Abstract
purpose. To examine the regulatory effects of interferon (IFN)-α, IFN-γ, transforming growth factor (TGF)-β, and tumor necrosis factor (TNF)-α on human fetal retinal pigment epithelial (HFRPE) cell–induced apoptosis of human Jurkat T (Jkt) cells.
methods. Pure cultures of HFRPE cells were isolated. The cells were precultured with medium alone or with addition of IFN-α, IFN-γ, TNF-α, or TGF-β for 72 hours. Thereafter, HFRPE cells were extensively washed before they were cocultured jointly with Jkt cells (standard) or cultured alone for another 48 hours to accumulate conditioned medium that is collected and added as cell-free conditioned medium to Jkt cell cultures (supernatant). Jkt cells were cocultured under the two culture conditions for 48, 72, and 96 hours. The rate of apoptosis in Jkt T cells was determined with annexin V staining and flow cytometry.
results. Both IFN-α and -γ upregulated HFRPE-induced apoptosis in Jkt T cells. However, the apoptosis induced by IFN-α–activated HFRPE cells was significant only in the absence of cell–cell contact (supernatant). The supernatant induced a higher rate of apoptosis in Jkt T cells when compared to the direct coculture of the cells. TGF-β and TNF-α did not upregulate HFRPE-induced apoptosis in Jkt T cells.
conclusions. These results indicate that type I and type II IFNs can upregulated HFRPE-induced apoptosis in Jkt T cells, IFN-γ being the more effective cytokine. Neither, TGF-β nor TNF-α upregulated the HFRPE-induced apoptosis in Jkt T cells. Although HFRPE-induced apoptosis was mediated in a cell–cell-contact–independent pathway, HFRPE cells may also express membrane-bound antiapoptotic molecules. These findings may help us to understand better the modulatory effects of pro- and anti-inflammatory cytokines on immune suppressive characteristics of RPE cells.
It is well established that the eye is an immune-privileged site, is protected from the spread of destructive inflammation.
1 Several explanations have been proposed for the phenomenon of immune privilege in the eye.
2 3 4 5 However, the mechanisms involved for maintaining the immune privilege in the subretinal space is not clearly known. We and others recently showed that human fetal retinal pigment epithelial (HFRPE) cells suppresses primary human CD4
+ and CD8
+ T-cell activation by inducing apoptosis.
6 7 8 9 The HFRPE-induced apoptosis in Jkt T cells was found to be cell–cell contact independent, initially involving the arrest of the cell cycle in T-cells followed by the loss of mitochondrial membrane potential, leading to their apoptosis.
10 The HFRPE-induced apoptosis, which is not mediated by Fas ligand (CD95 L) or TNF-related apoptosis-inducing ligand (TRAIL) pathways, may reflect a physiological mechanism for regulation of immune privilege in the subretinal space.
6 7 11
An increasing number of studies have described the potential role of pro- and anti-inflammatory cytokines in induction or prevention of apoptosis.
12 13 14 In addition, it has been shown that these cytokines may also modulate the immune privilege in the anterior segment of the eye.
15 16 In this study, we examined whether such cytokines can enhance or suppress the apoptotic effect of HFRPE cells on activated human T cells and therefore modulating their immunosuppressive characteristics. The human T-cell leukemia line Jurkat (Jkt) was used as a representative model for activated human T cells. Jkt T cells have been widely studied for signaling pathways and receptors representing activated human T cells.
17 18
Interferons (IFNs) were among the first identified cytokines and were discovered on the basis of their antiviral activities.
19 It is well known that a variety of cells produce IFNs in response to infection by various viruses, bacteria, and mycoplasmas.
20 21 IFNs can be classified into two groups: type I (IFN-α, and -β) and type II (IFN-γ).
19 In a large variety of cells, type I IFNs are produced in response to viral infection, where as type II IFN is produced only by activated T cells, natural killer cells, and macrophages.
22 Type I IFNs share the same cell surface receptor (IFNAR). In addition to their antiviral activities, IFNs exhibit pleiotropic biological properties, including antitumor and immunomodulatory effects.
21 We have shown that the type II IFN, IFN-γ, upregulates the HFRPE-induced apoptosis in T cells.
6 7 There are, however, conflicting reports on the regulatory effects of IFN-α on activated T-cell apoptosis.
12 23 24 In this study, we compared the regulatory effects of type I and type II IFNs on HFRPE-induced apoptosis.
Transforming growth factor (TGF)-β is a multifunctional cytokine that regulates cell growth, adhesion, and differentiation of a wide variety of cell types.
25 TGF-β is also a potent immunoregulatory molecule, which plays a critical role in maintaining the immune privilege in the anterior segment of the eye.
26 27 It has been reported that TGF-β also modulates apoptosis in T cells.
28 In this study, we analyzed the effect of TGF-β on HFRPE-induced apoptosis in Jkt T cells.
Tumor necrosis factor (TNF)-α is now recognized as a critical component of host inflammatory defenses that regulate many aspects of cellular immune response, including differentiation and proliferation and the induction of cell death.
