ABSTRACT
Complement activation correlates to rheumatoid arthritis disease activity, and increased amounts of the complement split product C5a is observed in synovial fluids from rheumatoid arthritis patients. Blockade of C5a or its receptor (C5aR) is efficacious in several arthritis models. The aim of this study was to investigate the role of C5a and C5aR in human rheumatoid arthritis and psoriatic arthritis-both with respect to expression and function. Synovial fluid, blood and synovial samples were obtained from rheumatoid arthritis, psoriatic arthritis and osteoarthritis patients as a less inflammatory arthritis type, and blood from healthy subjects. Cells infiltrating synovial tissue were analysed by immunohistochemistry and flow cytometry. SF and blood were analysed for biomarkers by flow cytometry or ELISA. The effect of a blocking anti-human C5aR mAb on leukocyte migration was determined using a Boyden chamber. Appropriate statistical tests were applied for comparisons. C5aR+ cells were detected in most rheumatoid arthritis, in all psoriatic arthritis, but not in non-inflammatory control synovia. C5aR+ cells were primarily neutrophils and macrophages. C5aR+ macrophages were mainly found in lymphoid aggregates in close contact with T cells. C5a levels were increased in both rheumatoid arthritis and psoriatic arthritis synovial fluid compared to osteoarthritis, and in blood from rheumatoid arthritis compared to healthy subjects. Neutrophil and monocyte migration to rheumatoid arthritis synovial fluid was significantly inhibited by anti-C5aR. The data support that the C5a-C5aR axis may be driving the infiltration of inflammatory cells into the synovial fluid and synovium in both rheumatoid and psoriatic arthritis, and suggest that C5a or C5aR may be a promising treatment target in both diseases.
Subject(s)
Arthritis, Psoriatic/metabolism , Arthritis, Rheumatoid/metabolism , Chemotaxis, Leukocyte , Complement C5a/metabolism , Leukocytes/pathology , Receptor, Anaphylatoxin C5a/metabolism , Synovial Fluid/metabolism , Enzyme-Linked Immunosorbent Assay , Flow Cytometry , Humans , ImmunohistochemistryABSTRACT
Blockade of the complement cascade at the C5a/C5a receptor (C5aR)-axis is believed to be an attractive treatment avenue in rheumatoid arthritis (RA). However, the effects of such interventions during the early phases of arthritis remain to be clarified. In this study we use the murine delayed-type hypersensitivity arthritis (DTHA) model to study the very early effects of a blocking, non-depleting anti-C5aR mAb on joint inflammation with treatment synchronised with disease onset, an approach not previously described. The DTHA model is a single-paw inflammatory arthritis model characterised by synchronised and rapid disease onset driven by T-cells, immune complexes and neutrophils. We show that a reduction in paw swelling, bone erosion, cartilage destruction, synovitis and new bone formation is apparent as little as 60 h after administration of a single dose of a blocking, non-depleting anti-mouse C5aR mAb. Importantly, infiltration of neutrophils into the joint and synovium is also reduced following a single dose, demonstrating that C5aR signalling during the early stage of arthritis regulates neutrophil infiltration and activation. Furthermore, the number of T-cells in circulation and in the draining popliteal lymph node is also reduced following a single dose of anti-C5aR, suggesting that modulation of the C5a/C5aR axis results in effects on the T cell compartment in inflammatory arthritis. In summary, these data demonstrate that blockade of C5aR leads to rapid and significant effects on arthritic disease development in a DTHA model strengthening the rationale of C5aR-blockade as a treatment strategy for RA, especially during the early stages of arthritis flare.
