Aleida M. Bakker

An IL-13 promoter polymorphism associated with increased risk of allergic asthma

IL-13 plays a crucial role in the development of allergic asthma by several mechanisms, including induction of IgE antibodies, airway eosinophilia and hyper-reactivity. We previously established a deregulated production of IL-13 by T cells from allergic asthma patients. In this report we describe the identification of a novel IL-13 promoter polymorphism (C to T exchange) at position −1055. The IL-13 −1055 TT genotype is associated with allergic asthma (P = 0.002), altered regulation of IL-13 production (P < 0.002), and increased binding of nuclear proteins to this region. we postulate that the presence of this polymorphism predisposes to the development of allergic asthma.

Role of Tumor Necrosis Factor–α and Interleukin-10 Promoter Gene Polymorphisms in Leprosy

Single-nucleotide polymorphismswithin thegenescodingfortumornecrosisfactor(TNF)‐a and interleukin (IL)‐10 have been associated with several infectious diseases. To determine whether such polymorphisms are associated with leprosy, genotyping was performed at the 308 and 238 positions of the promoter of the TNF-a gene in 210 and 191 patients with multibacillary (MB) leprosy, respectively; 90 and 79 patients with paucibacillary (PB) leprosy; and 92 control subjects. For the 592 and 819 positions within the promoter of the IL-10 gene, 143 patients with MB leprosy, 79 patients with PB leprosy, and 62 control subjects were included in the analysis. TNF2 allele frequency was significantly higheramong controlsubjects than among all patients with leprosy or in the MB group ( and ). For the ILP ! .05 P ! .01 10 gene, the frequency of the homozygous 819TT genotype was significantly higher among patients than among control subjects. These data indicate that a relationship exists between TNF-a and IL-10 promoter polymorphisms and the development of PB leprosy. The more benign paucibacillary (PB) forms of leprosy— borderline tuberculoid (BT) and tuberculoid tuberculoid (TT) leprosy—are characterized by the predominance of a Th1-type immune response, the presence of well-formed granulomas at the site of the lesion, and control of mycobacterial replication. In contrast, in the multibacillary (MB) forms—borderline borderline (BB) and borderline lepromatous (BL) leprosy and lepromatous leprosy (LL)—no granuloma is seen, and high bacterial load and antibody levels are detected. Cytokines evidently play a critical role in triggering host-pathogen interactions. On one hand, greater tumor necrosis factor (TNF)–a production,

Immunomodulatory Dendritic Cells Inhibit Th1 Responses and Arthritis via Different Mechanisms

Dendritic cells (DCs) are professional APCs which have the unique ability to present both foreign and self-Ags to T cells and steer the outcome of immune responses. Because of these characteristics, DCs are attractive vehicles for the delivery of therapeutic vaccines. Fully matured DCs are relatively well-defined and even used in clinical trials in cancer. DCs also have the potential to influence the outcome of autoimmunity by modulating the underlying autoimmune response. To gain a better appreciation of the abilities and mechanisms by which immunomodulatory DCs influence the outcome of T cell responses, we studied several immunomodulatory DCs (TNF-, IL-10-, or dexamethasone-stimulated bone marrow-derived DCs) side by side for their ability to modulate T cell responses and autoimmune diseases. Our data show that these differentially modulated DCs display a different composition of molecules involved in T cell activation. Although, all DC subsets analyzed were able to inhibit the induction of collagen-induced arthritis, the modulation of the underlying immune response was different. Vaccination with TNF- or IL-10-modulated DCs altered the Th1/Th2 balance as evidenced by the induction of IL-5- and IL-10-secreting T cells and the concomitant reduction of the IgG2a-IgG1 ratio against the immunizing Ag. In contrast, DCs modulated with dexamethasone did not affect the ratio of IL-5-producing vs IFN-γ-producing T cells and tended to affect the Ab response in a nonspecific manner. These data indicate that distinct mechanisms can be used by distinct DC subsets to change the outcome of autoimmunity.

