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(Investigative Ophthalmology and Visual Science. 2007;48:3246-3252.)
© 2007 by The Association for Research in Vision and Ophthalmology, Inc.
DOI:  10.1167/iovs.06-1444
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The Role of Soluble TNF Receptors for TNF-{alpha} in Uveitis

Sunao Sugita,1 Hiroshi Takase,1 Chikako Taguchi,2 and Manabu Mochizuki1

1From the Department of Ophthalmology and Visual Science, Tokyo Medical and Dental University Graduate School of Medicine, Tokyo, Japan; and the 2Department of Ophthalmology, Kurume University School of Medicine, Fukuoka, Japan.


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
PURPOSE. To investigate the presence of soluble tumor necrosis factor receptors (sTNF-Rs) and TNF-{alpha} in the ocular fluids of patients with uveitis and the capacity of sTNF-Rs to affect TNF-{alpha} production by intraocular T cells.

METHODS. Ocular fluid samples were collected from patients with active and inactive uveitis, as well as from control subjects without uveitis. The sTNF-Rs and TNF-{alpha} levels were measured by enzyme-linked immunosorbent assay (ELISA). T-cell clones (TCCs) were established from intraocular infiltrating cells, and the TCCs were cocultured with recombinant sTNF-Rs or TNF-{alpha}. The supernatants were measured by ELISA. The neutralization of sTNF-R production by TCCs was evaluated with an anti-human TNF-R antibody.

RESULTS. Significantly higher amounts of sTNF-R1 and -R2 were present in the ocular fluids of patients with active uveitis than in the ocular fluids of those with inactive uveitis and in control subjects. Significantly higher amounts of TNF-{alpha} were present in the ocular fluids of patients with active uveitis than in those with inactive uveitis. Recombinant sTNF-Rs enhanced TNF-{alpha} production by TCCs in a dose-dependent manner. Similarly, recombinant TNF-{alpha} enhanced sTNF-Rs production by the TCCs, and production was neutralized with anti-human TNF-R antibody.

CONCLUSIONS. sTNF-Rs are present in the ocular fluids of patients with uveitis. Intraocular levels of sTNF-Rs are significantly increased in patients with uveitis, particularly in those with active uveitis. The data suggest that intraocular sTNF-Rs may play a regulatory role in ocular inflammation such as occurs in uveitis.

Tumor necrosis factors (TNFs) are pleiotropic cytokines that are considered to be the primary modifiers of inflammatory and immune reactions. Two forms of TNF, designated TNF-{alpha} and -ß, have been reported to compete for binding to the same receptors. There are two distinct TNF receptors (TNF-Rs), TNF-R1 (CD120a/p55TNF-R), and TNF-R2 (CD120b/p75TNF-R).1 The members of the TNF-R family include TNF-R1, TNF-R2, CD27, CD30, CD40, and Fas (APO-1, CD95).1 2 3 4 Both TNF receptor types show high-affinity binding of either TNF-{alpha} or TNF-ß. TNF-R1 and -R2 are expressed on macrophages, neutrophils, T cells, B cells, and NK cells.5 6 In particular, TNF-R2 is expressed on activated T lymphocytes.6 It is known that both types of soluble receptors can bind to TNF-{alpha} in vitro and inhibit its biological activity by competing with cell surface receptors for TNF-{alpha} binding.7 Consequently, it has been suggested that shedding of soluble receptors in response to TNF-{alpha} release could serve as a mechanism for binding and inhibiting the TNF-{alpha} that is not immediately bound to surface receptors. This mechanism would protect other cells from the effects of TNF-{alpha} and would localize the inflammatory response.8 In contrast, at low TNF-{alpha} concentrations, binding to soluble receptors stabilizes TNF-{alpha} and augments some of its activities.9 Thus, it is possible that under some conditions, the pool of TNF bound to soluble receptors could represent a reservoir for the stabilization and controlled release of TNF-{alpha}. In fact, as a proinflammatory cytokine, TNF-{alpha} has an important role in the etiology of ocular inflammation.

