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1.
Exp Mol Pathol ; 93(3): 408-15, 2012 Dec.
Article in English | MEDLINE | ID: mdl-23000510

ABSTRACT

Mast cells play a key role in immunoglobulin E (IgE)-associated allergic disorders; however, the cellular effects of sensitization remain poorly understood. Using gene microarrays and the multiplexing ELISA method, we examined the effects of sensitization on RBL-CCR1 cell transcription and chemokine/cytokine secretion. Sensitization most prominently increased transcription of Trb3, the gene for tribbles homolog 3 (TRB3), and also increased the release of most of the cytokines and chemokines tested. Knockdown of TRB3 via RNAi significantly induced the production of tumor necrosis factor-α (TNF-α), interleukin-4 (IL-4), interleukin-6 (IL-6), and the chemokine mast cell protease-1 (MCP-1). TRB3 deficiency also induced IκBα phosphorylation. TRB3 may therefore serve as a negative regulator of pro-inflammatory cytokines and chemokines, controlling the extent of the inflammatory response.


Subject(s)
Immunoglobulin E/immunology , Mast Cells/immunology , Protein Serine-Threonine Kinases/antagonists & inhibitors , Animals , Cell Line, Tumor , Chemokines/biosynthesis , Enzyme-Linked Immunosorbent Assay , Gene Expression , Gene Expression Profiling , Gene Knockdown Techniques , Humans , Mast Cells/metabolism , Mice , Mice, Inbred BALB C , Protein Serine-Threonine Kinases/physiology , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , Rats
2.
Br J Ophthalmol ; 96(8): 1132-6, 2012 Aug.
Article in English | MEDLINE | ID: mdl-22694967

ABSTRACT

BACKGROUND/AIMS: Allergic conjunctivitis is characterised by early-phase clinical symptoms and late-phase inflammation in the conjunctiva. The role of different chemokine receptors in allergic conjunctivitis, especially during the early-phase reaction, is still unclear. We investigated the importance of CC chemokine receptor (CCR) 3 in a murine model of IgE-mediated allergic conjunctivitis using CCR3-deficient (CCR3(-/-)) mice. METHODS: Allergic conjunctivitis was initiated in wild-type (WT) and CCR3(-/-) mice by passive transfer of ragweed (RW)-specific IgE, followed by topical challenge with RW in eye drops. Early-phase reactions including clinical symptoms and vascular leakage, as well as late-phase eosinophil infiltration of the conjunctiva were evaluated. The expression of mRNAs in the conjunctiva was quantified by real-time PCR analysis. RESULTS: The number of infiltrated eosinophils in the conjunctiva following RW challenge, was significantly higher in RW-IgE-sensitised WT mice compared with those sensitised with phosphate-buffered saline for WT, but this was not observed in similarly treated CCR3(-/-) mice. The early-phase clinical symptoms and vascular leakage were also suppressed in CCR3(-/-) mice. The number of conjunctival mast cells were not different between CCR3(-/-) mice and WT mice, and the mRNAs for FcεRІα and the connective tissue-type mast cell proteases were detected in the conjunctiva of both WT and CCR3(-/-) mice. RW-IgE-sensitised CCR3(-/-) mice displayed significantly reduced expression of CCL2, CCL3, and IL-6 compared with WT mice. CONCLUSIONS: These results demonstrate a direct contribution of CCR3 to both the early-phase reaction and late-phase inflammation during ocular allergy.


Subject(s)
Conjunctivitis, Allergic/immunology , Disease Models, Animal , Eosinophilia/metabolism , Hypersensitivity, Immediate/metabolism , Immunoglobulin E/immunology , Receptors, CCR3/physiology , Ambrosia/immunology , Animals , Eosinophils/cytology , Leukocyte Count , Mice , Mice, Knockout , RNA, Messenger/metabolism , Real-Time Polymerase Chain Reaction
3.
J Biol Chem ; 287(29): 24516-24, 2012 Jul 13.
Article in English | MEDLINE | ID: mdl-22613718