Chronic obstructive pulmonary disease is associated with the -1055 IL-13 promoter polymorphism

IL-13 is strongly implicated in the development of asthma and chronic obstructive polymonary disease (COPD). We previously identified an IL-13 promoter polymorphism (−1055 C to T) that is associated with allergic asthma. We now report an increased frequency of the −1055 T allele in COPD patients compared to healthy controls (P=0.002) and compared to a second control group consisting of smoking individuals with normal lung function (P=0.01). A closely linked IL-13 exon polymorphism is present at normal allelic frequencies (P=0.3 and 0.4, respectively). In addition, we observed a normal distribution of two IL-4 polymorphisms at positions −590 and +33 (P=0.2 and 0.9, respectively). These results could implicate a functional role for the IL-13 promoter polymorphism in the enhanced risk to develop COPD.

Activation of human basophils by combined toll-like receptor- and FcεRI-triggering can promote Th2 skewing of naive T helper cells.

Basophils are mostly known for their involvement in allergic reactions. Recent studies in mice indicate a role for basophils in the induction of adaptive immunity, especially T helper 2 (Th2) responses. Therefore, it would be highly important to understand how basophils respond to pathogen‐associated molecules, such as ligands for toll‐like receptors (TLRs), and if the basophils could promote Th2 responses via these stimuli. To this end, the activation of basophils via TLRs in combination with activation via IgE was studied, as well as its effect on T helper cell skewing. Using quantitative PCR, we demonstrated the presence of mRNA for TLRs 1–8 in human basophils. Basophils responded to TLR triggering with differential cytokine production, but not with degranulation. Simultaneous triggering of TLRs and IgE led to synergy in production of IL‐4, IL‐8, IL‐13, and RANTES. Furthermore, the synergistic effects on basophils mediated by IgE and TLR‐4 triggering allowed robust Th2 skewing upon activation of naïve human CD4+ T cells.

Toll-like receptor triggering augments activation of human mast cells by anti-citrullinated protein antibodies

Objective Mast cells may play a role in rheumatoid arthritis (RA), but activation of human mast cells in autoimmune settings has been little studied. Toll-like receptors (TLR) and Fcγ receptors (FcγR) are important receptors for cellular activation in the joint, but expression and stimulation of these receptors in human mast cells or the functional interplay between these pathways is poorly understood. Here, we analysed triggering of human mast cells via these receptors in the context of anti-citrullinated protein antibody-positive (ACPA+) RA. Methods RNA and protein expression of TLRs and FcγR was quantified using PCR and flow cytometry, respectively. Mast cells were stimulated with TLR ligands (including HSP70) combined with IgG immune complexes and IgG-ACPA. Results Human mast cells expressed TLRs and produced cytokines in response to TLR ligands. Both cultured and synovial mast cells expressed FcγRIIA, and triggering of this receptor by IgG immune complexes synergised with activation by TLR ligands, leading to two- to fivefold increased cytokine levels. Mast cells produced cytokines in response to ACPA immune complexes in a citrulline-specific manner, which synergised in the presence of HSP70. Conclusions Our data show that synovial mast cells express FcγRIIA and that mast cells can be activated by IgG-ACPA and TLR ligands. Importantly, combined stimulation via TLRs and immune complexes leads to synergy in cytokine production. These findings suggest mast cells are important targets for TLR ligands and immune complexes, and that combined activation of mast cells via these pathways greatly enhances inflammation in synovial tissue of RA patients.

Genetic Variation of the Fc Gamma Receptor 3B Gene and Association with Rheumatoid Arthritis

