transforming-growth-factor-beta and Uveitis

The immune-privileged status of the anterior chamber of the eye is altered in experimentally induced intraocular inflammation and in the pigment dispersion syndrome of DBA/2J mice. However, the eye has developed multiple mechanisms to maintain ocular immune privilege even in the presence of intraocular inflammation.

Topics: Animals; Anterior Chamber; Autoimmune Diseases; Eye; Humans; Immune Tolerance; Mice; Ovalbumin; Transforming Growth Factor beta; Uveitis

"Idiopathic" is the most common category of uveitis, representing cases in which a specific diagnosis has not been established despite work-up. Sarcoidosis is a systemic granulomatous disorder affecting multiple organs including the lungs, skin, kidneys, and eyes. We used microRNA (miRNA) microarrays to investigate serum miRNA profiles of patients with ocular sarcoidosis as diagnosed by specific criteria (diagnosed ocular sarcoidosis), and patients with idiopathic uveitis characterized by ocular manifestations of sarcoidosis (suspected ocular sarcoidosis). Principal component analysis (PCA) and hierarchical clustering showed that serum miRNA profiles of diagnosed ocular sarcoidosis and suspected ocular sarcoidosis were both clearly distinguishable from healthy controls. Furthermore, comparative analysis of the miRNA profiles showed highly similar patterns between diagnosed ocular sarcoidosis and suspected ocular sarcoidosis. Pathway analysis revealed common pathways were involved in the two groups, including those of WNT signaling and TGF-beta signaling. Our study demonstrated a high overlap of differentially expressed serum miRNAs in patients with diagnosed ocular sarcoidosis and suspected ocular sarcoidosis, suggesting that these groups share a similar underlying pathology and may represent possible variants of the disease. Characterization of serum miRNA profiles may provide an opportunity for earlier diagnosis and treatment, and may inform more accurate clinical prognosis in patients with an ocular sarcoidosis phenotype.

Topics: Endophthalmitis; Eye; Humans; MicroRNAs; Sarcoidosis; Transforming Growth Factor beta; Uveitis

Induction of autoantigen-specific Treg cells that suppress tissue-specific autoimmunity without compromising beneficial immune responses is the holy-grail for immunotherapy to autoimmune diseases.. In a model of experimental autoimmune uveitis (EAU) that mimics human uveitis, ocular inflammation was induced by immunization with retinal antigen interphotoreceptor retinoid-binding protein (IRBP). Mice were given intraperitoneal injection of αCD4 antibody (Ab) after the onset of disease, followed by administration of IRBP. EAU was evaluated clinically and functionally. Splenocytes, CD4. The experimental approach resulted in remission of ocular inflammation and rescue of visual function in mice with established EAU. Mechanistically, the therapeutic effect was mediated by induction of antigen-specific Treg cells that inhibited IRBP-driven Th17 response in TGF-β and IL-10 dependent fashion. Importantly, the Ab-mediated immune tolerance could be achieved in EAU mice by administration of retinal autoantigens, arrestin but not limited to IRBP only, in an antigen-nonspecific bystander manner. Further, these EAU-suppressed tolerized mice did not compromise their anti-tumor T immunity in melanoma model.. We successfully addressed a specific immunotherapy of EAU by in vivo induction of autoantigen-specific Treg cells without compromising host overall T cell immunity, which should have potential implication for patients with autoimmune uveitis.. This study was supported by the Natural Science Foundation of Guangdong Province and the Fundamental Research Fund of the State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center.

Topics: Animals; Autoantigens; Autoimmune Diseases; Bystander Effect; Cell Line, Tumor; Cells, Cultured; Eye Proteins; Immunosuppression Therapy; Interleukin-10; Mice; Mice, Inbred C57BL; Retinol-Binding Proteins; T-Lymphocytes, Regulatory; Transforming Growth Factor beta; Uveitis

Regulatory T cells (Tregs) have been intensively studied in a myriad of autoimmune diseases. As for noninfectious uveitis (NIU), results have been contradictory, and studies have failed to demonstrate a consistent reduction in Treg cell frequency in patients with active disease. The present study aims to characterize T lymphocyte subsets, including naïve and memory Tregs as well as their respective CD39 expression, in the peripheral blood of NIU patients. Inflammatory as well as suppressive cytokine profiles were also evaluated.. T cell subpopulations were evaluated by multiparametric flow cytometry using anti-CD3, anti-CD4, anti-CD45, anti-CD45RA, anti-CD197, anti-CD25, anti-CD127, and anti-CD39. Treg cells were defined as CD3 + CD4. Twenty-nine patients with active NIU were included as well as 15 sex- and age-matched controls. There were no significant differences in T lymphocyte subsets, including Tregs, between patients and controls. However, patients with a lower grade of anterior chamber or vitreous inflammatory cellular reaction showed higher memory Treg counts than controls, with no respective increase in CD39+ expression, and a tendency for higher IL-17A levels (p = 0.06). This IL-17A elevation was present in the total NIU group (p = 0.08) as well as a positive correlation between IL-17A levels and the absolute counts of memory Tregs (p = 0.013; R = 0.465). Patients with higher IL-17A levels also showed higher serum concentrations of memory (p = 0.001) and naïve (p = 0.003) Tregs as well as elevated TNF-α (p < 0.0001) and IFN-ɣ (p = 0.016) levels. Negative correlations were observed between IL-10 and TGF-β levels and the percentages of memory (p = 0.030; R = - 0.411) and total CD39+ Tregs (p = 0.051; R = - 0.373) in the peripheral blood of NIU patients.. Our results showed that total Treg levels were not reduced in patients with NIU. Further characterization of Treg subsets, including memory Tregs and respective CD39 expression, may provide additional insight on the role of Treg cells in NIU. Consistent high levels of circulating IL-17A in NIU patients are in accordance with previous studies and reinforce this cytokine's vital role in uveitis pathogenesis and its possible use as a therapeutic target.

Topics: Adult; Aged; Aged, 80 and over; Female; Flow Cytometry; Humans; Immunoassay; Interferon-gamma; Interleukin-10; Interleukin-17; Male; Middle Aged; T-Lymphocyte Subsets; T-Lymphocytes, Regulatory; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha; Uveitis; Young Adult

Regulatory dendritic cells (DC

Topics: Animals; B7-1 Antigen; B7-2 Antigen; CD4 Antigens; Disease Models, Animal; Female; Flow Cytometry; Forkhead Transcription Factors; Indoleamine-Pyrrole 2,3,-Dioxygenase; Intercellular Adhesion Molecule-1; Interferon-gamma; Interleukin-10; Interleukin-2 Receptor alpha Subunit; Mice, Inbred C57BL; Transforming Growth Factor beta; Uveitis

To study the role of regulatory T cells (Tregs) in patients with tubercular uveitis.. Frequencies of peripheral Tregs, Th1, Th17 cells, and intracellular cytokines were determined in 17 tubercular uveitis patients and 18 disease controls. Function of Tregs, Th1, and Th17 cells was assessed in vitro. Simultaneously, ocular levels of IFN-γ, IL-17A, IL-4, and IL-10 were also measured.. Frequencies of peripheral Tregs in tubercular uveitis subjects were significantly lower compared with disease controls. Furthermore, expression of TGF-β and IL-2Rα, but not CTLA4, was reduced in Tregs of the tubercular uveitis group. The tubercular uveitis group demonstrated heightened Th1, Th17 responses following in vitro stimulation with phorbol myristate acetate (PMA)/ionomycin. Interestingly, Treg suppression assay did not show a significant difference between the two groups. Ocular levels of IFN-γ, IL-17A, and IL-10 were also elevated in tubercular uveitis group.. Low Treg frequency and hyporesponsive function contribute to proinflammatory responses manifesting at ocular level in tubercular uveitis.