29 TNF-α is produced by neutrophils, activated lymphocytes, and macrophages.
30 However, it can also be expressed by a variety of nonimmune cells.
31 In this study, we examined the effect of TNF-α on HFRPE-induced apoptosis in Jkt T cells.
The modulatory effect of the earlier-mentioned cytokines on HFRPE-induced apoptosis in Jkt T cells was evaluated in two different culture conditions: (1) preactivated HFRPE cells cocultured with Jkt T cells and (2) Jkt T cells incubated with the supernatant isolated from activated HFRPE cells (without HFRPE cells).
HFRPE cells were obtained from three independent human fetal eyes at 18 to 22 weeks of gestational age (Advanced Bioscience Resources, Alameda, CA) as described before.
7 Microdissection was performed under sterile conditions aided by a dissecting microscope. The eyes were opened by a circumferential incision just above the ora serrata near the limbus, and the anterior segment and lens were separated. The posterior segment of the eye was cut into four quadrants and placed in a Petri dish containing Dulbecco’s minimum essential medium (DMEM; Sigma-Aldrich, St. Louis, MO). The neural retina and any remaining vitreous were removed. Sheets of RPE cells were separated from the choroid using fine forceps and immediately placed into a Petri-dish containing phosphate buffered saline (PBS) without Ca
2+/Mg
2+ (BioWhittaker, Walkersville, MD). After the separation of all four quadrants, the sheets were trypsinized (0.25% trypsin; Sigma-Aldrich) for 15 minutes. Growth medium consisting of DMEM (Sigma-Aldrich), 15% fetal bovine serum (Sigma-Aldrich), and a 1% solution of antibiotics and
l-glutamine (Sigma-Aldrich) was added, and the content was centrifuged at 2000 rpm. The supernatant was discarded, and the cells isolated from each eye were resuspended with growth medium into one well of a 24-well plate (BD Labware, Bedford, MA) and incubated for 1 week in 95% air and 5% CO
2 at 37°C. The cells were trypsinized and resuspended into a larger culture flask. The cultures were examined on a daily basis, and the growth medium was changed twice a week. At confluence, the cells were subcultured by trypsinization. Fourth- to sixth-passage HFRPE cells were used in the experiments.
Cytokine-activated HFRPE cells were obtained by incubation of cells with 1000 U/mL IFN-α (Sigma-Aldrich), 1000 U/mL IFN-γ, 10 ng/mL TNF-α, or 2 ng/mL TGF-β (all from BD PharMingen, San Diego, CA) for 72 hours at 37°C. After activation, the cultures were washed twice with PBS, and then cells were incubated with Jkt T cells, as explained in the following section.
The human T-cell leukemia line Jurkat (Jkt) was used in this study. The Jkt T cells were cultured in standard culture medium containing RPMI 1640 (BioWhittaker) supplemented with 20 mM glutamine (BioWhittaker), 10% heat-inactivated fetal calf serum (Gibco-Life Technologies, Grand Island, NY), 100 IU/mL penicillin, 100 μg/mL streptomycin (BioWhittaker), and 20 mM HEPES (BioWhittaker). The cultures were incubated at 37°C in a humidified mixture of 95% air and 5% CO2. To maximize the number of viable cells, Jkt T cells were subcultured 72 hours before each assay at densities below 1 × 106 cells/mL.
Standard Coculture.
Nonactivated or cytokine-activated HFRPE cells (750,000) were washed twice with PBS and then cocultured with 1.5 × 106 Jkt cells in six-well plates (Falcon-BD Labware, Bedford, MA) for 48, 72, or 96 hours. Before the apoptosis analysis, the cultured Jkt cells were washed twice with PBS without Ca2+/Mg2+.
Supernatant.
In this assay, Jkt T cells were incubated with the conditioned culture supernatant isolated from either nonactivated or activated HFRPE cells. After incubation with culture medium or the cytokines for 72 hours, the medium was discarded, and cultures were washed twice with Ca2+/Mg2+-free PBS and then incubated with freshly prepared culture medium (without cytokines) for 48 hours. The supernatants from both nonactivated and activated HFRPE cells were collected and centrifuged for 5 minutes at 2000 rpm to separate the remaining debris. Jkt cells (1.5 × 106) were incubated with the isolated supernatants for 48, 72, or 96 hours. Before the apoptosis analysis Jkt T cells were centrifuged at 2000 rpm for 3 minutes and then washed twice with Ca2+/Mg2+-free PBS.
Apoptosis of Jkt T cells was determined by annexin V–FITC staining (R&D Systems, Minneapolis, MN). Annexin V binds to phosphatidyl serine (exposed on the cell membrane), which is one of the earliest indicators of cellular apoptosis. According to the manufacturer’s specifications, cells were collected, washed twice in bead-separation buffer, and resuspended in annexin V–binding buffer at 1 × 106/mL. Staining procedures were performed according to the manufacturer’s instructions (R&D). Samples were then diluted in annexin V binding buffer and analyzed on a flow cytometer with accompanying software (FACScan/Cell Quest; BD Biosciences, San Jose, CA).