Background Fc gamma receptors (FcγRs) play a crucial role in immunity by linking IgG antibody-mediated responses with cellular effector and regulatory functions. Genetic variants in these receptors have been previously identified as risk factors for several chronic inflammatory conditions. The present study aimed to investigate the presence of copy number variations (CNVs) in the FCGR3B gene and its potential association with the autoimmune disease rheumatoid arthritis (RA). Methodology/Principal Findings CNV of the FCGR3B gene was studied using Multiplex Ligation Dependent Probe Amplification (MLPA) in 518 Dutch RA patients and 304 healthy controls. Surprisingly, three independent MLPA probes targeting the FCGR3B promoter measured different CNV frequencies, with probe#1 and #2 measuring 0 to 5 gene copies and probe#3 showing little evidence of CNV. Quantitative-PCR correlated with the copy number results from MLPA probe#2, which detected low copy number (1 copy) in 6.7% and high copy number (≥3 copies) in 9.4% of the control population. No significant difference was observed between RA patients and the healthy controls, neither in the low copy nor the high copy number groups (p-values = 0.36 and 0.71, respectively). Sequencing of the FCGR3B promoter region revealed an insertion/deletion (indel) that explained the disparate CNV results of MLPA probe#1. Finally, a non-significant trend was found between the novel -256A>TG indel and RA (40.7% in healthy controls versus 35.9% in RA patients; P = 0.08). Conclusions/Significance The current study highlights the complexity and poor characterization of the FCGR3B gene sequence, indicating that the design and interpretation of genotyping assays based on specific probe sequences must be performed with caution. Nonetheless, we confirmed the presence of CNV and identified novel polymorphisms in the FCGR3B gene in the Dutch population. Although no association was found between RA and FCGR3B CNV, the possible protective effect of the -256A>TG indel polymorphism must be addressed in larger studies.

Allele-Specific Expression of the IL-1α Gene in Human CD4+ T Cell Clones

A number of reports have described the monoallelic expression of murine cytokine genes. Here we describe the monoallelic expression of the human IL-1α gene in CD4+ T cells. Analysis of peripheral blood T cell clones derived from healthy individuals revealed that the IL-1α gene shows predominantly monoallelic expression. Monoallelic expression was observed in Th0, Th1, and Th2 cell clones. In addition, we demonstrate monoallelic expression in T cell clones from rheumatoid arthritis patients derived from synovial fluid of the knee joint, suggesting that the occurrence of this phenomenon is not different from that in clones derived from healthy individuals. The finding of monoallelic expression of a cytokine gene in human CD4+ T cell clones provides evidence for allele-specific silencing/activation as another layer of regulation of IL-1α gene expression.

Analysis of allelic expression patterns of IL-2, IL-3, IL-4, and IL-13 in human CD4+ T cell clones

The occurrence of monoallelic expression of cytokine genes in single cells has been convincingly demonstrated, but there have been few reports of this phenomenon in T cell clones. Here we describe studies on the expression of alleles of the human genes encoding IL‐2, IL‐3, IL‐4, and IL‐13 in human CD4+ T cell clones. In contrast to the results reported in mouse T cell clones andsingle human T cells, we found no evidence for the monoallelic expression of the IL‐2, IL‐3, and IL‐13 genes. The gene for IL‐4 showed an imbalance in expression from each allele, indicating differential expression of IL‐4 alleles within or between IL‐4‐expressing cells.

Allele specific regulation of cytokine genes: Monoallelic expression of the IL-lA gene

The host immune response strongly controls the susceptibility and outcome of inflammatory and infectious diseases in humans. Cytokines play an important role in this response as crucial intercellular signaling molecules that are responsible for multidirectional communication among immune and inflammatory cells engaged in host defense. They exert their effects by binding to high-affinity receptors expressed on target cells thereby inducing biochemical signals within these cells which profoundly change their behaviour. It has become clear that cytokines not only play important roles in tissue homeostasis but also in the pathogenesis of many immune related disorders. A large body of evidence indicated that the cytokine profile, examplified by the balance between inflammatory and anti-inflammatory activities, is a determining factor in the progress of the immune response and the maintenance of tissue homeostasis and might provide an explanation for interindividual differences in the host immune response. A critical question to be answered is which molecular mechanism underlies the imbalance of pro-and anti-inflammatory activity in immune related disease. Clinically useful information should come from understanding the molecular regulatory steps underlying the imbalance in the cytokine profile that would allow the identification of people who display an imbalance in their cytokine profile enabling clinicians to predict outcomes and to tailor treatment.