ABSTRACT

Accumulating evidence points to cross-talk between FcεRI and CC chemokine receptor (CCR)-mediated signaling pathways in mast cells. Here, we propose that vimentin, a protein comprising type III intermediate filament, participates in such cross-talk for CCL2/monocyte chemotactic protein 1 (MCP-1) production in mast cells, which is a mechanism for allergic inflammation. Co-stimulation via FcεRI, using IgE/antigen, and CCR1, using recombinant CCL3/macrophage inflammatory protein-1α (MIP-1α), increased expression of phosphorylated, disassembled, and soluble vimentin in rat basophilic leukemia (RBL)-2H3 cells expressing human CCR1 (RBL-CCR1 cells) and bone marrow-derived murine mast cells, both models of mucosal type mast cells. Furthermore, co-stimulation enhanced production of CCL2 as well as phosphorylation of MAPK. Treating the cells with p38 MAPK inhibitor SB203580, but not with MEK inhibitor PD98058, reduced CCL2 production, suggesting that p38 MAPK, but not ERK1/2, plays a critical role in the chemokine production. Immunoprecipitation analysis showed that vimentin interacts with phosphorylated ERK1/2 and p38 MAPKs in the co-simulated cells. Preventing disassembly of the vimentin by aggregating vimentin filaments using ß,ß'-iminodipropionitrile reduced the interaction of vimentin with phosphorylated MAPKs as well as CCL2 production in the cells. Taken together, disassembled vimentin interacting with phosphorylated p38 MAPK could mediate CCL2 production in mast cells upon FcεRI and CCR1 activation.


Subject(s)
Mast Cells/metabolism , Mitogen-Activated Protein Kinases/metabolism , Receptors, CCR1/metabolism , Receptors, IgG/metabolism , Vimentin/metabolism , Animals , Cell Line , Cell Line, Tumor , Cells, Cultured , Chemokine CCL2/metabolism , Electrophoresis, Gel, Two-Dimensional , Enzyme-Linked Immunosorbent Assay , Humans , Imidazoles/pharmacology , Immunoprecipitation , Mast Cells/drug effects , Mice , Mice, Inbred BALB C , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3/metabolism , Mitogen-Activated Protein Kinases/antagonists & inhibitors , Organic Chemicals/pharmacology , Phosphorylation , Protein Binding , Pyridines/pharmacology , Rats , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , p38 Mitogen-Activated Protein Kinases/antagonists & inhibitors , p38 Mitogen-Activated Protein Kinases/metabolism
4.
J Autoimmun ; 33(3-4): 247-54, 2009.
Article in English | MEDLINE | ID: mdl-19846275

ABSTRACT

Autoantibody production is associated with a variety of ocular disorders, including autoimmune retinopathy (AIR) and age-related macular degeneration (AMD). A breakdown of immunologic tolerance (ocular immune privilege), including the blood-retinal barrier, anti-immune and anti-inflammatory proteins, and anterior chamber-associated immune deviation may play important roles in these disorders. Although the exact triggers for ocular autoimmunity are unknown, autoimmune targeting of retinal tissue is clearly associated with and may contribute to the pathogenesis of both AIR and AMD. Autoantibody production has long been associated with AIR, a collection of disorders that includes cancer-associated retinopathy, melanoma-associated retinopathy and non-paraneoplastic autoimmune retinopathy. A growing body of evidence indicates that AMD pathogenesis, too, involves ocular inflammation and autoimmunity. Identification and quantification of autoantibodies produced in patients with AIR and AMD may assist with diagnosis, prognosis, and choice of treatments. Animal models that allow investigation of ocular autoimmunity will also be needed to better understand the disease processes and to develop novel therapies. In this review we discuss ocular immune privilege and potential mechanisms of autoimmunity in the eye. We describe how autoimmunity relates to the pathogenesis of AIR and AMD. We explain how the antigen microarray technique is used to detect autoantibodies in patient serum samples, and discuss how current animal models for AMD can be used to investigate autoimmune pathogenesis. Finally, we outline unanswered questions and exciting areas of future study related to autoimmune retinal degeneration.


Subject(s)
Autoimmunity , Blood-Retinal Barrier/immunology , Macular Degeneration/immunology , Retinal Diseases/immunology , Animals , Autoantibodies/blood , Blood-Retinal Barrier/metabolism , Disease Models, Animal , Humans , Immune Tolerance/immunology , Protein Array Analysis , Retina/immunology , Retina/pathology
5.
Int Immunol ; 21(8): 991-1001, 2009 Aug.
Article in English | MEDLINE | ID: mdl-19592420

ABSTRACT

Chemokine receptors (CCRs) are important co-stimulatory molecules found on many blood cells and associated with various diseases. The expression and function of CCRs on mast cells has been quite controversial. In this study, we report for the first time that murine bone marrow-derived mast cells (BMMC) express messenger RNA and protein for CCR1. BMMC cultured in the presence of murine recombinant stem cell factor and murine IL-3 expressed CCR1 after 5-6 weeks. We also report for the first time that mBMMC(CCR1+) cells endogenously express neurokinin receptor-1 and intercellular adhesion molecule-1. To examine the activity of CCR1 on these BMMC, we simultaneously stimulated two receptors: CCR1 by its ligand macrophage inflammatory protein-1alpha and the IgE receptor FcepsilonRI by antigen cross-linking. We found that co-stimulation enhanced BMMC degranulation compared with FcepsilonRI stimulation alone, as assessed by beta-hexosaminidase activity (85 versus 54%, P < 0.0001) and Ca(2+) influx (223 versus 183 nM, P < 0.05). We also observed significant increases in mast cell secretion of key growth factors, cytokines and chemokine mediators upon CCR1-FcepsilonRI co-stimulation. These factors include transforming growth factor beta-1, tumor necrosis factor-alpha and the cytokine IL-6. Taken together, our data indicate that CCR1 plays a key role in BMMC function. These findings contribute to our understanding of mechanisms for immune cell trafficking during inflammation.


