ABSTRACT
Regulatory B cells (Breg cells) that secrete IL-10 or IL-35 (i35-Breg) play key roles in regulating immunity in tumor microenvironment or during autoimmune and infectious diseases. Thus, loss of Breg function is implicated in development of autoimmune diseases while aberrant elevation of Breg prevents sterilizing immunity, exacerbates infectious diseases, and promotes cancer metastasis. Breg cells identified thus far are largely antigen-specific and derive mainly from B2-lymphocyte lineage. Here, we describe an innate-like IL-27-producing natural regulatory B-1a cell (i27-Breg) in peritoneal cavity and human umbilical cord blood. i27-Bregs accumulate in CNS and lymphoid tissues during neuroinflammation and confers protection against CNS autoimmune disease. i27-Breg immunotherapy ameliorated encephalomyelitis and uveitis through up-regulation of inhibitory receptors (Lag3, PD-1), suppression of Th17/Th1 responses, and propagating inhibitory signals that convert conventional B cells to regulatory lymphocytes that secrete IL-10 and/or IL-35 in eye, brain, or spinal cord. Furthermore, i27-Breg proliferates in vivo and sustains IL-27 secretion in CNS and lymphoid tissues, a therapeutic advantage over administering biologics (IL-10, IL-35) that are rapidly cleared in vivo. Mutant mice lacking irf4 in B cells exhibit exaggerated increase of i27-Bregs with few i35-Bregs, while mice with loss of irf8 in B cells have abundance of i35-Bregs but defective in generating i27-Bregs, identifying IRF8/BATF and IRF4/BATF axis in skewing B cell differentiation toward i27-Breg and i35-Breg developmental programs, respectively. Consistent with its developmental origin, disease suppression by innate i27-Bregs is neither antigen-specific nor disease-specific, suggesting that i27-Breg would be effective immunotherapy for a wide spectrum of autoimmune diseases.
Subject(s)
Autoimmune Diseases/immunology , Central Nervous System Diseases/immunology , Interleukin-27/metabolism , Neuroinflammatory Diseases/immunology , Animals , B-Lymphocytes, Regulatory/immunology , Cell Differentiation , Encephalitis , Interferon Regulatory Factors , Interleukin-10 , Mice , Uveitis/immunologyABSTRACT
Experimental autoimmune uveitis (EAU), an animal model for severe intraocular inflammatory eye diseases, is mediated by both Th1 and Th17 cells. Here, we examined the capacity of TMP778, a selective inhibitor of RORγt, to inhibit the development of EAU, as well as the related immune responses. EAU was induced in B10.A mice by immunization with interphotoreceptor retinoid-binding protein (IRBP). Treatment with TMP778 significantly inhibited the development of EAU, determined by histological examination. In addition, the treatment suppressed the cellular immune response to IRBP, determined by reduced production of IL-17 and IFN-γ, as well as lower percentages of lymphocytes expressing these cytokines, as compared to vehicle-treated controls. The inhibition of IFN-γ expression by TMP778 is unexpected in view of this compound being a selective inhibitor of RORγt. The observation was further confirmed by the finding of reduced expression of the T-bet (Tbx21) gene, the transcription factor for IFN-γ, by cells of TMP778-treated mice. Thus, these data demonstrate the capacity of TMP778 to inhibit pathogenic autoimmunity in the eye and shed new light on its mode of action in vivo.
Subject(s)
Autoimmune Diseases/drug therapy , Heterocyclic Compounds, 4 or More Rings/pharmacology , Nuclear Receptor Subfamily 1, Group F, Member 3/antagonists & inhibitors , Th1 Cells/drug effects , Th17 Cells/drug effects , Uveitis/drug therapy , Animals , Autoimmune Diseases/metabolism , Cytokines/metabolism , Disease Models, Animal , Eye Proteins/metabolism , Female , Interferon-gamma/metabolism , Interleukin-17/metabolism , Mice , Retinol-Binding Proteins/metabolism , T-Box Domain Proteins/metabolism , Th1 Cells/metabolism , Th17 Cells/metabolism , Uveitis/metabolismABSTRACT
Inflammation and oxidative stress are involved in age-related macular degeneration (AMD) and possibly associated with an activation of neuronal apoptosis inhibitor protein/class II transcription activator of the Major Histocompatibility Complex (MHC)/heterokaryon incompatibility/telomerase-associated protein 1, leucine-rich repeat or nucleotide-binding domain, leucine-rich repeat-containing family, and pyrin domain-containing 3 (NLRP3) inflammasome. In the present study, we used a translational approach to address this hypothesis. In patients with AMD, we observed increased mRNA levels of NLRP3, pro-interleukin-1 beta (IL-1ß) and pro-IL-18 in AMD lesions of the retinal pigment epithelium (RPE) and photoreceptor. In vitro, a similar increase was evoked by oxidative stress or lipopolysaccharide (LPS) stimulation in the adult retinal pigment epithelium (ARPE-19) cell line, and the increase was reduced in siRNA transfected cells to knockdown NLRP3. Ultrastructural studies of ARPE-19 cells showed a swelling of the cytoplasm, mitochondrial damage, and occurrence of autophagosome-like structures. NLRP3 positive dots were detected within autophagosome-like structures or in the extracellular space. Next, we used a mouse model of AMD, Ccl2/Cx3cr1 double knockout on rd8 background (DKO rd8) to ascertain the in vivo relevance. Ultrastructural studies of the RPE of these mice showed damaged mitochondria, autophagosome-like structures, and cytoplasmic vacuoles, which are reminiscent of the pathology seen in stressed ARPE-19 cells. The data suggest that the NLRP3 inflammasome may contribute in AMD pathogenesis.
