TGF-β2 Inhibits Growth of Uveal Melanocytes at Physiological Concentrations

Exp. Eye Res. (1998) 67, 143–150 Article Number : ey980501

TGF-β2 Inhibits Growth of Uveal Melanocytes at Physiological Concentrations D A N-N I N G H U*, S T E V E N A. M  C O R M I CK, A L E X A N D E R Y. L I N    J E N N I F E R Y. L I N Tissue Culture Center, Department of Pathology and Laboratory Medicine, and Department of Ophthalmology, The New York Eye and Ear Infirmary, 310 E. 14th Street, New York, NY 10003 ; and New York Medical College, Valhalla, NY 10595, U.S.A. (Received Columbia 2 September 1997 and accepted in revised form 9 March 1998) The effect of TGF-β2 on growth of uveal melanocytes in vitro was studied and the dose-dependent inhibitory effect of TGF-β2 was compared with the known concentration of TGF-β2 in aqueous humor. Uveal melanocytes were isolated and cultured with medium supplemented with cAMP elevating agents and basic fibroblast growth factor. The uveal melanocytes were plated into multi-well plates. After 24 hr, TGF-β2 was added to the medium in various concentrations. After 5 days, the cells were detached, counted and compared to the controls. The effect of TGF-β2 on DNA synthesis (as evaluated by uptake of bromodeoxyuridine) were also tested. TGF-β2 inhibited growth and DNA synthesis of cultured uveal melanocytes in a dose-dependent manner at concentrations from 0±03–10±0 ng ml−". The growthinhibition of TGF-β was present even in serum-free medium. TGF-β had little or no effect on # # melanogenesis of cultured uveal melanocytes. The serum used for cultivation did not contain active TGFβ or TGF-β as measured by immunoassay. The known amount of active TGF-β2 in aqueous humor " # (0±2–0±4 ng ml−") is sufficient to inhibit the growth of uveal melanocytes. It indicates that TGF-β2 is a potent growth inhibit factor of uveal melanocytes and may play an important role in maintaining the non-proliferative, relatively quiescence status of uveal melanocytes in vivo. # 1998 Academic Press Key words : uveal melanocytes ; TGF-β ; growth inhibition ; bFGF ; in vitro ; melanogenesis ; melanin.

1. Introduction Human uveal melanocytes (UM) are normally in a relatively quiescent state during adulthood. Recent studies indicated that cultured human UM grow actively in the presence of serum, basic fibroblast growth factor (bFGF) and cAMP-elevating (Hu et al., 1993a ; 1993b ; 1995). There are two possible explanations for the quiescent state of UM in vivo : (1) Lack of these growth stimulating factors of UM in vivo. (2) Existence of growth inhibiting factors of UM in vivo. The absence of proliferation of UM present not only in physiological states, but also in many different pathological processes, such as inflammation and injury (Naumann and Apple, 1986), indicates that one or more growth inhibiting factors may exist in the eye. Although some growth stimulating factors of UM have been determined (Hu et al., 1993b), growth inhibiting factors of UM have not been previously reported. Transforming growth factor beta (TGF-β) is a group of cytokines with different effects on various cell types and cell stages (Roberts et al., 1985 ; Reiss and Sartorelli, 1987 ; Roberts and Sporn, 1991). TGF-β has recently been detected in various ocular tissues, and could be produced by various cell types in the eye * Address correspondence to : Dan-Ning Hu, Tissue Culture Center, Department of Pathology and Laboratory Medicine, The New York Eye and Ear Infirmary, 310 E. 14th Street, New York, NY 10003, U.S.A.

0014–4835}98}080143­08 $30.00}0

(Helbig et al., 1991 ; Knisely et al., 1991 ; Tanihara et al., 1993 ; Pfeffer et al., 1994 ; Tripathi et al., 1994b). TGF-β inhibits the growth of RPE, lens epithelial cells and corneal epithelial cells (Leschey et al., 1990 ; Kruse and Tseng, 1993 ; Kurosaka and Nagamoto, 1994), and also inhibits the growth of epidermal melanocytes (Pittelkow and Shipley, 1989 ; Rodeck et al., 1991). The TGF-β family is composed of at least five members (TGF-β1 through 5), with most evidence suggesting that they share similar biological activities. TGF-β2 is the major form in the eye. Both latent and active TGF-β2 are found in high concentrations in the aqueous humor (Jampel et al., 1990 ; Cousins et al., 1991 ; Tripathi et al., 1994c ; de Boer et al., 1994 ; Ie et al., 1994). Therefore, it is of interest to study the effect of TGF-β2 on the growth of UM in vitro. Comparing the dose-response inhibiting effect by TGFβ2 in vitro with the concentration of active TGF-β2 in aqueous humor may reveal the significance of TGF-β2 as a growth inhibitor of UM in vivo. 2. Materials and Methods Reagents F12 Nutrient Mixture (F12 medium), fetal bovine serum (FBS), -glutamine, gentamicin, trypsin solution, and trypsin ethylene diaminestetraacetic acid (trypsin-EDTA) solution were obtained from GIBCO BRL (New York, NY, U.S.A.). 12-0-tetradecanoylphorbol-13-acetate (TPA), cholera toxin (CT), isobutyl# 1998 Academic Press