Subject(s)
Bone Marrow Cells/metabolism , Interleukin-6/metabolism , Mast Cells/metabolism , Receptors, CCR1/biosynthesis , Transforming Growth Factor beta1/metabolism , Tumor Necrosis Factor-alpha/metabolism , Animals , Cells, Cultured , Female , Intercellular Adhesion Molecule-1/metabolism , Mice , Mice, Inbred BALB C , Receptors, CCR1/genetics , Receptors, CCR1/metabolism , Receptors, IgE/metabolism , Receptors, Neurokinin-1/metabolism , Signal Transduction
6.
J Pediatr Hematol Oncol ; 31(3): 209-14, 2009 Mar.
Article in English | MEDLINE | ID: mdl-19262251

ABSTRACT

Retinoblastoma (RB) is the commonest primary intraocular tumor in children. Overexpression of the high mobility group (HMG) A2 protein has been observed in a variety of malignant tumors and often correlates with poor prognosis. We studied the expression of HMGA2 in primary tumor samples and correlated with clinicopathologic features such as invasion, differentiation, and laterality of the tumors. Among 64 tumors, there were 29 tumors with invasion of the optic nerve, choroid, and/or orbit and 35 tumors without invasion. HMGA2 immunoreactivity was evaluated on archival paraffin sections and the results confirmed by Western blotting on 12 fresh tumor samples. Among 29 tumors with invasion, HMGA2 was strongly positive (++) in 10 tumors, moderately positive (+) in 11 tumors. Among 35 tumors without invasion, HMGA2 was strongly positive (++) in 6 tumors, moderately positive (+) in 6 tumors. Tumors with invasion showed significantly higher expression of HMGA2 compared with tumors without invasion (P<0.01). Non-neoplastic retina was negative for HMGA2. There was no correlation between HMGA2 expression and differentiation/laterality. Western blotting revealed that 7 tumors were strongly positive, 2 were moderately positive, and 1 was faintly positive for HMGA2. Our study has demonstrated the HMGA2 expression in a large cohort of primary retinoblastoma tumors and its correlation with invasiveness.


Subject(s)
HMGA2 Protein/biosynthesis , Retinal Neoplasms/metabolism , Retinal Neoplasms/pathology , Retinoblastoma/metabolism , Retinoblastoma/pathology , Biomarkers, Tumor/analysis , Blotting, Western , Child, Preschool , Female , Humans , Immunohistochemistry , Infant , Male , Neoplasm Invasiveness , Prognosis
7.
Int Immunol ; 21(2): 187-201, 2009 Feb.
Article in English | MEDLINE | ID: mdl-19147836

ABSTRACT

The immune response is regulated, in part, by effector cells whose activation requires multiple signals. For example, T cells require signals emanating from the T cell antigen receptor and co-stimulatory molecules for full activation. Here, we present evidence indicating that IgE-mediated hypersensitivity reactions in vivo also require cognate signals to activate mast cells. Immediate hypersensitivity reactions in the conjunctiva are ablated in mice deficient in eotaxin-1, despite normal numbers of tissue mast cells and levels of IgE. To further define the co-stimulatory signals mediated by chemokine receptor 3 (CCR3), an eotaxin-1 receptor, effects of CCR3 blockade were tested with an allergic conjunctivitis model and in ex vivo isolated connective tissue-type mast cells. Our results show that CCR3 blockade significantly suppresses allergen-mediated hypersensitivity reactions as well as IgE-mediated mast cell degranulation. We propose that a co-stimulatory axis by CCR3, mainly stimulated by eotaxin-1, is pivotal in mast cell-mediated hypersensitivity reactions.


