Berent J. Prakken

Simultaneous Detection of 15 Human Cytokines in a Single Sample of Stimulated Peripheral Blood Mononuclear Cells

ABSTRACT Cytokines secreted by cells of the immune system can alter the behavior and properties of immune or other cells. At a site of inflammation, sets of cytokines interact with immune cells, and their combined effect is often more important than the function of one isolated component. Conventional techniques, such as enzyme-linked immunosorbent assays, generally require large quantities of cells to characterize a complete cytokine profile of activated lymphocytes. The Bio-Plex system from Bio-Rad Laboratories combines the principle of a sandwich immunoassay with the Luminex fluorescent-bead-based technology. We developed a multiplex cytokine assay to detect different cytokines simultaneously in culture supernatant of human peripheral blood mononuclear cells stimulated with antigen and with mitogen. Fifteen human cytokines (interleukin 1α [IL-1α], IL-1β, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12p70, IL-13, IL-15, IL-17, IL-18, gamma interferon, and tumor necrosis factor alpha) were validated with a panel of healthy individuals, rheumatoid arthritis patients, and juvenile idiopathic arthritis patients. Comparing the multiplex assay with a regular enzyme-linked immunosorbent assay technique with this donor panel resulted in correlation coefficients for all cytokines ranging from 0.75 to 0.99. Intra-assay variance proved to be less then 10%, whereas interassay variability ranged between 10 and 22%. This multiplex system proved to be a powerful tool in the quantitation of cytokines. It will provide a more complete picture in differences between activated lymphocyte cytokine profiles from healthy individuals and those from patients with chronic inflammatory diseases.

Heat-shock proteins induce T-cell regulation of chronic inflammation.

Immune responses to certain heat-shock proteins (HSPs) develop in almost all inflammatory diseases; however, the significance of such responses is only now becoming clear. In experimental disease models, HSPs can prevent or arrest inflammatory damage, and in initial clinical trials in patients with chronic inflammatory disease, HSP-derived peptides have been shown to promote the production of anti-inflammatory cytokines, indicating that HSPs have immunoregulatory potential. In this Review, we discuss the unique characteristics of HSPs that endow them with these immunoregulatory qualities.

CD4+CD25bright regulatory T cells actively regulate inflammation in the joints of patients with the remitting form of juvenile idiopathic arthritis.

This study investigates the role of CD4+CD25+ regulatory T cells during the clinical course of juvenile idiopathic arthritis (JIA). Persistent oligoarticular JIA (pers-OA JIA) is a subtype of JIA with a relatively benign, self-remitting course while extended oligoarticular JIA (ext-OA JIA) is a subtype with a much less favorable prognosis. Our data show that patients with pers-OA JIA display a significantly higher frequency of CD4+CD25bright T cells with concomitant higher levels of mRNA FoxP3 in the peripheral blood than ext-OA JIA patients. Furthermore, while numbers of synovial fluid (SF) CD4+CD25bright T cells were equal in both patient groups, pers-OA JIA patients displayed a higher frequency of CD4+CD25int T cells and therefore of CD4+CD25total in the SF than ext-OA JIA patients. Analysis of FoxP3 mRNA levels revealed a high expression in SF CD4+CD25bright T cells of both patient groups and also significant expression of FoxP3 mRNA in the CD4+CD25int T cell population. The CD4+CD25bright cells of both patient groups and the CD4+CD25int cells of pers-OA JIA patients were able to suppress responses of CD25neg cells in vitro. A markedly higher expression of CTLA-4, glucocorticoid-induced TNFR, and HLA-DR on SF CD4+CD25bright T regulatory (Treg) cells compared with their peripheral counterparts suggests that the CD4+CD25+ Treg cells may undergo maturation in the joint. In correlation with this mature phenotype, the SF CD4+CD25bright T cells showed an increased regulatory capacity in vitro compared with peripheral blood CD4+CD25bright T cells. These data suggest that CD4+CD25bright Treg cells play a role in determining the patient’s fate toward either a favorable or unfavorable clinical course of disease.