144

D.-N. H U E T A L.

methylxanthine (IBMX), bromodeoxyuridine (BrdU) and human recombinant TGF-β2 (active form) were obtained from Sigma (St. Louis, MO, U.S.A.). Human recombinant basic fibroblast growth factor (bFGF) was obtained from Promega (Madison, WI, U.S.A.).

Melanin concentration was determined by measurement of optical density at 475 nm and compared with a standard curve determined using synthetic melanin. Melanin content was expressed as µg culture−" and ng cell−" (Hu et al., 1995 ; 1997).

Cell Culture

Effect of TGF-β2 on Uptake of BrdU on UM

UM were isolated and cultured from adult donor eyes as previously described (Hu et al., 1993a). The isolated UM were cultured with one of the following two culture media : (1) FIC medium : F12 medium supplemented with 10 % FBS, 2 m glutamine, 10 ng ml−" CT, 0±1 m IBMX, 20 ng ml−" bFGF and 50 µg ml−" gentamicin. (2) TIC medium : FIC medium prepared as above except that bFGF was replaced by 50 ng ml−" TPA. The medium was changed twice weekly. If contaminating cells persisted for more than 7 days in the culture, geneticin, a cytotoxic agent, was added (100 µg ml−") for 3–7 days to eliminate contaminating cells such as fibroblasts and pigment epithelial cells which are more sensitive to the effects of geneticin than UM (Hu et al., 1993a). After reaching confluence, the UM were detached by trypsin-EDTA solution, diluted, and plated for subculture. The UM used in the present studies consisted of three cell lines isolated from the iris, the ciliary body and the choroid. These cells had been in culture for no longer than 2 months, and had been passaged 3–5 times at a dilutional ratio of 1 : 4. The purity of the cell lines was demonstrated by immunocytochemical methods. UM display S-100 antigen but not cytokeratin, whereas pigment epithelial cells display both proteins ; fibroblasts display neither of these proteins (Hu et al., 1993a).

UM were plated into 4-well tissue culture chamber slides (Nunc, Naperville, IL, U.S.A.) with FIC medium at a density of 1¬10% cells per well. After 24 hr, TGFβ2 was added to the medium in various concentrations, each in three wells. UM cultured with FIC medium without TGF-β2 were used as controls. Forty three hours after adding TGF-β2 to the medium, BrdU (50 µg ml−") was added to the medium ; 5 hr later, the medium was withdrawn, the cells were washed, fixed, incubated in 0±15  trisodium citrate for 45 minutes at 70°C for partial denaturation of double stranded DNA, tested with monoclonal anti-bromodeoxyuridine antibody (DAKO, Carpinteria, CA, U.S.A.) using the immunoperoxidase-biotin-streptoavidin method, stained with 3,3«-diaminobenzendine (DAB), counterstained with Mayer’s hematoxylin, and examined by light microscopy. The nucleus of the cell exhibiting BrdU was dark brown in color (positive stain) in contrast to the blue colored nucleus without BrdU. Five hundred cells from each well were counted. The positive rate of UM cultured with different concentrations of TGF-β2 were compared with that of controls and were expressed as percentages of the control. For studying the effect of TGF-β2 on the uptake of BrdU in cultured UM in serum-free medium, UM were plated in 4-well chamber slides with FIC medium. After 24 hr, the cultures were washed with serum-free medium twice and then cultured with serum-free FIC medium. TGF-β2 (10 ng ml−") was added to the medium in three wells, and UM cultured without TGF-β2 were used as controls. Nineteen hr after culture in serumfree medium, BrdU (50 µg ml−") was added to the medium ; 5 hr later, the cultures were fixed and studied by immunocytochemistry to detect the BrdU as described above.

