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PubMed1994

Differences in transcription and promoters of Xmrk-1 and Xmrk-2 genes suggest a role for Xmrk-2 in pigment pattern development in the platyfish, Xiphophorus maculatus.

Pigment (macromelanophore) patterns in the platyfish Xiphophorus maculatus are due to a complex pigmentary locus; for example, the spotted-dorsal (Sd) fin pattern is due to the Sd locus. In interspecific backcross hybrids with the swordtail X. helleri, the Sd pattern changes into benign or malignant dorsal fin melanoma as a result of hemi- or homozygous loss of a platyfish regulatory (R) gene, the tumor suppressor gene Diff, that appears to play a role in the final differentiation of macromelanophores. Closely linked to the pigmentary locus is an epidermal growth factor receptor-like gene, Xmrk-2, that has arisen by duplication from the linked Xmrk-1. The transcriptional expression of the Xmrk genes was determined in various tissues including Sd pigment patterns and melanomas of various growth potential using reverse transcription-polymerase chain reaction. While Xmrk-1 expression was found in all tissues examined, Xmrk-2 expression correlated with pigment cell growth. Xmrk-2 was highly expressed in the dorsal fin exhibiting the Sd pattern but drastically reduced in a platyfish mutant which has lost the capacity to form these pigment cells in the dorsal fin. Most interestingly, Xmrk-2 expression increased with the proliferative capacity of the melanomas but declined once melanoma growth ceased. We conclude that Xmrk-2 plays a role in the formation of the pigment pattern cell type, perhaps in proliferation of precursor cells, which, in melanoma, are kept in a proliferative state due to loss of Diff.(ABSTRACT TRUNCATED AT 250 WORDS)

Alleles↗
PubMed1995

Comparative ocular pharmacokinetics of brimonidine after a single dose application to the eyes of albino and pigmented rabbits.

Brimonidine is a potent ocular hypotensive drug. The ocular pharmacokinetics of 14C-brimonidine in albino and pigmented rabbits were compared after ocular instillation of a 35-microliters drop of a 0.5% 14C-brimonidine solution. Ocular drug and metabolite concentrations were measured as total radioactivity and by a selective HPLC method. Rapid ocular absorption resulted in peak drug concentrations in aqueous humor of 2.16 +/- 0.75 micrograms/ml (mean +/- SD) and 1.52 +/- 0.38 micrograms/ml at 0.67 hr postdosing in albino and pigmented rabbits, respectively. Drug elimination from aqueous humor was rapid initially with a half-life of 1 hr in rabbits, followed by a slower decline phase in pigmented rabbits. Radioactivity concentration in iris-ciliary body of albino rabbit reached a peak of 5.04 micrograms-eq/g at 40 minutes and declined to 0.10 micrograms-eq/g at 6 hr postdosing with a half-life of 1 hr. The radioactivity concentrations in pigmented iris-ciliary body rose to a peak of 20.1 micrograms-eq/g at 1.5 hr and stayed relatively steady for at least 4 hr before declining slowly to 0.43 micrograms-eq/g 90 days postdose. The terminal half-life of brimonidine in pigmented iris-ciliary body was 160 hr. Three metabolites were detected in the conjuctiva and iris-ciliary body, and brimonidine was the major drug-related substance in aqueous humor and iris-ciliary body. The results indicate that brimonidine is absorbed rapidly into rabbit eyes, metabolized in ocular tissues, and has significant affinity for melanin-containing tissues.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenergic alpha-Agonists↗
PubMed1995

Ocular pigmentation protects the rabbit retina from gentamicin-induced toxicity.