Topics: Adolescent; Adult; Aged; CTLA-4 Antigen; Cytokines; Female; Humans; Immunophenotyping; Interleukin-2 Receptor alpha Subunit; Male; Middle Aged; T-Lymphocytes, Regulatory; Transforming Growth Factor beta; Tuberculosis, Ocular; Uveitis; Vitrectomy; Vitreous Body; Young Adult

The aim of this study is to explore the dynamic changes in IL-17-expressing T cells (Th17)/Treg expression in monophasic experimental autoimmune uveitis (mEAU). mEAU was induced in Lewis rats with IRBP1177-1191 peptide and evaluated clinically and pathologically on days 9, 13, 18, 23, 28, 35, and 48. Lymphocytes isolated from inguinal lymph nodes were subjected to flow cytometry to analyze the frequency of Th17/Treg cells. The levels of cytokines (IL-17, IL-6, IL-10, transforming growth factor (TGF)-β) in serum were detected by enzyme-linked immunosorbent assay (ELISA). Real-time quantitative PCR (RT-PCR) was used for measuring the levels of IL-17, IL-6, TGF-β, and Foxp3. Clinical and histopathologic assessment showed that mEAU began on day 9, peaked on day 13, and decreased to normal on day 18. The frequency of Th17 cells increased obviously on day 9, peaking on day 13, while the frequency of Treg cells increased on day 13, peaked on day 18, and remained at a high level until day 48. In the serum, the levels of IL-17 and IL-6 peaked on day 9 and gradually decreased to normal on day 28. The level of TGF-β increased on day 9, peaked on day 13, and decreased to normal on day 35. Meanwhile, the level of IL-10 increased on day 9 and stayed at a high level until day 48. Additionally, the above results were further confirmed by RT-PCR. The imbalance between Th17 and Treg cells contributes to the onset and progression of mEAU, and a compartmental imbalance of Treg over Th17 exists in the recovery phase of mEAU.

Topics: Animals; CD4 Lymphocyte Count; Enzyme-Linked Immunosorbent Assay; Eye; Female; Flow Cytometry; Forkhead Transcription Factors; Interleukin-17; Interleukin-6; Lymph Nodes; Peptide Fragments; Rats; Rats, Inbred Lew; Real-Time Polymerase Chain Reaction; Retinol-Binding Proteins; T-Lymphocytes, Regulatory; Th17 Cells; Transforming Growth Factor beta; Uveitis

To determine whether cultured corneal endothelial (CE) cells suppress interleukin 17 (IL-17)-producing effector T cells in vitro.. CE cell lines established from a normal mouse were used. Target bystander T cells were established from normal splenic T cells with anti-CD3 antibodies. Production of IL-17 by target T cells was evaluated by ELISA, flow cytometry and quantitative PCR. To abolish the CE-inhibitory function, transforming growth factor β (TGFβ)-small interfering RNA-transfected CE cells or transwell membrane inserts, which block cell-to-cell contact, were used.. Cultured CE cells greatly suppressed the activation of bystander target cells (pan-T, CD4 T, CD8 T, and B cells) in vitro, particularly inflammatory cytokine production by CD4 cells. Cultured CE cells significantly suppressed IL-17-producing T cells and fully suppressed polarised T helper 17 (Th17) cell lines that are induced by Th17-associated differentiation factors. However, CE cells failed to suppress Th17 cells if the CE cell lines were pretreated with TGFβ small interfering RNA or if direct contact with T cells was blocked with transwell membrane inserts.. CE cells impair the effector functions and activation of IL-17-producing helper T cells in a cell-contact-dependent mechanism. Thus, corneal endothelium may contribute to the maintenance of the privileged immune status in the eye by inducing peripheral immune tolerance.

Topics: Animals; Autoimmune Diseases; CD4-Positive T-Lymphocytes; CD8-Positive T-Lymphocytes; Cells, Cultured; Disease Models, Animal; Endothelium, Corneal; Enzyme-Linked Immunosorbent Assay; Flow Cytometry; Immune Tolerance; Interleukin-17; Lymphocyte Activation; Mice; Mice, Inbred C57BL; Peptide Fragments; Real-Time Polymerase Chain Reaction; Retinol-Binding Proteins; RNA, Small Interfering; Th17 Cells; Transfection; Transforming Growth Factor beta; Uveitis

Primary cultured retinal pigment epithelial (RPE) cells can convert T cells into T regulatory cells (Tregs) through inhibitory factor(s) including transforming growth factor β (TGFβ) in vitro. Retinoic acid (RA) enhances induction of CD4(+) Tregs in the presence of TGFβ. We investigated whether RA produced by RPE cells can promote generation of Tregs. We found that in vitro, RA-treated T cells expressed high levels of Foxp3 in the presence of recombinant TGFβ. In GeneChip analysis, cultured RPE cells constitutively expressed RA-associated molecules such as RA-binding proteins, enzymes, and receptors. RPE from normal mice, but not vitamin A-deficient mice, contained significant levels of TGFβ. RPE-induced Tregs from vitamin A-deficient mice failed to suppress activation of target T cells. Only a few Foxp3(+) T cells were found in intraocular cells from vitamin A-deficient experimental autoimmune uveitis (EAU) mice, whereas expression was higher in cells from normal EAU mice. RA receptor antagonist-pretreated or RA-binding protein-siRNA-transfected RPE cells failed to convert CD4(+) T cells into Tregs. Our data support the hypothesis that RPE cells produce RA, thereby enabling bystander T cells to be converted into Tregs through TGFβ promotion, which can then participate in the establishment of immune tolerance in the eye.

Topics: Animals; Autoimmune Diseases; CD4-Positive T-Lymphocytes; Cell Proliferation; Cells, Cultured; Coculture Techniques; Enzyme-Linked Immunosorbent Assay; Female; Flow Cytometry; Forkhead Transcription Factors; Lymphocyte Activation; Mice; Mice, Inbred C57BL; Oligonucleotide Array Sequence Analysis; Pregnancy; Real-Time Polymerase Chain Reaction; Receptors, Retinoic Acid; Retinal Pigment Epithelium; RNA, Messenger; RNA, Small Interfering; T-Lymphocytes, Regulatory; Transfection; Transforming Growth Factor beta; Tretinoin; Uveitis; Vitamin A; Vitamin A Deficiency

To assess the regulation of antigen specific Th17 cells differentiation in experimental autoimmune uveitis (EAU).. A randomized controlled trials research. EAU model was made through subcutaneous injection of interphotoreceptor retinoid-binding protein (IRBP) at backs and bellies. Mouse splenic CD4 T cells were collected on the fourteenth day and cultured in vitro in the cell culture plates containing IRBP antigen for 72 hours under different conditions: control group, TGF-beta group, IL-6 group, IL-23 group, TGF-beta + IL-6 group, TGF-beta + IL-6 + IL-23 group, IL-27 group, all transretinoic acid (ATRA) group. Cells and the clear liquid were collected. Then Th17 cells, IL-17 and other cells, cytokines were assessed by flow cytometry and ELISA. All data were analyzed by Student's t test.. Flow cytometric detection and ELISA experimental results showed:IRBP polypeptide alone mainly induced Th17 and Th1 response. Addition of TGF-beta and IL-6 induced the differentiation of antigen specific Th17 cells. Percentage of Th17 cells increased from 7.55% to 13.08% (t = -2.842, P = 0.048), and the concentration of IL-17 in cell culture fluid increased from 50.66 microg/L to 164.12 microg/L (t = -9.493, P = 0.009). Percentage of Th1 cells reduced from 6.33% to 3.43% (t = 6.059, P = 0.004). Percentage of Treg cells reduced from 4.96% to 1.52% (t = 5.683, P = 0.005); Furthermore, addition of IL-23 could enhance Th17 cells differentiation induced by TGF-beta and IL-6. Compared with IRBP polypeptide alone group, percentage of Th17 cells increased from 7.55% to 18.37% (t = -3.329, P = 0.029), and percentage of Th1 and Th2 cells reduced obviously (t = 7.410, P = 0.002; t = -3.863, P = 0.018). While IL-27 suppressed the differentiation of Th17 cells. Percentage of Th17 cells reduced from 7.55% to 1.92% (t = 4.425, P = 0.041), while percentage of Treg cells increased from 4.96% to 9.98% (t = -5.073, P = 0.015); In ATRA group, percentage of Th17 cells reduced from 7.55% to 4.06% (t = 2.163, P = 0.099).. Antigen specific Th17 differentiation is distinct from Th1 and Th2 cells. TGF-beta, IL-6 and IL-23 are the factors responsible for promoting the differentiation and development of Th17 subset, whereas IL-27 has inhibitory effects.