Effect of TGF-β2 on Cell Growth of UM The UM were plated into 24-well plates (Corning Glassworks, Corning, NY, U.S.A.) with FIC or TIC medium at a density of 1¬10% cells per well. After 24 hr, TGF-β2 was added to the medium. The doseeffect of TGF-β2 were tested at the following concentrations : 0±01, 0±03, 0±1, 0±3, 1, 3 and 10 ng ml−", each in three wells. Three wells of UM cultured with FIC or TIC medium without TGF-β2 were used as controls. The media were replaced every 3 days. Five days later, the cells were detached with trypsin-EDTA solution and counted. The results were compared to controls and expressed as a percentage of number of cells present in controls. Effect of TGF-β2 on Melanogenesis of UM The method was the same as described above, with the following exceptions : (1) Cells were plated in 12well plates at a density of 2¬10% cells per well. (2) Five days after adding TGF-β2, the cells were detached, centrifuged, and the pellet was dissolved in 1 N NaOH.

Statistical Analysis Student’s t-test was used in all experiments to assess the significance of differences between the mean of tested cultures and controls. Measurement of TGF-β in Serum The amounts of TGF-β1 and TGF-β2 in the FBS used for cultivation of UM were measured by a doubleantibody ‘ sandwich ’ enzyme-linked immunosorbent assay, using the TGF-β1 Quantikine kit and TGF-β2 Quantikine kit (R & D Systems, Minneapolis, MN, U.S.A.). The sensitivities of the ELISA kits were 5 pg ml−" (TGF-β1) and 2 pg ml−" (TGF-β2). The

T G F-β A N D U V E A L M E L A N O C Y T E S

standard TGFs and the test samples (F12 medium with 10 % FBS) were added to microtiter ELISA plates. The amounts of TGFs were measured following the manufacturers instructions and were assayed before and after acid activation. The amounts of TGFs before and after acid activation represent active and total TGFs in the FBS, respectively. The difference between the two values represents the amounts of latent TGFs. 3. Results UM grew very well in FIC medium, could be passaged for many generations, and divided 40–50 times. Cultured UM produced melanin in vitro and maintained their inherent capacity for melanogenesis. TPA, a protein kinase C activator, can be used as a substitute for bFGF (TIC medium). UM cultured with TIC medium also grew well and divided approximately 40 times. The UM selected for use in the present studies were in an actively growing state. UM cultured with FIC medium grew rapidly with a doubling time of 36–48 hr. TGF-β2 showed dose-dependent growth inhibition of UM at concentration of TGF-β2 from 0±03 to 10 ng ml−" in the UM cell line isolated from the iris (Fig. 1). TGF-β2 inhibited two other cell lines of UM isolated from the choroid and ciliary body in a similar dose-dependent manner (data not shown). Cell numbers of UM cultured with TGF-β2 for 5 days were significantly less than those cultured without TGF-β2 (0±05 " P " 0±01 at a concentration of 0±03 ng ml−", and P ! 0±01 at all concentrations above 0±03 ng ml−"). UM cultured with TIC medium also grew actively, with a doubling time ranging from 48 to 60 hr. TGF-

145

β2 inhibited the growth of UM cultured with TIC medium in a similar manner to UM cultured with FIC medium (Fig. 1). TGF-β2 increased melanin content per cell in a dose dependent manner [Fig. 2(A)]. Melanin content per cell increased significantly in TGF-β2 concentrations ranging from 0±03–10 ng ml−" (0±05 " P " 0±01 at concentrations of 0±03 ng ml−", P ! 0±01 at concentrations of 0±1 ng ml−" or more). However, no significant difference in the content of melanin per culture was demonstrated in TGF-β2 treated UM at concentrations of 0±01–3 ng ml−" (P " 0±05). Only a slight decrease of melanin content per culture was revealed in the highest concentration tested (0±01 ! P ! 0±05 at concentration of 10 ng ml−") [Fig. 2(B)]. BrdU uptake was demonstrated in 57–63 % of cells in FIC medium without TGF-β. UM cultured with TGFβ2 in concentrations of 0±03–10 ng ml−" demonstrated positively stained cells at levels of 47–89 % of the controls (Fig. 3). The inhibition of TGF-β2 on the uptake of BrdU by cultured UM was dose-dependent (0±05 " P " 0±01 at concentration of 0±03 and 0±1 ng ml−" and P ! 0±01 at concentration of 0±3 ng ml−" or more). In the UM cultured in serum-free FIC medium for 24 hr, BrdU uptake was demonstrated in 31–35 % of cells without TGF-β2 and in 10–15 % of cells with TGF-β2. The difference among cultures with and without TGF-β2 were statistically significant (P ! 0±01), indicating that TGF-β2 inhibited the growth of UM even in the absence of serum. TGF-β1 and TGF-β2 could not be detected before acid activation in the FBS using ELISA kits. The amounts of TGF-β1 and TGF-β2 in the sample containing 10 % FBS after acid activation were

F. 1. Dose-effect relationship of TGF-β2 on the growth of cultured UM. Cultured UM were plated in 24-well plates with FIC medium (——) or TIC medium (– – – – –). Various concentrations of TGF-β2 were added to the medium. UM cultured with FIC or TIC medium without TGF-β2 were used as controls. Five days later, the cells were detached and counted. The results are expressed as the percentages of the controls (average of 3 wells in each group, Mean³..).

