Tolerance to Melanin-Associated Antigen in Autoimmune Uveitis Is Mediated by CD4+CD25+ T-Regulatory Cells
2008; Elsevier BV; Volume: 173; Issue: 5 Linguagem: Inglês
10.2353/ajpath.2008.080150
ISSN1525-2191
AutoresBharati Matta, Purushottam Jha, Puran S. Bora, Nalini S. Bora,
Tópico(s)Systemic Lupus Erythematosus Research
ResumoExperimental autoimmune anterior uveitis (EAAU) serves as an animal model for human idiopathic AU, the most common form of intraocular inflammation of significant morbidity whose recurrence can lead to permanent vision loss. This study was undertaken to inhibit EAAU by inducing tolerance to melanin-associated antigen (MAA) and to investigate the underlying mechanisms responsible for tolerance induction. Intravenous administration of MAA both induced tolerance and inhibited EAAU in Lewis rats. Flow cytometric analysis revealed that the proliferation of lymph node cells in response to antigenic stimulation was drastically reduced in the state of tolerance both in vivo and in vitro. Our results from co-culture experiments demonstrated that intravenous administration of MAA led to the generation of T-regulatory cells that suppress T-cell proliferative responses and induce tolerance. Expression levels of both interleukin-10 and transforming growth factor-β2 were elevated whereas reduced levels of tumor necrosis factor-α, interferon-γ, and interleukin-2 were detected in tolerance-induced animals. Tolerance was reversed by replenishing these animals with recombinant interleukin-2. Tolerance could be adoptively transferred by removing lymph node cells from tolerance-induced donors and giving them to recipient rats. Interestingly, adoptive transfer of tolerance failed when lymph nodes cells were depleted of CD4+CD25+ T cells. In conclusion, T-cell nonresponsiveness because of active suppression mediated by T-regulatory cells facilitates the development of tolerance to MAA in EAAU. Experimental autoimmune anterior uveitis (EAAU) serves as an animal model for human idiopathic AU, the most common form of intraocular inflammation of significant morbidity whose recurrence can lead to permanent vision loss. This study was undertaken to inhibit EAAU by inducing tolerance to melanin-associated antigen (MAA) and to investigate the underlying mechanisms responsible for tolerance induction. Intravenous administration of MAA both induced tolerance and inhibited EAAU in Lewis rats. Flow cytometric analysis revealed that the proliferation of lymph node cells in response to antigenic stimulation was drastically reduced in the state of tolerance both in vivo and in vitro. Our results from co-culture experiments demonstrated that intravenous administration of MAA led to the generation of T-regulatory cells that suppress T-cell proliferative responses and induce tolerance. Expression levels of both interleukin-10 and transforming growth factor-β2 were elevated whereas reduced levels of tumor necrosis factor-α, interferon-γ, and interleukin-2 were detected in tolerance-induced animals. Tolerance was reversed by replenishing these animals with recombinant interleukin-2. Tolerance could be adoptively transferred by removing lymph node cells from tolerance-induced donors and giving them to recipient rats. Interestingly, adoptive transfer of tolerance failed when lymph nodes cells were depleted of CD4+CD25+ T cells. In conclusion, T-cell nonresponsiveness because of active suppression mediated by T-regulatory cells facilitates the development of tolerance to MAA in EAAU. Uveitis is broadly defined as inflammation of the uvea and is responsible for more than 2.8% of blindness in the US. Each year, 17.6% of active uveitis patients experience a transient or permanent loss of vision. A recent report from the Northern California Epidemiology Study suggested a higher disease rate for the older population in the US.1Gritz DC Wong IG Incidence and prevalence of uveitis in Northern California; the Northern California Epidemiology of Uveitis Study.Ophthalmology. 