Human S-antigen: peptide determinant recognition in uveitis patients

Experimental and Molecular Pathology 76 (2004) 122 – 128 www.elsevier.com/locate/yexmp

Human S-antigen: peptide determinant recognition in uveitis patients Parul Tripathi, a Sandeep Saxena, b Virendra S. Yadav, a Sita Naik, a and Vijay K. Singh a,* a

Department of Immunology, Sanjay Gandhi Post-Graduate Institute of Medical Sciences, Lucknow 226 014, India b Department of Ophthalmology, King George’s Medical College, Lucknow 226 003, India Received 10 October 2003

Abstract Uveitis is an inflammation of the uveal tract and is one of the major causes of visual impairment. Several lines of evidence suggest an important role for activated T lymphocytes in the perpetuation of posterior uveitis. In sequel to our preliminary observations with human Santigen, we have further investigated the proliferative response of peripheral blood lymphocytes of posterior uveitis patients against 20 linear and 9 overlapping peptides of retinal S-antigen. The expression of surface markers CD4, CD8, CD29, CD45RA in peripheral blood was detected by flow cytometry. We have also assessed the pattern of cytokines present in peripheral blood mononuclear cells (PBMCs) using ribonuclease protection assay (RPA). Nineteen out of 32 patients’ lymphocytes showed proliferative response to S-antigen, one or more of its 20 linear and nine overlapping synthetic peptides. Six patients showed significant lymphoproliferative response against various peptides. The maximum response was found to peptides from the 231 – 270 amino acid region of human S-antigen sequence. The percentage of CD29+ (memory cells) and CD45RA+ (naive cells) T-lymphocytes was higher in patients compared to healthy volunteers. There was a demonstrable difference in the percentage of CD4+ and CD8+ lymphocytes in the patients ( P V 0.05) as compared to controls. Higher message for interleukin (IL)-5, IL-10, IL-15, IL-9, IL-2, IL-13, and interferon (IFN)-g was observed in uveitis patients than in healthy individuals. In brief, our study suggests that a particular region of S-antigen plays an important role in idiopathic uveitis. D 2003 Elsevier Inc. All rights reserved. Keywords: Cytokines; mRNA; Retinal S-antigen peptides; Ribonuclease protection assay, T cell proliferation; Uveitis

Introduction Immune reactions against retinal autoantigens have been proposed to play an important role in the pathogenesis of uveitis. Several studies have suggested that the T-cellmediated autoimmune process plays a major role in the causation of the disease (de Smet and Chan, 2001). The retina contains several organ-specific antigens that can induce T-cell-mediated experimental ocular inflammation in susceptible animal strains. The clinical and histopathological appearance of uveitis occurring in animal models closely resembles uveitic conditions in man (Faure, 1980;

Abbreviations: BD, Behcet’s disease; CPM, mean count per minute; EAU, experimental autoimmune uveitis; IFN, interferon; IL, interleukin; PBMCs, peripheral blood mononuclear cells; PHA, phytohemagglutinin; PPD, purified protein derivative; RPA, ribonuclease protection assay; SD, standard deviation; SI, stimulation index. * Corresponding author. Radiation Medicine Department, Armed Forces Radiobiology Research Institute, 8901 Wisconsin Avenue, Bethesda, MD 20889, USA. Fax: +1-301-295-0292. E-mail address: [email protected] (V.K. Singh). 0014-4800/$ - see front matter D 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.yexmp.2003.10.007

Gery et al., 1986; Hirose et al., 1989; Singh et al., 1990; Wacker et al., 1977). One uveitogenic antigen, retinal Santigen, which plays a pivotal role in the visual process and has been extensively investigated (Gery et al., 1986; Pfister et al., 1985; Shinohara et al., 1987), is shown to be highly conserved among mammalian species (Mirshahi et al., 1985). Major uveitopathogenic epitopes of S-antigen have been investigated and identified in experimental models (de Smet et al., 1993; Donoso et al., 1987; Gregerson et al., 1990; Singh et al., 1988, 1989, 1992). Patients with uveitis show lymphoproliferative response to various retinal antigens in vitro (de Smet et al., 1990a,b; Doekes et al., 1987). We have also studied the cellular immune response of posterior uveitis patients to native S-antigen, 20 linear peptides spanning the entire sequence of the S-antigen, and two additional peptides known to be uveitopathogenic in animal models (Rai et al., 2001). Furthermore, CD4+ helper T cells have been shown to play an important role in the pathogenesis of experimental and clinical uveitis (Murray et al., 1999). The presence of T lymphocytes in the ocular fluid of patients with uveitis has also been demonstrated (Deschenes et al., 1986, 1988).