146

D.-N. H U E T A L.

F. 2. Dose-effect relationship of TGF-β2 on melanogenesis of cultured UM. Cultured UM were plated in 24-well plates with FIC medium. Various concentrations of TGF-β2 were added to the medium. UM cultured with FIC medium without TGF-β2 were used as controls. Five days later, the cells were detached and counted. Melanin content of UM was measured by spectrophotometer after treating cells with NaOH (1 N) and was expressed as melanin content per cell [ng cell−", (A)] and melanin content per culture [ µg culture−", (B)]. The results are expressed as the percentages of the controls (average of 3 wells in each group, Mean³..).

905 pg ml−" and 224 pg ml−", respectively. Therefore, no active TGF-β1 and TGF-β2 was present in the FBS used in the present studies. All TGF-β1 TGF-β2 in the FBS were in the latent form. 4. Discussion UM are normally in a dormant state in vivo during adulthood (Naumann and Apple, 1986). Our previous studies indicated that cultured UM are able to grow actively in the presence of certain growth stimulating factors (Hu et al., 1993a ; 1993b). Three classes of agents are required for the growth of UM in vitro, including : serum, bFGF or TPA, and cAMP-elevating

agents. Adult UM cultured in medium containing all three agents grew well and could divide as many as 50 times, in high contrast to the mitotically inactive status of UM in vivo (Hu et al., 1993a ; 1993b ; Naumann and Apple, 1986). Lack of these growth stimulating factors may explain the relatively inactive status of UM in vivo under normal physiologic conditions. Serum is absent in aqueous humor and vitreous due to the bloodaqueous and blood-retinal barriers. Serum components may be present in the uvea and may enter the aqueous humor when the blood-aqueous barrier is breached during pathologic states such as trauma and inflammation. bFGF is present in the normal aqueous

T G F-β A N D U V E A L M E L A N O C Y T E S

147

F. 3. Dose-effect relationship of TGF-β2 on uptake of BrdU of cultured UM. UM were plated in 4-well chamber slides with FIC medium. Various concentrations of TGF-β2 were added to the medium. UM cultured with FIC medium without TGF-β2 were used as controls. BrdU was added to the medium after 43 hr ; 5 hr later, the slides were stained using the immunoperoxidase method with anti-BrdU antibody. The positive rate (number of positively stained cells divided by the total) of UM cultured with TGF-β2 were compared with controls and expressed as percentages (average of 3 wells in each group, Mean³..).

humor at a concentration potentially capable of stimulating the growth of UM (1±07 ng ml−") (Tripathi et al., 1994a). cAMP-elevating agents used in our in vitro studies (IBMX, CT, and dbcAMP) are absent in the eye. However, many substances, such as neurotransmitters ( β-adrenergic agonists) and inflammatory mediators (prostaglandins) can similarly increase intracellular cAMP levels and may exist in the aqueous humor and various ocular tissues under physiologic and certain pathologic conditions (Hammerarstrom, 1982 ; Berman, 1991 ; Jumblatt et al., 1994 ; Bausher and Blake, 1995 ; Fleisher, Ferell and McGahan, 1995). Based on these facts, although these three growth stimulating factors may not be present under normal circumstances, they are possibly present in certain pathologic circumstances. However, even with such pathologic stimulation, reactive proliferation is still absent in the UM (Naumann et al., 1986). Therefore, it is reasonable to postulate the presence of growth inhibiting factor(s) in the aqueous humor and uvea as an explanation for the maintenance of relatively quiescent state of UM in vivo. TGF-β is a multipotent cytokine that is present in many tissues and modulates various aspects of cell growth and chemotaxis, extracellular matrix composition, and immune response (Roberts et al., 1985 ; Reiss and Sartorelli, 1987 ; Roberts and Sporn, 1991). In the aqueous humor and ocular tissues, TGF-β2 is the predominant form, and a large proportion of TGFβ2 is present in the latent form. The total amount of latent and active TGF-β2 in human aqueous humor ranged from 1–2 ng ml−", and the concentration of active TGF-β2 ranged from 0±2–0±36 ng ml−" in

