IL-5 production by resident mucosal allergen-specific T cells in an explant model of allergic rhinitis.

BACKGROUND: Seasonal allergic rhinitis is a chronic inflammation in the nasal mucosa triggered by inhaled aeroallergens. The inflammatory reaction is controlled by allergen-specific T cells, but where and how these T cells become activated is not fully understood. OBJECTIVES: We wanted to determine whether allergen-specific T-helper (Th) 2 cells are residing in the nasal mucosa under steady-state conditions outside of the pollen season and, if so, whether these cells are activated locally in response to allergen challenge. METHODS: Mucosal biopsies from the lower turbinate were obtained out of season from patients with either birch- or grass-pollen-allergic rhinitis and from healthy controls. Cultured explant samples were challenged with relevant pollen extract or with a mix of overlapping 20-mer peptides derived from the sequence of the major birch allergen, Betula verrucosa (Bet v) 1. After 24 h, culture medium was harvested for multiplex cytokine and tryptase analysis. RESULTS: Significant amounts of interleukin (IL)-5 were secreted from resident cells in response to ex vivo allergen challenge in the allergic group only. No increase was observed for the other cytokines measured. Production of IL-5 in response to both extract and the Bet v1-derived peptide mix strongly suggested that T cells were a major source of IL-5. CONCLUSION: Our explant model indicated that local presentation of antigen to resident allergen-specific Th2 cells is the early event in the pathogenesis of allergic rhinitis. These findings identify possible cellular targets for anti-inflammatory treatment.

Experimentally induced accumulation of Foxp3⁺ T cells in upper airway allergy.

BACKGROUND: It has been suggested that Foxp3(+) regulatory T (Treg) cells inhibit allergic inflammation in humans by suppressing the activation of allergen-specific effector T cells. Whether this occurs at the site of allergen exposure has not been determined. OBJECTIVE: To determine the occurrence of Foxp3(+) Treg cells in the nasal mucosa of allergic rhinitis (AR) patients and non-allergic controls after a nasal allergen challenge. METHODS: Pollen-allergic patients (n=18) and non-allergic volunteers (n=7) were challenged locally with pollen extract or placebo for 7 days outside the pollen season. Mucosal biopsies were obtained from the inferior turbinate on days 0, 1 and 7 and subjected to multi-colour immunofluorescence and blood was drawn for eosinophil counts on days 0, 2, 5 and 7. RESULTS: Only AR patients receiving pollen extract experienced typical allergic symptoms and demonstrated increased levels of eosinophils in peripheral blood and nasal mucosa. In allergic patients, a transient early increase (day 1) in CD3(+) T cells was observed in the nasal mucosa, followed by a significant increase of Foxp3(high) T cells at day 7. No changes were found in the control group. The majority of Foxp3(high) cells co-expressed CTLA-4, CD25 and CD4, and a substantial fraction expressed the proliferation marker Ki67. CONCLUSION AND CLINICAL RELEVANCE: Experimentally induced inflammation in AR patients leads to an early inflammatory response followed by accumulation of Foxp3(high) T cells in the nasal mucosa. Our findings are similar to that observed in allergic airways of experimental mice, which suggest that Treg cells are operative in allergic upper airway inflammation. It should be explored whether Treg cells accumulating in the nasal mucosa could be targets for therapeutic intervention.

Sun exposure induces rapid immunological changes in skin and peripheral blood in patients with psoriasis.