Topics: Animals; Autoimmune Diseases; Cell Differentiation; Disease Models, Animal; Female; Interleukin-23; Interleukin-6; Interleukins; Mice; Mice, Inbred C57BL; Random Allocation; Th1 Cells; Th17 Cells; Th2 Cells; Transforming Growth Factor beta; Uveitis

Chronic/recurrent autoimmune (idiopathic) uveitis is difficult to treat and they account for approximately 10% of legal blindness in the Western world. As it has been reported that anti-CD3 antibody can enhance T cell regulatory function, we investigated its effects in vivo on experimental autoimmune uveitis (EAU), a model for autoimmune uveitis in humans. B10RIII mice immunized with an uveitogenic peptide were treated with the F(ab')(2) fragment of anti-CD3 mAb either before or at clinical disease onset. Evaluation of EAU and cellular responses showed that disease was inhibited and the activation and expansion of pathogenic T cells selectively reduced, whereas functions of Treg in vivo were enhanced. Moreover, mice treated with anti-CD3 mAb were resistant to a second challenge with antigen and thus protected from recurrence of disease. Our results demonstrate that anti-CD3 mAb is a potent inhibitor of autoimmune uveitis.

Topics: Animals; Antibodies, Monoclonal; Autoimmune Diseases; CD3 Complex; Disease Models, Animal; Female; Interleukin-10; Lymphocyte Activation; Mice; T-Lymphocytes, Regulatory; Transforming Growth Factor beta; Uveitis

To determine whether retinal pigment epithelial (RPE) cells can inhibit cytokine production by activated T helper (Th) cells.. Primary RPE cells were cultured from normal C57BL/6 mice. Target bystander T cells were established from normal splenic T cells with anti-CD3 antibodies. T-cell activation was assessed for production of cytokines, determined by ELISA. Production of IL-17 on target T cells was evaluated using oligonucleotide microarray, RT-PCR and flow cytometry. TGFβ small interfering RNA was used to inhibit the RPE cells' inhibitory function.. The cultured RPE cells greatly suppressed the activation of bystander CD4(+) T cells in vitro, especially cytokine production by target T helper cells (Th1 cells, Th2 cells and Th17 cells, but not Th3 cells). The cultured RPE cells and RPE supernatants significantly suppressed the IL-17-producing CD4(+) T cells and fully suppressed the polarized Th17 cell lines that were induced by recombinant proteins IL-6 and TGFβ2. However, the RPE cells failed to suppress the IL-17-producing T cells in the presence of rIL-6. In addition, the TGFβ produced by the RPE cells suppressed the Th17 cells.. These results indicate that RPE cells have an immunosuppressive effect on Th17-type effector T cells, which highlights a role for ocular resident cells in establishing immune regulation in the eye.

Topics: Animals; Autoimmune Diseases; CD4-Positive T-Lymphocytes; Cells, Cultured; Coculture Techniques; Disease Models, Animal; Enzyme-Linked Immunosorbent Assay; Eye Proteins; Flow Cytometry; Immune Tolerance; Interleukin-17; Lymphocyte Activation; Mice; Mice, Inbred C57BL; Real-Time Polymerase Chain Reaction; Retinal Pigment Epithelium; Retinol-Binding Proteins; RNA, Small Interfering; Spleen; Th1 Cells; Th17 Cells; Th2 Cells; Transfection; Transforming Growth Factor beta; Uveitis

IRBPp-specific regulatory immunity is found in the spleens of mice recovered from experimental autoimmune uveoretinitis (EAU). Induction of this regulatory immunity is dependent on the expression of the melanocortin 5 receptor (MC5r). Therefore, the authors investigated whether dependence on the expression of MC5r was with the T cells or with the APCs mediating protective regulatory immunity in the EAU-recovered mouse spleen.. Wild-type and MC5r-/- mice were immunized to induce EAU. The IRBPp-stimulated T-cell response in spleens of wild-type and MC5r-/- mice were compared for surface markers and cytokine production. Spleen APC were isolated and used to stimulate cytokine production and regulatory activity in IRBP-specific T cells from wild-type or MC5r-/- mice assayed in culture by ELISA, by flow cytometry, and in vivo by adoptive transfer into EAU mice.. IRBPp-specific CD25+CD4+ T cells from spleens of EAU-recovered wild-type mice express a Treg cell phenotype of FoxP3 and TGF-β compared with the effector T-cell phenotype of IFN-γ and IL-17 production in EAU-recovered MC5r-/- mice. APCs from the spleens of wild-type mice recovering from EAU promoted regulatory T-cell activation in IRBP-specific effector T cells from the spleens of EAU-recovering MC5r-/- mice. Spleen APCs from EAU-recovering wild-type, but not MC5r-/-, mice induced TGF-β expression by primed IRBP-specific effector T cells.. Dependence on MC5r expression is with an APC that promotes or selectively activates IRBP-specific FoxP3+ TGF-β+ CD25+CD4+ Treg cells in the spleens of EAU-recovered mice.

Topics: Adoptive Transfer; Animals; Antigen-Presenting Cells; Cells, Cultured; Disease Models, Animal; Eye Proteins; Forkhead Transcription Factors; Interleukin-2 Receptor alpha Subunit; Mice; Mice, Inbred C57BL; Mice, Mutant Strains; Receptors, Melanocortin; Retinitis; Retinol-Binding Proteins; Spleen; T-Lymphocytes, Regulatory; Transforming Growth Factor beta; Uveitis

Murine retinal pigment epithelial (RPE) cells suppress T-cell activation by releasing soluble inhibitory factors and promote the generation of regulatory T cells in vitro. These T cells exposed to RPE supernatants (RPE-induced Treg cells) can suppress the activation of bystander effector T cells via the production of transforming growth factor-beta (TGFbeta). In the present study, we showed that human RPE-induced Treg cells are also able to acquire regulatory function when human RPE cell lines were pretreated with recombinant TGF beta 2. These RPE-induced Treg cells produced TGF beta 1 and IL-10 but not IFN gamma, and they significantly suppressed the activation of target cell lines and intraocular T-cell clones established from patients with active uveitis. Moreover, CD4(+)CD25(+) RPE-induced Treg cells expressed CTLA-4 and Foxp3 molecules, and the CD25(+) Treg cells profoundly suppressed the T-cell activation. Thus, in vitro manipulated Treg cells acquire functions that participate in the establishment of immune tolerance in the eye.