Subject(s)
Carrier Proteins/genetics , Macular Degeneration/genetics , Macular Degeneration/pathology , Retinal Pigment Epithelium/pathology , Up-Regulation , Adult , Aged, 80 and over , Animals , Carrier Proteins/immunology , Cell Line , Cells, Cultured , Humans , Inflammasomes/genetics , Inflammasomes/immunology , Inflammation/genetics , Inflammation/immunology , Inflammation/pathology , Macula Lutea/immunology , Macula Lutea/metabolism , Macula Lutea/pathology , Macular Degeneration/immunology , Male , Mice, Inbred C57BL , NLR Family, Pyrin Domain-Containing 3 Protein , Oxidative Stress , RNA Interference , RNA, Small Interfering/genetics , Retinal Pigment Epithelium/immunology , Retinal Pigment Epithelium/metabolismABSTRACT
The interaction between TLRs and their cognate ligands triggers both the innate and adaptive immune systems, and thus can play a pivotal role in the defense against pathogen invasion. This work investigates the differentiation of naive CD4 cells into Th1 or Th17 phenotypes in mice treated with different TLR ligands. We use a model system in which naive transgenic cells specific to hen egg lysozyme are adoptively transferred into recipients that express hen egg lysozyme in the lens of the eye. The transferred naive T cells induce ocular inflammation only in recipients treated with TLR ligands. Treatment with LPS preferentially stimulated IL-17 production, whereas CpG oligodeoxynucleotide and polyinosinic:polycytidylic acid primarily stimulated Th1 cells. Peptidoglycan stimulated the two Th subpopulations equally. The preferential induction of Th1 or Th17 by the four ligands was detected in the spleen (where a major portion of the adoptively transferred cells homed) and in the eyes, where activated Th cells initiate inflammation. Analysis of the cytokines present in recipient mice suggests that Th1 induction is elicited by IL-12 and/or IFN-α, whereas Th17 generation is preferentially mediated by IL-6. Importantly, we show in this article that treatment with LPS selectively promoted in the recipient mice the generation of IL-6-producing activated B cells. An inverse correlation was found between the level of regulatory T cells and severity of inflammation induced by the donor cells. Taken together, our data show that specific TLR ligands differentially activate the immune system as evidenced by the generation of distinct Th phenotypes from naive CD4 cells.
Subject(s)
Autoimmune Diseases/immunology , Th1 Cells/immunology , Th17 Cells/immunology , Toll-Like Receptors/metabolism , Animals , Autoimmune Diseases/metabolism , Autoimmune Diseases/pathology , Chickens , Ligands , Lipopolysaccharides/metabolism , Mice , Mice, Inbred C57BL , Mice, Transgenic , Muramidase/genetics , Peptidoglycan/metabolism , Poly I-C/metabolism , Th1 Cells/metabolism , Th1 Cells/pathology , Th17 Cells/metabolism , Th17 Cells/pathology , Toll-Like Receptor 2/metabolism , Toll-Like Receptor 3/metabolism , Toll-Like Receptor 4/metabolism , Toll-Like Receptor 9/metabolism , Toll-Like Receptors/physiologyABSTRACT
Regulatory T-cells (Tregs) are responsible for homeostasis of the immune system, as well as for inhibition of pathogenic autoimmune processes. Induced-(i)-Tregs, can be generated in vitro by activation of CD4 cells in the presence of TGF-ß. A commonly used activation mechanism is by antibodies against CD3 and CD28. The physiological-like activation of T-cells, however, is with the specific target antigen presented by antigen-presenting cells (APC). The two modes of activation have been considered to yield the same populations of iTregs. Here, we compared between iTreg populations generated by either one of the two methods and found differences between their capacities to inhibit T-lymphocyte proliferative response, their expression of cell surface antigens and particularly, in their transcript expression profiles of certain chemokines and chemokine receptors. Our data thus indicate that iTregs generated by activation with anti-CD3/CD28 antibodies cannot be considered identical to iTregs generated by antigen/APC.
Subject(s)
In Vitro Techniques/methods , Lymphocyte Activation/immunology , T-Lymphocyte Subsets/immunology , T-Lymphocytes, Regulatory/immunology , Animals , Antigen-Presenting Cells/immunology , CD28 Antigens/immunology , CD3 Complex/immunology , Cytokines/biosynthesis , Flow Cytometry , Mice , Mice, Transgenic , Polymerase Chain ReactionABSTRACT
Subpopulations of pathogenic or nonpathogenic Th17 cells were reported to develop when presensitized CD4 cells were activated with their target Ag during polarization by either IL-23 or IL-6 and TGF-ß, respectively. In this study, we generated two Th17 subpopulations by using a system in which naive CD4 cells from TCR transgenic mice specific to hen egg lysozyme (HEL) are polarized with IL-6/TGF-ß and, concurrently, are activated either with HEL presented by APCs, or with anti-CD3/CD28 Abs. Only the former cells were pathogenic, inducing inflammation in eyes expressing HEL. Naive CD4 cells activated by the anti-CD3/CD28 Abs acquired pathogenicity, however, when cocultured with HEL/APC. Importantly, the naive CD4 cells did not acquire pathogenicity when cocultured with APCs stimulated with LPS or when separated from the HEL-presenting cells by a semipermeable membrane. Unlike with presensitized Th17, soluble IL-23 does not participate in pathogenicity acquisition by naive CD4 cells; no pathogenicity was induced by adding IL-23 to cultures activated with anti-CD3/CD28 Abs. Furthermore, Abs against IL-23 or IL-23R did not inhibit acquisition of pathogenicity in cultures of naive CD4 cells activated by HEL/APC. Our data thus show that, unlike presensitized CD4 cells, naive CD4 cells polarized toward Th17 phenotype acquire pathogenicity only by direct interaction with APCs presenting the Ag, with no apparent involvement of soluble IL-23. We suggest that the Th17 lymphocytes derived from naive CD4 cells participate in pathogenic and other immune processes, along with the IL-23-dependent Th17 cells.