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Immunohistochemical staining of enucleated eyes of patients with Behcet’s disease (BD), intermediate uveitis, and sarcoidosis has revealed that CD4+ T-helper lymphocytes are present in the retina and choroid in active inflammatory lesions, whereas B-cells are only scarcely observed (Lightman and Chan, 1990). CD4+ cells have been divided into Th1 and Th2 subsets based on the pattern of cytokines they secrete (Romagnani, 1997; Singh et al., 1999). Cytokines appear to play an important role in the etiopathogenesis of posterior uveitis and its animal model, experimental autoimmune uveitis (EAU) (Sun et al., 1997, 1999). The exact contribution of these mediators to uveitis remains to be well defined. Recent studies suggest that the shift from a Th1 to a Th2-dominated response could be of therapeutic benefit (Caspi et al., 1996; Mosmann et al., 1986; Singh and Rai, 2001). Th1 and Th2 cytokines have also been investigated in serum samples and culture supernatant of peripheral blood mononuclear cells (PBMCs) of different categories of uveitis patients stimulated in vitro (Murray et al., 1999; Raziuddin et al., 1998). Numerous CD4+T cells and Th1 cytokine mRNAs have been documented in eyes with active Sympathetic Ophthalmia and BD (Chan and Li, 1998). Among various types of uveitis, BD is the most studied in relation to cytokines. In our present study, we investigated the cellular immune response of posterior uveitis patients to native S-antigen (404 amino acid long protein), 20 linear peptides (each peptide of approximately 20 amino acid length) spanning the entire sequence of the human S-antigen, as well as to nine overlapping peptides (overlapping to those linear peptides which had shown a significant proliferative response in an earlier study from our laboratory). To better define the role of lymphocytes in the pathogenesis of uveitis, the expression of memory and naive cell markers on peripheral blood T-lymphocytes and their cytokine patterns has been studied in these patients and compared to healthy controls.

Materials and methods Antigens and reagents Antigens used in the assay included native bovine Santigen, 20 linear synthetic peptides (numbered 1 – 20) spanning the entire sequence of S-antigen, and nine overlapping peptides (numbered A – I) selected from regions which showed a positive response for T cell proliferation in our previous study (Rai et al., 2001). All the peptides were synthesized by Chiron Technologies, Clayton Victoria, Australia, by conventional solid phase chemistry on a benzhydrylamine resin. The amino acid sequence and positions in human retinal S-antigen of the linear peptides have been published previously (Rai et al., 2001). The amino acid sequence and positions of the overlapping peptides are shown in Table 1.

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Table 1 Overlapping peptides of human S-antigen Peptides

Sequence

Sequence position

A B C D E F G H I

VDPDLVKGKKVYVTLTCAFR YGQEDVDVIGLTFRRDLYFS RVQVYPPVGAASTPTKLQES PCSVMLQPAPQDSGKSCGVD FEVKAFATDSTDAEEDKIPK KSSVRYLIRSVQHAPLEMGP VSLNREIYFHGEPIPVTVTV TNNTEKTVKKIKACVEQVAN VVLYSSDYYVKPVAMEEAQE

51 – 70 71 – 90 91 – 110 131 – 150 151 – 170 171 – 190 211 – 230 231 – 250 251 – 270