Subject(s)
Allergens/metabolism , Chemokine CCL11/immunology , Conjunctivitis, Allergic/immunology , Conjunctivitis, Allergic/prevention & control , Glycoproteins/metabolism , Mast Cells/metabolism , Receptors, CCR3/antagonists & inhibitors , Receptors, CCR3/metabolism , Allergens/immunology , Animals , Cats , Cell Degranulation/immunology , Chemokine CCL11/metabolism , Conjunctiva/immunology , Conjunctiva/pathology , Conjunctivitis, Allergic/genetics , Glycoproteins/immunology , Immunoglobulin E/blood , Mast Cells/immunology , Mast Cells/pathology , Mice , Mice, Inbred BALB C , Mice, Knockout , Receptors, CCR3/genetics , Receptors, CCR3/immunology , Signal Transduction/genetics , Signal Transduction/immunology , Skin/immunology , Skin/pathology , Vaccination
8.
Invest Ophthalmol Vis Sci ; 50(4): 1626-35, 2009 Apr.
Article in English | MEDLINE | ID: mdl-19060266

ABSTRACT

PURPOSE: To determine how CpG, an immunostimulatory sequence, affects experimental allergic conjunctivitis and to determine the mechanisms of its action. METHODS: Experimental allergic conjunctivitis was induced in mice to investigate the suppressive mechanism of CpG treatment. Cytokine profiling, fluorescence-activated cell sorting analyses, and adoptive transfer were used to analyze suppressive mechanisms after CpG treatment. RESULTS: Administration of the CpG oligonucleotide induced significant splenomegaly. Adoptive transfer of the splenocytes isolated from CpG-treated mice was able to confer resistance to allergen-induced inflammatory responses in recipient mice. CpG treatment led to a transient upregulation of IL-1ra, IL-18, IL-1alpha, and IL-12 in the spleen, draining lymph nodes, and conjunctiva. In contrast, IL-10 showed a marked and sustained induction in the inductive and effector tissues. Splenomegaly after CpG exposure was reduced in IL-10-deficient mice, indicating that IL-10 is required for immune remodeling of the spleen. Analyses of allergen-sensitized mice deficient in IL-10 exacerbated the late-phase inflammatory responses. Fluorescence-activated cell sorting analysis of the CpG-induced splenocyte subsets showed that the predominant source of IL-10 was B220(+) CD19(+) CD23(+)IgM(+)CD40(+)class II(high) follicular B cells. Adoptive transfer of IL-10-deficient B cells exacerbated eosinophilia. Transfer of an expanded population of cells after CpG treatment, including IL-10-secreting follicular B cells, protected against eosinophilia. CONCLUSIONS: CpG treatment provided B cell-mediated regulation of immune responses and B cell differentiation in CpG-induced immune remodeling with the use of IL-10.


Subject(s)
B-Lymphocytes/immunology , Conjunctivitis, Allergic/immunology , Lymphocyte Activation/physiology , Oligodeoxyribonucleotides/pharmacology , Acetates , Adoptive Transfer , Allergens/immunology , Ambrosia , Animals , Conjunctivitis, Allergic/metabolism , Cytokines/genetics , Disease Models, Animal , Female , Flow Cytometry , Hypersensitivity, Immediate/immunology , Lymphocyte Activation/drug effects , Mice , Mice, Inbred C57BL , Pollen/immunology , RNA, Messenger/metabolism , Spleen/cytology , Splenomegaly/chemically induced , Splenomegaly/immunology
9.
Exp Mol Pathol ; 85(3): 147-54, 2008 Dec.
Article in English | MEDLINE | ID: mdl-18952077

ABSTRACT

A promising cancer treatment strategy involves stimulation of anti-tumor immune responses. CD4(+) T cell responses are particularly desirable, as they enhance CD8(+) T cell activity and provide immune memory. The major histocompatibility complex (MHC) class II transactivator CIITA can be used to stimulate expression of MHC II on tumor cells, thereby promoting CD4(+) T cell activation. In this study, N2a neuroblastoma cells were stably transfected with CIITA. N2aCIITA cells displayed increased expression of MHC I, MHC II and invariant chain; CD80 and CD86 were expressed by neither the parental N2a cells nor by the N2aCIITA cells. All mice injected with N2aCIITA cells developed tumors. Furthermore, no increase in the numbers of T cells, natural killer cells, macrophages, or eosinophils was observed in the spleens or tumors of mice injected with N2aCIITA cells, compared to tissues from mice injected with the parental N2a cells. This absence of an anti-tumor immune response despite MHC II expression is likely due to the presence of invariant chain, in support of the MHCII(+)/Ii(-) paradigm.


Subject(s)
Antigens, Differentiation, B-Lymphocyte/immunology , Histocompatibility Antigens Class II/immunology , Immunity/immunology , Neuroblastoma/immunology , Nuclear Proteins/metabolism , Trans-Activators/metabolism , Animals , Biomarkers/metabolism , Cell Line, Tumor , Cell Proliferation , Flow Cytometry , Histocompatibility Antigens Class I/immunology , Kinetics , Mice , Neuroblastoma/pathology , Phenotype , Spleen/immunology , Spleen/pathology
10.
Ocul Surf ; 6(2): 70-8, 2008 Apr.
Article in English | MEDLINE | ID: mdl-18418504

ABSTRACT

The acquired immune response in health and disease is initiated when foreign antigens are processed and presented to T lymphocytes via antigen-presenting cells as peptides in the context of Class I and II major histocompatibility complex antigens. It is now clear that there are various types of antigen-presenting cells and that the phenotype of these cells (together with the milieu of the tissue or lymphoid organ) dictates the nature of the immune response to the antigen. Very little is known about the phenotype, distribution, and roles of dendritic cell subtypes that contribute to the pathophysiology of type I hypersensitivity reaction in the ocular surface. We review what has been learned from studies of both human ocular allergy and murine models and comment on how this compares to allergic reactions in other mucosal tissues.