Subject(s)
Antigen-Presenting Cells/immunology , CD4-Positive T-Lymphocytes/immunology , Cell Communication/immunology , Cell Lineage/immunology , Muramidase/metabolism , Th17 Cells/immunology , Animals , Antigen-Presenting Cells/metabolism , CD4-Positive T-Lymphocytes/enzymology , CD4-Positive T-Lymphocytes/pathology , Cells, Cultured , Coculture Techniques , Dendritic Cells/immunology , Eye Diseases/enzymology , Eye Diseases/immunology , Eye Diseases/pathology , Inflammation/enzymology , Inflammation/immunology , Inflammation/pathology , Interleukin-23/metabolism , Mice , Mice, Transgenic , Muramidase/adverse effects , Muramidase/immunology , Th17 Cells/enzymology , Th17 Cells/pathologyABSTRACT
PURPOSE: SWAP 70-like adaptor of T cells (SLAT; aka Def6) is a recently discovered guanine nucleotide exchange factor for Rho guanosine triphosphate (GTP)ases that has been previously shown to play a role in cluster of differentiation(CD)4+ T cell activation, T-helper (Th)1/Th2/Th17 differentiation and development of experimental autoimmune encephalomyelitis. Here, we investigated the role of SLAT/Def6 in the development of experimental autoimmune uveitis (EAU), an animal model for several uveitic conditions in humans. METHODS: SLAT/Def6 deficient ("KO") mice and C57BL/6 controls were immunized with interphotoreceptor retinoid-binding protein (IRBP), along with pertussis toxin. The development of ocular inflammation was determined by both fundoscopy and histological examination. Lymphoid cells from draining lymph nodes were cultured with IRBP to measure lymphocyte proliferation and release of cytokines. Purified dendritic cells were tested for their capacity to present antigen to responding lymphocytes. In addition, the lymphoid cells were tested for the expression of forkhead box P3 (FoxP3), using conventional methods, and the activity of T-regulatory cells was determined by their capacity to inhibit in vitro proliferative responses. Serum anti -IRBP antibody levels were measured by enzyme-linked immunosorbant assay (ELISA). quantitative polymerase chain reaction (qPCR) was used to determine the transcript levels of cytokines in inflamed eyes. RESULTS: SLAT/Def6 KO mice had significantly reduced EAU compared to controls. Cells isolated from draining lymph nodes of SLAT/Def6 KO mice exhibited impaired proliferation and production of Th1 and Th17 signature cytokines (interferon [IFN]-γ and interleukin [IL]-17, respectively) when compared with cells isolated from control mice. qPCR of inflamed eyes detected similar levels of IFN-γ transcript in control and SLAT/Def6 KO mice, whereas the IL-17 transcript levels in eyes of the SLAT/Def6 KO mice were lower than in eyes of the controls. The SLAT/Def6 KO mice resembled their wild type (WT) controls, however, in the levels of their serum antibody against IRBP, the antigen presenting capacity of their dendritic cells, the proportion of cells expressing Foxp3 and the immunosuppressive activity of their T-regulatory cells. CONCLUSIONS: SLAT/Def6 KO mice exhibit reduced capacity to develop ocular inflammation and cellular activity when immunized with IRBP. Our study provides new data showing that SLAT/Def6 plays a major role in the T cell-mediated autoimmune processes that bring about the inflammatory eye disease, EAU.