Subject(s)
Antibodies, Monoclonal/pharmacology , Antibodies, Neutralizing/pharmacology , Arthritis, Experimental/drug therapy , Edema/drug therapy , Hypersensitivity, Delayed/drug therapy , Receptor, Anaphylatoxin C5a/antagonists & inhibitors , Animals , Antigen-Antibody Complex/metabolism , Arthritis, Experimental/immunology , Arthritis, Experimental/pathology , Arthritis, Rheumatoid/drug therapy , Arthritis, Rheumatoid/immunology , Arthritis, Rheumatoid/pathology , Complement C5a/antagonists & inhibitors , Complement C5a/genetics , Complement C5a/immunology , Disease Models, Animal , Edema/immunology , Edema/pathology , Female , Gene Expression , Hindlimb , Humans , Hypersensitivity, Delayed/immunology , Hypersensitivity, Delayed/pathology , Lymphocyte Count , Mice , Mice, Inbred C57BL , Neutrophil Infiltration/drug effects , Neutrophils/drug effects , Neutrophils/immunology , Neutrophils/pathology , Receptor, Anaphylatoxin C5a/genetics , Receptor, Anaphylatoxin C5a/immunology , Signal Transduction , T-Lymphocytes/drug effects , T-Lymphocytes/immunology , T-Lymphocytes/pathologyABSTRACT
The Iddm4 gene controls diabetes in rats depleted of regulatory T cells (T reg) and immune-activated via treatment with the toll-like receptor 3 (TLR-3) ligand, polyI:C. Both diabetes-resistant (BBDR) and diabetes-prone (BBDP) BB rats carry dominant permissive alleles of Iddm4, while the recessive Wistar Furth (WF) rat allele is protective. Iddm4 is positioned close to Iddm2 on chromosome 4, but when we introgressed BBDP-derived parts of this region--either containing both genes or Iddm2 alone--into the WF genome, none of these congenic strains developed spontaneous diabetes. Although both strains harbor two copies of the recessive Iddm2 allele of the BBDP rat, making these animals devoid of T reg cells, immune activation in itself via polyI:C treatment did not induce overt diabetes. Interestingly, TLR-3 ligation without depletion of T regs resulted in diabetes and insulitis development in nonlymphopenic F1-offspring of mating the Iddm4+Iddm2 congenic strain to WF. This demonstrates that the diabetogenic allele of Iddm4 is able to confer diabetes susceptibility even in a nonlymphopenic host with a full T reg pool, and that homozygosity for Iddm2--although responsible for an almost total lack of T regs-delays the disease process. Finally, we have confirmed the position of Iddm4 in truly congenic strains.
Subject(s)
Diabetes Mellitus/genetics , Diabetes Mellitus/immunology , Poly I-C/pharmacology , T-Lymphocytes/immunology , T-Lymphocytes/metabolism , Animals , Biomarkers , Diabetes Mellitus/chemically induced , Diabetes Mellitus/metabolism , Genome/genetics , Leukocyte Common Antigens/metabolism , Rats , Receptors, Antigen, T-Cell/metabolismABSTRACT
Gimap5, a member of the GTPase of the immunity-associated protein family (Gimap), regulates T cell survival. A strong indication of this is found in the diabetes-prone BioBreeding rat (BBDP), where a frameshift mutation in Gimap5 results in T-cell lymphopenia. We have investigated the function of human Gimap5 in T cells. We found that reduction of Gimap5 by RNA interference in Jurkat cells did not affect the number of apoptotic cells whereas transient over-expression of Gimap5 resulted in a major increase in the number of apoptotic cells. The same effect of over-expression was found in naive human T cells purified from blood but not in activated human T cells. This suggests that the apoptosis-inducing effect of Gimap5 over-expression is dependent on the activation status of the cells. Since the apoptosis-inducing effect of Gimap5 was contrary to the expected function of Gimap5 based on the phenotype of BBDP rats, we over-expressed rat wt Gimap5 and Gimap5 with the mutation found in BBDP (Gimap5-lyp). Both versions of rat Gimap5 induced apoptosis when expressed in the rat T-cell line C58(NT)D.1.G.OVAR.1, however, Gimap5-lyp greatly exacerbated cell death. Finally, we detected the subcellular localization of Gimap5 to be at the endoplasmic reticulum and by quantitative PCR, we found that endogenous Gimap5 mRNA is up-regulated in activated T cells.