BACKGROUND: Ultraviolet (UV) radiation has immunosuppressive effects and heliotherapy is a well-described treatment modality for psoriasis. OBJECTIVES: To characterize early sun-induced immunological changes both local and systemic in patients with psoriasis. METHODS: Twenty patients with moderate to severe psoriasis were subjected to controlled sun exposure on Gran Canaria, Canary Islands, Spain. Psoriasis Area and Severity Index (PASI) scores were evaluated. Skin biopsies were obtained from lesional and nonlesional skin in 10 patients at baseline and on day 16 and from five additional patients on day 2. Specimens were examined with immunohistochemistry and polymerase chain reaction. Blood samples were obtained from all patients at the same time points and were examined for T-cell subsets and cytokine production. RESULTS: Significant clinical improvement was achieved during the study period. CD4+ and CD8+ T cells in lesional skin were significantly reduced in both the epidermis and dermis. In contrast, dermal FOXP3+ T cells were relatively increased. In the peripheral blood skin homing cutaneous lymphocyte-associated antigen (CLA)+ T cells were significantly decreased after only 1 day in the sun and in vitro stimulated peripheral blood mononuclear cells demonstrated reduced capacity to secrete cytokines after 16 days. CONCLUSIONS: Our data show that clinical improvement of psoriasis following sun exposure is preceded by a rapid reduction in local and systemic inflammatory markers, strongly suggesting that immune modulation mediated the observed clinical effect. We cannot completely rule out that other mechanisms, such as stress reduction, may contribute, but it is extensively documented that UV irradiation is a potent inducer of immunosuppression and we therefore conclude that the observed effect was primarily due to sun exposure.

Absence of epithelial immunoglobulin A transport, with increased mucosal leakiness, in polymeric immunoglobulin receptor/secretory component-deficient mice.

Mucosal surfaces are protected specifically by secretory immunoglobulin A (SIgA) and SIgM generated through external translocation of locally produced dimeric IgA and pentameric IgM. Their active transport is mediated by the epithelial polymeric Ig receptor (pIgR), also called the transmembrane secretory component. Paracellular passive external transfer of systemic and locally produced antibodies also provides mucosal protection, making the biological importance of secretory immunity difficult to assess. Here we report complete lack of active external IgA and IgM translocation in pIgR knockout mice, indicating no redundancy in epithelial transport mechanisms. The knockout mice were of normal size and fertility but had increased serum IgG levels, including antibodies to Escherichia coli, suggesting undue triggering of systemic immunity. Deterioration of their epithelial barrier function in the absence of SIgA (and SIgM) was further attested to by elevated levels of albumin in their saliva and feces, reflecting leakage of serum proteins. Thus, SIgA did not appear to be essential for health under the antigen exposure conditions of these experimental animals. Nevertheless, our results showed that SIgA contributes to maintenance of mucosal homeostasis. Production of SIgA might therefore be a variable in the initiation of human immunopathology such as inflammatory bowel disease or gluten-sensitive enteropathy.

Hypersensitivity and oral tolerance in the absence of a secretory immune system.

BACKGROUND: Mucosal immunity protects the epithelial barrier by immune exclusion of foreign antigens and by anti-inflammatory tolerance mechanisms, but there is a continuing debate about the role of secretory immunoglobulins (SIgs), particularly SIgA, in the protection against allergy and other inflammatory diseases. Lack of secretory antibodies may cause immune dysfunction and affect mucosally induced (oral) tolerance against food antigens. METHODS: We used polymeric Ig receptor (pIgR) knockout (KO) mice, which cannot export SIgA or SIgM, to study oral tolerance induction by ovalbumin (OVA) feeding and for parenteral antigen sensitization in the same animal. RESULTS: Remarkable systemic hyperreactivity was observed in pIgR KO mice, as 50% died after intradermal OVA challenge, which was not seen in similarly sensitized and challenged wild-type (WT) mice. Oral tolerance induced by OVA completely protected the sensitized pIgR KO mice against anaphylaxis and suppressed antibody levels (particularly IgG1) as well as delayed-type hypersensitivity (DTH) to OVA. Delayed-type hypersensitivity to a bystander antigen, human serum albumin, was also suppressed and T-cell proliferation against OVA in vitro was reduced in tolerized compared with non-tolerized pIgR KO mice. This effect was largely mediated by CD25+ T cells. Adoptive transfer of splenic putative regulatory T cells (CD4+ CD25+) obtained from OVA-fed pIgR KO mice to naïve WT mice mediated suppression of DTH against OVA after sensitization of the recipients. CONCLUSION: Compensatory regulatory T-cell function becomes critical in pIgR-deficient mice to avoid the potentially catastrophic effects of systemic immune hyperreactivity, presumably resulting from defective secretory antibody-mediated immune exclusion of microbial components.