Topics: Antibodies, Monoclonal; CD4-Positive T-Lymphocytes; Cell Line; Cell Proliferation; Culture Media, Conditioned; Dinoprostone; Epithelial Cells; Eye; Forkhead Transcription Factors; Gene Expression; Humans; Immune Tolerance; Interferon-gamma; Interleukin-10; Interleukin-2 Receptor alpha Subunit; Lymphocyte Activation; Receptors, Transforming Growth Factor beta; Retinal Pigment Epithelium; RNA, Small Interfering; T-Lymphocytes; T-Lymphocytes, Regulatory; Th1 Cells; Thrombospondin 1; Transforming Growth Factor beta; Transforming Growth Factor beta1; Transforming Growth Factor beta2; Uveitis

Pigment epithelium isolated from the eye possesses immunosuppressive properties such as regulatory T (Treg) cell induction; e.g., cultured retinal pigment epithelium (RPE) converts CD4(+) T cells into Treg cells in vitro. RPE constitutively expresses a novel immunosuppressive factor, CTLA-2alpha, which is a cathepsin L (CathL) inhibitor, and this molecule acts via RPE to induce Treg cells. To clarify CTLA-2alpha's role in the T cell response to RPE in ocular inflammation, we used the experimental autoimmune uveitis (EAU) animal model to examine this new immunosuppressive property of RPE. In EAU models, TGF-beta, but not IFN-gamma inflammatory cytokines, promotes the up-regulation of the expression of CTLA-2alpha in RPE. Similarly, CTLA-2alpha via RPE was able to promote TGF-beta production by the CD4(+) T cells. The RPE-exposed T cells (RPE-induced Treg cells) greatly produced TGF-beta and suppressed bystander effector T cells. There was less expression of CathL by the RPE-exposed T cells, and CathL-inhibited T cells were able to acquire the Treg phenotype. Moreover, CathL-deficient mice spontaneously produced Treg cells, with the increase in T cells potentially providing protection against ocular inflammation. More importantly, CD4(+) T cells from EAU in CathL knockout mice or rCTLA-2alpha from EAU animals were found to contain a high population of forkhead box p3(+) T cells. In both EAU models, there was significant suppression of the ocular inflammation. These results indicate that RPE secretes CTLA-2alpha, thereby enabling the bystander T cells to be converted into Treg cells via TGF-beta promotion.

Topics: Animals; Antigens, Differentiation; Cathepsin L; Cathepsins; Cysteine Endopeptidases; Disease Models, Animal; Eye; Eye Proteins; Forkhead Transcription Factors; Immune Tolerance; Interferon-gamma; Mice; Mice, Inbred C57BL; Mice, Knockout; Retinal Pigment Epithelium; Retinol-Binding Proteins; T-Lymphocytes, Regulatory; Transforming Growth Factor beta; Uveitis

Interleukin (IL)-21 has recently been shown to play a vital role in the development of many autoimmune diseases. Our study is designed to investigate the alteration and possible function of IL-21 in the development of an experimental autoimmune uveitis (EAU) model.. EAU was induced in B10.RIII mice by subcutaneous injection of interphotoreceptor retinoid-binding protein (IRBP) 161-180 emulsified with complete Freund's adjuvant (CFA) and evaluated by clinical and histopathologic observation. IL-21 and IL-21R mRNA expressions in cells of draining lymph node (DLN) and spleen in EAU and control mice were determined by reverse transcription-PCR. The frequencies of interleukin-21 receptor positive cells were also examined using flow cytometry. IL-17 levels in the supernatant of the cell culture upon IL-21 stimulation were assayed by enzyme-linked immunosorbent assay.. Results showed that EAU was successfully induced by IRBP161-180. Expression of IL-21 mRNA was significantly increased in cells of DLN and spleen in EAU compared with recovery phase mice and normal controls. IL-21R was also found upregulated in DLN and spleen cells of EAU mice by reverse transcription-PCR and flow cytometry. Cells in EAU cultured with IL-21 combined with transforming growth factor-beta induced increased production of IL-17.. The findings revealed that increased IL-21 and IL-21R expression may be involved in the development of EAU, possibly by promoting IL-17 secretion.

Topics: Animals; Autoimmune Diseases; Cell Count; Disease Models, Animal; Humans; Interleukin-17; Interleukins; Lymph Nodes; Mice; Receptors, Interleukin-21; Recombinant Proteins; Reverse Transcriptase Polymerase Chain Reaction; Spleen; Transforming Growth Factor beta; Up-Regulation; Uveitis

Transforming growth factor-beta2 is known to be present at elevated levels in the aqueous humor of patients with primary open angle glaucoma (POAG) and diabetes but not in uveitis-related secondary glaucoma. We investigated total TGF-beta2 levels and levels of the active form of TGF-beta2 in the aqueous humor of eyes with different types of glaucoma.. The concentration of the total and active form of TGF-beta2 was measured in 63 patients with primary open angle glaucoma, neovascular glaucoma complicated with diabetes (NVG), and secondary open angle glaucoma complicated with uveitis (SOAG) using a double antibody 'sandwich-indirect' ELISA method.. The levels of total TGF-beta2 in the aqueous samples of POAG, NVG, and SOAG were elevated. The levels of active TGF-beta2 in the aqueous samples of POAG, and NVG were also elevated, whereas the level of active TGF-beta2 was within the normal range in the aqueous samples of SOAG.. These results suggest that the level of TGF-beta2 may play a role in the pathology of various types of glaucoma.

Topics: Adult; Aged; Aged, 80 and over; Aqueous Humor; Biomarkers; Diabetic Retinopathy; Enzyme-Linked Immunosorbent Assay; Glaucoma, Angle-Closure; Glaucoma, Open-Angle; Humans; Middle Aged; Severity of Illness Index; Transforming Growth Factor beta; Uveitis

The role of thrombospondin (TSP)-1 in TGF-beta activation and T-cell suppression was studied in the retinal pigment epithelial (RPE) cells, a monolayer of pigmented cells that line the subretinal space, an immune-privileged site in the eye.. Posterior eyecups were prepared by excising the anterior segment, lens, and retina from enucleated eyes of C57BL/6, thrombospondin-1 knockout (TSP-1KO), and TGF-beta2 receptor II double-negative (TGF-beta2 RII DN) mice, leaving behind a healthy monolayer of RPE resting on choroid and sclera. Serum-free medium was added to these RPE eyecups, and, after various time intervals, supernatants (SNs) were removed and tested.. SNs of an ex vivo culture of RPE cells from C57BL/6 mice were shown to inhibit both antigen and anti-CD3 activation of T cells, partially due to constitutive production of TGF-beta and to the ability of RPE to activate the latent form of TGF-beta. Activation of TGF-beta was entirely dependent on TSP-1, also produced by RPE. SNs of RPE from TSP-1KO mice failed to inhibit T-cell activation. Ovalbumin (OVA)-specific delayed hypersensitivity (DH) was not impaired when OVA was injected either into the subretinal space or into the anterior chamber of TSP-1KO mice before OVA immunization. Moreover, experimental autoimmune uveoretinitis was significantly more intense in eyes of TSP-1KO mice and failed to undergo spontaneous resolution unlike wild-type mice.. Production of both TSP-1 and active TGF-beta by RPE is essential to the creation and maintenance of immune privilege in the subretinal space and that the immune privilege limits the severity and duration of retinal inflammation due to autoimmunity.

Topics: Animals; Autoimmune Diseases; Enzyme-Linked Immunosorbent Assay; Immune System; Interferon-gamma; Lymphocyte Activation; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Pigment Epithelium of Eye; Retinitis; Reverse Transcriptase Polymerase Chain Reaction; T-Lymphocytes; Thrombospondin 1; Transforming Growth Factor beta; Transforming Growth Factor beta2; Uveitis

Pregnancy and the postpartum period are associated with the activity of autoimmune diseases including uveitis. Although the exact mechanism is unknown, hormones are reported to alter inflammatory cytokines and influence disease activity. The authors studied ocular inflammation, female hormones, and serum cytokine levels during and after pregnancy.. A prospective, observational case study was conducted. Four pregnant women in their first trimester with chronic non-infectious uveitis were followed monthly until 6 months after delivery. Serum female hormones (oestrogen, progesterone, prolactin) and various cytokines (IL-2, IL-4, IL-5, IL-6, IL-10, IFN-gamma, and TGF-beta) were measured by ELISA.. The four patients had five full term pregnancies. Uveitis activity decreased after the first trimester but flared in the early postpartum period. Serum female hormones, highly elevated during pregnancy, drastically dropped post partum. Cytokine levels except TGF-beta were mostly undetectable.. Female hormones and TGF-beta may contribute to the activity of uveitis during pregnancy and the postpartum period.

