Rhodamine 123 phototoxicity in laser-irradiated MGH-U1 human carcinoma cells studied in vitro by electron microscopy and confocal laser scanning microscopy.

Rhodamine 123 (R123) is a permeant, cationic, fluorescent dye that localizes preferentially within mitochondria of living carcinoma cells. MGH-U1 human bladder carcinoma cells incubated in vitro with 10 microM R123 for 30 min and then irradiated at 514.5 nm with an argon ion laser underwent selective, phototoxic injury to mitochondria. Ultrastructurally, treatment with R123 plus irradiation with 10 J/cm2 caused selective, progressive mitochondrial alterations consisting of disruption of cristae, vacuolization, swelling, increasing numbers of ring-shaped and angulated mitochondria at 4 to 8 h after irradiation, and obliteration of many mitochondria at 24 to 48 h. Confocal laser scanning microscopy after treatment with R123 plus irradiation with 10 to 30 J/cm2 demonstrated altered uptake and localization of subsequently administered R123, accompanied by striking mitochondrial fragmentation. Irradiation caused a dose-dependent depletion of extractable R123, due to a photosensitized efflux that began immediately and progressed by 4 h after irradiation with 10 to 30 J/cm2; further uptake after reincubation in the presence of R123 was also quantitatively impaired in cells previously irradiated with 30 J/cm2.

Wavelength and fluence effect on vascular damage with photodynamic therapy on skin.

Normal skin phototoxicity is clinically predictable during photodynamic therapy with light at 690 and 458 nm wavelengths, in the first 5 h after intravenous bolus infusion of benzoporphyrin derivative mono-acid ring A. This study goal was to determine histologic milestones that lead to tissue necrosis with exposure to red (690 nm) and blue (458 nm) light. The threshold doses for skin necrosis on rabbits were equal at both wavelengths. Lower, equal to, and higher than threshold fluences were delivered in duplicates at hourly intervals, with 40% increments, at constant irradiance. Pathology specimens from irradiated and control sites, were collected at 0, 2, 7, 24, 48 h, and 2 wk after treatment and were paired to equivalent treated sites for clinical evaluation. Immediately after irradiation, at 690 and 458 nm thresholds, light microscopy showed stasis and inflammatory infiltrate in the papillary dermis, respectively; electron microscopy demonstrated pericyte and endothelial cell damage - greater at 690 than 458 nm. At day 1, vascular stasis in the dermis showed a steeper dose-response with red than blue light, and led to necrosis of skin appendages (day 1) and epidermis (days 1-2) at both wavelengths. Sub-threshold fluences induced similar, but significantly milder (p < 0.05) changes and epidermis recovered. Skin necrosis, at threshold fluences in photodynamic therapy with benzoporphyrin derivative mono-acid ring A, was primarily due to vascular compromise to a depth potentially reaching the subcutaneous muscle at 690 nm, whereas at 458 nm vascular damage was confined to upper dermis. This system facilitates selective destruction of skin vasculature, sparing normal epidermis.

Morphologic and ultrastructural examination of I-A+ cells in the murine iris.

The surface membrane expression of major histocompatibility (MHC) class II antigens is an important prerequisite for presentation of foreign antigens to the immune system. Because particular antigens that are placed within the anterior chamber of the eye elicit a deviant form of immunity in which effector delayed-type hypersensitivity responses are suppressed, it has been proposed that novel MHC class II antigen-bearing cells exist in the tissues that line the anterior chamber. Class II MHC antigen expression has been identified within the iris, but the detailed morphologic description of these cells is incomplete. With the use of in situ immunoperoxidase and immunoelectron microscopic techniques, we examined the morphologic and ultrastructural characteristics of resident MHC-positive class II (I-A+) cells in murine irises. A significant number of these cells was found in the connective tissue of BALB/c irises. The majority showed extensive dendritic morphologic characteristics and formed a network throughout the iris that did not overlap. Ultrastructurally, I-A+ cells had an indented nucleus, some vacuoles, lysosomes, mitochondria, and an occasional phagosome within their cytoplasm and an absence of desmosomes or other intercellular junctions. Based on these features, it is unlikely that these cells are epithelial or endothelial in origin, but rather are similar to cells of the monocyte/macrophage/dendritic cell lineage. These results show the presence of an I-A+ dendritic cell population, within the murine iris, distributed in a pattern that is similar to that of Langerhans cells in the skin. Due to their compartmentalization within the eye, this cell population may represent a novel antigen-presenting cell that contributes to the immunologic privilege of the anterior chamber.

Immunohistochemical staining of the human anterior segment. Evidence that resident cells play a role in immunologic responses.

We examined human corneoscleral tissue for cells that are phenotypically similar to known antigen-presenting cell (APC) populations. Antigen-presenting cells are involved in the uptake and processing of antigen for presentation to T lymphocytes, thereby playing a central role in induction of the immune response. The recognition of antigen by T lymphocytes requires that an APC express major histocompatibility complex class II molecules. Using immunoperoxidase staining techniques, the presence of cells expressing class II glycoproteins and T-cell subsets were determined. The staining patterns of the trabecular meshwork, ciliary body, cornea/sclera, and conjunctive are described for monoclonal antibodies OKT6, OKM1, HLA-DR, and HLA-DQ, and T-cell markers OKT8, Leu-3a, and Leu-4. The results of the present study demonstrate that the anterior chamber contains a network of immunocompetent cells. The presence of a subpopulation of cells within the anterior chamber that express class II glycoproteins of the major histocompatibility complex suggests this tissue may play an important role in immune regulation within the eye.