RPMI-1640, HEPES, phytohemagglutinin (PHA), and TriReagant were purchased from Sigma (St Louis, MO, USA). Lymphoprep was obtained from Nycomed Pharma (Oslo, Norway); purified protein derivative (PPD) was a gift from Span diagnostics (Mumbai, India). Ribonuclease protection assay (RPA) kit was procured from Pharmingen (hCK-1; San Diego, CA, USA). Monoclonal antibodies and FACS lysing solution for flow cytometry were purchased from Becton Dickinson Immunocytometry Systems (Palo Alto, CA, USA). Patients Thirty-two uveitis patients (16 males and 16 females) were selected for this study. Various ophthalmologists of Lucknow and Kanpur referred these uveitis patients to our laboratory for evaluation. Informed consent was obtained from all patients before their inclusion in the study. There was no bias for inclusion in respect of their current medical therapy or degree of disease activity. The ages of the patients ranged between 8 and 75 years (median: 33.5 years). The classical signs of uveitis, viz circumcilliary congestion, loss of luster and pattern of iris, irregularity of pupil, posterior synechiae, and pigment on lens surface, were present in most of the patients. The disease was of acute onset in nine patients, subacute in five, and chronic in 18 patients. Seventeen patients had choroiditis, two were diagnosed to have serpiginious choroiditis, three had pars planitis, two had iridiocyclitis, and eight were suffering from posterior uveitis. Among these patients, five had rheumatoid arthritis and one had a history of intestinal tuberculosis. Five cases had a history of ocular trauma preceding the onset of uveitis. Pupillary reaction was present in 12 patients. On slit lamp examination, eight patients had fine, old keratic precipitates (KPs) and 13 had cells. Fifteen normal healthy volunteers who did not handle retinal S-antigen or peptides served as the control group. Lymphocyte proliferation assay Mononuclear cells were separated from venous blood by density gradient centrifugation using commercial lympho-

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prep. PBMCs were resuspended at a concentration of 1  105 cells per well in RPMI-1640 medium supplemented with 25 mM HEPES, L-glutamine (2 mM), penicillin (100 U/ml), streptomycin (100 Ag/ml), and 10% heat-inactivated fetal calf serum. Cultures were set up as described earlier (Bajpai et al., 1995). PPD, native retinal S-antigen, 20 linear, and nine overlapping peptides were used at a dose of 20 Ag/ml in triplicate wells for stimulation. Wells without any stimulant served as controls. The cultures were pulsed with 0.5 ACi [3H]-thymidine (6 Ci/mmol, BARC, Bombay, India) per well during the last 18 h of incubation and harvested on glass fiber filters (Advance Microdevice, Ambala, India) using a multiharvester (Skatron, Lier, Norway). Thymidine uptake was determined by liquid scintillation counter (LKB, Fullerton, CA, USA). The mean count per minute (CPM) of triplicate cultures was calculated for each set of replicate cultures. The standard deviation (SD) of the mean CPM of triplicates was routinely <20%. The stimulation index (SI) was derived by dividing the mean CPM for each of the antigen-stimulated cultures by the mean CPM of the control cultures. The SI of 2 and above was taken as positive in this study. Results are presented both as CPM F SD and SI. Flow cytometric analysis for cell surface markers Whole blood (100 Al) was stained with anti-IgG1 FITC/ IgG2aPE (isotype control), anti-CD29 (memory cell marker), anti-CD45RA (naive cell marker), anti-CD4 (helper or inducer T cell), and anti-CD8 (cytotoxic or suppressor T cell) antibodies labeled with FITC or PE. The samples were then incubated in the dark for 30 min followed by lysis of erythrocytes with FACS lysing solution as per the manufacturer’s instructions. The samples were analyzed with FACScalibur flow cytometer using Cell Quest software as

described earlier (Mehrotra et al., 2002). The cell surface marker expression was analyzed in the gated population for total lymphocytes. Ribonuclease protection assay Total RNA was isolated from PBMCs using TriReagent as described elsewhere (Narayan et al., 2002). In vitro transcription was performed for the preparation of [32P]labeled anti-sense RNA probe using multiprobe RPA template set for human cytokines following manufacturer instructions. In brief, T7 RNA polymerase-directed synthesis of a high specific activity RNA probe was carried out using [a-32P] UTP, GACU nucleotide pool, DTT, 5 transcription buffer, and RPA template set followed by its purification using phenol and chloroform, and quantitation in liquid scintillation counter. Thereafter, the probe set was hybridized in excess to target RNA in solution for 16 h at 56jC, after which free probe and other single-stranded RNA were digested with RNAase. The remaining ‘‘RNAaseprotected’’ hybridized products were purified as described earlier and resolved on 5% sequencing denaturing ureaPAGE. A dilution of the probe set in loading buffer (4000 CPM/lane) was also loaded to serve as size markers. The gel was run at 50 W constant power until the leading edge of the front dye reached 30 cm. Detection of specific bands of protected mRNA of various cytokines was carried out by autoradiography and quantitated by spot densitometry using ALPHA Imager v5.5 software. Statistical analysis Data were analyzed using statistical software SPSS 9.0. The significance of the T cell proliferation response was assessed by Z test for proportions. Data are expressed as

Fig. 1. T cell response of uveitis patients to linear and overlapping peptides of S-antigen. Linear peptides are numbered 1 – 20, and overlapping peptides as A to I. For each peptide, number of responders are shown (out of 19 patients who showed response to one or other peptide).