Topics: Administration, Oral; Adult; Anti-Inflammatory Agents; Cytokines; Estrogens; Female; Hormones; Humans; Interleukins; Postpartum Period; Prednisone; Pregnancy; Pregnancy Complications; Pregnancy Trimester, First; Progesterone; Prolactin; Prospective Studies; Transforming Growth Factor beta; Uveitis

Oral administration of the uveitogenic peptide (aa 336-351) derived from human HSP60 induced clinical and histological manifestations of uveitis in 65.8% (48/73) of Lewis rats. Uveitis was significantly decreased to 16.7% (11/66) in parallel experiments with the peptide linked to recombinant cholera toxin B subunit (rCTB), also given by mouth (chi(2)=34.2, p<0.0001). The protective efficacy between tolerized and immunized animals was 74.7%. Adoptive transfer of mesenteric lymph node cells from tolerized rats prevented the development of uveitis. A significantly higher proportion of regulatory CD4(+)CD45RC(low)RT6(+) subset of Th2 memory cells were found in the mesenteric lymph nodes (p<0.005) and spleens (p

Topics: Adoptive Transfer; Animals; CD4-Positive T-Lymphocytes; Chaperonin 60; Cholera Toxin; Flow Cytometry; Immune Tolerance; Interferon-gamma; Interleukin-10; Interleukin-12; Peptide Fragments; Rats; Rats, Inbred Lew; RNA, Messenger; Transforming Growth Factor beta; Uveitis

TGF-beta exerts suppressive effects on immunity, but its potential applications in therapy of ocular autoimmunity have not been widely explored. In the present study, the effects of TGF-beta on uveitogenic T cells were examined.. The effects of TGF-beta on newly primed cells from mice given a uveitogenic regimen of interphotoreceptor retinoid-binding protein (IRBP) were compared with the effects on fully polarized Th1 cells from a long-term uveitogenic T-cell line. The parameters measured were T-cell proliferation, IFN-gamma production, induction of IL-12R expression, triggering of pathogenicity, and expression of costimulatory molecules on antigen-presenting cells (APCs) during in vitro exposure to antigen.. TGF-beta suppressed B7.1 expression on APCs in cultures of lymph node cells from immunized mice. It also suppressed T-cell proliferation, IFN-gamma production, IL-12 receptor accumulation, and the IL-12-promoted acquisition of uveitogenic function. In contrast, the polarized Th1 cells were either resistant to suppression or were enhanced by TGF-beta.. The results suggest that TGF-beta suppresses acquisition of effector functions by autopathogenic T cells, in part by interfering with their response to IL-12 through downregulation of IL-12R expression and in part through inhibition of APC function. The data suggest that although TGF-beta may effectively inhibit activation and recruitment of new T cells into the effector pool, it may be less effective in suppressing the reactivation of already polarized memory T cells that are less dependent on IL-12 and costimulation.

Topics: Animals; Antigen-Presenting Cells; Autoimmune Diseases; B7-1 Antigen; Cells, Cultured; Enzyme-Linked Immunosorbent Assay; Eye Proteins; Female; Flow Cytometry; Immunologic Memory; Interferon-gamma; Lymphocyte Activation; Male; Mice; Receptors, Interleukin; Receptors, Interleukin-12; Retinitis; Retinol-Binding Proteins; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; T-Lymphocytes, Regulatory; Th1 Cells; Transforming Growth Factor beta; Uveitis

Recently, we have reported that the cytokines alpha-melanocyte-stimulating hormone (alpha-MSH) and transforming growth factor-beta2 (TGF-beta2) work in synergy to induce the activation of regulatory T (Treg) cells. When we used alpha-MSH and TGF-beta2 to generate ocular autoantigen-specific Treg cells and adoptively transferred them into mice susceptible to experimental autoimmune uveoretinitis (EAU), there was suppression in the incidence and severity of EAU. Specificity to a retinal autoantigen was required for the Treg cells to suppress EAU. When stimulated, these Treg cells produced TGF-beta1, and their production of interferon-gamma, interleukin (IL)-10, and IL-4 was suppressed. Also, the Treg cells are suppressed in their proliferative response. Our results demonstrate that alpha-MSH with TGF-beta2 induce Treg cells that can subdue a tissue-specific autoimmune response. This also promotes the possibility of using these immunomodulating cytokines to purposely induce antigen-specific Treg cells to prevent and suppress autoimmune disease.

Topics: Adoptive Transfer; alpha-MSH; Animals; Autoantigens; Autoimmune Diseases; CD4-Positive T-Lymphocytes; Cells, Cultured; Eye Diseases; Eye Proteins; Female; Immunosuppressive Agents; Interferon-gamma; Lymphokines; Mice; Retinitis; Retinol-Binding Proteins; RNA, Messenger; Transforming Growth Factor beta; Transforming Growth Factor beta1; Transforming Growth Factor beta2; Uveitis

To determine whether clinical characteristics are correlated with increased levels of transforming growth factor-beta 2 (TGF-beta 2) in aqueous humor in glaucomatous eyes.. Aqueous humor samples were collected from 91 glaucomatous eyes. Included were samples from primary open-angle glaucoma (POAG) in 40 eyes, (pseudo)exfoliation syndrome (EXS) in 18 eyes, primary angle-closure glaucoma (PACG) in 26 eyes and uveitis-related secondary glaucoma (SG) in 7 eyes. TGF-beta 2 in aqueous humor was assessed with a specific-capture ELISA.. The mean concentration (+/- standard error) of mature (biologically active) TGF-beta 2 in the aqueous humor of eyes with POAG was 293.6 +/- 33.6 pg/ml, significantly higher than that in eyes with PACG, EXS and SG: 147.5 +/- 28.1, 135.8 +/- 30.2 and 41.0 +/- 10.7 pg/ml, respectively (P = 0.0006, P = 0.0010 and P = 0.0003; analysis of variance). The mean concentration (+/- standard error) of total TGF-beta 2 in the aqueous humor of eyes with POAG was 1647.6 +/- 124.5 pg/ml, not significantly different from that in eyes with PACG, EXS and SG: 1482.9 +/- 148.2, 1442.7 +/- 187.8 and 1929.0 +/- 367.6 pg/ml, respectively. A multivariate analysis using logistic regression showed significant correlations between mature TGF-beta 2 concentration and history of cataract surgery (P = 0.0225) and the use of carbonic anhydrase inhibitors (P = 0.0143).. Our results indicate that increased levels of TGF-beta 2 may play an important role in the pathogenesis of POAG.

Topics: Aged; Aqueous Humor; Enzyme-Linked Immunosorbent Assay; Exfoliation Syndrome; Glaucoma, Angle-Closure; Glaucoma, Open-Angle; Humans; Intraocular Pressure; Middle Aged; Transforming Growth Factor beta; Transforming Growth Factor beta2; Uveitis

Messenger RNAs for six cytokines (IL-12p40, IFN-gamma, IL-10, IL-4, TNF-alpha and TGF-beta1) expressed in vivo during development of experimental autoimmune uveitis (EAU) were quantitated by PCR in (uncultured) peripheral lymphoid cells and in the eyes of EAU-susceptible Lewis and EAU resistant F344 rats. Disease was induced by immunization with the R16 peptide of IRBP (in RT1B haplotype rats) or with whole IRBP (in all haplotypes). In the periphery, both Lewis and F 344 expressed similar cytokine patterns. In ocular tissues, however, only Lewis expressed elevated type 1 and inflammatory cytokines (IL-12p40, IFN-gamma and TNF-alpha), coincident with onset and peak of disease. Interestingly, naive F344 rats expressed higher basal levels of IL-10 mRNA in the eyes. To examine the possible involvement of this phenomenon in resistance, basal levels of IL-10 vs susceptibility to IRBP were compared in Lewis, BN, DA. F344 and ACI strains. Lewis, BN and DA were susceptible and had low levels of IL-10 mRNA in eyes. F344 and ACI were resistant and expressed high basal levels of IL-10 mRNA. In an in vitro study, recombinant rat IL-10 (but not human or mouse IL-10) suppressed lymphocyte proliferation and IFN-gamma production by primed lymph node cells of R16 immunized rats, but did not suppress uveitogenic long-term T-cell lines polarized to the Thl phenotype, suggesting that mature effector lymphocytes in the rat may lose their ability to be suppressed by IL-10. We propose that higher expression of the IL-10 gene in ocular tissues in some rat strains may represent a mechanism that contributes to a higher threshold of resistance to EAU, but this threshold may be overcome by a more mature Thl effector with a reduced sensitivity to IL-10.