PURPOSE: This study was designed to investigate the possibility that gentamicin-induced retinal toxicity is dependent on ocular pigmentation by comparing the effects of the drug on the functional and morphologic integrity of the retina in albino and pigmented rabbits. METHODS: In each rabbit, a solution of gentamicin sulfate was injected into the vitreous of one eye, and saline was injected into the other eye. Retinal function was assessed by electroretinogram (ERG) at different time intervals after injection. Retinal structure was examined at the light microscopic level. RESULTS: In albino and pigmented rabbits, functional retinal damage developed to a maximal level within the first week after gentamicin injection. Thereafter, gradual recovery could be seen in eyes that suffered less than 80% maximal reduction in the ERG b-wave. For each dose >0.1 mg studied, retinal damage was more severe in the albino rabbits than in the pigmented ones. The degree of damage was not affected by the level of ambient illumination, nor was it reduced by the administration of N-acetylcystein, a free radical scavenger, together with gentamicin. CONCLUSIONS: Ocular pigmentation partially protects the rabbit retina from the toxic action of gentamicin. This protection probably reflects binding of the drug by the melanin, which thereby reduces the concentration of the free gentamicin. When the initial gentamicin-induced retinal damage is expressed in < 80% reduction in the ERG, substantial recovery may occur in both strains of rabbits.

Albinism↗
PubMed1995

[Histopathological studies on the rds mouse retina--relationship between photoreceptor and pigment epithelial cells].

We used light and electron microscopy to examine detailed ultrastructural changes in the course of the development and degeneration of the rds mouse (rds/rds) retina with particular reference to the relationship between the photoreceptor and retinal pigment epithelial cells. During the first 6 postpartum days, the mutant retina showed the same developmental pattern as in normal mice. After that, however, rds mouse retinas failed to develop the photoreceptor outer segments. After 14 days, the photoreceptor inner segments and the cilia were all arranged parallel in the same direction in which the pigment epithelial cells extend their long microvilli. The inner segments and the cilia were very long and surrounded by the microvilli of the retinal pigment epithelial cells in the mutant retinas 21 days postpartum or latter. Some inner segments and cilia showed cytoplasmic degeneration in the part surrounded by the microvilli, while the other part appeared normal. There were pigment epithelial cells that contained fragments of the inner segments. The photoreceptor nucleus and inner segment were reduced in number after 21 days. These findings indicate that the pigment epithelial cells play a role in photoreceptor degeneration in rds mice.

Animals↗
PubMed1994

Natriuretic peptides and their receptors in human neural retina and retinal pigment epithelium.

Atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) play a role in transepithelial fluid movement in the kidney and at the blood-aqueous barrier. We sought a similar natriuretic peptide-mediated regulatory system at the level of the blood-retinal barrier by investigating human neural retina and retinal pigment epithelium for the presence of ANP, BNP and their receptors. ANP and BNP binding to receptors could be demonstrated autoradiographically in all layers of the retina inclusive of the retinal pigment epithelium. Competitive preincubation with unlabeled peptides blocked the binding of the respective radioactive peptide. ANP and BNP could also be demonstrated immunohistochemically in both the neural retina and the retinal pigment epithelium. Our results suggest a role of these peptides both in the regulation of intraretinal fluid movement and--by analogy with other peptides--as possible neutrotransmittors. The localisation of ANP and BNP in the retinal pigment epithelium suggests that these peptides may influence ocular fluid homeostasis at the outer blood-retinal barrier by modulating pigment epithelial function.

Atrial Natriuretic Factor↗
PubMed1976

Mechanism of recurrence of pigmented nevi following dermabrasion.