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Table 2 Lymphocyte proliferative response of uveitis patients to linear fragments of S-antigen Treatment

Patient number 10

11

Control PPD Peptide 1 Peptide 2 Peptide 3 Peptide 4 Peptide 5 Peptide 6 Peptide 7 Peptide 8 Peptide 9 Peptide 11 Peptide 13 Peptide 15 Peptide 16 Peptide 17 Peptide 18

1202 F 126

252 F 26 526 F 28 (2.0)

17

22

557 F 20

26

459 F 18 1553 F 25 (3.4)

29

260 F 40

1036 F 24

1141 F 82 (2.0) 2273 F 311 (4.9) 1319 F 185 (2.3) 648 F 22 (2.6) 2733 F 230 (2.2)

1589 F 33 (3.4) 1231 F 30 (2.7) 1158 F 16 (2.5)

8153 F 62 (14.6)

946 F 14 (3.8) 676 F 28 (2.7)

1153 F 32 (2.0) 7758 F 1421 (13.9) 1144 F 72 (2.0)

1118 F 27 (2.4) 1208 F 82 (2.6) 1453 F 66 (3.1) 977 F 5 (2.1)

1093 1706 1879 1243

F F F F

94 (4.2) 263 (6.5) 89 (7.2) 70 (4.7)

2759 F 264 (2.6)

1326 F 75 (5.1) 1308 F 73 (5.0) 719 F 19 (2.7)

1486 F 19 (5.7) 584 F 16 (2.2) 925 F 7 (3.5)

2377 F 9 (2.3)

2503 F 117 (2.0)

Values are given in CPM F SD for patients showing positive response (SI z 2). Actual SI values are given in parenthesis.

mean F SD. Comparison between results from the patients and control group was done using the Mann – Whitney test. The P value of V0.05 was considered significant.

Results Lymphocyte proliferation response of patients and controls Nineteen out of 32 patients (approximately 59%) with posterior uveitis showed significant lymphocyte proliferative response against at least one or more of both the linear and the overlapping fragments of S-antigen. Of the 19 responders, 10 were females and 9 were males. Eight patients showed T cell response to PPD. None among the 15 healthy volunteers (data not presented) showed any significant proliferative response (SI more than 2) to retinal

S-antigen or its peptides. Compared to controls, the frequency of responders in the patient group was significant ( P V 0.05). The overlapping fragments of S-antigen had 10 amino acid overlaps of the linear fragments, which had shown response in our earlier study (Rai et al., 2001). The frequency of responders to the individual peptides (linear fragments and their corresponding overlapping peptides) is shown in Fig. 1. Six patients showed a response to both linear (Table 2) and overlapping peptides (Table 3). The salient clinical features of these six patients and the peptides against which these patients have shown response are given in Table 4. Previous study from our laboratory has reported that our uveitis patients from India respond most frequently to linear peptides 4, 5, 8, 9, 12, and 13 of the human S-antigen (Rai et al., 2001). In the present study also, we observed significant response against the above

Table 3 Lymphocyte proliferative response of uveitis patients to overlapping fragments of S-antigen Treatment

Patient number 10

11

Control PPD Peptide A Peptide B Peptide C Peptide D Peptide E Peptide F Peptide G Peptide H Peptide I

279 F 42

150 F 27 883 F 14 (5.9)

17 473 F 98 1311 F 224 (2.7)

571 F 49 (2.0)

1540 F 28 (3.2)

623 F 4 (2.2)

1115 F 99 (2.3) 1189 F 47 (2.5) 411 F 12 (2.7)

634 F 68 (2.2) 646 F 108 (2.3)

22

26

184 F 16 983 F 9 (5.3) 527 F 28 (2.8)

183 F 10

29 154 F 17 339 F 34 (2.2)

1421 F 35 (7.8) 706 F 14 (3.9) 478 735 656 658

F F F F

16 19 14 28

(2.6) (4.0) (3.6) (3.6)

780 F 42 (4.3) 1631 F 185 (8.9)

468 F 35 (3.1)

Values are given in CPM F SD for patients showing positive response (SI z 2). Actual SI values are given in parenthesis.