Topics: Animals; Autoimmune Diseases; Cell Division; Cytokines; Eye; Immunity, Innate; Interleukin-10; Interleukin-12; Interleukin-18; Interleukin-4; Lymphocytes; Polymerase Chain Reaction; Rats; Rats, Inbred ACI; Rats, Inbred BN; Rats, Inbred F344; Rats, Inbred Lew; RNA, Messenger; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha; Uveitis

Protection from the development of experimental autoimmune uveitis (EAU) can be induced by feeding mice interphotoreceptor retinoid binding protein before uveitogenic challenge with the same protein. Two different regimens are equally effective in inducing protective tolerance, although they seem to do so through different mechanisms: one involving regulatory cytokines (IL-4, IL-10, and TGF-beta), and the other with minimal involvement of cytokines. Here we studied the importance of IL-4 and IL-10 for the development of oral tolerance using mice genetically engineered to lack either one or both of these cytokines. In these animals we were able to protect against EAU only through the regimen inducing cytokine-independent tolerance. When these animals were fed a regimen that in the wild-type animal is thought to predominantly induce regulatory cells and is associated with cytokine secretion, they were not protected from EAU. Interestingly, both regimens were associated with reduced IL-2 production and proliferation in response to interphotoreceptor retinoid binding protein. These findings indicate that both IL-4 and IL-10 are required for induction of protective oral tolerance dependent on regulatory cytokines, and that one cytokine cannot substitute for the other in this process. These data also underscore the fact that oral tolerance, manifested as suppression of proliferation and IL-2 production, is not synonymous with protection from disease.

Topics: Animals; Autoimmune Diseases; Eye Proteins; Female; Immune Tolerance; Interleukin-10; Interleukin-4; Lymphocyte Activation; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Retinol-Binding Proteins; Transforming Growth Factor beta; Uveitis

Female patients suffering from autoimmune uveitis are reported to experience a temporary remission during pregnancy. Experimental autoimmune uveitis (EAU) is a model for human uveitis. Here we examine the effect of pregnancy on the development of EAU and its associated immunological responses. Susceptible C57BL/6 mice were immunized with interphotoreceptor retinoid-binding protein (IRBP). EAU scores and Ag-specific responses were evaluated 21 days later. Mice immunized during pregnancy developed significantly less EAU than nonpregnant controls. Their lymph node cells and splenocytes produced a distinct pattern of cytokines in response to IRBP: reduced IFN-gamma and IL-12 p40, but unchanged levels of TNF-alpha, IL-4, IL-5, and IL-10. Anti-IRBP Ab isotypes revealed an up-regulation of IgG1, indicating a possible Th2 bias at the humoral level. Ag-specific proliferation and delayed hypersensitivity, as well as mitogen-induced IFN-gamma production, remained undiminished, arguing against an overall immune deficit. Interestingly, pregnant mice that received an infusion of IRBP-primed lymphoid cells from nonpregnant donors also developed reduced EAU, suggesting that pregnancy suppresses not only the generation, but also the function of mature uveitogenic effector T cells. Pregnant mice at the time of immunization exhibited elevated levels of TGF-beta, but not of IL-10, in the serum. We suggest that protection from EAU during pregnancy is due primarily to a selective reduction of Ag-specific Th1 responses with only marginal enhancement of Th2 function, and that these effects may in part be secondary to elevated systemic levels of TGF-beta.

Topics: Adoptive Transfer; Animals; Autoimmune Diseases; Female; Mice; Mice, Inbred C57BL; Pregnancy; Pregnancy Complications; Retinol-Binding Proteins; Transforming Growth Factor beta; Uveitis

To compare cell types and cytokines in aqueous humor from patients with uveitis either occurring in association with a systemic disease or apparently isolated and not associated with a systemic disease.. Cells were collected by centrifugation of fresh aqueous humor from uveitis and controls, and immunofluorescence techniques were performed with markers for T cells, B cells, and monocytes. Cytokines were measured in the aqueous supernatants, and serum samples were assayed for soluble interleukin-2 receptors.. When aqueous samples from idiopathic uveitis were compared with those from uveitis associated with a systemic disease, there were increases in CD3+, CD4+ (p = 0.001), and activated CD4+ T cells (p = 0.02) and a decrease in B cells (p = 0.0013). This was not reflected in the peripheral blood where there were no differences in the cell types or in soluble interleukin-2 receptor levels. No cells were obtainable from control aqueous. Interleukins-10 and -12, interferon-gamma, and transforming growth factor-beta2 were detected in aqueous supernatants. Interleukin-10 was reduced (p = 0.024) in uveitis in comparison with controls.. The results suggest a selective recruitment of CD4+ T cells within aqueous humor but only in idiopathic uveitis. In both disease groups there was a decrease in the immunoregulatory cytokine interleukin-10, which might enable an immune response to occur in an otherwise highly immunosuppressive microenvironment. Increases in activated CD4+ T cells combined with depressed interleukin-10 levels could partially explain why, for example, in acute anterior uveitis, the inflammatory disease is often more severe.

Topics: Adult; Aged; Antigens, CD; Aqueous Humor; B-Lymphocytes; CD4-Positive T-Lymphocytes; Flow Cytometry; Humans; Interferon-gamma; Interleukin-10; Interleukin-12; Lymphocyte Activation; Transforming Growth Factor beta; Uveitis

Experimental autoimmune uveoretinitis (EAU) was induced in Lewis rats and B10.A mice by immunization with S-antigen (S-Ag) to study the potential roles of vascular endothelial growth factor (VEGF) and the beta1 and beta2 isoforms of transforming growth factor (TGFbeta1 and TGFbeta2) during the progression of the disease. VEGF has been implicated as an angiogenic factor in ischemic retinopathies; however, Lewis rats developing EAU have high levels of VEGF in the retina, but no neovascularization. In the present study, immunohistochemical staining for VEGF, TGFbeta1 and TGFbeta2 was performed on the retinas of Lewis rats developing EAU or with oxygen-induced ischemic retinopathy. In rats immunized with S-antigen, a marked upregulation of VEGF was immunohistochemically visualized from the inner nuclear layer to the inner limiting membrane prior to blood-retinal barrier (BRB) failure and lymphocytic infiltration. VEGF is normally induced by hypoxia and its induction leads to neovascularization. Coincident with the increase in VEGF, there was increased immunoreactivity for TGFbeta1 and TGFbeta2 within the same layers of the retina. In contrast, rats with ischemic retinopathy and retinal neovascularization showed only a modest increase in VEGF immunoreactivity, which is largely confined to retinal ganglion cells and inner retinal vessels, and little or no increase in TGFbeta1 or TGFbeta2. In addition, in mice developing EAU, which does not have an abrupt onset as it does in rats and may involve neovascularization, a comparable upregulation of VEGF in the inner retina to that seen in rats developing EAU occurs with no increase in TGFbeta1 or TGFbeta2. Since TGFbeta can inhibit endothelial cell proliferation, it is likely that an increase in TGFbeta may prevent VEGF from exerting its endothelial growth activity in the rat EAU model, but VEGF may be operative in inducing BRB failure. These data suggest that there is a complex interaction among growth factors in the retina and that retinal neovascularization may require an imbalance between stimulatory and inhibitory factors.