Soluble (s)TNF-Rs have biological activities. However, the presence and possible function of sTNF-Rs in the eye have not yet been clarified. Therefore, the purpose of the present study was to determine the extent to which soluble forms of TNF-Rs and TNF-{alpha} are present in the eye, by measuring their levels in the ocular fluid of patients with uveitis and the capacity of soluble forms of the receptors against TNF-{alpha} production by intraocular T cells.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Subjects
Samples of aqueous humor (AH, n = 21) and vitreous fluid (VF; n = 17) were collected from patients with uveitis, either active or inactive, associated with Behçet’s disease (n = 10), sarcoidosis (n = 16), Vogt-Koyanagi-Harada (VKH) disease (n = 1), human T-lymphotropic virus type 1 (HTLV-I) uveitis (n = 3), acute retinal necrosis (ARN; n = 4), cytomegalovirus retinitis (CMVR; n = 2), toxoplasmosis (n = 1), and toxocariasis (n = 1). At the time of AH sampling, the patients with uveitis had active intraocular inflammation, but they were not being treated with systemic therapy (n = 12). From patients having had no inflammation for more than 6 months (n = 9), we also obtained AH samples of inactive uveitis. In patients with uveitis who were undergoing vitreous surgery, VF samples were collected during the surgery. At the time of surgery, none of the patients was on systemic corticosteroids, and uveitis was active in 10 of them (2 with Behçet’s disease, 3 with sarcoidosis, 2 with ARN, 2 with CMVR, and 1 with toxocariasis) and inactive in 7. In the two patients with Behçet’s disease, vitrectomy was performed, although the patients had active uveitis because they had retinal detachment associated with a macular hole. In the two patients with sarcoidosis, vitrectomy was performed to obtain a cytological diagnosis due to clinical suspicion of malignancy, though no malignancy was found. In the remaining patients with active uveitis, therapeutic vitrectomy was performed. The severity of the uveitis was graded according to the classification of posterior uveitis developed by Nussenblatt et al.10 All patients with active uveitis had vitreous haze of grade 2 to 4. The control samples consisted of the AH of patients with age-related cataracts (n = 17) and the VF of patients with idiopathic macular holes (n = 16); the fluids were obtained during surgery. These control patients had no clinical history of uveitis or systemic diseases.

Approximately 0.1 mL of AH was drawn into tuberculin syringes. Blood-tinged samples were excluded from the study. After the AH samples were centrifuged at 3000 rpm for 5 minutes and the VF (~0.5 mL) was centrifuged at 10,000 rpm for 5 minutes, the supernatants were collected and stored in separate tubes at –80°C until used. The samples used in this study were collected between 1995 and 2001, and assayed from 2001 to 2005. The samples were thawed only once and then assayed. All samples were obtained after informed consent was obtained from the patients. The research adhered to the tenets of the Declaration of Helsinki. The Institutional Ethics Committees of Tokyo Medical and Dental University and Kurume University approved the study.

Establishment of T-Cell Clones
T-cell clones (TCCs) were established by the limiting dilution method as previously described.11 12 13 The cells were all CD4+ T cells (Th1 type cells) obtained from patients with uveitis who had Behçet’s disease (B3-4), sarcoidosis (S1-2), or VKH disease (VKH3-1).13 As the control, TCCs were also established from peripheral blood mononuclear cells (PBMCs) obtained from healthy donors.

Enzyme-Linked Immunosorbent Assay
The sTNF-Rs and TNF-{alpha} levels were determined by enzyme-linked immunosorbent assay (ELISA). The supernatant levels of sTNF-R1 and -R2 were measured by ELISA (R&D Systems, Minneapolis, MN). The sensitivity of the two assay kits was >7.8 pg/mL. The TNF-{alpha} levels were evaluated by two ELISA systems. A human TNF-{alpha} ELISA kit (Quantikine; R&D Systems) was used to evaluate in vitro the TNF-{alpha} levels in the supernatant of TCCs. The sensitivity of the kit was >4.4 pg/mL. The same kit was used to evaluate the supernatant levels of TNF-{alpha} in the ocular fluids (AH and VF). This kit can measure small amounts of TNF-{alpha}; the sensitivity of the assay kit was >0.18 pg/mL.

Flow Cytometry
We used the double-color immunofluorescence technique to examine the expression of TNF-Rs in ocular infiltrates obtained from AH. The expression of TNF-Rs was also examined in TCCs. The cells were incubated at 4°C for 30 minutes with FITC-conjugated anti-human TNF-R1 or anti-human TNF-R2 monoclonal antibody (R&D Systems) and PE-conjugated anti-human CD4 monoclonal antibody (NU-Th/i; Nichirei, Tokyo, Japan). These samples were then examined by double-color fluorescence flow cytometry (FACSCalibur; BD Biosciences, San Jose, CA).