Subject(s)
Apoptosis/physiology , Frameshift Mutation , GTP-Binding Proteins/physiology , T-Lymphocytes/physiology , Animals , Cell Line , Endoplasmic Reticulum/metabolism , Flow Cytometry , GTP-Binding Proteins/genetics , GTP-Binding Proteins/metabolism , Gene Expression , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Humans , Interleukin-2/pharmacology , Jurkat Cells , Lymphocyte Activation/drug effects , Microscopy, Confocal , Nuclear Envelope/metabolism , Phytohemagglutinins/pharmacology , RNA Interference , Rats , Rats, Mutant Strains , T-Lymphocytes/drug effects , TransfectionABSTRACT
To understand the ability of regulatory T-cells to control diabetes development in clinically relevant situations, we established a new model of accelerated diabetes in young DP-BB rats by transferring purified T-cells from DR-BB rats made acutely diabetic. Transfer of 3, 5, 10, or 23 million pure in vitro-activated T-cells accelerated diabetes onset in >90% of the recipients, with the degree of acceleration being dosage dependent. Cotransfer of unfractionated leukocytes from healthy donors prevented diabetes. Full protection was achieved when protective cells were transferred 3-4 days before diabetogenic cells, whereas transfer 2 days before conferred only partial protection. Protection resided in the CD4(+) fraction, as purified CD4(+) T-cells prevented the accelerated diabetes. When CD25(+) cells were depleted from these cells before they were transferred, their ability to prevent diabetes was impaired. In contrast, two million CD4(+)CD25(+) cells (expressing Foxp3) prevented the accelerated diabetes when transferred both before and simultaneously with the diabetogenic T-cells. In addition, 2 million CD4(+)CD25(+) T-cells prevented spontaneous diabetes, even when given to rats age 42 days, whereas 20 million CD4(+)CD25(-) cells (with low Foxp3 expression) were far less effective. We thus demonstrated that CD4(+)CD25(+) cells exhibit powerful regulatory potential in rat diabetes.
Subject(s)
CD4 Antigens/physiology , DNA-Binding Proteins/physiology , Diabetes Mellitus, Type 1/prevention & control , Receptors, Interleukin-2/physiology , T-Lymphocyte Subsets/physiology , Transcription Factors/physiology , Adoptive Transfer , Aging , Animals , Diabetes Mellitus, Type 1/immunology , Forkhead Transcription Factors , Gene Expression , Ionomycin , Lymphocyte Activation/physiology , Prediabetic State , Rats , T-Lymphocyte Subsets/transplantation , Tetradecanoylphorbol AcetateABSTRACT
Rat immune-associated nucleotide 4-like 1 (Ian4l1) encodes an antiapoptotic protein, which is essential for T-cell survival. A frameshift mutation at codon 85 in the biobreeding diabetes-prone (BBDP) rat is the cause of their life-long T-cell lymphopenia, which includes lack of regulatory T-cells--a prerequisite for spontaneous autoimmune destruction of their beta-cells. This study reports the identification of seven Ian4l1 mRNA variants. The genomic organization of the exons indicates three promoter regions. The promoter of two of the mRNAs was characterized. Rapid amplification of cDNA ends (RACE) and ribonuclease protection assay (RPA) demonstrated multiple transcription start sites (TSS) with two major sites. The localization of the core promoter and regulatory regions was identified by a luciferase assay of the 2.7-kb upstream of the TSS. The regulatory regions functioned similarly in two cell lines--one expressing Ian4l1 and one not expressing it. This indicates that the cell-specific expression is controlled by regions outside the 2.7-kb region, or by the chromatin structure or chromatin methylation level. The core promoter is TATA-less and initiator element-less, and contains putative binding sites for YY1, Sp1, and MED-1, the latter being an element believed to be important for transcription from TATA-less promoters.
Subject(s)
Apoptosis/genetics , GTP-Binding Proteins/genetics , Promoter Regions, Genetic/genetics , Alternative Splicing , Animals , Base Sequence , Binding Sites/genetics , Cell Line , DNA, Complementary/chemistry , DNA, Complementary/genetics , Exons , Genes/genetics , Introns , Luciferases/genetics , Luciferases/metabolism , Molecular Sequence Data , RNA, Messenger/genetics , RNA, Messenger/metabolism , Rats , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Sequence Analysis, DNA , Transcription Initiation Site , TransfectionABSTRACT
Type 1 diabetes is an autoimmune disease with a complex polygenic inheritance. Until recently, only three susceptibility genes had been reproducibly identified, namely HLA, INS-VNTR, and CTLA4. During the past 7 years, a number of new putative susceptibility genes have been isolated from both human and animal models of the disease. We present eight genes implicated in type 1 diabetes etiology and discuss them in relation to the pathogenesis of the disease: VDR, IL6, IL12B, AIRE, FOXP3, B2m, Cblb, and Lyp/Ian4l1.