Topics: Animals; Blood-Brain Barrier; Endothelial Growth Factors; Eye Proteins; Female; Immunization; Ischemia; Lymphokines; Mice; Mice, Inbred Strains; Neovascularization, Pathologic; Rats; Rats, Inbred Lew; Retina; Retinal Artery; Retinitis; Retinol-Binding Proteins; Transforming Growth Factor beta; Up-Regulation; Uveitis; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

To determine changes in the levels of nitric oxide metabolites and transforming growth factor-beta (TGF-beta) in the rabbit aqueous humour during ocular inflammation.. Active experimental uveitis was induced by injection of porcine lens protein (PLP) in three rabbits and of human serum albumin (HSA) in three rabbits; three control rabbits received an injection of saline.. Degree of inflammation, antibody titres (determined with the enzyme-linked immunosorbent assay), and aqueous humour levels of nitric oxide metabolites and TGF-beta. A modified Griess assay for nitrites and nitrates (NO2- and NO3-) was used as a measure of nitric oxide generation, and a modification of the CCL-64 mink lung epithelial cell bioassay was used to quantify TGF-beta levels.. Following the primary immunologic challenge both experimental groups initially showed a two- to fourfold increment in aqueous levels of nitric oxide metabolites and TGF-beta compared with baseline values. At the peak of the clinically observed inflammation there was a significant increase in the mean nitric oxide metabolite level compared with the control value (p < or = 0.005) (432 nmol/mL for the PLP group and 112 nmol/mL for the HSA group) and a significant decrease (p < or = 0.03) in the mean TGF-beta level (3.1 ng/mL and 0.3 ng/mL respectively).. Nitric oxide may be used as a marker for intraocular inflammation. The increased production of nitric oxide may reflect the loss of immunologic privilege of the ocular microenvironment that occurs during inflammation.

Topics: Animals; Aqueous Humor; Biomarkers; Crystallins; Disease Models, Animal; Enzyme-Linked Immunosorbent Assay; Female; Follow-Up Studies; Nitric Oxide; Rabbits; Serum Albumin; Transforming Growth Factor beta; Uveitis

The authors have previously reported that an injection of S-antigen (S-Ag) into rat testes prior to immunization induces systemic tolerance (designated orchidic tolerance) and protects the animals from experimental autoimmune uveoretinitis (EAU) and that the signal for orchidic tolerance induction emigrates from the testis within a few hours after antigen priming of the testis. In order to understand the mechanism by which the signal or signal carrier is generated, they determined in this study changes in immunoreactivity for transforming growth factor-beta (TGF-beta), IFN-gamma, IL-2, Fas and Fas ligand in the testis following an injection of S-Ag. Immunoreactivity for TGF-beta increased with time, reaching a maximum in six hours and declining thereafter. The time required for the maximum expression of TGF-beta coincided well with the time-dependent profile of orchidic tolerance signal generation within the testis. Little or no immunoreactivity was observed for IFN-gamma and IL-2 in normal (control) and S-Ag-injected testes. Immunoreactivity for Fas and Fas ligand was detected both in control and experimental testes and did not change appreciably with time following Ag-priming of the testis. Fas immunoreactivity was found in spermatids and virtually absent in the interstitial tissue, while Fas ligand immunoreactivity was primarily associated with the interstitial cells such as Leydig cells. Fas ligand immunoreactivity was very weak, if any, in the germ cells and Sertoli cells. These results suggest that TGF-beta and Fas ligand expressed in MHC-positive interstitial cells may play an important role in the generation of orchidic tolerance induction signal. A preliminary study showed that splenocytes preincubated with testis extracts and S-Ag, when transferred to naive rats, induced systemic tolerance in recipient animals. Inclusion of anti-TGF-beta or a carboxyl terminal peptide of Fas in the testis extract reduced the potency of incubated splenocytes to induce systemic tolerance in recipient rats. These results indicate that generation of the orchidic tolerance signal does not require the anatomical structure of the testis but is mediated by molecular entities such as TGF-beta and Fas ligand.

Topics: Amino Acid Sequence; Animals; Arrestin; Autoimmune Diseases; Fas Ligand Protein; fas Receptor; Immunization; Immunoenzyme Techniques; Immunosuppression Therapy; Male; Membrane Glycoproteins; Molecular Sequence Data; Peptide Fragments; Rabbits; Rats; Rats, Inbred Lew; Retinitis; Testis; Transforming Growth Factor beta; Uveitis

Experimental autoimmune uveoretinitis (EAU) in mice, an organ specific autoimmune disease, has been investigated as an animal model for human endogenous uveitis. In this study, we report on the immunosuppressive effect of transforming growth factor-beta 1 (TGF-beta 1) on the development of EAU in mice. Inhibition by TGF-beta 1 of proliferation of interphotoreceptor retinoid-binding protein (IRBP)-specific T cell lines in B10.A mice against IRBP antigen was dose-dependent. However, when spleen cells used as the antigen presenting cell were first cultured with TGF-beta 1, this anti-proliferation effect was abolished. When IRBP-immunized mice were injected intraperitoneally with TGF-beta 1, dose-dependent suppression of EAU was obtained. The proliferation response of lymph node cells from TGF-beta 1 injected mice with IRBP-induced EAU was suppressed compared with phosphate buffered saline (PBS)-injected mice. These findings suggest that TGF-beta 1 may be a cytokine that plays a role in suppressing IRBP induced EAU in mice.

Topics: Animals; Antigen-Presenting Cells; Autoimmune Diseases; Cells, Cultured; Disease Models, Animal; Eye Proteins; Female; Immunosuppressive Agents; Mice; Mice, Inbred Strains; Retinitis; Retinol-Binding Proteins; Specific Pathogen-Free Organisms; Transforming Growth Factor beta; Uveitis

Intratesticular injection of a retinal protein (S-antigen) into Lewis rats induces systemic immunotolerance (designated orchidic tolerance) and renders animals refractory to experimental autoimmune uveoretinitis (EAU) produced by S-antigen immunization. We demonstrated in this work that the immunotolerance could be transferred to syngeneic naive rats by both CD4+ and CD8+ regulatory cells. Attempts were then made to characterize the cytokines involved in the immunosuppressive activity of these regulatory cells. Using the in vitro lymphoproliferation assay, the inhibitory effect of CD4+ cells on effector cells was found to be reversed by antibodies against IL-4 and IL-10 but not by anti-TGF-beta antibody. IL-4 (IC50 = 1.6 ng/10(6) cells) and IL-10 (IC50 = 0.6 ng/10(6) cells) added to the assay medium were potent inhibitors of effector cell proliferation. Increased immunoreactivity and mRNA expression for IL-4 and IL-10 was observed for CD4+ regulatory cells. The inhibitory effect of CD8+ regulatory cells was reversed by anti-TGF-beta antibody but not by antibodies against IL-4 and IL-10. Compared with control CD8+ cells, CD8+ cells from tolerized rats demonstrated higher immunoreactivity for TGF-beta but did not show an enhanced expression of mRNA for TGF-beta. TGF-beta 1 and TGF-beta 2 added to effector cells showed dichotomous effects; both isoforms stimulated cell proliferation at 2.5 ng/10(6) cells and inhibited at lower or higher concentrations. These results led us to conclude that IL-4 and IL-10 are important cytokines for the immunosuppressive effect of CD4+ regulatory cells generated in orchidic tolerance. TGF-beta is an important immunosuppressive cytokine for CD8+ regulatory cells but further studies will determine whether other cytokines are also involved.