Subject(s)
Autoimmune Diseases/immunology , DNA-Binding Proteins/immunology , Eye Proteins/immunology , Inflammation/immunology , Nuclear Proteins/immunology , Retinol-Binding Proteins/immunology , Uveitis/immunology , Animals , Antibodies/blood , Antibodies/immunology , Autoimmune Diseases/chemically induced , Autoimmune Diseases/genetics , Cell Proliferation/drug effects , DNA-Binding Proteins/deficiency , DNA-Binding Proteins/genetics , Disease Models, Animal , Eye Proteins/administration & dosage , Forkhead Transcription Factors/biosynthesis , Forkhead Transcription Factors/immunology , Guanine Nucleotide Exchange Factors , Inflammation/chemically induced , Inflammation/genetics , Interferon-gamma/biosynthesis , Interferon-gamma/immunology , Interleukin-17/biosynthesis , Interleukin-17/immunology , Lymph Nodes/drug effects , Lymph Nodes/immunology , Lymph Nodes/pathology , Mice , Mice, Knockout , Nuclear Proteins/deficiency , Nuclear Proteins/genetics , Pertussis Toxin/administration & dosage , Retinol-Binding Proteins/administration & dosage , T-Lymphocytes, Regulatory/drug effects , T-Lymphocytes, Regulatory/immunology , T-Lymphocytes, Regulatory/pathology , Th1 Cells/drug effects , Th1 Cells/immunology , Th1 Cells/pathology , Th17 Cells/drug effects , Th17 Cells/immunology , Th17 Cells/pathology , Th2 Cells/drug effects , Th2 Cells/immunology , Th2 Cells/pathology , Uveitis/chemically induced , Uveitis/geneticsABSTRACT
Recently reported lines of Th9 cells, producing IL-9 and IL-10, were generated by polarization with IL-4 and TGF-ß and activation with Abs against CD3 and CD28. In this paper, we analyzed features of Th9 lines similarly polarized but activated by the "natural mode" (i.e., exposure of CD4 cells to their target Ag, hen egg lysozyme [HEL] and APCs). Main observations are the following: 1) both IL-9 and IL-10 were expressed by the line cells, but with strikingly different kinetics, with IL-9 being produced rapidly, reaching a peak on day 3 in culture and declining sharply thereafter, whereas IL-10 production increased gradually, resembling IL-4 and IL-17 production by their corresponding lineage cells; 2) reactivation of Th9, following expansion, triggered faster and higher production of both IL-9 and IL-10; 3) incubating Th9 cells in polarizing media specific for other phenotypes stimulated moderate levels of phenotype switching to Th1 or Th17 but a massive switching to Th2; 4) Th9 cells induced moderate inflammation in HEL-expressing recipient eyes but only when producing high levels of IL-9; and 5) IL-9-producing donor cells were detected in the blood of Th9 recipients but not in their inflamed eyes, suggesting that similar to findings in culture, exposure to HEL in these eyes arrested the IL-9 production in Th9 cells. Collectively, these data provide new information concerning Th9 cells and reveal their uniqueness, in particular with regard to the unusual production kinetics of IL-9 and the short retention of these cells in affected target tissues.
Subject(s)
Eye Proteins/metabolism , Inflammation Mediators/metabolism , Interleukin-9/biosynthesis , T-Lymphocytes, Helper-Inducer/immunology , T-Lymphocytes, Helper-Inducer/metabolism , Animals , Cell Line , Chickens , Dose-Response Relationship, Immunologic , Epitopes, T-Lymphocyte/biosynthesis , Epitopes, T-Lymphocyte/immunology , Eye Proteins/physiology , Inflammation/immunology , Inflammation/metabolism , Inflammation/pathology , Inflammation Mediators/physiology , Interleukin-10/biosynthesis , Interleukin-10/metabolism , Interleukin-9/metabolism , Kinetics , Mice , Mice, Inbred C57BL , Mice, Transgenic , Muramidase/biosynthesis , Muramidase/genetics , Muramidase/immunology , Organ Specificity/immunology , Receptors, Antigen, T-Cell/biosynthesis , T-Lymphocytes, Helper-Inducer/pathology , Time FactorsABSTRACT
Th1 cells are remarkably more susceptible to activation induced cell death than Th17. Here, we compared cultures of these two cell subpopulations for their expression of apoptosis-related molecules when re-exposed to their specific antigen. We also compared the expression of apoptosis-related molecules in the mouse eye with inflammation induced by Th1 or Th17 cells. Using qPCR we found that the mRNA transcript levels of the majority of tested apoptosis-related molecules were higher in the Th1 cultures, and in eyes with Th1-induced inflammation. Apoptotic intrinsic pathway molecules played minor roles in the processes in vitro or in vivo, whereas extrinsic pathway molecules, as well as PD-1, its ligands and Tim3, were heavily involved.
Subject(s)
Apoptosis/immunology , Th1 Cells/immunology , Th1 Cells/metabolism , Th17 Cells/immunology , Th17 Cells/metabolism , Adoptive Transfer , Animals , Cell Line , Eye Diseases/etiology , Eye Diseases/immunology , Inflammation/etiology , Inflammation/immunology , Lymphocyte Activation , Mice , Mice, TransgenicABSTRACT
Both Th1 and Th17 T cell subsets can mediate inflammation, but the kinetics of the pathogenic processes mediated by these two subsets have not been investigated. Using an experimental system in which TCR-transgenic Th1 or Th17 cells specific for hen egg lysozyme induce ocular inflammation in recipient mice expressing eye-restricted hen egg lysozyme, we found important differences in the in vivo behavior of these two subsets. Th1 cells initially proliferated considerably faster and invaded the eye more quickly than their Th17 counterparts, but then disappeared rapidly. By contrast, Th17 cells accumulated and remained the majority of the infiltrating CD4(+) cells in the eye for as long as 25 days after transfer, mediating more long-lasting pathological changes. Unlike Th1, Th17 cells were highly resistant to restimulation-induced apoptosis, a major pathway by which autoimmune and chronically restimulated Th1 cells are eliminated. Th17 cells had reduced Fas ligand production and resistance to Fas-induced apoptosis, relative to Th1 cells, despite similar surface expression of Fas. Th17-induced ocular inflammation also differed from Th1-induced inflammation by consisting of more neutrophils, whereas Th1-induced disease had higher proportions of CD8 cells. Taken together, our data show that pathogenic processes triggered by Th17 lag behind those induced by Th1, but then persist remarkably longer, apparently due to the relative resistance of Th17 cells to restimulation-induced cell death. The long-lasting inflammation induced by Th17 cells is in accord with these cells being involved in chronic conditions in humans.