2004; 111: 491-500Abstract Full Text Full Text PDF PubMed Scopus (654) Google Scholar, 2Bora NS Kaplan HJ Intraocular diseases—anterior uveitis.Chem Immunol Allergy. 2007; 92: 213-220Crossref PubMed Scopus (20) Google Scholar Anterior uveitis (AU) is a term that refers to inflammation within the anterior segment of the eye; retinal involvement is not a component of AU.1Gritz DC Wong IG Incidence and prevalence of uveitis in Northern California; the Northern California Epidemiology of Uveitis Study.Ophthalmology. 2004; 111: 491-500Abstract Full Text Full Text PDF PubMed Scopus (654) Google Scholar, 2Bora NS Kaplan HJ Intraocular diseases—anterior uveitis.Chem Immunol Allergy. 2007; 92: 213-220Crossref PubMed Scopus (20) Google Scholar, 3Bloch-Michel E Nussenblatt RB International Uveitis Study Group recommendations for the evaluation of intraocular inflammatory disease.Am J Ophthalmol. 1987; 103: 234-235Abstract Full Text PDF PubMed Scopus (617) Google Scholar Idiopathic AU is the most common form of intraocular inflammation in humans and the recurrent nature of the disease can lead to permanent visual loss.1Gritz DC Wong IG Incidence and prevalence of uveitis in Northern California; the Northern California Epidemiology of Uveitis Study.Ophthalmology. 2004; 111: 491-500Abstract Full Text Full Text PDF PubMed Scopus (654) Google Scholar, 2Bora NS Kaplan HJ Intraocular diseases—anterior uveitis.Chem Immunol Allergy. 2007; 92: 213-220Crossref PubMed Scopus (20) Google Scholar, 3Bloch-Michel E Nussenblatt RB International Uveitis Study Group recommendations for the evaluation of intraocular inflammatory disease.Am J Ophthalmol. 1987; 103: 234-235Abstract Full Text PDF PubMed Scopus (617) Google Scholar Idiopathic AU is a disease that is associated with significant morbidity and can only be treated symptomatically, but not cured. Treatment options available to uveitis patients have limitations because uveitis is generally treated symptomatically with topical, periocular, or systemic administration of corticosteroids. Uveitis patients with severe unresponsiveness to corticosteroid may need immunosuppressive therapy.1Gritz DC Wong IG Incidence and prevalence of uveitis in Northern California; the Northern California Epidemiology of Uveitis Study.Ophthalmology. 2004; 111: 491-500Abstract Full Text Full Text PDF PubMed Scopus (654) Google Scholar, 2Bora NS Kaplan HJ Intraocular diseases—anterior uveitis.Chem Immunol Allergy. 2007; 92: 213-220Crossref PubMed Scopus (20) Google Scholar, 3Bloch-Michel E Nussenblatt RB International Uveitis Study Group recommendations for the evaluation of intraocular inflammatory disease.Am J Ophthalmol. 1987; 103: 234-235Abstract Full Text PDF PubMed Scopus (617) Google Scholar Unfortunately, these therapies are associated with serious side effects. 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The present study was undertaken to inhibit EAAU by intravenously-induced immune tolerance to MAA. Additionally, in vivo and in vitro methods were used to elucidate the underlying mechanism(s) of intravenously-induced immune tolerance to MAA that leads to the inhibition of EAAU. Inhibition of EAAU by inducing immunological tolerance to MAA via oral and intranasal routes is currently being explored in our laboratory. Pathogen-free male Lewis rats (5 to 6 weeks old) were obtained from Harlan Sprague Dawley (Indianapolis, IN). This study was approved by the Institutional Animal Care and Use Committee, University of Arkansas for Medical Sciences, Little Rock, AR. MAA was purified from bovine iris and ciliary body as previously described by us.11Bora NS Sohn JH Kang SG Cruz JM Nishihori H Suk HJ Wang Y Kaplan HJ Bora PS Type I collagen is the autoantigen in experimental autoimmune anterior uveitis.J Immunol. 2004; 172: 7086-7094Crossref PubMed Scopus (33) Google Scholar Male Lewis rats were immunized with 100 μl of stable emulsion containing 75 μg of MAA emulsified (1:1) in CFA (Sigma-Aldrich, St. Louis, MO) using a single-dose induction protocol in the hind footpad as previously described by us.11Bora NS Sohn JH Kang SG Cruz JM Nishihori H Suk HJ Wang Y Kaplan HJ Bora PS Type I collagen is the autoantigen in experimental autoimmune anterior uveitis.J Immunol. 