Dendritic cells engineered to express defined allo-HLA peptide complexes induce antigen-specific cytotoxic T cells efficiently killing tumour cells.

Most tumour-associated antigens (TAA) are non-mutated self-antigens. The peripheral T cell repertoire is devoid of high-avidity TAA-specific cytotoxic T lymphocytes (CTL) due to self-tolerance. As tolerance is major histocompatibility complex-restricted, T cells may be immunized against TAA presented by a non-self human leucocyte antigen (HLA) molecule and transferred to cancer patients expressing that HLA molecule. Obtaining allo-restricted CTL of high-avidity and low cross-reactivity has, however, proven difficult. Here, we show that dendritic cells transfected with mRNA encoding HLA-A*0201, efficiently present externally loaded peptides from the antigen, Melan-A/MART-1 to T cells from HLA-A*0201-negative donors. CD8(+) T cells binding HLA-A*0201/MART-1 pentamers were detected already after 12 days of co-culture in 11/11 donors. The majority of cells from pentamer(+) cell lines were CTL and efficiently killed HLA-A*0201(+) melanoma cells, whilst sparing HLA-A*0201(+) B-cells. Allo-restricted CTL specific for peptides from the leukaemia-associated antigens CD33 and CD19 were obtained with comparable efficiency. Collectively, the results show that dendritic cells engineered to express defined allo-HLA peptide complexes are highly efficient in generating CTL specifically reacting with tumour-associated antigens.

Comparison of the Agilent, ROMA/NimbleGen and Illumina platforms for classification of copy number alterations in human breast tumors.

BACKGROUND: Microarray Comparative Genomic Hybridization (array CGH) provides a means to examine DNA copy number aberrations. Various platforms, brands and underlying technologies are available, facing the user with many choices regarding platform sensitivity and number, localization, and density distribution of probes. RESULTS: We evaluate three different platforms presenting different nature and arrangement of the probes: The Agilent Human Genome CGH Microarray 44 k, the ROMA/NimbleGen Representational Oligonucleotide Microarray 82 k, and the Illumina Human-1 Genotyping 109 k BeadChip, with Agilent being gene oriented, ROMA/NimbleGen being genome oriented, and Illumina being genotyping oriented. We investigated copy number changes in 20 human breast tumor samples representing different gene expression subclasses, using a suite of graphical and statistical methods designed to work across platforms. Despite substantial differences in the composition and spatial distribution of probes, the comparison revealed high overall concordance. Notably however, some short amplifications and deletions of potential biological importance were not detected by all platforms. Both correlation and cluster analysis indicate a somewhat higher similarity between ROMA/NimbleGen and Illumina than between Agilent and the other two platforms. The programs developed for the analysis are available from http://www.ifi.uio.no/bioinf/Projects/. CONCLUSION: We conclude that platforms based on different technology principles reveal similar aberration patterns, although we observed some unique amplification or deletion peaks at various locations, only detected by one of the platforms. The correct platform choice for a particular study is dependent on whether the appointed research intention is gene, genome, or genotype oriented.

Depletion of CD4+CD25+CD127lo regulatory T cells does not increase allergen-driven T cell activation.