Subject(s)
Apoptosis/immunology , Autoimmune Diseases/immunology , CD4-Positive T-Lymphocytes/immunology , Inflammation/immunology , T-Lymphocyte Subsets/immunology , Adoptive Transfer , Animals , CD8-Positive T-Lymphocytes , Cell Division , Eye/immunology , Fas Ligand Protein/immunology , Fas Ligand Protein/metabolism , Flow Cytometry , Interleukin-17/biosynthesis , Interleukin-17/immunology , Mice , Mice, Transgenic , Muramidase/immunology , Th1 Cells/immunology , fas Receptor/immunology , fas Receptor/metabolismABSTRACT
Th1 and Th17 cells are characterized by their expression of IFN-gamma or IL-17, respectively. The finding of Th cells producing both IL-17 and IFN-gamma suggested, however, that certain Th cells may modify their selective cytokine expression. In this study, we examined changes in cytokine expression in an experimental system in which polarized Th1 or Th17 cells specific against hen egg lysozyme induce ocular inflammation in recipient mice expressing hen egg lysozyme in their eyes. Whereas only IFN-gamma was expressed in eyes of Th1 recipient mice, substantial proportions of donor cells expressed IFN-gamma or both IFN-gamma and IL-17 in Th17 recipient eyes. The possibility that nonpolarized cells in Th17 preparations were responsible for expression of IFN-gamma or IFN-gamma/IL-17 in Th17 recipient eyes was contradicted by the finding that the proportions of such cells were larger in recipients of Th17 preparations with 20-25% nonpolarized cells than in recipients of 35-40% preparations. Moreover, whereas incubation in vitro of Th1 cells with Th17-polarizing mixture had no effect on their phenotype, incubation of Th17 with Th1-polarizing mixture, or in the absence of cytokines, converted most of these cells into IFN-gamma or IFN-gamma/IL-17-expressing cells. In addition, Th17 incubated with the Th1 mixture expressed T-bet, whereas no ROR-gamma t was detected in Th1 incubated with Th17 mixture. Thus, polarized Th1 cells retain their phenotype in the tested systems, whereas Th17 may switch to express IFN-gamma or IFN-gamma/IL-17 following activation in the absence of cytokines, or exposure to certain cytokine milieus at the inflammation site or in culture.
Subject(s)
Inflammation/immunology , Interferon-gamma/biosynthesis , Interleukin-17/biosynthesis , T-Lymphocyte Subsets/immunology , Th1 Cells/immunology , Animals , Eye Diseases/immunology , Eye Diseases/metabolism , Flow Cytometry , Immunophenotyping , Inflammation/metabolism , Lymphocyte Activation/immunology , Mice , Mice, Transgenic , Muramidase/immunology , Phenotype , Receptors, Antigen, T-Cell/immunology , Reverse Transcriptase Polymerase Chain Reaction , T-Lymphocyte Subsets/metabolism , Th1 Cells/metabolismABSTRACT
PURPOSE: Microbial products stimulate the immune system by interacting with Toll-like receptors (TLR) on antigen-presenting cells. This study examined the hypothesis that microbial products, which function as TLR ligands, are playing a major role in triggering pathogenic autoimmunity. METHODS: An experimental system was developed in which microbial TLR ligands were tested in vivo for their capacity to stimulate naïve CD4 cells specific against hen egg lysozyme (HEL) to become effector cells capable of inducing inflammation in eyes in which HEL is expressed. The ligands' mode of action was analyzed by determining their effects on the proliferation, acquisition of tissue-invading capacity, i.e. elevated CD49d and decreased CD62L expression, and production of interferon-gamma by the HEL-specific cells. RESULTS: All the 7 tested TLR ligands triggered ocular inflammation in the experimental system used here, with pertussis toxin surpassing all other ligands in its activities. A correlation was found between the capacity of the ligands to trigger pathogenic immunity and to stimulate the proliferation, modification of cell surface and interferon-gamma production by T cells. CONCLUSIONS: This study provides direct evidence to support the notion that microbial products are capable of triggering pathogenic autoimmunity.
Subject(s)
Autoantigens/immunology , Autoimmune Diseases/immunology , Bacteria/metabolism , Bacterial Toxins/toxicity , Polysaccharides, Bacterial/toxicity , Toll-Like Receptors/metabolism , Uveitis/immunology , Animals , Autoimmune Diseases/chemically induced , Autoimmune Diseases/pathology , CD4-Positive T-Lymphocytes/immunology , CD4-Positive T-Lymphocytes/metabolism , Disease Models, Animal , Integrin alpha2/biosynthesis , Interferon-gamma/biosynthesis , L-Selectin/biosynthesis , Ligands , Mice , Mice, Transgenic , Muramidase/immunology , Toll-Like Receptors/immunology , Uveitis/chemically induced , Uveitis/pathologyABSTRACT
AIMS: The aim of this study was to investigate the effect of arsenic trioxide (ATO) on the growth of cultured uveal melanoma cells (OCM-1 cell line). METHODS: Cultured OCM-1 cells were treated with 0.75-24 microM of ATO for 4-96 h. Cell viability was evaluated with the methylthiazoletetrazolium (MTT) assay as proliferation test. Apoptotic and necrotic cells were quantified using flow cytometry following Annexin-V/PI double stain. The cell morphology alteration was examined by light and electron transmission microscopy. To investigate the underlying mechanism of ATO-induced apoptosis and necrosis, glutathione peroxidase activity was measured, and mitochondrial membrane potentials were quantified using confocal microscopy. RESULTS: In the MTT assays, OCM-1 cell growth was inhibited at ATO concentrations of 1.5-24 microM. The 50% inhibitory concentration of ATO was 16.8 microM. At ATO concentrations of 12 and 24 microM, apoptosis and necrosis were induced after 24 h of incubation as shown by light and transmission electron microscopy. At ATO concentrations of 12 and 24 microM, the glutathione peroxidase activity was significantly (p < 0.05) reduced after 24 h of incubation, and the mitochondrial membrane potentials were significantly (p < 0.01) decreased after 4, 12 and 24 h of incubation. CONCLUSION: ATO inhibited ocular melanoma cell line growth at concentrations of 1.5-24 microM of ATO in a dose- and time-dependent manner by inducing apoptosis and necrosis of the tumor cells. The concurringly decreased glutathione peroxidase activity and the reduced mitochondrial membrane potentials may be possible underlying mechanisms for the apoptosis and necrosis of the tumor cells.