2004; 172: 7086-7094Crossref PubMed Scopus (33) Google Scholar Depending on the experiment, animals were examined daily between days 3 and 30 after injection for the clinical signs of uveitis using slit lamp biomicroscopy. EAAU was scored by an observer unaware of the experimental design. Intensity of uveitis was scored in a masked manner on the arbitrary scale of 0 to 6, as follows: 0, normal; 1, dilated iris vessels and thickened iris and ciliary body; exudates in the anterior chamber with protein, a few scattered inflammatory cells, or both; 2, moderate infiltration of inflammatory cells in the iris, ciliary body, or both; moderate number of inflammatory cells within the anterior chamber; 3, heavy infiltration of inflammatory cells within the iris and ciliary body and within the anterior chamber; 4, heavy exudation of cells with dense protein aggregation in the anterior chamber; inflammatory cell deposits on the corneal endothelium; 5, presence of hemorrhage with extremely heavy infiltration of inflammatory cells within the iris, the ciliary body, and the anterior chamber as well as dense inflammatory cell deposits on the corneal endothelium; 6, extreme hemorrhage with very dense deposits on corneal endothelium. Eyes were also harvested at various time points for histological analysis to assess the course and severity of inflammation using the criteria previously reported.6Bora NS Kim MC Kabeer NH Simpson SC Tandhasetti MT Cirrito TP Kaplan AD Kaplan HJ Experimental autoimmune anterior uveitis. Induction with melanin-associated antigen from the iris and ciliary body.Invest Ophthalmol Vis Sci. 1995; 36: 1056-1066PubMed Google Scholar, 29Thornton AM Shevach EM CD4+CD25+ immunoregulatory T cells suppress polyclonal T cell activation in vitro by inhibiting interleukin 2 production.J Exp Med. 1998; 188: 287-296Crossref PubMed Scopus (2182) Google Scholar To induce tolerance, each Lewis rat received a total of three intravenous injections of MAA at the following three time points: 7 days before (day −7), 4 days before (day −4), and 1 day before (day −1) immunization with MAA to induce EAAU. MAA was dissolved 400 μl of phosphate-buffered saline (PBS) and three different doses (100, 200, and 400 μg) of MAA were used separately at these time points. Lewis rats injected similarly with 400 μg of ovalbumin (Sigma-Aldrich) or PBS (400 μl) were used as control. Twenty-four hours after the last intravenous injection (day 0), Lewis rats were immunized subcutaneously with MAA to induce EAAU as described above. A separate group of animals tolerized by intravenous administration of MAA (400 μg in 400 μl of PBS) received a single injection of recombinant IL-2 (rIL-2; R&D Systems, Minneapolis, MN) intravenously (2 μg/rat in 400 μl of reconstitution solution) on days 8, 10, and 12 after immunization with MAA. Control animals received 400 μl of reconstitution solution. Reconstitution solution was prepared according to the manufacturer's specifications (R&D Systems). Animals were monitored for the development and severity of EAAU as described above. Freshly enucleated rat eyes were fixed in neutral buffered 10% formalin solution (Sigma-Aldrich) for 24 hours at room temperature, dehydrated in ethanol through ascending series of ethanol concentrations and embedded in paraffin. Four-μm sections were stained with hematoxylin and eosin (H&E) purchased from Fisher Scientific (Fair Lawn, NJ). Sections were examined using an Axioskop microscope (Carl Zeiss Meditec, Inc., Thornwood, NY). Single cell suspension from spleen, popliteal lymph nodes (LNs), and eye was prepared by mashing the tissues with frosted slides (Fisher Scientific) followed by filtration through the cell strainer (BD Biosciences, San Jose, CA). Red blood cells were removed from LN cells by treating the cells with red blood cell lysis buffer (2.07 g NH4Cl, 0.25 g NaHCO3, 9.3 mg ethylenediaminetetraacetic acid, in 100 ml H2O). Total lymphocytes were purified by passing the cells through Histopaque