In Vitro Assay with Recombinant Cytokines
The established TCCs were washed twice with PBS and then resuspended at 2 x 105 cells/mL in RPMI-1640 with 10% fetal bovine serum (FBS; Bioserum, Parkville, Victoria, Australia) in triplicate in 96-well culture plates, with or without human recombinant sTNF-R1 or -R2 (R&D Systems)Z in a dose-dependent manner. The TNF-{alpha} levels in the supernatant of TCCs obtained from the AH of patients with Behçet’s disease (B3-4), sarcoidosis (S1-2), and VKH disease (VKH3-1) were measured after coculturing with each of the recombinant sTNF-Rs: rTNF-R1 and -R2.

TCCs (B3-4) were examined by using methods similar to those used for the recombinant sTNF-R in vitro assay. The TCCs were washed, then resuspended at 2 x 105 cells/mL in RPMI-1640+10% FBS in triplicate, with or without human recombinant TNF-{alpha} (R&D Systems) in a dose-dependent manner.

Neutralization with Anti-human TNF-R2 Antibody
The TCCs (B3-4) were cocultured with or without anti-human TNF-R2 monoclonal antibody (R&D Systems) and incubated for 1 hour at 37°C. As an isotype control, mouse IgG was used (Ancell Corp., Bayport, MN). The T cells were also cultured with human recombinant TNF-{alpha} (100 pg/mL). After 24 hours, the supernatant of each of the TCCs was assayed to determine its effect on TCC sTNF-R2 production.

Statistical Analysis
Statistical analysis was performed with the Mann-Whitney test or a paired t-test. The difference between the two groups was considered to be statistically significant at P < 0.05.


   Results
Top
 Abstract
 Materials and Methods
 Results
Discussion
 References
 
sTNF-R1 and -R2 Concentrations in the Ocular Fluids of Patients with Uveitis
The sTNF-Rs and TNF-{alpha} levels in the ocular fluids of control subjects and patients with uveitis were quantified by ELISA. Representative results are shown in Table 1 . The sTNF-R1 and -R2 levels in the AH of patients with active uveitis (n = 12) were 1258 pg/mL and 2134 pg/mL, respectively, whereas in the control samples, the levels were very low (Table 1) . Significantly higher amounts of sTNF-R1 and -R2 were seen in the AH of patients with active uveitis than in patients with inactive uveitis (n = 9) and control subjects (n = 17). The VF levels of sTNF-R1 and -R2 in patients with uveitis were 2769 pg/mL and 3103 pg/mL, respectively; those in the control patients were low. In contrast, the VF levels of sTNF-R1 and -R2 were not significantly different between patients with active uveitis (n = 10) and those with inactive uveitis (n = 7). However, there was a statistically significant difference in the VF levels of sTNF-R1 and -R2 between the control subjects (n = 16) and patients with active uveitis (Table 1) . These results indicate that ocular fluids of patients with active uveitis contain high levels of soluble forms of TNF-Rs.


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  		TABLE 1. Concentration of sTNF-R1 and sTNF-R2 in Ocular Fluids

 
TNF-{alpha} Concentrations in Ocular Fluids of Patients with Uveitis
We attempted to measure the TNF-{alpha} concentration of normal ocular fluids using a TNF-{alpha} ELISA kit (Quantikine human TNF-{alpha} ELISA kit; R&D Systems), but all 20 samples had undetectable levels (data not shown). Therefore, the HS human TNF-{alpha} immunoassay kit, which can measure small amounts of TNF-{alpha} (≤0.18 pg/mL), was used. In the control group of 17 patients with age-related cataract with no history of ocular inflammation, AH TNF-{alpha} levels were detectable in 15 patients (range, 0.6–28.0 pg/mL), whereas AH TNF-{alpha} levels were undetectable in the other two patients (Table 2) . The mean AH TNF-{alpha} level was 6.8 pg/mL. Similar to the AH results, all patients with a macular hole had detectable VF TNF-{alpha} (9.7 pg/mL). These results indicate that almost all the patients in the control group with no inflammation had significant TNF-{alpha} levels. As for the patients with inactive uveitis, significant TNF-{alpha} levels were detected in 7 of 10 (mean AH level, 1.5 pg/mL). Furthermore, significant VF TNF-{alpha} levels (mean, 4.5 pg/mL) were detected in all patients with inactive uveitis. The difference in the ocular fluid TNF-{alpha} levels between the control and the inactive uveitis groups was not statistically significant (P = 0.18). The AH TNF-{alpha} level in patients with active uveitis was 19.6 pg/mL, and the VF was 29.8 pg/mL (Table 2) . There was a statistically significant difference in ocular fluid TNF-{alpha} levels between the active uveitis and the inactive uveitis groups, as well as between the active uveitis and control groups (Table 2) . As expected, the ocular fluids in patients with active uveitis had higher levels of TNF-{alpha}, which is a proinflammatory cytokine. In addition, the levels of TNF-{alpha}, sTNF-R1, and sTNF-R2 correlated in individual samples. For example, in the case of severe uveitis, such as acute retinal necrosis, ocular fluids including high TNF-{alpha} production contained significant amounts of both soluble receptors.