Subject(s)
Conjunctivitis, Allergic/immunology , Dendritic Cells/immunology , Animals , Conjunctivitis, Allergic/therapy , Disease Models, Animal , Humans
11.
Exp Mol Pathol ; 84(2): 90-101, 2008 Apr.
Article in English | MEDLINE | ID: mdl-18255057

ABSTRACT

Here we report the discovery of and phenotypic characterization of a retinal disorder of unknown origin in adults using clinical, electrophysiological and psychophysical techniques, and to seek the presence of circulating retinal autoantibodies in the sera of these patients. Sixteen patients were identified with progressive bilateral visual loss over a period of months. Ten of the patients were male, and the average age was 55.3 years (range from 43 to 76 years). Known causes such as carcinoma-associated retinopathy, acute zonal occult outer retinopathy and hereditary cone dystrophy appeared unlikely. Investigations included electrophysiology, fundus autofluorescence imaging and psychophysical tests. The sera of these patients were analyzed with indirect immunocytochemistry and Western immunoblot analysis on murine (BALB/c) retinal tissue for the presence of retinal autoantibodies. Bilateral visual loss and photophobia progressed over a period of months to years (average 28.7 months, range 3-67) and subsequently stabilized. No abnormality was observed by biomicroscopy, angiography or autofluorescence imaging. Electrophysiology indicated predominant cone-system dysfunction, either macular or generalized, and post-phototransduction involvement in 9 patients (56%). Photopic and scotopic visual fields and dark adaptation kinetics showed both cone and rod system involvement in all cases. Heterogeneous immunohistochemical staining patterns were seen with the sera of these patients as compared with controls. A majority of the affected patients (9/15) stained with an antinuclear pattern. The retinal autoantibodies from the sera of most patients reacted with the retinal proteins of molecular weight between 34 and 40 kDa. The aetiology of this distinctive retinal disorder therefore appears to be mediated through an autoimmune mechanism.


Subject(s)
Autoantibodies/blood , Autoantigens/immunology , Autoimmune Diseases/immunology , Fundus Oculi , Macula Lutea/immunology , Retinal Diseases/immunology , Adult , Aged , Animals , Autoimmune Diseases/physiopathology , Electroretinography , Female , Fluorescent Antibody Technique, Indirect , Humans , Macula Lutea/physiopathology , Male , Mice , Mice, Inbred BALB C , Microarray Analysis , Middle Aged , Ophthalmoscopy , Psychophysics/methods , Retinal Diseases/physiopathology , Visual Fields/physiology
12.
Exp Mol Pathol ; 83(2): 216-23, 2007 Oct.
Article in English | MEDLINE | ID: mdl-17560570

ABSTRACT

Dendritic cells (DCs) are a subset of antigen-presenting cells (APCs) that are involved in the initiation and control of the immune response to antigens present at the interface with the environment. A limited number of groups have studied DCs in human and animal conjunctiva but no data is available concerning the different DC subsets present in the conjunctival tissue. The aims of this study are to characterize the phenotypes and numbers of DCs present in the murine model of allergic conjunctivitis using the technique of immunohistochemistry so as to aid the understanding of the mechanisms involved in allergic eye disease. A double immunofluorescence method was used to analyze the phenotypic distribution and density of DC subsets in the mouse conjunctival tissues of the allergic model using a panel of antibodies: CD11c, as a general marker of DCs, coupled with another DC subset marker such as Langerin for Langerhans cells (LCs), CD11b for myeloid DCs (mDCs) and mPDCA-1 for plasmacytoid DCs (pDCs). In the naïve conjunctiva, mDCs were consistently detected in the subepithelial layer and substantia propria. In the epithelium and the subepithelial layer, very few LCs and virtually no pDCs were observed. Following allergen challenge, there was a marked influx of mDCs and pDCs, but no LCs, into the subepithelial layer and throughout the substantia propria. These results indicate that conjunctival DC subsets may play an important role in the immune-regulatory processes involved in the inflammatory component of allergic conjunctivitis.