A study was made of pigment freckles which recur after skin abrasion of spotted grouped pigmented nevi, according to the lapse of time, chiefly by means of the fluorescence method of Falck and Hillarp. The mechanism of recurrence is summarized as follows. Nearly simultaneously with the epidermal regeneration, dendritic melanin-producing cells derived from hair follicles and eccrine sweat ducts appear in the basal and prickle-cell layers of epidermis. These cells then create junction activity in the basal layer of epidermis, in the hair follicles and in the eccrine sweat duct walls. Finally, these nevus cells drop off into the underlying layer of scar tissue along the epidermal appendages. The dendritic melanin-producing cells seen in the early regenerative pigment freckles were thought to be incompletely differentiated nevus cells in the pre-stage of junction nevus formation. Appearance of dendritic melanin-producing cells and formation of junction activity in the eccrine sweat duct walls suggest the following possibilities. (1) In the cases of spotted grouped pigmented nevus which we studied, nevus cell proliferation also occurred eccrine-centrically on pathogenesis. (2) An important role is played by the eccrine sweat ducts in the recurrence of ordinary pigmented nevi after incomplete removal. (3) Eccrine-centered nevus cells are derived from nevoblasts in the eccrine sweat duct walls.

Adult↗
PubMed1994

Plasmodium falciparum pigment induces monocytes to release high levels of tumor necrosis factor-alpha and interleukin-1 beta.

We show that high levels of tumor necrosis factor-alpha (TNF-alpha) activity were consistently detected when monocytes were cocultured with Plasmodium falciparum schizont stage-parasitized erythrocytes that subsequently ruptured. Isolated pigment recovered from ruptured schizonts was found to specifically induce monocyte release of high levels of TNF-alpha and interleukin-1 beta (IL-1 beta). Particulate free-culture supernatant that contained various soluble parasite macromolecules induced relatively low levels of TNF-alpha and IL-1 beta. When isolated pigment was treated with protease, the monokine inducing-activity was abolished. Isolated pigment prepared from different natural isolates of P. falciparum stimulated variable levels of monokine production. We propose that in vivo, malaria pigment from parasites sequestered in the host microvasculature is a physiologically relevant moiety that interacts with monocytes and stimulates the release of TNF-alpha and IL-1 beta. These observations suggest that malaria pigment may be a virulence factor in the monokine-mediated induction of organ-specific and systemic pathophysiology in falciparum malaria.

Animals↗
PubMed1994

[Pigmentation on the anterior chamber angle in retinal detachment associated with atopic dermatitis].

We reported pigmentation on the anterior chamber angle in twenty-two patients suffering from atopic dermatitis. Dense pigmentation on the anterior chamber angle was observed in all eight eyes with retinal detachment and was evident before clinical recognition of detached retina. Minimal or slight pigmentation was also evident in eyes without retinal detachment. Increased pigmentation on the anterior chamber angle is a characteristic and predictive sign of retinal detachment associated with atopic dermatitis. This pigment appeared to migrate from latent breaks because of inflammation of the peripheral retina and ciliary body.

Adolescent↗
PubMed1994

Treatment of epidermal pigmented lesions with the frequency-doubled Q-switched Nd:YAG laser. A controlled, single-impact, dose-response, multicenter trial.

BACKGROUND AND DESIGN: The removal of benign, aesthetically important, pigmented lesions can be effectively treated with multiple modalities. Selective removal of the pigment by lasers is becoming increasingly popular. A three-center trial evaluated the effectiveness of the frequency-doubled Q-switched neodymium (Nd):YAG laser (532 nm, 2.0-mm spot size, 10 nanoseconds) in removing benign epidermal pigmented lesions with a single treatment. Forty-nine patients were treated for multiple lentigines (n = 37), for cafe au lait macules (n = 7), and for miscellaneous lesions (n = 5). Treatment areas were divided into four quadrants, irradiated with fluences of 2, 3, 4, or 5 J/cm2 and evaluated at 1- and 3-month intervals following treatment. RESULTS: For lentigines, response was related to dose with a greater than 75% pigment removal achieved in 60% of those lesions treated at higher energy fluences. Responses were more variable with other lesions, with fair-to-good improvement noted in most cases. Mild, transient erythema; hypopigmentation; and hyperpigmentation were noted in several patients, but resolved spontaneously within 3 months. No other textural changes, scarring, or other side effects were noted. CONCLUSION: The frequency-doubled Q-switched Nd: YAG laser (532 nm) safely and effectively treats benign epidermal pigmented lesions.