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  		TABLE 2. Concentration of TNF-{alpha} in Ocular Fluids

 
sTNF-R2 and TNF-{alpha} Production by TCCs
Ocular infiltrating T cells were studied to determine whether they could produce soluble forms of TNF-Rs and TNF-{alpha}. We measured soluble forms of TNF-Rs receptor type 2 (sTNF-R2) in supernatants from T cells. In our preliminary experiment using ELISA, TCCs established from active uveitis produced large amounts of sTNF-R1. However, control T cells established from the PBMCs of healthy donors also produced sTNF-R1 (no significant difference between control and uveitis T cells). Thus, we decided to use only the sTNF-R2 data. The TCCs were established from the AH obtained from patients with representative ocular disorders associated with active uveitis. Significant sTNF-R2 and TNF-{alpha} levels were detected in the culture supernatant of TCCs established from intraocular infiltrating cells obtained from various disorders associated with uveitis (Table 3) . The mean sTNF-R2 levels in the culture supernatant of TCCs obtained from Behçet’s disease, sarcoidosis, and VKH disease were 144, 72, and 46 pg/mL, respectively; the mean TNF-{alpha} levels were 99, 55, and 45 pg/mL, respectively. The levels of sTNF-R2 and TNF-{alpha} production were significantly higher in the TCCs obtained from patients with Behçet’s disease, sarcoidosis, and VKH disease than in those obtained from healthy donors (P < 0.05 or P < 0.005).


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  		TABLE 3. sTNF-R2 and TNF-{alpha} Production in Supernatants of TCCs

 
The levels of these cytokines in patients with active and inactive uveitis were then compared. Ocular fluid levels of sTNF-R2 and TNF-{alpha} were measured in patients with sarcoidosis (n = 20). In these patients, eye that had shown no signs and symptoms of uveitis for the past 3 months were considered to have inactive disease (n = 10; 5 AH and 5 VF). Eyes with inflammation in the anterior segment, vitreous, or posterior segment at the time of sample collection were classified as having active uveitis (n = 10; 5 AH and 5 VF). Significant levels of sTNF-R2 and TNF-{alpha} were detected in the ocular fluids in all patients with sarcoidosis and active uveitis, compared with those with inactive uveitis (Fig. 1) ; the differences between the two groups were statistically significant (P < 0.05 or P < 0.005). Taken together, these results indicate that ocular fluids with active inflammation contain large amounts of these cytokines.


Figure 1
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  		FIGURE 1. TNF-{alpha} (left) and sTNF-R2 (right) concentrations in ocular fluids. The levels of these cytokines were evaluated by ELISA. AH and VF samples (n = 5) were obtained from patients with active uveitis and AH and VF samples (n = 5) from patients with inactive uveitis (no signs and symptoms of uveitis) who had sarcoidosis. Bars, mean ± SD. TNF-{alpha} or sTNF-R2 production by TCCs. *P < 0.05, **P < 0.005, compared with two groups.

 
Detection of TNF-R on Fresh Ocular Infiltrating Cells and TCCs
The expression of TNF-R on ocular infiltrating cells that were freshly obtained from patients with uveitis was determined. As shown in Figure 2A , ocular infiltrating cells, such as CD4+ T cells, in the AH expressed TNF-R2 on their surface. The level of double-positive cells was 57% (Fig. 2A) . The stained cells show TNF-R1 and -R2 expression on CD4+ TCCs obtained from patients with VKH disease (VKH3-1, Fig. 2B ). Similarly, CD4+ TCCs of patients with Behçet’s disease and sarcoidosis clearly expressed these receptors (data not shown).