normal or cataract subjects (Cousins et al., 1991 ; de Boer et al., 1994 ; Tripathi et al., 1994c). The concentration was slightly higher in glaucoma patients (0±36–0±45 ng ml−") (de Boer et al., 1994 ; Tripathi et al., 1994c). TGF-β2 has also been detected in the stroma of the iris, ciliary body and choroid (Knisely et al., 1991 ; Lutty et al., 1993 ; SchlotzerSchrehardt and Dorfler, 1993 ; Pasquale et al., 1993 ; Pfeffer et al., 1994 ; Planck et al., 1994 ; Tripathi, 1994b ; Seku, Shimokawa and Tokoro, 1995). Two parameters have been used in the present study for evaluating the effect of TGF-β on the growth of UM : Cell counts and uptake of BrdU. Uptake of BrdU is one of the most common methods for studying DNA synthesis (Doyle, Griffiths and Newell, 1996), and has the advantage of being more sensitive. Changes in cell number result from the accumulative effect of cell growth during the entire test period. DNA synthesis reflects the growth capacity of cells (Baserga, 1989 ; Doyle et al., 1996). Therefore, it has been used in the present study, especially in the short term experiment of UM cultured in serum-free medium to recognize the early changes of cell division. The present studies indicate that TGF-β2 is a potent growth inhibitor of UM in vitro. Concentrations of TGF-β2 as low as 0±03 ng ml−", which is lower than the normal concentration of TGF-β2 in the aqueous humor, significantly decreased the number of cells and inhibited the growth of UM in vitro. Concentrations of TGF-β2 from 0±03 to 10 ng ml−" significantly decreased cell number in UM cultured with FIC medium in a dose-dependent manner. The inhibiting effects of TGF-β2 on the growth of UM at physiologic

148

concentrations were further demonstrated by uptake of BrdU studies. Most of our experiments were performed in culture media containing 10 % serum, because UM cultured in serum-free medium do not grow well (Hu et al., 1993b). The possible effect of TGF-β in the FBS contained in the culture media should be considered. However, based on the enzyme-linked immunosorbent assay, no active TGF-β1 and TGF-β2 were present in the serum used in the present studies ; TGF-βs were present in the serum only in the latent form. The latent form of TGF-β can be activated by acid and by some proteases such as cathepsin (Lyons, Keski-Oja and Moses, 1988 ; Danielpour et al., 1989). In the uveal tract, cathepsin has been found only in the pigment epithelium and endothelium, but not in the UM (Yamada, Hara and Tamai, 1990). Acid activation only occurs with extreme fluctuation of the pH (pH 1±5) (Lyons et al., 1988). The pH of culture media is stabilized by the buffer (within pH 6±5–7±5). Therefore, acid activation of TGF-β is impossible to occur in the culture media, the presence of latent TGF-β contained in the serum in the culture media would not influence the results of these studies. Furthermore, in a short-term study, the growth-inhibiting effect of TGF-β2 was also demonstrated in UM cultured in serum-free medium. Under certain circumstances, the effects of TGF-β on cells are related on the presence of other growth factors (Reiss and Sartorelli, 1987 ; Bryckaert et al., 1988 ; Massague, 1990). In the present studies, the growth inhibiting effect of TGF-β2 was present not only in UM cultured with medium supplemented with bFGF), but also in UM cultured with medium using TPA as a substitute for bFGF. This indicates that the inhibiting effect of TGF-β2 on UM is non-specific and does not depend on the presence of other growth factors such as bFGF. In the present studies, two parameters (melanin content per cell and melanin content per culture) were used to evaluate the effect of TGF-β2 on melanogenesis in cultured UM. Melanin content per culture is more important than melanin content per cell and is used as the main parameter for evaluating the effect of various substances on melanogenesis of UM in vitro (Hu et al., 1995 ; 1997). TGF-β2 had a marked growth inhibiting effect on cultured UM, followed by a secondary increase in melanin content per cell, because in stationary cells, the melanin produced accumulates within the cell and results in a rapid increase of melanin content per cell (accumulation effect). No significant difference of melanin content per culture has been observed in UM cultured with TGF-β2 at concentrations ranging from 0±01–3 ng ml−", indicating that TGF-β2 may not affect melanogenesis in UM. Aqueous humor contains a high level of TGF-β2 relative to serum, which contains large amounts of TGF-β1 (Cousins et al., 1991 ; Tripathi et al., 1994a ;

D.-N. H U E T A L.