Subject(s)
Conjunctiva/immunology , Conjunctivitis, Allergic/immunology , Dendritic Cells/immunology , Animals , Antigen-Presenting Cells/immunology , Antigen-Presenting Cells/pathology , Conjunctiva/pathology , Conjunctivitis, Allergic/pathology , Dendritic Cells/pathology , Disease Models, Animal , Eosinophils/immunology , Eosinophils/pathology , Female , Immunoenzyme Techniques , Mice , Mice, Inbred A , Microscopy, Fluorescence
13.
Immunol Rev ; 217: 96-104, 2007 Jun.
Article in English | MEDLINE | ID: mdl-17498054

ABSTRACT

Chemokines have a clearly defined role in mobilizing the recruitment of leukocytes to both healthy and inflamed tissues. This review details work from our and other laboratories, indicating that beta-chemokines may play important roles (i) in driving the terminal differentiation of mast cell precursors in mucosal tissues and (ii) in providing priming or costimulatory signals required for mast cell activation, leading to an antigen-driven inflammatory response. These data stem from in vivo, ex vivo, and in vitro studies. Data are also presented that suggest that Fc epsilon RI:chemokine receptor cross talk may involve spatiotemporal dynamics that may control the strength and nature of the complex activating signals controlling mast cell effector function.


Subject(s)
Chemokines, CC/metabolism , Conjunctiva/immunology , Hypersensitivity/immunology , Mast Cells/immunology , Receptors, IgE/immunology , Animals , Cell Membrane/chemistry , Chemokines, CC/analysis , Chemokines, CC/genetics , Humans , Mast Cells/ultrastructure
14.
Exp Mol Pathol ; 82(1): 68-76, 2007 Feb.
Article in English | MEDLINE | ID: mdl-16650406

ABSTRACT

Most cells with an intact interferon-gamma receptor and signaling pathway are able to express MHC class II molecules when treated with cytokines such as interferon-gamma and tumor necrosis factor-a. Interestingly, primary uveal melanocytes and most ocular melanoma cells are resistant to interferon-gamma mediated induction of class II MHC genes. This unusual phenotype is hypothesized to be germane to the immune-privileged status to the eye. Via a series of experiments, we have probed the molecular basis of this class II MHC resistant phenotype. We have analyzed the methylation status of the gene encoding the class II transactivator (CIITA), and asked whether treatment of class II MHC resistant ocular melanoma cells with the demethylating agent 5'-azacytidine can restore interferon-gamma inducibility of these class II MHC genes in these cells. The data obtained suggest that the specific blockade in cytokine-induced class II MHC gene expression is due to a suppression of the gene encoding the class II transactivator (CIITA). Treatment with 5' azacytidine restores the ability of these cells to express class II MHC genes upon interferon-gamma treatment. Whilst this is reminiscent of what occurs in another immune-privileged tissue--the placental trophoblast--we show here that silencing of the CIITA gene in uveal melanocytes either involves methylation of distinct nucleotides from those detected in trophoblasts, or involves an upstream activator of CIITA gene expression.


Subject(s)
DNA Methylation , Gene Expression Regulation/physiology , Genes, MHC Class II , Melanoma/genetics , Nuclear Proteins/genetics , Trans-Activators/genetics , Uveal Neoplasms/genetics , Azacitidine/pharmacology , Base Sequence , Blotting, Southern , Cell Line, Tumor , DNA Methylation/drug effects , Eye/immunology , Eye/metabolism , Gene Expression/drug effects , Gene Expression/physiology , Gene Expression Profiling , Gene Expression Regulation/drug effects , HeLa Cells , Humans , Interferon-gamma/metabolism , Molecular Sequence Data , Nuclear Proteins/metabolism , Promoter Regions, Genetic , Reverse Transcriptase Polymerase Chain Reaction , Trans-Activators/metabolism
15.
Expert Rev Clin Immunol ; 3(3): 351-64, 2007 May.
Article in English | MEDLINE | ID: mdl-20477679

ABSTRACT

Marked inflammatory infiltration by activated leukocytes is a characteristic feature of allergic diseases. Elucidation of the mechanisms of leukocyte trafficking in allergic diseases would identify targets to establish novel anti-inflammatory strategies for treatment of these diseases. Leukocyte trafficking is controlled by tissue-specific expression of chemokines and chemokine receptor expression on the leukocyte surface. Here, we review the role of chemokines and their receptors in leukocyte trafficking to inflammatory sites in allergic diseases and discuss therapeutic strategies targeting chemokine networks for treatment of these diseases.