Subject(s)
Arsenicals/pharmacology , Cell Proliferation/drug effects , Growth Inhibitors/pharmacology , Melanoma/pathology , Oxides/pharmacology , Apoptosis/drug effects , Arsenic Trioxide , Arsenicals/administration & dosage , Cell Line, Tumor , Dose-Response Relationship, Drug , Flow Cytometry , Glutathione Peroxidase/antagonists & inhibitors , Glutathione Peroxidase/metabolism , Growth Inhibitors/administration & dosage , Humans , Melanoma/enzymology , Melanoma/physiopathology , Membrane Potentials/drug effects , Microscopy, Electron , Mitochondria/drug effects , Necrosis , Osmolar Concentration , Oxides/administration & dosageABSTRACT
PURPOSE: Provide a reproducible method for culturing confluent monolayers of hfRPE cells that exhibit morphology, physiology, polarity, and protein expression patterns similar to native tissue. METHODS: Human fetal eyes were dissected on arrival, and RPE cell sheets were mechanically separated from the choroid and cultured in a specifically designed medium comprised entirely of commercially available components. Physiology experiments were performed with previously described techniques. Standard techniques were used for immunohistochemistry, electron microscopy, and cytokine measurement by ELISA. RESULTS: Confluent monolayers of RPE cell cultures exhibited epithelial morphology and heavy pigmentation, and electron microscopy showed extensive apical membrane microvilli. The junctional complexes were identified with immunofluorescence labeling of various tight junction proteins. The mean transepithelial potential (TEP) was 2.6 +/- 0.8 mV, apical positive, and the mean transepithelial resistance (R(T)) was 501 +/- 138 Omega . cm(2) (mean +/- SD; n = 35). Addition of 100 microM adenosine triphosphate (ATP) to the apical bath increased net fluid absorption from 13.6 +/- 2.6 to 18.8 +/- 4.6 microL . cm(-2) per hour (mean +/- SD; n = 4). In other experiments, VEGF was mainly secreted into the basal bath (n = 10), whereas PEDF was mainly secreted into the apical bath (n = 10). CONCLUSIONS: A new cell culture procedure has been developed that produces confluent primary hfRPE cultures with morphological and physiological characteristics of the native tissue. Epithelial polarity and function of these easily reproducible primary cultures closely resemble previously studied native human fetal and bovine RPE-choroid explants.
Subject(s)
Cell Polarity/physiology , Eye Proteins/metabolism , Fetus/cytology , Pigment Epithelium of Eye/cytology , Pigment Epithelium of Eye/physiology , Blotting, Western , Cell Culture Techniques , Cell Separation , Cytokines/metabolism , Electrophysiology , Enzyme-Linked Immunosorbent Assay , Fluorescent Antibody Technique, Indirect , Humans , Membrane Potentials , Microscopy, Electron , Nerve Growth Factors/metabolism , Serpins/metabolism , Vascular Endothelial Growth Factor A/metabolismABSTRACT
PURPOSE: Digoxin, a major medication for heart disease, was recently reported to have immunosuppressive capacity. Here, we determined the immunosuppressive capacity of digoxin on the development of experimental autoimmune uveitis (EAU) and on related immune responses. METHODS: The B10.A mice were immunized with interphotoreceptor retinoid-binding protein (IRBP) and were treated daily with digoxin or vehicle control. On postimmunization day 14, the mouse eyes were examined histologically, while spleen cells were tested for cytokine production in response to IRBP and purified protein derivative. The immunosuppressive activity of digoxin was also tested in vitro, by its capacity to inhibit development of Th1 or Th17 cells. To investigate the degenerative effect of digoxin on the retina, naïve (FVB/N × B10.BR)F1 mice were similarly treated with digoxin and tested histologically and by ERG. RESULTS: Treatment with digoxin inhibited the development of EAU, as well as the cellular response to IRBP. Unexpectedly, treatment with digoxin suppressed the production of interferon-γ to a larger extent than the production of interleukin 17. Importantly, digoxin treatment induced severe retinal degeneration, determined by histologic analysis with thinning across all layers of the retina. Digoxin treatment also induced dose-dependent vision loss monitored by ERG on naïve mice without induction of EAU. CONCLUSIONS: Treatment of mice with digoxin inhibited the development of EAU and cellular immune response to IRBP. However, the treatment induced severe damage to the retina. Thus, the use of digoxin in humans should be avoided due to its toxicity to the retina.