1295 F 1 (8.4) 1299 F 7 (8.4)

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Table 4 Important features of uveitis patients showing significant T-cell response in vitro

Table 5 Lymphocyte surface markers in posterior uveitis patients Cell surface markers

Sr. no.

Patient no.

Important clinical features

Linear peptides showing response

Overlapping peptides showing response

CD4 cells (mean % F SD) CD8+ cells (mean % F SD) CD29+ cells (mean % F SD) CD45RA+ cells (mean % F SD)

1

10

6, 18

C, F, H, I

* p < 0.05 as compared to control.

2

11

Acute, pupillary reaction, pigmented lens Chronic, posterior synechiae, pupillary reaction, pigmented lens, PPD positive Acute, fine Kps, cells in vitreous chamber Chronic, pars planitis, history of intestinal tuberculosis, PPD positive Acute, pars planitis, cells in vitreous chamber Chronic, iridiocyclitis, associated rheumatoid arthritis, reduction of joint spaces, persistent pain with redness and watering from eyes.

5, 11, 13

G, I

3

17

4

22

5

26

6

29

1, 4, 6, 11, 13, 15

A, C, E, F

3, 6, 7, 8, 13, 15, 16, 17

A, D, E, F, G

2, 3, 4, 5, 7, 8, 9, 15, 16, 17

B, C, E, F

5, 17

A, H, I

peptides in addition to peptides 6, 7, 15, 16, 17, and 18 of the human S-antigen. Among overlapping peptides, the maximum response was observed against peptides A, E, F, and I (Fig. 1). As it is evident from Fig. 1, the maximum response (five patients) was against linear peptide 13. Its corresponding overlapping fragment, H and I, also elicited a response to three and five patients, respectively. The observation with linear peptides 5, 6, 7, and 8 (four patients each) was similar with their overlapping fragments B, C, D, and E (two, three, one, and four patients), respectively. Among overlapping peptides, the maximum response (six patients) was found to be against the overlapping peptide F. Its linear peptides 9 and 10 showed response in one and three patients, respectively. Thus, the region lying between amino acid sequences 41 – 270 showed frequent responses. The maximum response was found to be against peptides in the region of amino acid position 231– 270.

+

Controls (n = 15)

Patients (n = 34)

56.2 42.1 85.2 66.2

46.1 32.2 76.8 57.2

F F F F

17.4 13.2 13.8 10.5

F F F F

18.1* 12.9* 16.1 21.1

lation of total lymphocytes (T and B) were 46.1 F 18.1 and 32.2 F 12.9 for patients and 56.2 F 17.4 and 42.1 F 13.2 for controls, respectively, showing a significant decrease in uveitis patients ( P < 0.05). The CD4/CD8 ratio was 2.6 for controls against 1.7 for patients, reflecting a decrease in CD4+ cells (Table 5). The percentage of CD29 + and CD45RA+ T lymphocytes in patients was higher than controls, but differences were not significant. These results show an abnormal distribution of T-lymphocytes in patients with an associated uveitis with or without an underlying systemic disease. Cytokine profile in peripheral blood Six uveitis patients and two controls were evaluated for the presence of cytokine mRNAs (interleukin (IL)-4, IL-5, IL-10, IL-14, IL-15, IL-9, IL-2, IL-13, and interferon (IFN)g) by RPA using RNA isolated from PBMCs that were cultured in vitro in the presence of PHA for 24 h. L32 and GAPDH were used as housekeeping genes. The data were expressed as a ratio of the sum intensities of cytokine mRNA to that of L32. There was a marginal decrease in the expression of IL-5, IL-15, IL-9, IL-2, IL-13, and IFN-g in patients. IL-10 level was not altered while there was no expression of IL-4 and IL-14 (Fig. 2).

Lymphocyte subsets The frequency of lymphocyte subsets in uveitis patients and controls is shown in Table 5. There was a demonstrable difference in the percentage of CD4+ and CD8+ lymphocytes in the patient group as compared to controls ( P V 0.05). The percentages of CD4+ and CD8+cells out of the gated popu-

Fig. 2. Densitometric analysis of different cytokine mRNA in PHA stimulated patients and control PBMCs after correcting for L-32 expression as detected by RPA. Results are expressed as ratio of the sum intensity of cytokine mRNA and L-32 mRNA.