Adult↗
PubMed1994

Physiological variations in the pigmentation of newborn infants.

Skin pigmentation is a physiological feature of great value and usefulness from both an anthropological point of view and a clinical point of view. This article is a first approach to the study of the skin pigmentation of 126 full-term newborn infants (37-42 weeks gestational age) at the Maternity Hospital "La Paz" (Madrid, Spain) using the reflectance method. We provide the normal patterns, which have to be taken into account when applying this method to detect transcutaneous bilirubin and some physiopathological conditions, such as hyperbilirubinemia, anemia, plethora, and hormonal disturbances. Measurements of the reflectance were taken at the forehead, the internal and external aspects of the arm, the thorax, and the gluteus with blue, green, and amber tristimulus filters. With all filters the least pigmented zone was the thorax, and the most pigmented zone was the forehead; the internal aspect of the arm, the gluteus, and the external aspect of the arm were intermediate in increasing order. Our data confirm a higher pigmentation at birth than during the first year. We think that the idea of cephalocaudal progression of hyperbilirubinemia must be rejected. Our results also indicate that the thickening of the skin with gestational age is one of the most important factors in the skin color changes of newborn infants with an apparently different rhythm in males than in females.

Analysis of Variance↗
PubMed1993

Binding of antiglaucomatous drugs to synthetic melanin and their hypotensive effects on pigmented and nonpigmented rabbit eyes.

The binding of ocular hypotensive drugs to synthetic melanin was studied spectrophotometrically in vitro. The ocular hypotensive effects of the drugs, namely, timolol, befunolol, carteolol, pilocarpine, epinephrine, prostaglandin A2, F2 alpha and E2, also were compared in vivo on eyes of pigmented and albino rabbits. At an initial concentration of 10(-4) M, each of the three beta-blockers exhibited a binding rate of 80-85% as compared to only 40% for pilocarpine and 50% for epinephrine. Almost none of the prostaglandins were found to bind to synthetic melanin. Topically applied, 0.5% timolol and 3% pilocarpine significantly lowered the intraocular pressure in albino but not in pigmented rabbits. Epinephrine (1%) caused a significant reduction in the intraocular pressure both in albino and pigmented rabbits; however, the maximum reduction was greater in albino than in pigmented rabbits. Intraocular pressure was reduced to the same extent and with a similar time-course in both albino and pigmented rabbits by 0.02% prostaglandin A2, F2 alpha and E2. These findings show that several ocular hypotensive drugs bind to melanin and suggest that this process can modify the extent of their pharmacological effects when tested in a single dose, or the time-course of their effects when used to treat chronic conditions.

Adrenergic beta-Antagonists↗
PubMed1994

Cytokines and serum cause alpha 2 beta 1 integrin-mediated contraction of collagen gels by cultured retinal pigment epithelial cells.

PURPOSE: Integrins, heterodimeric cell surface glycoproteins, are involved in cell-substratum and cell-cell interactions. The role of these molecules in cytokine-mediated contraction of extracellular matrix by retinal pigment epithelial cells has been investigated in a model system that may mimic epiretinal membrane contraction during retinal detachment in proliferative vitreoretinopathy. METHODS: Retinal pigment epithelial cells were cultured on three-dimensional collagen gels that they cause to contract. The involvement of new protein synthesis and C-kinase-mediated signal transduction were studied using specific inhibitors. Cell surface integrins synthesized by pigment epithelial cells were identified using immunofluorescence, and the same antibodies were used in gel contraction assays to identify the integrin species responsible for force transduction. RESULTS: Contraction of collagen type I gels was small without cytokines but was greatly enhanced in the presence of serum or IL1 plus TGF beta. It was dependent on new protein synthesis. Contraction induced by the combination of cytokines was dependent on active protein kinases, whereas that induced by serum was not because only the former was inhibited by staurosporine, a C-kinase inhibitor. Pigment epithelial cells were found to produce beta 1, alpha 2, and alpha 5 integrins, but only alpha 2 and beta 1 appeared to participate in the contraction process because only antibodies against these integrins inhibited contraction. CONCLUSIONS: Retinal pigment epithelial cells contract an extracellular matrix in vitro in a manner similar to epiretinal membrane contraction in vivo. This contraction is mediated via cell surface glycoproteins of the integrin family that bind directly to extracellular matrix molecules.