16.
Immunology ; 115(3): 422-30, 2005 Jul.
Article in English | MEDLINE | ID: mdl-15946260

ABSTRACT

Age-related macular maculopathy (ARM) and age-related macular degeneration (AMD) are the leading causes of blindness in the Western world. Despite the magnitude of this clinical problem, very little is known about the pathogenesis of the disease. In this study, we analysed the sera (using indirect immunohistochemistry and Western blot analysis) from a very large cohort of such patients and normal age-matched controls to detect circulating anti-retinal antibodies. Patients with bilateral drusen (n = 64) and with chorioretinal neovascularization (CNV) (n = 51) were recruited in addition to age-matched control subjects (n = 39). The sera were analysed for anti-retinal immunoglobulins on retinal sections. The data were then correlated with the clinical features graded according to the International Classification and Grading System of ARM and AMD. The sera of patients with drusen (93.75%) and CNV (82.27%) were found to have a significantly (P = 0.02) higher titre of autoantibodies to the retina in comparison with controls (8.69%), indicating significant evidence of involvement of the immune process in early stages of AMD. Subsequent statistical analysis of the drusen group showed significant progressive staining (P = 0.0009) in the nuclei layers from early to late stages of ARM. Western blotting confirmed the presence of anti-retinal immunoglobulins to retinal antigens. As anti-retinal immunoglobulins are present in patients with bilateral drusen and exudative AMD, these antibodies could play a significant role in the pathogenesis of AMD. Whilst we do not have evidence that these antibodies precede disease onset, the possibility that their presence might contribute to disease progression needs to be investigated. Finally, the eventual identification of the target antigens detected by these antibodies may permit the future development of new diagnostic methods for ARM and AMD.


Subject(s)
Autoantibodies/immunology , Macular Degeneration/immunology , Retina/immunology , Aged , Antigens/immunology , Autoantibodies/blood , Biomarkers/blood , Cells, Cultured , Cohort Studies , Female , HeLa Cells , Humans , Immunoglobulins/blood , Immunoglobulins/immunology , Immunohistochemistry/methods , Jurkat Cells , Macular Degeneration/blood , Male , Microscopy, Confocal/methods , Middle Aged , Pigment Epithelium of Eye/immunology , Retinal Drusen/immunology , Retinal Neovascularization/immunology
17.
Int Immunol ; 17(3): 297-306, 2005 Mar.
Article in English | MEDLINE | ID: mdl-15710911

ABSTRACT

We report for the first time that IFNG gene expression requires high mobility group (HMG)A1, the architectural transcription factor mediating enhanceosome formation. This finding is supported by our direct studies of T cells isolated from the HMGA1-transgenic mice displaying an up-regulation of IFN-gamma production and of HMGA1-deficient mice exhibited a decreased IFN-gamma induction. In parallel transfection studies in EL4 cells, we observed elevated IFNG gene promoter activity in cells stably over-expressing HMGA1 and a reduction of such activity in cells expressing dominant-negative HMGA1. In vitro binding assays further demonstrated a specific interaction of HMGA1 to defined regions of the IFNG gene proximal promoter.


Subject(s)
Gene Expression Regulation , HMGA Proteins/physiology , Interferon-gamma/genetics , Transcription Factors/physiology , Animals , Base Sequence , HMGA Proteins/genetics , Interferon-gamma/metabolism , Lymphocyte Activation , Mice , Molecular Sequence Data , Promoter Regions, Genetic , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , T-Lymphocytes/immunology , Transcription Factors/genetics , Transcription, Genetic
18.
J Allergy Clin Immunol ; 115(1): 118-22, 2005 Jan.
Article in English | MEDLINE | ID: mdl-15637556

ABSTRACT

Allergic conjunctivitis is in actuality a group of diseases affecting the ocular surface and is usually associated with type 1 hypersensitivity reactions. Two acute disorders, seasonal allergic conjunctivitis and perennial allergic conjunctivitis, exist, as do 3 chronic diseases, vernal keratoconjunctivitis, atopic keratoconjunctivitis, and giant papillary conjunctivitis. The ocular surface inflammation (usually mast cell driven) results in itching, tearing, lid and conjunctival edema-redness, and photophobia during the acute phase and can lead to a classic late-phase response (with associated eosinophilia and neutrophilia) in a subset of individuals. As is the case in other allergic diseases, a chronic disease can also develop, accompanied by remodeling of the ocular surface tissues. In severe cases the patient experiences extreme discomfort and sustains damage to the ocular surface. For such cases, there is no highly effective and safe treatment regimen. Topical administration of corticosteroids is used in severe cases but is associated with an increased risk for the development of cataracts and glaucoma. Thus there is a worldwide search for new biotargets for the treatment of these diseases. Here we provide a brief update of the clinical symptoms associated with these diseases, the rationale for disease classification, recent advances in our understanding of the pathogenesis of the diseases, and an update on both preclinical and clinical advances toward refined therapies for these diseases.