1994b). This indicates that TGF-β2 in the aqueous humor is synthesized locally in ocular cells which border the anterior and posterior chambers of the eye and}or by a diffusional exchange with the vitreous humor (Connor et al., 1989 ; Jampel et al., 1990 ; Cousins et al., 1991 ; Ie et al., 1994 ; Tripathi et al., 1994a ; 1994b). It has been reported that cultured RPE, ciliary epithelial cells, and trabecular cells are capable of secreting TGF-β2 into the culture medium (Schlotzer-Schrehardt and Dorfler, 1993 ; Peress and Perillo, 1994 ; Pfeffer et al., 1994 ; Tripathi et al., 1994b ; Khaliq et al., 1995). mRNA of TGF-β2 has been detected in RPE, trabecular cells, corneal epithelial cells and fibroblasts (Wilson, He and Lloyd, 1992 ; Tripathi et al., 1994b ; Jaffe et al., 1995). Immunocytochemical studies indicated that the choriocapillaris endothelium, hyalocytes, and retinal vessel cells may produce TGF-β1 (Lutty et al., 1993). The ciliary body and iris as a whole, has been reported to contain mRNA transcript of TGF-β2 (Knisely et al., 1991 ; Planck et al., 1994). A large proportion of TGF-β produced by cultured cells is present in the latent form. The mechanism of latent TGF-β activation in the eye is still unclear. It is thought that activation in vivo takes place by some proteases, such as cathepsin D and plasmin (Lyons et al., 1988). In the uveal tract, cathepsin has been found in the iris pigment epithelium, sphincter muscles, ciliary epithelium and endothelium of various vessels. RPE also contain cathepsin D and plasmin (Yamada et al., 1990). The previous discussion indicates that UM normally exist in an environment with a high level of TGF-β. Our study demonstrates that the amount of active TGF-β in human aqueous humor (0±2–0±4 ng ml−") is sufficient to inhibit the growth of UM in vitro. Therefore, we concluded that TGF-β2 may be an important, if not exclusive, growth inhibitory factor of UM in vivo, and therefore plays an important role in maintaining the relatively quiescent status of UM in vivo. Furthermore, the significance of negative control of TGF-β on the growth of UM may also relate to some pathologic processes in the eye. For example, it has been proposed that loss of response to the growth inhibiting effect to TGF-β or loss of ability to produce TGF-β or activate endogenous TGF-β in transformed cells may result in loss of a normal growth-regulatory mechanism and lead to uncontrolled growth of transformed cells (Roberts et al., 1985 ; 1987 ; Arteaga et al., 1990 ; Pelton and Moses, 1990). It is possible that loss of inhibitory growth pathways could be a factor in malignant transformation of the UM and development of uveal melanoma.

Acknowledgements This work was supported by The New York Eye and Ear Infirmary Pathology Research Fund, and the Department of Ophthalmology Research Fund.

T G F-β A N D U V E A L M E L A N O C Y T E S

References Arteaga, C. L., Coffey, Jr. R. J., Dugger, T. C., McCutchen, C. M., Moses, H. L. and Lyons, R. M. (1990). Growth stimulation of human breast cancer cells with antitransforming growth factor β antibodies : Evidence of negative autocrine regulation by transforming growth factor β. Cell Growth and Diff. 1, 367–74. Baserga, R. (1989). Cell growth and division : A practical approach. Pp. 1–16. IRL Press : Oxford, U.S.A. Bausher, L. P. and Blake. H. (1995). Regulation of cyclic AMP production in adult human ciliary processes. Exp. Eye Res. 60, 43–8. Berman, E. R. (1991). Biochemistry of the eye. Pp. 153–65. Plenum Press : New York, U.S.A. de Boer, J. H., Limpens, J., Orengo-Nania, S., de Jong, P. T. V. M., Herj, E. J. and Kijlstra, A. (1994). Low mature TGF-β2 levels in aqueous humor during uveitis. Invest. Ophthalmol. Vis. Sci. 35, 3702–10. Bryckaert, M. C., Lindroth, M., Lonn, A., Tobelem, G. and Wateson, A. (1988). Transforming growth factor (TGFβ) decreases the proliferation of human bone marrow fibroblasts by inhibiting the platelet-derived growth factor (PDGF) binding. Exp. Cell Res. 179, 311–21. Connor, J. T. B., Roberts, A. B., Sporn, M. B., Danielpour, D., Dart, L. L., Michels, R. G., de Bustros, S. Enger, G., Kato, H., Lansing, M. et al. (1989). Correlation of fibrosis an transforming growth factor-β type 2 levels in the eye. J. Clin. Invest. 83, 1661–6. Cousins, S. W., McCabe, M. M., Danielpour, D. and Streilein, J. W. (1991). Identification of transforming growth factor-beta as an immunosuppressive factor in aqueous humor. Invest. Ophthalmol. Vis. Sci. 32, 2201–11. Danielpour, D., Kim, K., Dart, L. L., Watanabe, S., Roberts, A. B. and Sporn, M. B. (1989). Sandwich enzymelinked immunosorbent assays (SELISAs) quantitate and distinguish two forms of transforming growth factorbeta (TGF-β1 and TGF-β2) in complex biological fluids. Growth Factors 2, 61–71. Doyle, A., Griffiths, J. B. and Newell, D. G. (1996). Cell and tissue culture : Laboratory procedures. Pp. 10E : 1.1–1.8. John Wiley & Sons : Chichester, U.K. Fleisher, L. N., Ferell, J. B. and McGahan, C. (1995). Inflammation induced changes in adenosine 3«,5«-cyclic monophosphate production by ciliary epithelial cell bilayers. Exp. Eye Res. 60, 165–71. Hammerarstrom, S. (1982). Endogenous prostaglandin production and cell replication in vitro. In : Prostaglandins and Cancer : First International Conference. (Ed. Power, T. C. et al.) Pp. 297–307. Alan R Liss : New York. Helbig, H., Kittredge, K. L., Coca-Prados, M., Davis, J., Palestine, A. G. and Nussenblatt, R. B. (1991). Mammalian ciliary body epithelial cells in culture produce transforming growth factor-beta. Graefe’s Arch. Clin. Exp. Ophthalmol. 229, 84–7. Hu, D. N., McCormick, S. A., Ritch, R. and Pelton-Henrion, K. (1993a). Studies of human uveal melanocytes in vitro : Isolation, purification and cultivation of human uveal melanocytes. Invest. Ophthalmol. Vis. Sci. 34, 2210–29. Hu, D. N., McCormick, S. A. and Ritch, R. (1993b). Studies of human uveal melanocytes in vitro : Growth regulation of cultured human uveal melanocytes. Invest. Ophthalmol. Vis. Sci. 34, 2220–7. Hu, D. N., McCormick, S. A., Orlow, S. J., Rosemblat, S., Lin, A. Y. and Wo, K. (1995). Melanogenesis by human uveal melanocytes in vitro. Invest. Ophthalmol. Vis. Sci. 36, 931–8. Hu, D. N., McCormick, S. A., Orlow, S. J., Rosemblat, S. and