Topics: Animals; Arrestin; Autoimmune Diseases; Base Sequence; CD4-Positive T-Lymphocytes; CD8-Positive T-Lymphocytes; Cytokines; Disease Models, Animal; DNA Primers; Immune Tolerance; In Vitro Techniques; Interleukin-10; Interleukin-4; Lymphocyte Activation; Male; Rats; Rats, Inbred Lew; Retinitis; RNA, Messenger; Testis; Transforming Growth Factor beta; Uveitis

Transforming growth factor-beta (TGF-beta), a multifunctional cytokine which has been identified in normal and inflamed ocular fluids, may play a role in the evolution of inflammatory ocular lesions. In this study we utilized a rabbit model of LPS-induced uveitis to determine if exogenous TGF-beta 2 could alter its course. Recombinant TGF-beta 2 (1-2000 ng), LPS (10 or 20 ng), or TGF-beta 2 (100 ng) plus LPS (10 ng) were injected intravitreally in one eye of a New Zealand white rabbit and the contralateral eye served as a paired control which received an equal volume of vehicle. The uveitic response was assessed by biomicroscopic examination of the anterior uvea and analysis of protein and cells in the aqueous humor. Ocular tissues were processed for histologic, immunohistochemical and in situ hybridization analyses. Rabbits injected with doses of TGF-beta 2 > or = 500 ng developed a mild uveitic response, compared to LPS alone, accompanied by expression of IL-1 beta mRNA and protein in the anterior uvea. Interestingly, rabbits coinjected with LPS (10 ng) and a nonuveitic dose (100 ng) of TGF-beta 2 exhibited a similar increase in ocular vascular permeability, but a decrease in inflammatory cell infiltration into the anterior uvea and aqueous humor (1185 +/- 117 versus 2465 +/- 176; p < 0.05). No evidence of inflammation was observed in eyes injected with 100 ng TGF-beta 2 alone. Similar to other models of inflammation, TGF-beta may interrupt the cascade of events leading to ocular inflammation, thereby suggesting therapeutic potential.

Topics: Animals; Anterior Eye Segment; Body Fluids; Cell Movement; Injections; Interleukin-1; Lipopolysaccharides; Male; Rabbits; Transforming Growth Factor beta; Uveitis; Vitreous Body

Uveitis is induced in Lewis rats by immunization with bovine melanin protein (BMP) derived from the uvea and retinal pigment epithelium. Recurrence of this experimental melanin protein-induced uveitis (EMIU) develops after footpad injection of a minimal amount of Salmonella typhimurium endotoxin (LPS) following the remission of EMIU. To investigate the effect of transforming growth factor beta1 (TGFbeta1) on the recurrence of EMIU, 5 micrograms LPS booster was given to Lewis rats by footpad injection on Day 45 after BMP immunization. Daily TGFbeta1 or phosphate-buffered saline was administered either from Day 0 to 7 (group 1) or from Day 7 to 13 (group 2) after LPS booster. Delayed-type hypersensitivity (DTH) ear test was conducted on Day 12 after LPS booster and eye and blood were collected on Day 14. The incidence and severity of recurrent uveitis markedly decreased in both groups of TGFbeta1-treated rats. A lower level of serum BMP antibody was also observed by agglutination in these groups. There was no statistical difference in DTH responses between the treated and control groups. Ocular cytokine mRNA of group 1 and controls was analyzed by RT-PCR. Interleukin (IL)-2 and interferon-gamma were not detectable. IL-4 was identified at a similar level in both groups. A higher level of IL-10 was observed in group 1 rats. We conclude that TGFbeta1 suppresses recurrent EMIU, probably through upregulation of IL-10.

Topics: Animals; Base Sequence; Cattle; Disease Models, Animal; DNA Primers; Eye Proteins; Female; Hemagglutination Tests; Hypersensitivity, Delayed; Immunization; Interleukin-10; Lipopolysaccharides; Melanins; Molecular Sequence Data; Polymerase Chain Reaction; Rats; Rats, Inbred Lew; Recurrence; RNA, Messenger; Transforming Growth Factor beta; Up-Regulation; Uveitis

To investigate whether transforming growth factor-beta 2 (TGF-beta 2), a strong immunosuppressive factor normally present in aqueous humor, is involved in the inflammatory process of clinical uveitis.. Mature TGF-beta 2 levels were determined in aqueous humor samples of 9 patients with Fuchs' heterochromic cyclitis, aqueous humor samples of 21 patients with other uveitis entities, and vitreous fluid samples of 19 patients with uveitis by using a commercially available sandwich ELISA: Total TGF-beta 2 levels in ocular fluids were measured after heat activation. Aqueous humor samples from patients with cataract and glaucoma and vitreous fluid samples from eye bank eyes were tested as controls. Albumin levels, determined by radial immunodiffusion, were used as a measure of the disruption of the blood aqueous barrier.. Significantly lower mature TGF-beta 2 levels were detected in aqueous humor samples of patients with uveitis, compared to the two control groups without intraocular inflammation. Samples of patients with uveitis without detectable mature TGF-beta 2 did contain latent TGF-beta 2 levels (504 to 6024 pg/ml). In aqueous humor, there was a significant negative correlation between mature TGF-beta 2 and albumin levels. No mature TGF-beta could be detected in vitreous fluid. Total TGF-beta 2 levels in vitreous fluid were significantly lower in samples from patients with uveitis than in samples from eye bank eyes.. These results indicate that the mature TGF-beta 2 levels in aqueous humor and the total TGF-beta 2 levels in vitreous fluid are reduced during ocular inflammation. In aqueous humor, this might be caused by binding of mature TGF-beta to serum proteins, for instance, alpha 2-macroglobulin, or by a disturbance in the activation process of latent TGF-beta 2.

Topics: alpha-Macroglobulins; Aqueous Humor; Cataract; Enzyme-Linked Immunosorbent Assay; Glaucoma; Glucocorticoids; Humans; Serum Albumin; Transforming Growth Factor beta; Uveitis; Vitreous Body

Experimental autoimmune uveoretinitis was induced in female Lewis rats with bovine retinal soluble antigen (S-antigen). Tissue changes and immunoreactivities of transforming growth factor-beta (TGF-beta), epidermal growth factor (EGF), and extracellular matrix compounds in the anterior segment (ciliary body) were investigated by immunocytochemical methods. Control animals received adjuvant only. The immunized animals were sacrificed at day 0, 3, 7, 14, 20, and 30 postimmunization. Tissue changes that occurred at the peak of inflammation (day 14) included destruction of the inner basement membrane, epithelial cell loss, distortion of the ciliary stroma, and loss of epithelial basal infoldings. Ciliary body architecture was regenerated almost completely by day 30. Basement membrane laminin and collagen type IV levels did not change much during the inflammatory process. Fibronectin labeling level peaked at day 14 postimmunization. Collagen type V level was low at day 14 and elevated at day 20 and day 30. TGF-beta immunoreactivity peaked at day 14 and remained elevated thereafter. EGF labeling did not increase until day 20 and was maximal at day 30. Labeling of both growth factors was principally confinded to the stromal regions. The presence of TGF-beta and EGF in the ciliary stroma at well defined intervals suggests a coordinated effect upon the synthesis and reorganization of the extracellular matrix and possibly upon the inflammatory cell population in the anterior tissue.

Topics: Animals; Antigens; Arrestin; Autoimmune Diseases; Ciliary Body; Disease Models, Animal; Epidermal Growth Factor; Extracellular Matrix Proteins; Eye Proteins; Female; Immunohistochemistry; Phosphodiesterase Inhibitors; Rats; Rats, Inbred Lew; Transforming Growth Factor beta; Uveitis

The immunoreactivity of types I, III and V collagen, fibronectin and transforming growth factor-beta (TGF-beta) was studied by an immunogold labeling method in retinal vessels of rats with experimental autoimmune uveoretinitis (EAU) induced by retinal S-antigen. The basal lamina of retinal capillaries in normal rats showed low immunoreactivities for the extracellular matrix components and TGF-beta. However, at the peak of inflammation (day 13-16 postimmunization), labeling of all three types of collagen and fibronectin increased considerably in the basal lamina of retinal vessels. TGF-beta immunoreactivity was detected mainly in the vascular endothelium and the pericytes. The results suggest that TGF-beta synthesized by the endothelial cells and pericytes may regulate the synthesis and composition of extracellular matrix components in the vascular basal lamina during the course of EAU development.

Topics: Animals; Antigens; Arrestin; Autoantigens; Autoimmune Diseases; Collagen; Endothelium, Vascular; Eye Proteins; Female; Fibronectins; Immunohistochemistry; Membrane Proteins; Microscopy, Immunoelectron; Phosphodiesterase Inhibitors; Rats; Rats, Inbred Lew; Retinal Vessels; Retinitis; Transforming Growth Factor beta; Uveitis