Subject(s)
Diabetes Mellitus, Type 1 , Digoxin/pharmacology , Immunity, Cellular/drug effects , Retinal Degeneration/prevention & control , Uveitis/drug therapy , Animals , Disease Models, Animal , Enzyme Inhibitors/pharmacology , Female , Mice , Retinal Degeneration/etiology , Retinal Degeneration/immunology , Severity of Illness Index , Uveitis/complications , Uveitis/immunologyABSTRACT
PURPOSE: The inflammatory process plays a major role in the pathogenesis of AMD, and recent data indicate the involvement of inflammasomes. Inflammasomes are intracellular structures that trigger inflammation by producing mature interleukin-(IL)-1ß and IL-18. This study examined the capacity of 7-ketocholesterol (7KCh), an oxysterol that accumulates in the retinal pigmented epithelium (RPE) and choroid, to initiate inflammasome formation in RPE and bone marrow-derived cells. METHODS: Tested cells included fetal human RPE (fhRPE), human ARPE-19 cells, primary human brain microglia cells, and human THP-1 monocyte cells. 7-Ketocholesterol and other compounds were added to the cell cultures, and their stimulatory effects were determined by quantitative PCR and release of cytokines, measured by ELISA and Western blotting. RESULTS: 7-Ketocholesterol efficiently induced inflammasome formation by all primed cell populations, but secreted cytokine levels were higher in cultures of bone marrow-derived cells (microglia and THP-1 cells) than in RPE cultures. Interestingly, inflammasomes formed in cells of the two populations differed strikingly in their preferential production of the two cytokines. Thus, whereas bone marrow-derived cells produced levels of IL-1ß that were higher than those of IL-18, the opposite was found with RPE cells, which secreted higher levels of IL-18. Importantly, Western blot analysis showed that IL-18, but not IL-1ß, was expressed constitutively by RPE cells. CONCLUSIONS: 7-Ketocholesterol efficiently stimulates inflammasome formation and is conceivably involved in the pathogenesis of AMD. In contrast to bone marrow-derived cells, RPE cells produced higher levels of IL-18 than IL-1ß. Further, IL-18, a multifunctional cytokine, was expressed constitutively by RPE cells. These observations provide new information about stimuli and cells and their products assumed to be involved in the pathogenesis of AMD.
Subject(s)
Bone Marrow Cells/drug effects , Bone Marrow Cells/physiology , Inflammasomes/drug effects , Inflammasomes/physiology , Interleukin-18/metabolism , Interleukin-1beta/metabolism , Ketocholesterols/pharmacology , Retinal Pigment Epithelium/drug effects , Retinal Pigment Epithelium/physiology , Blotting, Western , Cells, Cultured , Enzyme-Linked Immunosorbent Assay , HumansABSTRACT
BACKGROUND: Mutations in LRRK2 are related to certain forms of Parkinson's disease and, possibly, to the pathogenesis of Crohn's disease. In both these diseases inflammatory processes participate in the pathogenic process. LRRK2 is expressed in lymphoid cells and, interestingly, Lrrk2 (-/-) mice were reported to develop more severe experimental colitis than their wild type (WT) controls. Here, we examined the possible involvement of LRRK2 in the pathogenesis of experimental autoimmune uveitis (EAU), an animal model for human uveitis, by testing Lrrk2 (-/-) mice for their capacity to develop this experimental eye disease and related immune responses. METHODS: Lrrk2 (-/-) mice and their WT controls (C57Bl/6) were immunized with interphotoreceptor retinoid-binding protein (IRBP) and compared for their development of EAU, delayed type hypersensitivity (DTH) by skin tests, production of cytokines in culture, and expression of interferon (IFN)-γ, interleukin (IL)-17 and FoxP3 by spleen cells, using flow cytometry. Peritoneal macrophages were examined for their production of cytokines/chemokines in culture following stimulation with LPS or the oligodeoxynucleotide CpG. The Lrrk2 (-/-) and WT mice were also compared for their response to bovine serum albumin (BSA). RESULTS: The Lrrk2 (-/-) mice developed lower levels of EAU, DTH responses and cytokine production by lymphocytes than did their WT controls. Intracellular expression of IFN-γ and IL-17, by spleen cells, and secretion of cytokines/chemokines by activated peritoneal macrophages of Lrrk2 (-/-) mice trended toward diminished levels, although variabilities were noted. The expression levels of FoxP3 by Lrrk2 (-/-) spleen cells, however, were similar to those seen in WT controls. Consistent with their low response to IRBP, Lrrk2 (-/-) mice responded to BSA less vigorously than their WT controls. CONCLUSIONS: Lrrk2 deficiency in mice diminished the development of EAU and the related adaptive immune responses to IRBP as compared to the WT controls.