149

Lin, A. Y. (1997). Regulation of melanogenesis by human uveal melanocytes in vitro. Exp. Eye Res. 64, 397–404. Ie, D., Gordon, L. W., Glaser, B. M. and Pena, R. A. (1994). Transforming growth factor-beta 2 levels increase following retinal laser photocoagulation. Curr. Eye Res. 13, 743–6. Jaffe, G. J., Roberts, L. R., Wong, H. L., Yurochko, A. D. and Cianciolo, G. J. (1995). Monocyte-induced cytokine expression in cultured human retinal pigment epithelial cells. Exp. Eye Res. 60, 533–43. Jampel, H. D., Roche, N., Stark, W. J. and Roberts, A. B. (1990). Transforming growth factor-β in human aqueous humor. Curr. Eye. Res. 9, 963–9. Jumblatt, M. M., Neltner, A. A., Coca-Prados, M. and Paterson, C. A. (1994). EP -receptor stimulated cyclic # Amp synthesis in cultured human non-pigmented ciliary epithelium. Exp. Eye Res. 58, 563–6. Khaliq, A., Patel, B., Jarvis-Evans, J., Moriarity, P., McLeod, D. and Boulton, M. (1995). Oxygen modulates production of bFGF and TGF-β by retinal cells in vitro. Exp. Eye Res. 60, 415–24. Knisely, T. L., Bleicher, P. A., Vibbard, C. A. and Granstein, R. D. (1991). Production of latent transforming growth factor-beta and other inhibitory factors by cultured murine iris and ciliary body cells. Curr. Eye Res. 10, 761–71. Kruse, F. E. and Tseng, S. C. G. (1993). Growth factors modulate clonal growth and differentiation of cultured rabbit lumbal and corneal epithelium. Invest. Ophthalmol. Vis. Sci. 34, 1963–76. Kurosaka, D. and Nagamoto, T. (1994). Inhibitory effect of TGF-β2 in human aqueous humor on bovine lens epithelial cell proliferation. Invest. Ophthalmol. Vis. Sci. 35, 3408–12. Leschey, K. H., Hackett, S. F., Singer, J. H. and Compochiaro, P. A. (1990). Growth factors responsiveness of human retinal pigment epithelial cells. Invest. Ophthalmol. Vis. Sci. 31, 839–46. Lutty, G. A., Merges, C., Threlkeld, A. B., Crone, S. and McLeod, D. S. (1993). Heterogeneity in localization of isoforms of TGF-β in human retina, vitreous, and choroid. Invest. Ophthalmol. Vis. Sci. 34, 477–87. Lyons, J. M., Keski-Oja, J. and Moses, H. L. (1988). Proteolytic activation of latent transforming growth factorβ from fibroblast-conditioned medium. J. Cell. Biol. 106, 1659–65. Massague, J. (1990). The transforming growth factor-β family. Ann. Rev. Cell. Biol. 6, 597–641. Naumann, G. O. H. and Apple, D. J. (1986). Pathology of the eye. Pp. 430. Springer-Verlag : New York, U.S.A. Pasquale, L. R., Dorman-Pease, M. E., Lutty, G. A., Quigley, H. A. and Jampel, H. D. (1993). Immunolocalization of TGF-β1, TGF-β2, TGF-β3 in the anterior segment of the human eye. Invest. Ophthalmol. Vis. Sci. 34, 23–30. Pelton, R. W. and Moses, H. L. (1990). The transforming growth factor β family of growth regulatory peptides. In : Contributions to Oncology. (Eds Bartram, C. R., Munk, K. and Schwab, M.) 39, 94–114. Karger : Basel. Peress, N. S. and Perillo, E. (1994). TGF and TGFβ3 immunoreactivity within the ciliary epithelium. Invest. Ophthalmol. Vis. Sci. 35, 453–7. Pfeffer, B. A., Flanders, K. C., Guerin, C. J., Danielpour, D. and Anderson, D. H. (1994). Transforming growth factor beta 2 is the predominant isoform in the neutral retina, retinal pigment epithelium, choroid, and vitreous of the monkey eye. Exp. Eye Res. 59, 323–33. Pittelkow, M. R. and Shipley, G. D. (1989). Serum-free culture of normal human melanocytes : Growth kinetics and growth factor requirements. J. Cell Physiol. 140, 565–76.