Statistical analysis was performed with the paired t-test. P < 0.01 was accepted as significant.
In this system, HFRPE cells were cocultured directly with Jkt T cells. Before the coculture, HFRPE cells were activated with various cytokines. The rate of Jkt T-cell apoptosis after 48, 72, or 96 hours of incubation was evaluated with annexin V–FITC staining (
Fig. 1 ,
Table 1 ). Nonactivated HFRPE cells induced apoptosis in Jkt T cells at 72 and 96 hours (
P < 0.01). Although, IFN-α increased the proapoptotic activity of HFRPE cells at all incubation intervals, this upregulatory effect was not significant. IFN-γ was the strongest apoptosis-inducing cytokine in this group. It significantly upregulated HFRPE-induced apoptosis at 48, 72, and 96 hours (
P < 0.01). Neither TGF-β nor TNF-α had any significant upregulatory or inhibitory effect on HFRPE-induced Jkt T-cell apoptosis.
In this assay, Jkt T cells were incubated with the supernatant isolated from nonactivated or cytokine-activated HFRPE cells in a HFRPE-free system. The rate of apoptosis was analyzed after 48, 72, and 96 hours of incubation (
Fig. 1 ,
Table 1 ). The supernatant of nonactivated HFRPE cells showed a significant induction of apoptosis in Jkt T cells in comparison to the culture medium at 48, 72, and 96 hours (
P < 0.01). The supernatant of IFN-α–activated HFRPE cells significantly upregulated the apoptosis of Jkt T cells at 48 (
P < 0.01), 72 (
P < 0.05) and 96 (
P < 0.01) hours. IFN-γ, also upregulated the apoptosis in Jkt T cells at 48, 72, and 96 hours (at all time points,
P < 0.01). The supernatants of TGF-β– and TNF-α–activated HFRPE cells did not have any significant effect on Jkt T-cell apoptosis.
We and others have shown that RPE cells can induce apoptosis in human T cells.
6 7 8 10 11 It is well known that specific pro- and anti-inflammatory cytokines can modulate the proapoptotic activity of various cell types.
12 13 14 15 16 Soluble factors, including cytokines, are known to play a role in the immune privilege of the eye.
32 33 34 35 The regulatory effects of pro- and anti-inflammatory cytokines on expression of cell surface molecules in the RPE has been studied extensively.
36 37 38 39 However, whether these cytokines can modulate the immune privilege in the subretinal space by regulating the proapoptotic effect of RPE cells on T cells is not known.
We found that both IFN-α and -γ upregulated HFRPE-induced apoptosis in Jkt T cells. IFN-α induced apoptosis, but the rate was significant only in the absence of cell–cell contact (supernatant system). It may be postulated that IFN-α–activated HFRPE cells also express a cell-contact–mediated antiapoptotic signal to the Jkt T cells, preventing their apoptosis. In comparison to IFN-α, IFN-γ possessed a much stronger proapoptotic activity on HFRPE cells. It upregulated the HFRPE-induced apoptosis at 48, 72, and 96 hours. Although the upregulatory effect of IFN-γ was effective in the presence or absence of cell–cell contact, it was again clearly stronger in the latter condition, suggesting also the potential for the expression of a cell-contact–dependent antiapoptotic signal on HFRPE cells.
TGF-β is a potent immunoregulatory molecule capable of modulating the life and death fate of T-lymphocytes.
25 28 In the anterior segment of the eye, TGF-β is mainly an immune-suppressive cytokine.
26 27 This inhibitory cytokine is also found in both normal and pathologic vitreous and is capable of suppressing antigen presentation.
40 41 In our study, TGF-β did not upregulate the HFRPE-induced apoptosis in Jkt T cells. This may indicate that the immune suppressive characteristics of TGF-β in the subretinal space is mediated by direct suppression of T-cell activation, rather than modulation of the immune-suppressive characteristics of RPE.
TNF-α has been recognized as the prototype of a superfamily of immunoregulatory and effector molecules. It can be produced by macrophages, T cells, and nonlymphoid cells.
29 30 Depending on the cell type, TNF-α has the capability of inducing or inhibiting apoptosis. In HFRPE cells, however, TNF-α did not have any consistent significant up- or downregulatory effect on induction of apoptosis in Jkt T cells.
In summary, we conclude that whereas the cytokines IFN-α and -γ are generally known as prototypic proinflammatory cytokines they may in contrast promote an anti-inflammatory response in the subretinal space through the upregulation of RPE-mediated T-cell apoptosis. This anti-inflammatory effect of both type I and type II IFNs may play an important role in the maintenance of the immune privilege in the subretinal space.