gradient (Sigma Aldrich) according to the manufacturer's protocol. The cells were suspended in complete RPMI 1640 culture medium with l-glutamine (Mediatech, Herndon, VA) containing 1% (v/v) minimum essential medium (Life Technologies, Rockville, MD), NEAA (BioWhittaker, Allendale, NJ), mixture of antibiotics (100 U/ml penicillin, 100 U/ml streptomycin, and 0.25 μg/ml Amphotericin B) purchased from BioWhittaker, and 10% (v/v) fetal calf serum (Mediatech). Total lymphocytes harvested from the popliteal LNs of tolerized and nontolerized animals were plated with MAA (10 μg/ml) for 3 days and nonadherent cells were collected. For depletion of CD25+ cells nonadherent cells were labeled with biotin-labeled CD25 antibody (BD Biosciences). CD25+ cells were then removed using anti-biotin microbeads (Miltenyi Biotec, Auburn, CA) according to the manufacturer's instructions. For co-culture experiments, the cells were separated using a BD FACSAria cell sorter (BD Biosciences). Briefly, total lymphocytes from LNs of tolerized and nontolerized animals were labeled with anti-CD4-APC and anti-CD25-PE. The CD4+CD25−, CD4+CD25+, and CD4− cells were sorted in different tubes using BD FACSAria. CD4+CD25− cells were labeled with carboxyfluorescein succinimidyl ester (CFSE). The cells were cultured with MAA (10 μg/ml) for 6 days as follows: well 1 received CD4− (rest of the cells after separation of CD4+ cells), CD4+CD25+ and CFSE-labeled CD4+CD25− cells from the popliteal LNs of the tolerized animal. Well 2 received CD4− (rest of the cells after separation of CD4+ cells) and CFSE-labeled CD4+CD25− cells from tolerized animal along with CD4+CD25+ cells from the nontolerized animal. Well 3 received CD4− (rest of the cells after separation of CD4+ cells), CD4+CD25+ cells and CFSE-labeled CD4+ CD25− cells from the popliteal LNs of the nontolerized animal. Well 4 received CD4− (rest of the cells after separation of CD4+ cells) and CFSE-labeled CD4+ CD25− cells from popliteal LNs of nontolerized animal along with CD4+CD25+ cells from the tolerized animal. In a separate set of experiment wells 1 and 2 received CD4− (rest of the cells after separation of CD4+ cells) and CFSE-labeled CD4+CD25− cells from the popliteal LNs of the tolerized animal. Wells 3 and 4 received CD4− (rest of the cells after separation of CD4+ cells) and CFSE-labeled CD4+CD25− cells from the popliteal LNs of the nontolerized animal. MAA (10 μg/ml) was added to wells 1 and 3 whereas wells 2 and 4 did not receive MAA. In this set of experiment wells 1, 2, 3, and 4 did not receive CD4+CD25+ cells. On the 7th day the cells were collected and CFSE-positive cells were analyzed on FACSCalibur. The peaks for each generation and data were obtained by analyzing the raw data on the ModFit Proliferation Wizard program Verity Software, Topsham, ME. One million cells were treated with anti-rat CD32 antibody (BD Biosciences) for 15 minutes at 4°C to block the Fc receptors. Appropriately diluted primary antibody (anti- rat CD25-PE, anti-rat CD4-APC, and anti-rat CD8a-APC from Biolegend (San Diego, CA) was added and incubated for 30 minutes at 4°C in dark. The cells were then washed with stain buffer (BD Biosciences). Cells were resuspended in 500 μl of stain buffer for flow analysis on BD FACSCalibur (BD Biosciences). The data were then analyzed on WinMDI 2.8 J. Trotter, The Scripps Research Institute, La Jolla, CA. Total lymphocyte harvested from popliteal LNs were cultured overnight with MAA (10 μg/ml) and Golgi stop (BD Biosciences). The Fc receptor was blocked and stained for surface markers as described above. The cells were washed two times with stain buffer and were resuspended in 100 μl of cytofix/cytoperm solution (BD Biosciences) containing optimal concentration of primary antibody IL-2 (R&D Systems) for 20 minutes at 4°C. The cells were then washed two times with Perm/Wash solution (BD Biosciences) and incubated with secondary antibody for 20 minutes in the dark at 4°C. The cells were washed again two times with Perm/Wash solution (BD Biosciences) and resuspended in 500 μl of stain