Photodynamic destruction of lysosomes mediated by Nile blue photosensitizers.

Previous studies have established that a number of Nile blue derivatives are potent photosensitizers and that they are localized primarily in the lysosomes. The present study examines whether the lysosome is a main target of the photocytotoxic action mediated by these sensitizers. Chosen for this study were NBS-6I and sat-NBS, which represented, respectively, derivatives with high and moderate degrees of lysosomal. This is indicated by the light-and drug-dose-dependent losses of acid phosphatase staining particles, reduction of hexosaminidase in the lysosome-containing subcellular fraction, and impairment of the lysosomes to take up and sequester acridine orange. Ultrastructurally, swollen and ruptured lysosomes were seen as one of the first evidences of cell damage mediated by these photosensitizers. However, the study also showed that sat-NBS, which is less lysosomal selective, was less effective in mediating lysosomal destruction. Also, the degree of lysosomal destruction mediated by sat-NBS did not parallel the degree of cytotoxicity generated. This implies that for derivatives that are not exclusively localized in the lysosome, other subcellular sites may also be damaged by the photodynamic action and may play a role in the photocytotoxic process.

Phagocytosis by trabecular meshwork cells: sequence of events in cats and monkeys.

Trabecular meshwork cells have well-established phagocytic properties. However, the sequential stages of this process have not been studied in detail. Zymosan particles, yeast cell walls 3 micron in diameter, were either injected or perfused into the anterior chambers (AC) of cats and monkeys. Glutaraldehyde (3%) was then infused into the AC at 1-, 6-, 17-, 30- and 60 min and the fate of the particles observed by transmission electron microscopy. After contact with zymosan particles, trabecular meshwork cells demonstrated short cytoplasmic extensions at contact sites and an increased number of cytoplasmic organelles. Internalization of the particles occurred by 6 min in the cat and by 17 min in the monkey. In both species similar events occurred following internalization of zymosan: trabecular cells, laden with particles, rounded up, detached from their underlying collagenous beams and migrated towards Schlemm's canal or the aqueous plexus. By 1 hr the individual trabecular cells completed the entire sequence of phagocytic events although some cells were always observed in earlier stages of the process. These data document for the first time the timing of the cellular events that take place during zymosan particle phagocytosis by trabecular meshwork cells.

Apoptosis and morphologic changes in drug-treated trabecular meshwork cells in vitro.

Using an in vitro culture system, we investigated whether bovine trabecular meshwork cells undergo apoptosis (programmed cell death) following exposure to anti-glaucoma medications (timolol, pilocarpine and epinephrine) and known inducers of apoptosis (5-fluorouracil, mitomycin-C and dexamethasone). Third to fifth passage bovine trabecular meshwork cells were grown to confluence and incubated for 1-12 days in growth media with timolol (1-1000 microM), pilocarpine (15-15,000 microM), epinephrine (5-5000 microM), 5-fluorouracil (10-100 micrograms ml-1), mitomycin-C (0.01-100 micrograms ml-1) and dexamethasone (0.01-100 microM). The cultures were evaluated for apoptosis by phase-contrast microscopy, transmission electron microscopy and in situ apoptosis labeling. 5-Fluorouracil (10-100 micrograms ml-1), mitomycin-C (0.1-100 micrograms ml-1) and epinephrine (500-5000 microM) induced apoptosis in a dose and time-dependent manner. Timolol, pilocarpine, and dexamethasone-treated specimens did not show evidence of apoptosis at any of the concentrations tested. Trabecular meshwork cells incubated in timolol (100-1000 microM) developed cytoplasmic granules, and specimens treated with pilocarpine (15,000 microM) developed cytoplasmic vacuoles. These granules and vacuoles have the appearance of secondary lysosomes. Dexamethasone-treated cells developed an increased number of mitochondria. This study suggests that the trabecular meshwork may undergo apoptosis following exposure to 5-fluorouracil, mitomycin-C and epinephrine. Timolol, pilocarpine and dexamethasone did not induce apoptosis. However, these drugs can incite characteristic morphologic changes in cultured trabecular meshwork cells.

Establishment of calf trabecular meshwork cell cultures.

Trabecular meshwork cell cultures have been grown from excised calf aqueous outflow pathway tissue using two different methods. In one method, tissue explants were placed directly in culture dishes and primary cultures were established by those cells migrating from the tissue. In the second method, a cell suspension was generated by incubating trabecular tissue strips with 0.1% pronase, and the washed cells were used to seed primary cultures. The cell cultures generated by each method appeared to progress through morphological and biochemical changes characteristic of the cell type, although at different respective rates. Comparisons of the cell morphologies, biosynthetic activities, and growth characteristics of the respective subcultures suggested that similar cell populations were obtained by each method. These studies further suggested that sufficient quantities of calf trabecular cells could be grown in culture to permit biochemical and pharmacological studies of trabecular-cell metabolism.