Regulation of Treg functionality by acetylation-mediated Foxp3 protein stabilization

Regulatory T cells (Tregs) are a specific subset of lymphocytes that are critical for the maintenance of self-tolerance. Expression levels of the transcription factor Foxp3 have been causally associated with Treg differentiation and function. Recent studies show that Foxp3 can also be transiently expressed in effector T cells; however, stable Foxp3 expression is required for development of a functional Treg suppressor phenotype. Here, we demonstrate that Foxp3 is acetylated, and this can be reciprocally regulated by the histone acetyltransferase p300 and the histone deacetylase SIRT1. Hyperacetylation of Foxp3 prevented polyubiquitination and proteasomal degradation, therefore dramatically increasing stable Foxp3 protein levels. Moreover, using mouse splenocytes, human peripheral blood mononuclear cells, T cell clones, and skin-derived T cells, we demonstrate that treatment with histone deacetylase inhibitors resulted in significantly increased numbers of functional Treg cells. Taken together, our data demonstrate that modulation of the acetylation state of Foxp3 provides a novel molecular mechanism for assuring rapid temporal control of Foxp3 levels in T cells, thereby regulating Treg numbers and functionality. Manipulating Foxp3 acetylation levels could therefore provide a new therapeutic strategy to control inappropriate (auto)immune responses.

Blood and synovial fluid cytokine signatures in patients with juvenile idiopathic arthritis: a cross-sectional study

Background: Juvenile idiopathic arthritis (JIA) consists of a heterogeneous group of disorders with, for the most part, an unknown immunopathogenesis. Although onset and disease course differ, the subtypes of JIA share the occurrence of chronic inflammation of the joints, with infiltrations of immunocompetent cells that secrete inflammatory mediators. Objective: To identify a panel of cytokines specifically related to the inflammatory process in JIA. Methods: Using a new technology, the multiplex immunoassay, 30 cytokines were measured in plasma of 65 patients with JIA, of which 34 were paired with synovial fluid. These data were compared with plasma of 20 healthy controls and 9 patients with type I diabetes, a chronic inflammatory disease. Results: Patients with JIA had, irrespective of their subclassification, significantly higher levels of tumour necrosis factor α, macrophage inhibitory factor (MIF), CCL2, CCL3, CCL11, CCL22 and CXCL9 in plasma than controls. In paired plasma and synovial fluid samples of patients with JIA, significantly higher levels of interleukin (IL)6, IL15, CCL2, CCL3, CXCL8, CXCL9 and CXCL10 were present in synovial fluid. Cluster analysis in all patients with JIA revealed a predominant pro-inflammatory cytokine cluster during active disease and a regulatory/anti-inflammatory-related cytokine cluster during remission. Whether a discrimination profile of various cytokines could help in the determination of disease classification was tested. Conclusion: It is suggested that several cytokines (IL18, MIF, CCL2, CCL3, CCL11, CXCL9 and CXCL10) may correspond to the activation status during inflammation in JIA and could be instrumental in monitoring disease activity and outcomes of (new) immunotherapies.

Prerequisites for cytokine measurements in clinical trials with multiplex immunoassays

BackgroundGrowing knowledge about cellular interactions in the immune system, including the central role of cytokine networks, has lead to new treatments using monoclonal antibodies that block specific components of the immune system. Systemic cytokine concentrations can serve as surrogate outcome parameters of these interventions to study inflammatory pathways operative in patients in vivo. This is now possible due to novel technologies such as multiplex immunoassays (MIA) that allows detection of multiple cytokines in a single sample. However, apparently trivial underappreciated processes, (sample handling and storage, interference of endogenous plasma proteins) can greatly impact the reliability and reproducibility of cytokine detection.Therefore we set out to investigate several processes that might impact cytokine profiles such as blood collecting tubes, duration of storage, and number of freeze thawing cycles.ResultsSince under physiological conditions cytokine concentrations normally are low or undetectable we spiked cytokines in the various plasma and serum samples. Overall recoveries ranged between 80-120%. Long time storage showed cytokines are stable for a period up to 2 years of storage at -80°C. After 4 years several cytokines (IL-1α, IL-1β, IL-10, IL-15 and CXCL8) degraded up to 75% or less of baseline values. Furthermore we show that only 2 out of 15 cytokines remained stable after several freeze-thawing cycles. We also demonstrate implementation of an internal control for multiplex cytokine immunoassays.ConclusionAll together we show parameters which are essential for measurement of cytokines in the context of clinical trials.