Subject(s)
Conjunctivitis, Allergic/drug therapy , Conjunctivitis, Allergic/physiopathology , Hydroxycorticosteroids/therapeutic use , Administration, Topical , Animals , Conjunctiva/pathology , Conjunctivitis, Allergic/etiology , Edema/pathology , Eyelids/pathology , Humans , Photophobia/pathology , Pruritus/pathology , Seasons , Tears/metabolism
19.
J Clin Invest ; 115(2): 434-42, 2005 Feb.
Article in English | MEDLINE | ID: mdl-15650768

ABSTRACT

Regulation of the immune response requires the cooperation of multiple signals in the activation of effector cells. For example, T cells require signals emanating from both the TCR for antigen (upon recognition of MHC/antigenic peptide) and receptors for costimulatory molecules (e.g., CD80 and CD60) for full activation. Here we show that IgE-mediated reactions in the conjunctiva also require multiple signals. Immediate hypersensitivity reactions in the conjunctiva were inhibited in mice deficient in macrophage inflammatory protein-1alpha (MIP-1alpha) despite normal numbers of tissue mast cells and no decrease in the levels of allergen-specific IgE. Treatment of sensitized animals with neutralizing antibodies with specificity for MIP-1alpha also inhibited hypersensitivity in the conjunctiva. In both cases (MIP-1alpha deficiency and antibody treatment), the degranulation of mast cells in situ was affected. In vitro sensitization assays showed that MIP-1alpha is indeed required for optimal mast cell degranulation, along with cross-linking of the high-affinity IgE receptor, FcepsilonRI. The data indicate that MIP-1alpha constitutes an important second signal for mast cell degranulation in the conjunctiva in vivo and consequently for acute-phase disease. Antagonizing the interaction of MIP-1alpha with its receptor CC chemokine receptor 1 (CCR1) or signal transduction from CCR1 may therefore prove to be effective as an antiinflammatory therapy on the ocular surface.


Subject(s)
Cell Degranulation/physiology , Conjunctivitis, Allergic/immunology , Macrophage Inflammatory Proteins/immunology , Mast Cells/physiology , Signal Transduction/immunology , Animals , Antibodies, Monoclonal/administration & dosage , Antibodies, Monoclonal/immunology , B7-1 Antigen/immunology , Cell Degranulation/drug effects , Cell Degranulation/genetics , Chemokine CCL3 , Chemokine CCL4 , Conjunctiva/immunology , Conjunctiva/pathology , Conjunctivitis, Allergic/chemically induced , Conjunctivitis, Allergic/drug therapy , Conjunctivitis, Allergic/genetics , Conjunctivitis, Allergic/pathology , Histocompatibility Antigens/immunology , Immunoglobulin E/immunology , Lymphocyte Activation/genetics , Lymphocyte Activation/immunology , Macrophage Inflammatory Proteins/genetics , Mice , Mice, Inbred BALB C , Mice, Knockout , Peptides/administration & dosage , Peptides/immunology , Receptors, Antigen, T-Cell/immunology , Receptors, CCR1 , Receptors, Chemokine/immunology , Receptors, IgE/immunology , Signal Transduction/drug effects , Signal Transduction/genetics , T-Lymphocytes/immunology , T-Lymphocytes/pathology
20.
Novartis Found Symp ; 271: 131-40; discussion 140-51, 2005.
Article in English | MEDLINE | ID: mdl-16605132

ABSTRACT

Apart from the FcepsilonRI-mediated mechanism, mast cells are activated by chemokines. Evidence has accumulated indicating that there is cross-talk between the FcepsilonRI-mediated signalling pathway and CC chemokine receptor (CCR)-mediated signalling pathways in mast cells. We have found that costimulation with IgE/antigen and CC chemokine ligand 3 (CCL3) enhances degranulation but inhibits chemotaxis of rat basophilic leukaemia (RBL)-2H3 cells expressing human CCR1 (RBL-CCR1 cells). We hypothesize that this signalling cross-talk in mast cells may play important roles in the orchestration and focusing of the allergic response. In this study, we have sought information about global protein networks either enhanced or inhibited following cross-talk between the FcepsilonRI-mediated and CCR-mediated signalling pathways in mast cells. We believe this information may be useful for providing an understanding of mast cell function and in the establishment of new anti-inflammatory drugs for allergic diseases. Proteomics is a promising tool for studying protein profiles within biological samples and facilitates an understanding of the complex responses of an organism to a stimulus. Here, we show comparative data of protein profiles derived from FcepsilonRI-engaged and/or CCR1-engaged RBL-CCR1 cells using protein chip array technology, a proteomic technology. We also discuss our view of the role of CC chemokines and CCRs in regulating multiple aspects of mast cell biology.


Subject(s)
Chemokines, CC/immunology , Mast Cells/immunology , Receptors, Chemokine/immunology , Receptors, IgE/immunology , Animals , Antigens/immunology , Cell Movement , Humans , Immunoglobulin E/immunology , Mast Cells/cytology , Mice , Protein Array Analysis , Rats , Receptors, CCR1 , Receptors, CCR3 , Signal Transduction/physiology , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
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