Cell Communication↗
PubMed1976

Studies on café au lait spots in neurofibromatosis and pigmented macules of nevus spilus.

Café au lait spots from 14 Japanese patients with neurofibromatosis and nevus spilus from 9 Japanese patients were subjected to the studies on the differences in nature of their melanocytes. When the number of melanocytes of the pigmented lesions was compared with that of the surrounding normal skin, the former was always increased and that of café au lait spot was higher than that of nevus spilus. Giant pigment granules were recognized only in 6 patients out of 14 with neurofibromatosis but not in nevus spilus examined. 2 days after UV irradiation at 4 MED, the number of melanocytes was increased in both surrounding normal skin and pigmented lesion, and the rates of increase were lower in the pigmented lesion. Under the electron microscope, melanocytes in café au lait spots which received an ultraviolet light irradiation showed various changes in their cytoplasm; a development of dendrites containing many mature melanosomes, an increased number of cytoplasmic vacuoles and mitochondria, a development of Golgi apparatus in their cytoplasm, appearances of some dense-bodies and of autophagosomal melanosome-complexes. In nevus spilus, the same kind of changes occurred, but they were moderate compared with those developed in café au lait spots. Melanosomes in the keratinocytes of café au lait spots tended to come together around the nucleus and to form melanosome-complexes; while, melanosomes in the keratinocytes of nevus spilus seemed to be single-dispersed after irradiation. The causative factors of the hyperpigmentation and the different reactivity of melanocytes against UV irradiation in these two pigmented macules were discussed.

Adult↗
PubMed1994

Characterization of glucose transporter in cultured human retinal pigment epithelial cells: gene expression and effect of growth factors.

PURPOSE: To investigate gene and protein expression of glucose transporter isoforms in cultured human retinal pigment epithelial cells. To investigate growth factor-dependent stimulation of glucose transport and the effect of growth factors on the gene and protein expression in retinal pigment epithelial cells. METHODS: Glucose transport activity was analyzed by [3H]2-deoxyglucose uptake studies. Gene and protein expression of glucose transporter isoforms were analyzed by polymerase chain reaction, Northern blot analysis, and Western blot analysis. RESULTS: Polymerase chain reaction, nucleotide sequencing, and Southern blot analyses revealed that the retinal pigment epithelial cells express GLUT-1, -3, and -5 genes. Northern and Western blot analysis detected only GLUT-1 transcripts and protein. A 24-hour exposure to fetal bovine serum (15%), basic fibroblast growth factor (50 ng/ml), platelet-derived growth factor (10 ng/ml), epidermal growth factor (50 ng/ml), and insulin-like growth factor-1 (50 ng/ml) significantly stimulated [3H]2-deoxyglucose uptake in cultured human retinal pigment epithelial cells. Western blot analysis showed that serum and platelet-derived growth factor induced an increase of GLUT-1 protein in the membrane preparation in the cells. Serum, fibroblast growth factor, platelet-derived growth factor, and insulin-like growth factor-1 did not increase GLUT-1 gene expression to an appreciable level, as shown by Northern blot analysis. CONCLUSION: Cultured human retinal pigment epithelial cells dominantly express GLUT-1 gene and protein with minor expression of GLUT-3 and -5 genes. Fetal bovine serum, fibroblast growth factor, platelet-derived growth factor, epidermal growth factor, and insulin-like growth factor-1 significantly, although modestly, increase glucose transport activity of the cells.

