Label-Free Proteomics Reveals Decreased Expression of CD18 and AKNA in Peripheral CD4 T Cells from Patients with Vogt-Koyanagi-Harada Syndrome

University of Miami, United States of America
PLoS ONE (Impact Factor: 3.23). 01/2011; 6(1):e14616. DOI: 10.1371/journal.pone.0014616
Source: PubMed


Vogt-Koyanagi-Harada (VKH) syndrome is a systemic autoimmune disease. CD4(+) T cells have been shown to be involved in autoimmune diseases including VKH syndrome. To screen aberrantly expressed membrane proteins in CD4(+) T cell from patients with active VKH syndrome, blood samples were taken from five patients with active VKH syndrome and five healthy individuals. A label-free quantitative proteomic strategy was used to identify the differently expressed proteins between the two groups. The results revealed that the expression of 102 peptides was significantly altered (p<0.05) between two groups and matched amino acid sequences of proteins deposited in the international protein index (ipi.HUMAN.v3.36.fasta). The identified peptides corresponded to 64 proteins, in which 30 showed more than a 1.5-fold difference between the two groups. The decreased expression of CD18 and AKNA transcription factor (AKNA), both being three-fold lower than controls in expression identified by the label-free method, was further confirmed in an additional group of five active VKH patients and six normal individuals using the Western blot technique. A significantly decreased expression of CD18 and AKNA suggests a role for both proteins in the pathogenesis of this syndrome.

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    • "Interferon (IFN)-γ was found elevated in the aqueous humor of VKH patients with uveitis [13]. A few differences between blood T cells from VKH patients and control donors have been reported: a decreased expression of CD18 and AKNA transcription factors in VKH patients [14], a higher expression of transcription factor T-bet [15], and less apoptosis of T cells from VKH patients after in vitro stimulation with phytohemagglutinin [16]. Upon ex vivo nonspecific stimulation, blood CD4 T lymphocytes of VKH patients secreted slightly more IFN-γ and interleukin (IL)-2 than did cells obtained from control individuals, whereas IL-4 secretion was similar in both groups [17]. "
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    ABSTRACT: Purpose Vogt-Koyanagi-Harada (VKH) syndrome is an autoimmune disease characterized by inaugural uveomeningitidis and hearing loss and at late stages a depigmentation in eyes and skin. Melanocytes are the cells common to the four affected tissues, namely eye, brain, inner ear, and skin. Melanocytes are therefore considered as the source of self-antigens. The melanocytic proteins tyrosinase-related protein-1 (TRP1), TRP2, tyrosinase, and gp100 have been proposed as the proteins targeted by autoreactive T cells from VKH patients bearing human leukocyte antigen (HLA)-DRB1*04:05, the HLA allele classically associated with VKH disease. The objective of this work was to determine the antigens recognized by a large number of potentially autoreactive CD4 T lymphocytes obtained from the cerebrospinal fluid of one VKH patient who did not express HLA-DRB1*04:05. Methods T cells were isolated from the cerebrospinal fluid of a newly diagnosed HLA-DRB1*14:01,*15:03;-DPB1*01:01,*04:02 patient in the acute phase of the VKH disease and cloned by limiting dilution. Each of the 107 T cell clones, of which 90% were CD4+, was tested for its ability to secrete cytokines upon contact with autologous antigen-presenting cells loaded with either of the melanocytic proteins TRP1, TRP2, tyrosinase, gp100, Melan-A and KU-MEL-1. The sensitivity of our recombinant bacteria-based approach was validated with a CD4 T cell clone with known antigen specificity. The ability of each of the 107 clones to secrete cytokines upon nonspecific stimulation was verified. Results None of the 107 T cell clones was able to secrete tumor necrosis factor-α, interferon-γ, interleukin (IL)-5, or IL-17 upon contact with autologous B cells loaded with any of the six common melanocytic proteins. Nine clones secreted high-level IL-17 upon stimulation with beads coated with antibodies. Conclusions The self-antigens that triggered the VKH disease in this patient probably derive from proteins other than the six melanocytic proteins mentioned above. Further study of antigens that are recognized by potential autoreactive T cells from VKH patients is likely to benefit from testing a broader set of melanocytic proteins.
    Full-text · Article · Jul 2014 · Molecular vision
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    • "Identification of globally dysregulated proteins in systemic and local inflammatory diseases became possible recently with a significant advance of technologies such as mass spectrometry, multi-parameter flow cytometry, and protein arrays [149]. Both ocular fluids and serum proteins from patients with Behcet's disease [150, 151] and Vogt–Koyanagi–Harada disease [152] have been surveyed in multiple studies using mass spectrometry-based technologies. However, it is unclear how these proteins are related to intraocular inflammation and disease pathology. "
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    ABSTRACT: The eye, as currently viewed, is neither immunologically ignorant nor sequestered from the systemic environment. The eye utilises distinct immunoregulatory mechanisms to preserve tissue and cellular function in the face of immune-mediated insult; clinically, inflammation following such an insult is termed uveitis. The intra-ocular inflammation in uveitis may be clinically obvious as a result of infection (e.g. toxoplasma, herpes), but in the main infection, if any, remains covert. We now recognise that healthy tissues including the retina have regulatory mechanisms imparted by control of myeloid cells through receptors (e.g. CD200R) and soluble inhibitory factors (e.g. alpha-MSH), regulation of the blood retinal barrier, and active immune surveillance. Once homoeostasis has been disrupted and inflammation ensues, the mechanisms to regulate inflammation, including T cell apoptosis, generation of Treg cells, and myeloid cell suppression in situ, are less successful. Why inflammation becomes persistent remains unknown, but extrapolating from animal models, possibilities include differential trafficking of T cells from the retina, residency of CD8(+) T cells, and alterations of myeloid cell phenotype and function. Translating lessons learned from animal models to humans has been helped by system biology approaches and informatics, which suggest that diseased animals and people share similar changes in T cell phenotypes and monocyte function to date. Together the data infer a possible cryptic infectious drive in uveitis that unlocks and drives persistent autoimmune responses, or promotes further innate immune responses. Thus there may be many mechanisms in common with those observed in autoinflammatory disorders.
    Full-text · Article · May 2014 · Seminars in Immunopathology
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    • "This example points to the potential importance of profiling vitreous proteins to determine the best therapy, especially to avoid targeting proteins that are absent. Other potential biomarkers for uveitides have been identified [42] [43] [44]. "
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    ABSTRACT: The vitreous is an extracellular matrix that is still poorly understood. Although many constituents and characteristics have been previously determined, there are many attributes still being discovered. Currently, using protein arrays, mass spectrometry, and bioinformatics, the vitreous provides a wealth of knowledge regarding ocular diseases and potential targets for personalized therapeutics. This article is protected by copyright. All rights reserved.
    Full-text · Article · Apr 2014 · PROTEOMICS - CLINICAL APPLICATIONS
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