Subject(s)
Autoimmunity/genetics , Diabetes Mellitus, Type 1/genetics , Animals , DNA-Binding Proteins/genetics , Forkhead Transcription Factors , Genetic Predisposition to Disease , Humans , Interleukin-12/genetics , Interleukin-12 Subunit p40 , Interleukin-6/genetics , Receptors, Calcitriol/genetics , Transcription Factors/genetics , AIRE ProteinABSTRACT
Iddm4 is one of several susceptibility genes that have been identified in the BB rat model of type 1 diabetes. The BB rat allele of this gene confers dominant predisposition to diabetes induction by immune perturbation in both the diabetes-prone and the diabetes-resistant substrains, whereas the Wistar Furth (WF) allele confers resistance. We have positioned the gene in a 2.8-cM region on rat Chromosome (Chr) 4, proximal to Lyp/Ian4l1. We have produced a radiation hybrid map of the Iddm4-region that includes a number of rat genes with their mouse and human orthologs. We present a comparative map of the rat Iddm4 region in rat, human, and mouse, assigning the gene to a 6.3-Mb segment between PTN and ZYX at 7q32 in the human genome, and to a 5.7-Mb segment between Ptn and Zyx in the mouse genome.
Subject(s)
Carrier Proteins/genetics , Chromosome Mapping , Diabetes Mellitus, Type 1/genetics , Animals , Chromosomes/genetics , Diabetes Mellitus, Type 1/pathology , Genetic Markers , Genetic Predisposition to Disease , Genome , Humans , Lod Score , Mice , Microsatellite Repeats , Radiation Hybrid Mapping , Rats , Rats, Inbred BB , Rats, Inbred WFABSTRACT
Apoptosis is a regulated cell death program controlled by extrinsic and intrinsic signaling pathways. The intrinsic pathway involves stress signals that activate pro-apoptotic members of the Bcl-2 family, inducing permeabilization of mitochondria and release of apoptogenic factors. These proteins localize to the outer mitochondrial membrane. Ian4, a mitochondrial outer membrane protein with GTP-binding activity, is normally present in thymocytes, T cells, and B cells. We and others have recently discovered that a mutation in the rat Ian4 gene results in severe T cell lymphopenia that is associated with the expression of autoimmune diabetes. The mechanism by which Ian4 controls T cell homeostasis is unknown. Here we show that the absence of Ian4 in T cells causes mitochondrial dysfunction, increased mitochondrial levels of stress-inducible chaperonins and a leucine-rich protein, and T cell-specific spontaneous apoptosis. T cell activation and caspase 8 inhibition both prevented apoptosis, whereas transfection of T cells with Ian4-specific small interfering RNA recapitulated the apoptotic phenotype. The findings establish Ian4 as a tissue-specific regulator of mitochondrial integrity.
Subject(s)
GTP-Binding Proteins/physiology , Membrane Proteins/physiology , Mitochondria/physiology , Mitochondrial Proteins/physiology , T-Lymphocytes/physiology , Animals , Apoptosis , Caspases/physiology , Cell Survival , DNA Fragmentation , Female , Male , Membrane Potentials , RNA, Small Interfering/pharmacology , Rats , Rats, Inbred WFABSTRACT
Diabetes-prone (DP) BB rats spontaneously develop insulin-dependent diabetes resembling human type 1 diabetes. They also exhibit lifelong T-cell lymphopenia. Functional and genetic data support the hypothesis that the gene responsible for the lymphopenia, Lyp, is also a diabetes susceptibility gene, named Iddm1. We constructed a 550-kb P1-derived artificial chromosome contig of the region. Here, we present a corrected genetic map reducing the genetic interval to 0.2 cM and the physical interval to 150-290 kb. A total of 13 genes and six GenomeScan models are assigned to the homologous human DNA segment on HSA7q36.1, 8 of which belong to the family of immune-associated nucleotides (Ian genes). Two of these are orthologous to mouse Ian1 and -4, both excellent candidates for Iddm1. In normal rats, they are expressed in the thymus and T-cell regions of the spleen. In the thymus of lymphopenic rats, Ian1 exhibits wild-type expression patterns, whereas Ian4 expression is reduced. Mutational screening of their coding sequences revealed a frameshift mutation in Ian4 among lymphopenic rats. The mutation results in a truncated protein in which the COOH-terminal 215 amino acids-including the anchor localizing the protein to the outer mitochondrial membrane-are replaced by 19 other amino acids. We propose that Ian4 is identical to Iddm1.