Base Sequence↗
PubMed1994

Role of retinal pigment epithelium in the development of experimental autoimmune uveitis.

PURPOSE: To determine the role of retinal pigment epithelium in the induction of S-antigen-induced uveitis by administration of sodium iodate (NaIO3) to selectively damage the retinal pigment epithelium. METHODS: Forty-four Lewis rats were injected with 60 micrograms of S antigen in complete Freund's adjuvant. On postimmunization day 9 the rats were separated into four groups: three groups received NaIO3 at doses of 50, 25, and 10 mg/kg body weight, respectively, and the fourth group (control) received diluent. In addition, separate groups of animals (three in each group) received various doses of NaIO3 or diluent. All of the animals were killed on day 6 after NaIO3 injection, and the eyes were enucleated and submitted for light and electron microscopic examination. In addition, two groups of Lewis rats (6 in each group) were immunized with 0.5 ml of guinea pig spinal cord homogenate in complete Freund's adjuvant to induce experimental allergic encephalomyelitis. On postimmunization day 7, one group received NaIO3 at a dose of 50 mg/kg body weight, whereas the other group received diluent. All animals were killed between days 12 and 14, and spinal cord sections were obtained for microscopic examination. RESULTS: In the control group immunized with S antigen, severe (2+ to 4+) uveoretinitis developed in 70% of the animals. In contrast, only 18% of the animals injected with NaIO3 at a dose of 50 mg/kg body weight exhibited disease, and this was a mild (1+) form. The groups injected with 25 mg/kg (1+ to 2+) and with 10 mg/kg (2+ to 3+) of NaIO3 showed a mild to moderate degree of uveoretinitis in 27% and 50% of the animals, respectively. In the remainder of the animals there was no evidence of uveoretinitis. All of the NaIO3-treated animals showed selective necrosis of the retinal pigment epithelium; this was extensive in the higher dose group and focal in the lower dose groups. In the experimental allergic encephalomyelitis model there was no significant difference in incidence or histologic appearance of demyelinating disease in NaIO3- vs diluent-treated groups. CONCLUSIONS: These results indicate that the retinal pigment epithelium may play a role in the initiation and perpetuation of uveitis after sensitization with S antigen. The effect of NaIO3 appears to be localized to the retinal pigment epithelium; it had no effect on immune reactive cells, as evidenced by the development of experimental allergic encephalomyelitis in animals treated with NaIO3.

Animals↗
PubMed1993

Visualization of [3H]docosahexaenoic acid trafficking through photoreceptors and retinal pigment epithelium by electron microscopic autoradiography.

PURPOSE: [3H]docosahexaenoic (DHA) acid was followed through the retinal pigment epithelial cells and photoreceptors for up to 5 days after injection to specifically determine which membrane systems of the retinal pigment epithelial cells are used in the handling of [3H]DHA after shedding and phagocytosis of rod tips. METHODS: Frogs (Rana pipiens) were injected with [3H]DHA in the dorsal lymph sacs, and maintained for up to 5 days. Retinas were processed for electron microscopic autoradiography, stored for various periods of time, and then analyzed by transmission electron microscopy. RESULTS: After 1 day, [3H]DHA had accumulated within photoreceptor ellipsoids, and had begun to appear as dense label in newly formed discs. By day 5, the basal region of dense label had expanded apically. Newly shed rod outer segment tips were diffusely labeled; but occasionally after several hours, they acquired additional label as they moved near Bruch's membrane. Retinal pigment epithelial cytoplasm maintained a constant level of label, with myeloid bodies sometimes slightly labeled. Oil droplets of the retinal pigment epithelium accumulated dense label throughout this study. CONCLUSIONS: When [3H]DHA enters the retinal pigment epithelium, some is retained within oil droplets, whereas the rest is passed on to the photoreceptors. [3H]DHA is initially taken up by inner segments and then dispersed to photoreceptor synaptic terminals as well as to ellipsoids where discs are assembled. Phagosomal labeling exactly matches rod outer segment tips, but occasionally increases as degradation occurs near Bruch's membrane. Normally, density of label remains constant throughout the degradation process.