BACKGROUND: It has been suggested that allergic diseases are caused by defective suppression of allergen-specific Th2 cells by CD4(+)CD25(+) regulatory T cells. However, such studies have been hampered by the difficulty in distinguishing regulatory T cells from CD25-expressing activated T cells. Recently, it was shown that conventional T cells expressed high levels of CD127, whereas regulatory T cells were CD127(lo), allowing discrimination between these distinct T cell subpopulations. OBJECTIVE: The aim of this study was to study whether the putative regulatory subset defined as CD4(+)CD25(+)CD127(lo) was involved in grass pollen-reactive T cell responses. METHODS: Peripheral blood mononuclear cells (PBMCs) were obtained from allergic donors and non-atopic controls out of season. Grass pollen-induced cytokine production and proliferation were compared in cultures of undepleted cells and cells depleted of CD4(+)CD25(+), CD4(+)CD25(+)CD127(hi) or CD4(+)CD25(+)CD127(lo) T cells. RESULTS: Undepleted cell cultures from allergic patients showed significantly increased proliferation and Th2 cytokine production compared with non-atopic controls. Depletion of all CD25(+) T cells did not increase cytokine production or proliferation, and more importantly, no increase in Th2 cytokine production or proliferation was observed in cell cultures depleted of CD4(+)CD25(+)CD127(lo) cells (putative regulatory T cells) compared with undepleted PBMCs in both the allergic and the non-atopic group. CONCLUSION: Our study showed that T cells from grass pollen-allergic patients and non-atopic controls responded very differently to grass pollen extract, but this difference could not be explained by differences in regulatory T cell function. Further studies are needed to understand the importance of regulatory T cells in allergy.

Two pathways for serum regulation of the c-fos serum response element require specific sequence elements and a minimal domain of serum response factor.

The c-fos serum response element (SRE) is necessary and sufficient for induction of the c-fos gene in response to serum and growth factors. This activation is dependent upon serum response factor (SRF), a transcriptional activator which binds the SRE. A factor, p62TCF, which binds in conjunction with SRF to the SRE and which is activated by mitogen-activated protein kinase, has also been implicated in c-fos regulation. By using a reporter gene system with weak SRE mutations that is dependent upon overexpression of SRF for serum induction, we have found that there are at least two pathways for serum induction that converge on the SRE. Loss of TCF binding by mutations in SRF and the SRE did not reduce serum induction of the reporter genes. We have found a pathway for serum induction that is sensitive to mutations in the A/T-containing central sequence of the SRE and which is independent of TCF. When this pathway was mutated, activation was dependent upon TCF binding, demonstrating that TCF can also function in serum induction. Both of the signalling pathways required a minimal domain of SRF. This domain, spanning SRF's DNA binding domain, was sufficient for serum induction when fused to a heterologous transcriptional activation domain.

Identification of transcriptional activation and inhibitory domains in serum response factor (SRF) by using GAL4-SRF constructs.

The binding of serum response factor (SRF) to the c-fos serum response element has been shown to be essential for serum and growth factor activation of c-Fos. Since SRF is ubiquitously expressed, it has been difficult to measure the activity of SRF introduced into cells. To assay for functions of SRF in cells, we have changed its DNA binding specificity by fusing it to the DNA binding domain of GAL4. Transfection of GAL4-SRF constructs into cells has allowed us to identify SRF's transcriptional activation domain as well as domains which inhibit this activity. First, we found that the transcriptional activation domain maps to between amino acids 339 and 508 in HeLa cells and to between amino acids 414 and 508 in NIH 3T3 cells. Second, we show that in the context of GAL4-SRF constructs, there are two separate domains of SRF that can inhibit its activation domain. Although these domains overlap the DNA binding and dimerization domains of SRF, these functions were not required for inhibition. Finally, we show that one of the inhibitory domains is modular in that it can also inhibit activation when it is moved amino terminal to GAL4's DNA binding domain in an SRF-GAL4-SRF construct. The implications of these inhibitory domains for SRF regulation are discussed.

Interaction of ATF6 and serum response factor.

Serum response factor (SRF) is a transcription factor which binds to the serum response element (SRE) in the c-fos promoter. It is required for regulated expression of the c-fos gene as well as other immediate-early genes and some tissue-specific genes. To better understand the regulation of SRF, we used a yeast interaction assay to screen a human HeLa cell cDNA library for SRF-interacting proteins. ATF6, a basic-leucine zipper protein, was isolated by binding to SRF and in particular to its transcriptional activation domain. The binding of ATF6 to SRF was also detected in vitro. An ATF6-VP16 chimera activated expression of an SRE reporter gene in HeLa cells, suggesting that ATF6 can interact with endogenous SRF. More strikingly, an antisense ATF6 construct reduced serum induction of a c-fos reporter gene, suggesting that ATF6 is involved in activation of transcription by SRF. ATF6 was previously partially cloned as a member of the ATF family. The complete cDNA of ATF6 was isolated, and its expression pattern was described.