buffer (BD Biosciences) for flow analysis. For FoxP3 intracellular staining, total lymphocyte cell suspension was prepared from the popliteal LNs harvested at different time points during EAAU. The cells were labeled with surface markers and were then labeled intracellularly with fluorescein isothiocyanate-conjugated FoxP3 antibody (eBiosciences, San Diego, CA) as described above. Total lymphocytes from popliteal LNs were labeled with CFSE using the Cell Trace CFSE cell proliferation kit according to the manufacturer's protocol (Molecular Probes, Invitrogen, Carlsbad, CA) and were cultured with MAA (10 μg/ml) for 6 days. CFSE-labeled total lymphocytes from tolerized and nontolerized rats cultured in the absence of MAA served as control. The cells were then collected, labeled for surface markers, and used for flow analysis. The raw data from FACSCalibur was analyzed using the ModFit Proliferation Wizard Program. An equal number of CFSE-labeled total lymphocytes from popliteal LNs of tolerized and nontoleralized animal were intravenously injected into naïve Lewis rats. The rats were immunized with MAA 24 hours after the cell transfer. CFSE-positive cells harvested from spleen, popliteal LNs, and eyes at day 12 after immunization were analyzed using FACSCalibur. The data were further analyzed using the ModFit Proliferation Wizard program. Total lymphocytes harvested from popliteal LNs were cultured in the absence and presence of MAA (10 μg/ml) for 24 hours. Supernatants were collected after 24 hours for quantitative ELISA for interferon (IFN)-γ, tumor necrosis factor (TNF)-α, IL-10 (BD PharMingen, San Diego, CA), and IL-2 (R&D Systems). ELISA was performed using paired mAbs according to the manufacturer's recommendations. The concentration of each cytokine was calculated by computer software using the standard curves obtained from known concentrations. The values were then normalized by the amount of total protein present in the culture supernatant. Equal amounts of the total RNA were used to detect the mRNA levels of β-actin, TNF-α, IFN-γ, IL-10, TGF-β2, and IL-2 by RT-PCR using a RNA-PCR kit (Bio-Rad, Hercules, CA). Total RNA was extracted using the SV total RNA isolation kit (Promega, Madison, WI) according to the manufacturer's protocol. The sense and anti-sense oligonucleotide primers for rat proteins were synthesized at IDT (Coralville, IA). The primer sequences as well as the predicted sizes of amplified cDNA are presented in the Table 1. The cDNA was synthesized using MuLV reverse transcriptase (Bio-Rad) with 1 μg of total RNA in a total volume of 20 μl (pH 8.3). Reverse transcription reaction was performed according to the manufacturer's manual. The cDNA (2 μl per reaction) was then amplified by PCR using 20, 25, 30, and 35 cycles. The thermal cycle profile used in this study is as follows: i) an initial denaturing at 95°C for 5 minutes, then 30 seconds in each cycle; ii) annealing the primer with DNA at 55°C for 30 seconds; and iii) extending the primer at 72°C for 30 seconds. All reactions were normalized for β-actin expression. The negative controls consisted of omission of RNA template or reverse transcriptase from the reaction mixture. PCR products were analyzed on a 2% agarose gel and visualized by using the Gel Doc XR and Quantity One program (Bio-Rad). These experiments were repeated three times with similar results.Table 1Primer Sequences Used in RT-PCR for Each ProteinGenePrimerSequenceAmplified PCR product size (bp)β-ActinForward5′-GCGCTCGTCGTCGACAACGG-3′335Reverse5′-GTGTGGTGCCAAATCTTCTCC-3′IFN-γForward5′-ATCTGGAGGAACTGGCAAAAGGACG-3′288Reverse5′-CCTTAGGCTAGATTCTGGTGACAGC-3′TNF-αForward5′-ATGATCCGAGATGTGGAACTGGCA-3′295Reverse5′-GCTCCTCTGCTTGGTGGTTTGCTA-3′IL-10Forward5′-AAGGACCAGCTGGACAACAT-3′292Reverse5′-AGACACCTTTGTCTTGGAGCTTA-3′TGF-β2Forward5′-ATCGTCCGCTTCGATGTCTCAACA-3′274Reverse5′-ATCCCAGGTTCCTGTCTTTGTGGT-3′IL-2Forward5′-TTGCACTGACGCTTGTCCTCCTT-3′398Reverse5′-CCATCTCCTCAGAAATTCCACCAC-3′The sequences of all oligonucleotides are shown in 5′ to 3′ d
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