Figure 2
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  		FIGURE 2. Expression of surface TNF receptors on ocular infiltrating CD4+ T cells. (A) Expression of TNF-R2 was evaluated with flow cytometry of infiltrating cells in the fresh AH of a patient with Vogt-Koyanagi-Harada disease. Briefly, these cells were incubated with FITC-labeled anti-human TNF-R2 monoclonal antibody and PE-labeled anti-human CD4 antibody. (B) TCCs (1 x 106 cells) were assessed with the same method. The stained cells were CD4+ TCC (VKH3-1) established from a patient with VKH disease. TCCs were incubated with FITC-labeled anti-TNF-R1 or R2 antibody and PE-labeled anti-CD4 antibody. These samples were assayed by double-color fluorescence flow cytometry. The number indicates percentage of double-positive cells.

 
Capacity of Recombinant sTNF-R against TNF-{alpha} Production by TCCs
As described in previous experiments, significant levels of sTNF-Rs and TNF-{alpha} were detected in ocular fluids with active uveitis. In addition, TCCs established from ocular infiltrating cells produced significant levels of these cytokines. Therefore, we evaluated the in vitro effect of recombinant sTNF-Rs on TNF-{alpha}. CD4+ TCCs (B3-4, S1-2, and VKH3-1) were used in this experiment. As shown in Figure 3A , recombinant sTNF-R1 significantly enhanced TNF-{alpha} production by the TCCs that were used in a dose-dependent manner between 1 and 100 ng/mL (P < 0.05, P < 0.005, respectively). Similarly, recombinant sTNF-R2 enhanced TNF-{alpha} production by these TCCs (Fig. 3B) . These results suggest that T cells stimulated with soluble TNF-Rs begin to synthesize TNF-{alpha}.


Figure 3
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  		FIGURE 3. The effect of recombinant sTNF-R1 (A) and sTNF-R2 (B) on TNF-{alpha} production by TCCs established from infiltrating cells obtained from patients with uveitis. Target cells established from AH of patients with Behçet’s disease (B3-4), sarcoidosis (S1-2), or VKH disease (VKH3-1). Bars, mean ± SD. TNF-{alpha} production by TCCs. Probabilities are mean values significantly higher than control medium only ({square}, the absence of recombinant sTNF-R1or -R2): *P < 0.05, **P < 0.005.

 
Effect of Recombinant TNF-{alpha} on TNF-R2 Production by TCCs
Finally, the effects of recombinant TNF-{alpha} on sTNF-R2 production by TCCs were evaluated. In preliminary experiments, it was found that high concentrations of recombinant TNF-{alpha} induced T-cell death (data not shown). Therefore, a concentration range of recombinant TNF-{alpha} that produced no T cell toxicity was used. Similar to the results obtained with recombinant sTNF-R, recombinant TNF-{alpha} significantly increased sTNF-R2 production by the TCCs that were tested in a dose-dependent manner between 1 and 500 pg/mL (P < 0.05; Fig. 4A ). In further experiments, sTNF-R2 production by TCCs was neutralized by coculturing with anti-human TNF-R2 monoclonal antibody compared with coculturing with control isotype antibody (P < 0.05, Fig. 4B ). Similarly, sTNF-R2 production by TCCs was neutralized by anti-TNF-R2 antibody when the TCCs were pretreated with rTNF-{alpha} in vitro. Together, these results suggest that TNF-{alpha} produced by ocular infiltrating cells binds to TNF-R on T cells, and then the stimulated T cells are promoted to synthesize soluble forms of TNF-Rs to produce TNF-{alpha}. Thus, soluble forms of TNF-Rs, as well as TNF-{alpha}, function as proinflammatory cytokines.


