-treated rabbits.

Lavaged lung cells from normal rabbits and rabbits given hydrochloric acid intratracheally were analyzed using flow cytometry techniques. The rabbit alveolar macrophage was characterized as a cell with relatively high light scatter and high intrinsic autofluorescence compared with the lung neutrophil with its lower light scatter and autofluorescence. The flow cytometry characteristics of these 2 cell populations were confirmed with light microscopy after cell sorting. A flow cytometry analysis of alveolar macrophage phagocytosis using fluorescent latex particles was applied to normal rabbit alveolar macrophages. These studies showed that flow cytometry can be used to study the morphologic features and function of rabbit lavage lung cell populations and may be ultimately useful in the analysis of human lung cells.