Subject(s)
Autoimmune Diseases/pathology , Protein Serine-Threonine Kinases/genetics , Uveitis/pathology , Adaptive Immunity , Animals , Autoimmune Diseases/metabolism , Chemokines/metabolism , Disease Models, Animal , Eye Proteins/immunology , Forkhead Transcription Factors/metabolism , Hypersensitivity, Delayed/etiology , Hypersensitivity, Delayed/immunology , Interferon-gamma/metabolism , Interleukin-17/metabolism , Leucine-Rich Repeat Serine-Threonine Protein Kinase-2 , Macrophages, Peritoneal/immunology , Macrophages, Peritoneal/metabolism , Mice , Mice, Inbred C57BL , Mice, Knockout , Protein Serine-Threonine Kinases/deficiency , Retinol-Binding Proteins/immunology , Skin Tests , Th1 Cells/cytology , Th1 Cells/immunology , Th1 Cells/metabolism , Th17 Cells/cytology , Th17 Cells/immunology , Th17 Cells/metabolism , Uveitis/metabolismABSTRACT
OBJECTIVE: To study the effects and mechanism of hydrogen peroxide (H2O2) of low concentration on dynamic changes of intracellular free calcium contents ([Ca2+]i) in cultural rat liver oval cells (WB-F344 cells). METHODS: Using Fluo-3/Am as fluorescent indicator of [Ca2+]i and it was measured by laser scanning confocal microscope system. RESULTS: The results showed that: (1) A rapid transient spiking of [Ca2+]i occurred after the stimulation of H2O2 of low concentration (800 nmol/L). (2) The [Ca2+]i increase was abolished by pretreated with catalase (CAT) or by incubated in D-Hank's solution containing EGTA, the chelate of extracellular Ca2+. (3) The [Ca2+]i increase was not inhibited by pretreated nifedipine, Ca2+ channel blocker, but was abolished by pretreated with anthracere-9-cardoxylic acid (A9C), the Cl-channel blocker and which also blocked calcium activated non-selective cation channel (CAN). CONCLUSIONS: These results suggest that the increase of [Ca2+]i induced by H2O2 of low concentration may be due to the influx of extracellular Ca2+ through CAN.
Subject(s)
Calcium/metabolism , Hepatocytes/metabolism , Hydrogen Peroxide/pharmacology , Animals , Cells, Cultured , Ion Channels/drug effects , Microscopy, Confocal , RatsABSTRACT
T-helper (Th) lineages have been generated in vitro by activating CD4 cells with anti-CD3/CD28 antibodies during polarization. Physiologically, however, the generation of Th lineages is by activation with the specific antigen presented by antigen-presenting cells (APC). Here, we used T-cell receptor (TCR)-transgenic mice to compare the phenotypes of Th1, Th9 and Th17 lineages when generated by either one of the two activation modes. Lineage Th cells specific against hen egg lysozyme (HEL), were adoptively transferred into recipient mice transgenically expressing HEL in their lens. Remarkable differences were found between lineages of Th1, Th9 or Th17, generated by either one of the two modes in their capacities to migrate to and proliferate in the recipient spleen and, importantly, to induce inflammation in the recipient mouse eyes. Substantial differences were also observed between the lineage pairs in their transcript expression profiles of certain chemokines and chemokine receptors. Surprisingly, however, close similarities were observed between the transcript expression profiles of lineages of the three phenotypes, activated by the same mode. Furthermore, Th cell lineages generated by the two activation modes differed considerably in their pattern of gene expression, as monitored by microarray analysis, but exhibited commonality with lineages of other phenotypes generated by the same activation mode. This study thus shows that (i) Th lineages generated by activation with anti-CD3/CD28 antibodies differ from lineages generated by antigen/APC; and (ii) the mode of activation determines to a large extent the expression profile of major transcripts.
Subject(s)
Antibodies/immunology , CD28 Antigens/immunology , CD3 Complex/immunology , Cell Lineage/immunology , Lymphocyte Activation/immunology , T-Lymphocytes, Helper-Inducer/cytology , T-Lymphocytes, Helper-Inducer/immunology , Animals , Antigen-Presenting Cells/drug effects , Antigen-Presenting Cells/immunology , Biomarkers/metabolism , Cell Movement/drug effects , Cell Proliferation/drug effects , Chemokines/genetics , Chemokines/metabolism , Chickens , Gene Expression Regulation/drug effects , Lymphocyte Activation/drug effects , Mice , Muramidase/immunology , Oligonucleotide Array Sequence Analysis , Phenotype , RNA, Messenger/genetics , RNA, Messenger/metabolism , Receptors, Chemokine/genetics , Receptors, Chemokine/metabolism , Spleen/cytology , Spleen/immunologyABSTRACT
PURPOSE: Ligands for aryl hydrocarbon receptor (AHR), such as dioxins, are highly toxic. One such ligand, TCDD, was found to exert potent immunosuppressive capacities in mice developing pathogenic autoimmune processes, including EAU, but its toxicity makes it unusable for humans. A recently identified endogenous AHR ligand, ITE, is also immunosuppressive, but is nontoxic and could therefore be useful for therapy in humans. Here, we tested ITE for its capacity to inhibit EAU and related immune responses. METHODS: EAU was induced in B10.A mice by immunization with interphotoreceptor retinoid-binding protein (IRBP; 40 µg) in CFA. Treatment with ITE was by daily intraperitoneal injection of 0.2 mg. Disease severity was assessed by both fundoscopy and histological examination. Draining lymph node cells were tested for proliferation by thymidine uptake and for cytokine production and release by ELISA. In addition, the intracellular expression of cytokines and Foxp3 was determined by flow cytometry. Serum antibodies were measured by ELISA. RESULTS: Treatment with ITE efficiently inhibited the development of EAU in mice, as well as the cellular immune responses against IRBP and PPD. ITE treatment inhibited the expansion of both Th1 and Th17 subpopulations, as well as their release of the signature cytokines, IFN-gamma and IL-17. The treatment moderately increased, however, the proportion of Foxp3 expressing T-regulatory cells. Antibody production was not affected by the treatment. CONCLUSIONS: ITE, an endogenous AHR ligand, efficiently inhibits EAU development and related cellular immune responses. Being nontoxic, ITE may be considered for treatment of pathogenic immunity in humans.