Animals↗
PubMed1993

Retinoids increase urokinase-type plasminogen activator production by human retinal pigment epithelial cells in culture.

PURPOSE: To examine the effect of two retinoids, all-trans-retinoic acid (tretinoin) and all-trans-9-(4-methoxy-2,3,6- trimethylphenyl)-3,7-dimethyl- 2,4,6,8-nonatetraenoic acid (acitretin) on the production of plasminogen activators and plasminogen activator inhibitors by human retinal pigment epithelial cells in culture. METHODS: Cultures of human retinal pigment epithelial cells were incubated with either of the retinoids at a concentration of 1 microM for 24-72 hours. The media were assayed using solid-phase immunocapture assays and zymography. RESULTS: Both retinoids caused a twofold to sevenfold increase in urokinase-type plasminogen activator in the medium. The effect was seen after 24 hours in culture and was further augmented up to 72 hours. No significant amounts of tissue-type plasminogen activator were detected. The plasminogen activator inhibitor activity was unaffected by the retinoids. Proliferation and morphology of retinal pigment epithelial cells were also unaffected by the retinoids in incubations for up to 72 hours. CONCLUSIONS: Retinoids profoundly affect the extracellular proteolysis of retinal pigment epithelial cells in culture. This effect may be related to the differentiation-inducing activity of retinoids seen in other cell types, often connected with changes in extracellular proteolysis. It is possible that retinoids may modulate dedifferentiation, proliferation, and migration of retinal pigment epithelial cells seen in vitro, as well as in the pathogenesis of retinal disease.

Acitretin↗
PubMed1996

Kinetics of rod outer segment phagocytosis by cultured retinal pigment epithelial cells. Relationship to cell morphology.

PURPOSE: To study phenotypic variation in primary cultures of rat retinal pigment epithelium (RPE) and to correlate cell morphology with rates of binding and ingestion of rod outer segments (ROS). METHOD: Replicate cultures were prepared using RPE cell sheets isolated with Dispase from Royal College of Surgeons normal (RCS rdy+ p+) and dystrophic (RCS p+) rats. Retinal pigment epithelial morphology was analyzed, and phagocytosis was assessed by fluorescence microscopy in cultures fixed at 2-hour intervals from 3 to 19 hours after continuous incubations with fluorescein isothiocyanate (FITC)-stained ROS. RESULTS: A wide range of RPE cell size, shape, and pigmentation was present at confluence; however, distinct morphologic subtypes were recognized, defined as types 1 to 3, and studied separately. In both normal and dystrophic cultures, the extent and rate of ROS binding varied with RPE phenotype. In normal cultures, highly spread pigmented binucleate cells (type 3) bound and rapidly ingested multiple ROS per cell starting at 3 hours and reached a peak at 9 hours. Lightly pigmented daughter cells (type 2) bound and ingested far fewer ROS per cell than did type 3 RPE, which had not divided. Patches of hexagonally packed cells with in vivo morphology (type 1) bound large numbers of ROS per cell only after prolonged (9- to 11-hour) incubations and ingested them synchronously. Comparison of normal versus dystrophic RPE subtypes 1 to 3 revealed the known ingestion defect in all three mutant phenotypes but indicated delayed ROS binding in type 2 and type 3 cells as well. CONCLUSIONS: Kinetics of ROS binding and ingestion differ markedly among phenotypic variants of RPE cells typically found in primary cultures at confluence. Thus, accurate quantitation requires comparison of equivalent microscopic fields or like RPE subtypes, and the heterogeneous responses of various RPE subtypes should be considered when interpreting phagocytic data obtained from entire cultures at a particular time.

Animals↗

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