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Discussion Posterior uveitis is considered to be a T-cell-mediated autoimmune disease, although its immunopathogenic mechanisms remain unclear (Calder et al., 1999). Several studies have demonstrated that cellular proliferative responses to Santigen are present in acute inflammatory posterior uveitis (Doekes et al., 1987; Nussenblatt et al., 1980, 1982; Rajasingh et al., 1996). In the present study, we observed significant lymphocyte proliferative responses not only to linear peptides (reported to be consistently proliferative from patient to patient) but also to their corresponding overlapping fragments and a few additional linear peptides (Rai et al., 2001). Lymphocyte responses to 40 peptides of human S-antigen have been studied in several ocular inflammatory conditions (de Smet et al., 1990a,b, 2001) and CD4+ T cell lines (Soylu et al., 1998). Uveitis patients have been tested for proliferative responses against 39 overlapping peptides that span the entire sequence of the human Santigen. Their cell lines recognized a few peptides with individual profiles of specificity being exhibited by each line. They observed a response against peptides 2, 7, 8, 10 14, 16, 17, and 18, and overlapping fragments of peptides 8 and 9. In our study also, we found responses against the above peptides as well as to additional overlapping fragments of peptides 4, 5, 8, 9, 12, 13. The overlapping peptides showing significant responses are peptides A, E, F, and I. A number of patients have shown in vitro lymphocyte proliferation against multiple peptides (de Smet et al., 1990a,b, 2001; Hirose et al., 1989). This is in contrast to animal models where cells respond only to the antigen against which they are immunized (de Smet et al., 1993). It has been reported that with each disease recurrence, the immune response appears to shift to a new determinant, which is called ‘determinant spreading’. There were some patients who showed a response only to the whole S-antigen and not to any of the peptides of the S-antigen, while many other patients elicited T-cell proliferative responses to one or more peptides of the S-antigen but not to the native Santigen. In the present study, patients showing responses to linear peptides also show a response to adjoining overlapping fragments. Hence, we may assume that these fragments represent the immunodominant sites for a subgroup of patients and this may help elucidate certain aspects of the mechanism involved in posterior uveitis. We have observed a decrease in CD4+ and CD8+ cell populations in uveitis patients along with a substantial decrease in CD4+/CD8+ ratio, which is consistent with the earlier report (Deschenes et al., 1986). This decrease of CD+ cells may have been due to immunosuppressive therapy. Furthermore, a larger proportion of T-lymphocytes were CD29+ and CD45RA+ in patients than in controls (Table 5). In an earlier report, the percentage of CD4+CD29+ lymphocytes was high in all patients with uveitis while CD4+CD45RA+ was low in patients with BD and Vogt – Koyanagi Harada syndrome, although the difference was

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not statistically significant (Ohta et al., 1997). These results suggest an abnormal distribution of T lymphocytes in patients with uveitis from India and call for further in-depth evaluation. The mRNA levels of the immunoregulatory cytokines (IL-2, IL-5, IL-10, IL-15, IL-9, IL-13, and IFN-g) were marginally lower in patient samples as compared to controls; mRNA level of IL-10 expression varied a lot from patient to patient, and expression was more prominent than other cytokines. However, it must be borne in mind that these patients were on immunosuppressive therapy and this may account, in part, for decreased levels of the above cytokines. It is also important to mention that in our study cytokine messages have been estimated after stimulating PBMCs in vitro with T cell mitogen (PHA), and not with antigens or peptides. Th1 (IFN-g, IL-2) and Th2 cytokines (IL-10, IL-13) were found in the same ocular fluid samples from patients (Feys et al., 1994). There is no previous data on mRNA expression of cytokines produced by PHA-stimulated PBMCs, and hence it is difficult to draw any firm conclusions. The role of IL-10 and other cytokines needs to be investigated in depth if cytokine immunotherapy is to be applied in the future. Our study confirms earlier observations that retinal Santigen definitely plays a role in the etiopathogenesis of a subset of idiopathic human uveitis. Several peptides have been recognized that may be involved in the initiation and perpetuation of the disease. Our data also show a perturbation of the systemic T-helper cells in patients of idiopathic uveitis along with a deviation in their ability to produce both Th1 and Th2 cytokines in mitogen-stimulated cells in vitro. Further studies are necessary to delineate the relative importance of these mechanisms as well as to determine the relative contribution of intraocular versus systemic lymphocyte activation in uveitis.

Acknowledgments We are thankful to Dr. S.K. Sachan for his interest in this study. Parul Tripathi is a PhD student supported by SGPGI. The laboratory infrastructure was provided by a JICA grantin-aid to the SGPGI project.

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