Figure 4
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  		FIGURE 4. Effect of human recombinant TNF-{alpha} on sTNF-R2 production by TCCs. The target cells, B3-4 TCCs, were established from a patient with Behçet’s disease. (A) The effect of recombinant TNF-{alpha} on TNF-R2 production by TCCs was evaluated by ELISA. Bars, mean ± SD of sTNF-R2 production by TCCs. Probabilities are mean values significantly higher than the control (no rTNF-{alpha}, {square}): *P < 0.05. (B) The T cells were cocultured with anti-human TNF-R2 monoclonal antibody (bar 2). Before this, T cells were precultured with ({blacksquare}) or without ({square}) human recombinant TNF-{alpha} (final concentration 100 pg/mL). Isotype antibody of mouse IgG was used for the control (bar 3). Bars, mean ± SD of sTNF-R2 production by TCCs. Probabilities are for values significantly higher than the control (no antibodies, bar 1): *P < 0.05.

 

   Discussion
Top
 Abstract
 Materials and Methods
 Results
 Discussion
References
 
The present study showed for the first time that sTNF-Rs are present in the ocular fluids of eyes with uveitis. Intraocular levels of sTNF-Rs were significantly increased in uveitis, particularly in active uveitis. Both types of soluble receptors, TNF-R1 and -R2, are capable of binding TNF-{alpha} and thus promote the biological activities of TNF-{alpha}. These data suggest that intraocular sTNF-Rs may play a regulatory role in ocular inflammation.

TNF-{alpha} and TNF-Rs have a significant role in certain types of inflammation, including ocular inflammation. It has been reported that AH TNF-{alpha} levels were increased in patients with uveitis with active inflammation,14 and that the serum TNF-{alpha}14 15 and sTNF-R216 levels were higher in patients with Behçet’s disease. Torun et al.17 recently reported that serum sTNF-R1 levels appeared to be higher in patients with posterior and intermediate uveitis compared with control subjects. Our group has found that TCCs established from the ocular infiltrating cells in Behçet’s disease,12 sarcoidosis,18 and VKH disease19 produced significant TNF-{alpha} levels. In animal studies, TNF-{alpha} has been shown to play an important role in pathogenic mechanisms. TNF-{alpha} induced experimental autoimmune uveoretinitis (EAU) in mice20 ; anti-TNF-{alpha} antibody therapy suppressed the induction of EAU in mice.21 Recently, we reported that primary cultured ocular pigment epithelial (PE) cells, iris PE, ciliary body PE, and retina PE constitutively expressed TNF-R1 and -R222 ; this suggests that ocular resident cells, such as PE cells, can express TNF receptors under normal conditions. In other reports, sTNF-Rs were detected in the vitreous from normal human subjects23 and from patients with vitreoretinal proliferative disease.24 However, there have been no reports dealing with TNF-Rs and eye-derived soluble receptor forms in patients with uveitis. In the present study, ocular fluids of patients with active uveitis contained significantly higher sTNF-R1 and -R2 levels than the ocular fluids of inpatients with active uveitis and controls without uveitis. In contrast to the results of patients with uveitis, sTNF-R1 and -R2 were undetectable or at minimal levels in the ocular fluids of patients without uveitis. These results are consistent with the fact that immunocytes are absent in the ocular fluids of normal eyes. Several groups have found that sTNF-R-binding proteins in human serum and urine can neutralize the biological activities of TNF-{alpha}.7 The soluble receptor forms apparently arise as a result of shedding of the receptors’ extracellular domains; concentrations of approximately 1 to 2 ng/mL are found in the serum and urine of healthy subjects.8 In contrast, another group reported that, at low concentrations, TNF-{alpha} bind sTNF-Rs, resulting in stabilization and augmentation of TNF-{alpha} activity.9 In the present study, recombinant sTNF-R1 and -R2 (1–100 ng/mL) enhanced TNF-{alpha} production by ocular infiltrating T cells in vitro. However, sTNF-R at physiologic concentrations (i.e., in the picograms per milliliter range) did not promote TNF production. In contrast, most ocular fluids from active uveitis contained sTNF-Rs in the nanogram per milliliter range. We believe that the aqueous or vitreous humor itself increases TNF-{alpha} production in inflammatory conditions, but not in normal conditions. Thus, intraocular sTNF-Rs may augment the intraocular TNF-{alpha} that is produced by ocular infiltrating T cells.