150

Planck, S. R., Huang, X. N., Robertson, J. E. and Rosenbaum, J. T. (1994). Cytokine mRNA levels in rat ocular tissues after systemic endotoxin treatment. Invest. Ophthalmol. Vis. Sci. 35, 924–30. Reiss, M. and Sartorelli, A. C. (1987). Regulation of growth and differentiation of human keratinocytes by type β transforming growth factor and epidermal growth factor. Cancer Research 47, 6705–9. Roberts, A. B. and Sporn, M. B. (1991). The transforming growth factor-βs. In : Peptide growth factors and their receptors. (Eds Sporn, M. B. and Roberts, A. B.) Pp. 435–47. Springer-Verlag : New York, U.S.A. Roberts, A. B., Anzano, M. A., Wakefield, L. M., Roche, N. S., Stern, D. F. and Sporn, M. B. (1985). Type β transforming growth factor : A bifunctional regulator of cellular growth. Proc. Natl Acad. Sci. U.S.A. 82, 119–23. Rodeck, U., Melber, K., Kath, R., Menssen, H. D., Varello, M., Atkinson, B. and Herlyn, M. (1991). Constitutive expression of multiple growth factor genes by melanoma cells but not normal melanocytes. J. Invest. Dermatol. 97, 20–6. Schlotzer-Schrehardt, U. and Dorfler, S. (1993). Immunolocalization of growth factors in the human ciliary body epithelium. Curr. Eye Res. 12, 893–905.

D.-N. H U E T A L.

Seku, Y., Shimokawa, H. and Tokoro, T. (1995). Expression of bFGF and TFG-β in experimental myopia of chicks. Invest. Ophthalmol. Vis. Sci. 36, 1183–7. Tanihara, H., Yoshida, M., Matsumoto, M. and Yoshimura, N. (1993). Identification of transforming growth factorβ expressed in cultured human retinal pigment epithelial cells. Invest. Ophthalmol. Vis. Sci. 34, 413–19. Tripathi, R. C., Borisuth, N. S. C., Li, J. and Tripathi, B. J. (1994a). Growth factors in the aqueous humor and their clinical significance. J. Glaucoma 3, 248–58. Tripathi, R. C., Chan, W. F. A., Li, J. and Tripathi, B. J. (1994b). Trabecular cells express the TGF-β2 gene and secrete the cytokine. Exp. Eye Res. 58, 523–8. Tripathi, R. C., Li, J., Chan, W. F. A. and Tripathi, B. J. (1994c). Aqueous Humor in glaucomatous eyes contains an increased level of TGF-β2. Exp. Eye Res. 59, 723–8. Wilson, S. E., He, Y. G. and Lloyd, S. A. (1992). EGF, EGF receptor, basic FGF, TGF Beta-1, and IL-1 Alpha mRNA in human corneal epithelial cells and stromal fibroblasts. Invest. Ophthalmol. Vis. Sci. 33, 1756–65. Yamada, T., Hara, S. and Tamai, M. (1990). Immunohistochemical localization of cathepsin D in ocular tissues. Invest. Ophthalmol. Vis. Sci. 31, 1217–23.