Epitope-specific immunotherapy induces immune deviation of proinflammatory T cells in rheumatoid arthritis

Modulation of epitope-specific immune responses would represent a major addition to available therapeutic options for many autoimmune diseases. The objective of this work was to induce immune deviation by mucosal peptide-specific immunotherapy in rheumatoid arthritis (RA) patients, and to dissect the related immunological mechanisms by using a technology for the detection of low-affinity class II-restricted peptide-specific T cells. A group of patients with early RA was treated for 6 months orally with dnaJP1, a peptide that induces proinflammatory T cell responses in naive RA patients. Immunological analysis at initial, intermediate and end treatment points showed an intriguing change from proinflammatory to regulatory T cell function. In fact, dnaJP1-induced T cell production of IL-4 and IL-10 increased significantly when initial and end treatment points were compared, whereas dnaJP1-induced T cell proliferation and production of IL-2, IFN-γ, and tumor necrosis factor-α decreased significantly. The total number of dnaJP1-specific cells did not change over time, whereas expression of foxP3 by CD4+CD25bright cells increased, suggesting that the treatment affected regulatory T cell function. Thus, rather than clonal deletion, the observed change in immune reactivity to dnaJP1 was the outcome of treatment-induced emergence of T cells with a different functional phenotype. This study contributes to our knowledge of mechanisms and tools needed for antigen-specific immune modulation in humans, thus laying the foundation for exploitation of this approach for therapeutic purposes.

A Conserved Mycobacterial Heat Shock Protein (hsp) 70 Sequence Prevents Adjuvant Arthritis upon Nasal Administration and Induces IL-10-Producing T Cells That Cross-React with the Mammalian Self-hsp70 Homologue

Immunization with Mycobacterium tuberculosis heat shock protein (hsp) 60 has been shown to protect rats from experimental arthritis. Previously, the protection-inducing capacity was shown to reside in the evolutionary conserved parts of the molecule. Now we have studied the nature of the arthritis suppressive capacity of a distinct, antigenically unrelated protein, M. tuberculosis hsp70. Again, a conserved mycobacterial hsp70 sequence was found to be immunogenic and to induce T cells that cross-reacted with the rat homologue sequence. However, in this case parenteral immunization with the peptide containing the critical cross-reactive T cell epitope did not suppress disease. Upon analysis of cytokines produced by these peptide-specific T cells, high IL-10 production was found, as was the case with T cells responsive to whole hsp70 protein. Nasal administration of this peptide was found to lead to inhibition of subsequent adjuvant arthritis induction. The data presented here shows the intrinsic capacity of conserved bacterial hsp to trigger self-hsp cross-reactive T cells with the potential to down-regulate arthritis via IL-10.

Tolerogenic immune responses to novel T-cell epitopes from heat-shock protein 60 in juvenile idiopathic arthritis

BACKGROUND Juvenile idiopathic arthritis is a heterogeneous autoimmune disease characterised by chronic inflammation of one or more joints. In patients with this disease, T-cell reactivity to autologous heat-shock protein 60 (HSP60) is associated with a favourable prognosis. We sought to identify HSP60 T-cell epitopes to find potential targets for HSP60 immunotherapy and to assess whether immune responses to these epitopes contribute to the distinct clinical outcome of this disease. METHODS We identified eight potential epitopes using a computer algorithm from both self and microbial HSP60 binding to many HLA-DR molecules. We analysed the pattern of T-cell responses induced by these HSP60 peptides in peripheral-blood mononuclear cells (PBMC) of 57 patients with juvenile idiopathic arthritis, 27 healthy controls, and 20 disease controls. We undertook in-vitro MHC binding studies with the identified peptides, and HLA class II typing of a subset of patients with juvenile idiopathic arthritis. FINDINGS Five of the eight peptides identified yielded proliferative T-cell responses in 50-70% of PBMC from patients with juvenile idiopathic arthritis irrespective of MHC genotype, but not in PBMC from healthy or disease controls. Although PBMC from both patients with juvenile idiopathic arthritis and healthy controls produced interferon gamma in response to these peptides, only PBMC from patients with the disease produced interleukin 10. INTERPRETATION The recorded T-cell-induction in juvenile idiopathic arthritis is tolerogenic. In patients with oligoarticular disease, the immune responses to the HSP60 epitopes identified could contribute to disease remission. RELEVANCE TO PRACTICE The broad recognition of these HSP60 epitopes in a population of patients with polymorphic MHC genotypes opens the way for HSP60-peptide immunotherapy, representing a novel treatment option to specifically modulate the immune system in patients with juvenile idiopathic arthritis.

Do heat shock proteins control the balance of T-cell regulation in inlammatory diseases?

Abstract Heat shock proteins (Hsps) are remarkably immunogenic, despite their high degree of evolutionary conservation. Experimental and clinical observations on autoimmune diseases indicate that immune responses to Hsps arise spontaneously during the disease process. Based on current evidence, Willem van Eden and colleagues argue that such immunity to Hsps is part of a normal immunoregulatory T-cell response with disease controlling potential.