Regulation of the formation and external transport of secretory immunoglobulins.

Secretory IgA (SIgA) is the best defined effector component of the mucosal immune system. Generation of SIgA and secretory IgM (SIgM) in exocrine glands and mucous membranes depends on a fascinating cooperation between local plasma cells that produce polymeric IgA (pIgA, mainly dimers and some larger polymers) and pentameric IgM, and secretory epithelial cells that express the polymeric Ig receptor (pIgR)--also known as transmembrane secretory component. After release from the local plasma cells and diffusion through the stroma, pIgA and pentameric IgM become readily bound to pIgR, and are then actively transported across secretory epithelial cells for extrusion into external secretions after cleavage of pIgR. Much knowledge has recently been obtained at the molecular level about the regulation of pIgR-mediated transport of antibodies. This mechanism is of considerable biological interest because SIgA and SIgM form the first line of specific immunological defense against infectious agents and other harmful substances that may enter the body through the mucosae.

A role for CCL28-CCR3 in T-cell homing to the human upper airway mucosa.

Lymphocyte recruitment to peripheral tissues is fundamental for immune surveillance and homeostasis, but the chemokines and chemokine receptors responsible for tissue-specific homing of T cells to the upper airway mucosa have not been determined. To address this, we analyzed the chemokines expressed in the normal human nasal mucosa and found that CCL28 is preferentially expressed at a high level on the lumenal face of vascular endothelial cells in the mucosa. Analysis of the cognate chemokine receptors revealed that close to 50% of the CD4(+) T cells in the human nasal mucosa expressed the CCL28 receptor CCR3, whereas CCR3 was hardly detectable on T cells in the small intestine and skin. In the circulation, CCR3(+) T cells comprised a small subset that did not express homing receptors to the intestine or skin. Moreover, depletion of CCR3(+)CD4(+) T cells abrogated the proliferative response of human blood CD4(+) T cells against the opportunistic nasopharyngeal pathogen Haemophilus influenzae, indicating that the CCR3(+)CD4(+) T-cell subset in the circulation contains antigen specificities relevant for the upper airways. Together, these findings indicate that CCL28-CCR3 interactions are involved in the homeostatic trafficking of CD4(+) T cells to the upper airways.

Measurements of cytokine mRNA expression by quantitative polymerase chain reaction in studies of celiac disease.

Cytokines are known to play a key regulatory role in immune responses. The onset or progression of immunopathology in various diseases is often associated with aberrant production of one or more cytokines. It is therefore of considerable interest to characterize cytokine "profiles" associated with disease processes. Many methods employed for identification and quantification of cytokines produced by different cell types rely on the responsiveness of indicator cell lines. Such bioassays are technically restrictive owing to the time required for performance and because of sensitivity and specificity problems. Enzyme-linked immunosorbent assays (ELISAs), on the other hand, detect both biologically active and inactive cytokines without discrimination. These assays are easy to use, but the commercial kits are usually expensive. Both bioassays and ELISAs are unable to identify actual cytokine production and do not account for cytokines consumed by cells. In addition, the minute amounts of cytokine protein often produced in autocrine or paracrine microenvironments may not be easily detectable in a sample, especially when tissue or cells are available in only small quantities (1). Furthermore, although cells producing cytokine protein may be detected by immunocyto/histochemistry, only a limited number of antibodies with good performance are available (2), and the possibility of confusing synthesis with cellular uptake of cytokines exists.

Regulation of the polymeric immunoglobulin receptor and IgA transport: new advances in environmental factors that stimulate pIgR expression and its role in mucosal immunity.