Our study also demonstrated significant TNF-{alpha} levels in the ocular fluids of patients with age-related cataract who had no history of ocular inflammation. Under almost normal conditions (in patients with cataracts), ocular resident cells in the iris-ciliary body25 and retina26 have been found to express TNF-{alpha} constitutively. The extracellular domain of TNF is thought to be processed by specific matrix metalloproteinases (MMPs) and then released in soluble form. The MMPs facilitate the secretion of TNF-{alpha} by cleaving the membrane-bound form that is present on cells.27 28 In fact, MMPs were found in normal human AH and in patients with uveal inflammation.29 Consequently, MMPs may promote the secretion of TNF-{alpha} by ocular infiltrating cells and ocular resident cells. Therefore, it is likely that the TNF-{alpha} detected in the ocular fluids of patients without uveitis is released from the resident cells that surround the ocular fluids. Ocular fluids contain immunosuppressive factors, such as {alpha}-melanocyte-stimulating hormone,30 vasoactive intestinal peptide,31 and TGF-ß.32 Soluble FasL (sFasL) is present in the AH of nonuveitic eyes with age-related cataract.33 It is known that TNF-{alpha} is involved in autoregulatory apoptosis through a different mechanism than FasL.34 TNF-{alpha} can mediate mature T-cell receptor–induced apoptosis through TNF-R2. TNF-R2-deficient mice show normal T-cell development and activity, but they have increased resistance to TNF-induced death.35 In addition, TNF-R-deficient mice have been found to have decreased inflammation in uveitis animal models.36 Therefore, TNF-{alpha}, as well as sFasL, in the ocular fluids may be one of the factors that play a role in inducing apoptotic cell death in infiltrating cells, thereby regulating ocular inflammation.

TNF-{alpha} functions as a proinflammatory cytokine; however, it is not always proinflammatory, and blocking it in some animal models exacerbates disease.37 Similarly, TNF-{alpha} is required for the induction of anterior chamber–associated immune deviation (ACAID).38 Chronic stimulation of T cells by TNF-{alpha} leads to hyporesponsiveness, whereas acute stimulation leads to cytokine activation. In another interesting report by Ehrenstein et al.,39 it was demonstrated that during anti-TNF therapy, decreasing TNF-{alpha} levels are a surrogate marker for the effects on regulatory T cells. This reaction suggests that TNF-{alpha} itself may not be an effector in uveitis, but may simply inhibit regulatory T-cell function.

Ohno et al.40 recently reported the use of anti-human TNF-{alpha} as a therapy in patients with Behçet’s disease. The administration of anti-TNF-{alpha} antibodies inhibited EAU in animal studies.21 41 Moreover, some groups have reported that patients with rheumatoid arthritis42 and Crohn’s disease43 had profoundly decreased systemic and local inflammation; compared with steroid treatment, antibody treatment appeared to be safe and well tolerated in these diseases. The present study showed that recombinant TNF-{alpha} increased sTNF-R production by T cells obtained from patients with Behçet’s disease. The assay was neutralized with anti-human TNF-R antibody in vitro. In addition to anti-human TNF-{alpha} antibody therapy, anti-human TNF-R antibody may be a useful therapy in some patients with active uveitis.

Therefore, soluble forms of TNF-Rs are among the inflammatory cytokines found in uveitis. In the pathogenic mechanisms of uveitis, sTNF-Rs are produced by intraocular infiltrating cells, such as macrophages, neutrophils, T lymphocytes, and ocular resident cells. Consequently, sTNF-Rs produced by these cells may be promoted by TNF-{alpha}, one of the proinflammatory cytokines. Thus, intraocular sTNF-Rs may play a major role in ocular inflammation, including uveitis.


   Acknowledgements
 
The authors thank Naofumi Hikita and Kouichi Yoshimura (Department of Ophthalmology, Kurume University School of Medicine, Fukuoka, Japan) for obtaining samples of ocular fluids and Tomoko Yoshida for expert technical assistance.


   Footnotes
 
Supported by Grants-in-Aid for Encouragement of Young Scientists (A) 13771015 and Scientific Research (B) 1437055 of the Ministry of Education, Culture, Sports, Science and Technology, Japan.

Submitted for publication December 5, 2006; revised January 15 and 30, 2007; accepted April 18, 2007.

Disclosure: S. Sugita, None; H. Takase, None; C. Taguchi, None; M. Mochizuki, None.

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Corresponding author: Sunao Sugita, Department of Ophthalmology and Visual Science, Tokyo Medical and Dental University Graduate School of Medicine, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8519, Japan; sunaoph{at}tmd.ac.jp.


   References
Top
 Abstract
 Materials and Methods
 Results
 Discussion
 References
 

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