Secretory IgA (SIgA) antibodies represent the first line of antigen-specific immune defense protecting the mucosal surfaces against environmental pathogens and antigens, and maintaining homeostasis with the commensal microbiota. The polymeric immunoglobulin receptor (pIgR) has the dual role of transporting locally produced dimeric IgA across mucosal epithelia, and serving as the precursor of secretory component, a glycoprotein that enhances the immune functions of SIgA. The complex regulation of pIgR expression and transcytosis by host and microbial factors is finely tuned to optimize the role of SIgA in mucosal immunity. Disruption of this regulatory network in disease states similar to inflammatory bowel disease can result in profound consequences for mucosal homeostasis and systemic sequelae. Future research into the function and regulation of pIgR and SIgA may offer new insights into the prevention and treatment of infectious and inflammatory diseases that originate at mucosal surfaces.

Regulation of the polymeric immunoglobulin receptor by the classical and alternative NF-κB pathways in intestinal epithelial cells.

The polymeric immunoglobulin receptor (pIgR) transports IgA antibodies across intestinal epithelial cells (IECs). Expression of pIgR is upregulated by proinflammatory signaling pathways via activation of nuclear factor-κB (NF-κB). Here, we examined the contributions of the RelA-dependent classical and RelB-dependent alternative pathways of NF-κB to pIgR regulation in the HT-29 human IEC line following stimulation with tumor necrosis factor (TNF), lipopolysaccharide (LPS; Toll-like receptor 4 (TLR4) ligand), and polyinosinic: polycytidylic acid (pIC; TLR3 ligand). Whereas induction of proinflammatory genes such as interleukin-8 (IL-8) required only RelA, pIgR expression was regulated by complex mechanisms that involved both RelA and RelB. Upregulation of pIgR expression by ligation of the lymphotoxin-ß receptor suggested a direct role for the alternative NF-κB pathway. Inhibition of mitogen-activated protein kinases reduced the induction of IL-8, but enhanced the induction of pIgR by TNF and TLR signaling. Regulation of pIgR through unique signaling pathways could allow IECs to sustain high levels of IgA transport while limiting the proinflammatory responses.

Cholera toxin B suppresses allergic inflammation through induction of secretory IgA.

In healthy individuals, humoral immune responses to allergens consist of serum IgA and IgG4, whereas cellular immune responses are controlled by regulatory T (Treg) cells. In search of new compounds that might prevent the onset of allergies by stimulating this type of immune response, we have focused on the mucosal adjuvant, cholera toxin B (CTB), as it induces the formation of Treg cells and production of IgA. Here, we have found that CTB suppresses the potential of dendritic cells to prime for Th2 responses to inhaled allergen. When we administered CTB to the airways of naïve and allergic mice, it strongly suppressed the salient features of asthma, such as airway eosinophilia, Th2 cytokine synthesis, and bronchial hyperreactivity. This beneficial effect was only transferable to other mice by transfer of B but not of T lymphocytes. CTB caused a transforming growth factor-beta-dependent rise in antigen-specific IgA in the airway luminal secretions, which was necessary for its preventive and curative effect, as all effects of CTB were abrogated in mice lacking the luminal IgA transporting polymeric Ig receptor. Not only do these findings show a novel therapeutic avenue for allergy, they also help to explain the complex relationship between IgA levels and risk of developing allergy in humans.

The immune geography of IgA induction and function.

The production of immunoglobulin A (IgA) in mammals exceeds all other isotypes, and it is mostly exported across mucous membranes. The discovery of IgA and the realization that it dominates humoral mucosal immunity, in contrast to the IgG dominance of the systemic immune system, was early evidence for the distinct nature of mucosal immunology. It is now clear that IgA can function in high-affinity modes for neutralization of toxins and pathogenic microbes, and as a low-affinity system to contain the dense commensal microbiota within the intestinal lumen. The basic map of induction of IgA B cells in the Peyer's patches, which then circulate through the lymph and bloodstream to seed the mucosa with precursors of plasma cells that produce dimeric IgA for export through the intestinal epithelium, has been known for more than 30 years. In this review, we discuss the mechanisms underlying selective IgA induction of mucosal B cells for IgA production and the immune geography of their homing characteristics. We also review the functionality of secretory IgA directed against both commensal organisms and pathogens.