Age-specific Metabolomic profiles in children with food allergy.

BACKGROUND: Food allergy (FA) in young children is often associated with eczema, frequently directed to egg/cow milk allergens and has a higher chance of resolution, while FA that persists in older children has less chance of resolution and is less clearly associated with atopy. METHODS: Children with FA (n = 62) and healthy controls (n = 28) were categorized into "younger" (≤5 years) and "older" (>5 years). Mass spectrometry-based untargeted metabolomic profiling as wells as cytokine profiling were performed on plasma samples in FA children in each age group. RESULTS: Younger FA children manifested unique alterations in bile acids, polyamine metabolites and chemokines associated with Th2 responses, while older FA children displayed pronounced changes in long chain fatty acids, acylcarnitines and proinflammatory cytokines. CONCLUSIONS: FA children of different ages manifest unique metabolic changes which may reflect at least in part pathogenic mechanisms and environmental influences operative at different time points in the disease course.

Oral immunotherapy with omalizumab reverses the Th2 cell-like programme of regulatory T cells and restores their function.

BACKGROUND: Oral immunotherapy (OIT) successfully desensitizes patients with food allergies, but the immune mechanisms mediating its efficacy remain obscure. OBJECTIVES: We tested the hypothesis that allergen-specific regulatory T (Treg) cell function is impaired in food allergy and is restored by anti-IgE antibody (omalizumab)-supplemented OIT. METHODS: Peanut-specific T effector (Teff) and Treg cell proliferative responses, activation markers and cytokine expression were analysed by flow cytometry in 13 peanut-allergic subjects before the start of omalizumab-supplemented OIT and periodically in some subjects thereafter for up to 2 years. Peripheral blood regulatory T cells (Treg cells) were analysed for their peanut-specific suppressor function before and at 1 year following OIT. This study was registered on ClinicalTrials.gov (NCT01290913). RESULTS: Proliferation of allergen-specific Teff and Treg cells precipitously declined following the initiation of omalizumab therapy prior to OIT, followed by partial recovery after the initiation of OIT. At baseline, peanut-specific Treg cells exhibited a Th2 cell-like phenotype, characterized by increased IL-4 expression, which progressively reversed upon OIT. Peanut-specific Treg cell suppressor activity was absent at the start of omalizumab/OIT therapy but became robust following OIT. Absent peanut-specific Treg cell function could also be recovered by the acute blockade of IL-4/IL-4R receptor signalling in Treg cells, which inhibited their IL-4 production. CONCLUSIONS AND CLINICAL RELEVANCE: OIT supplemented by omalizumab promotes allergen desensitization through an initial omalizumab-dependent step that acutely depletes allergen-reactive T cells, followed by an increase in allergen-specific Treg cell activity due to the reversal of their Th2 cell-like programme. Improved Treg cell function may be a key mechanism by which OIT ameliorates food allergy.

Direct effects of IL-4 on mast cells drive their intestinal expansion and increase susceptibility to anaphylaxis in a murine model of food allergy.

Interleukin (IL)-4 has critical roles in allergic disorders, including food hypersensitivity. The direct effects of the cytokine on the survival and function of mast cells, the key effectors of food anaphylaxis, have not been established. In this study, we demonstrate that IL-4 induces a marked intestinal mastocytosis in mice. This phenotype is reproduced in animals expressing Il4rαF709, an activating variant of the IL-4 receptor α-chain (IL-4Rα). Il4rαF709 mice exhibit enhanced anaphylactic reactions but unaltered physiological responses to vasoactive mediators. IL-4 induces Bcl-2 and Bcl-X(L) and enhances survival and stimulates proliferation in cultured bone marrow-derived mast cells (BMMC). These effects are STAT6 (signal transducer and activator of transcription factor 6)-dependent and are amplified in Il4rαF709 BMMC. In competitive bone marrow chimeras, Il4rαF709 mast cells display a substantial competitive advantage over wild-type mast cells, which, in turn, prevail over IL-4Rα⁻/⁻ mast cells in populating the intestine, establishing a cell-intrinsic effect of IL-4 in intestinal mast cell homeostasis. Our results demonstrate that IL-4-signaling is a key determinant of mast cell expansion in food allergy.

Requirement for Ca2+/calmodulin-dependent kinase type IV/Gr in setting the thymocyte selection threshold.

The outcome of thymocyte selection is influenced by the nature of Ca2+ signals transduced by the TCR. Robust Ca2+ responses characterize high-affinity, negatively selecting peptide/TCR interactions, while modest responses typify lower-affinity, positively selecting interactions. To elucidate mechanisms by which thymocytes decode distinct Ca2+ signals, we examined selection events in mice lacking Ca2+/calmodulin-dependent protein kinase type IV/Gr (CaMKIV/Gr), which is enriched in thymocytes. CaMKIV/Gr-deficient thymocytes exhibited impaired positive selection and defective Ca2+-dependent gene transcription. Significantly, CaMKIV/Gr deficiency raised the selection threshold of peptide/TCR interactions such that a peptide that normally induced weak negative selection instead promoted positive selection. These results demonstrate an important role for CaMKIV/Gr in sensitizing thymocytes to selection by low-affinity peptides.

CaMKIV/Gr is dispensable for spermatogenesis and CREM-regulated transcription in male germ cells.

The calcium/calmodulin-dependent protein kinase type IV/Gr (CaMKIV/Gr) is expressed in male germ cells and spermatids and has been implicated in controlling the differentiation of germ cells into mature spermatozoa. The function of CaMKIV/Gr in spermatogenesis was investigated using CaMKIV/Gr-deficient mice generated by targeted gene disruption. CaMKIV/Gr-deficient males exhibited normal spermatogenesis, and their fertility was similar to that of wild-type littermates. Notwithstanding the function of CaMKIV/Gr as an activator of cAMP response element (CRE)-dependent transcription, mRNA levels of several testis-specific CRE modulator (CREM)-regulated genes were unaltered. These results indicate that CaMKIV/Gr is not essential for spermatogenesis or for CRE-regulated gene transcription in the testis.

JM2, encoding a fork head-related protein, is mutated in X-linked autoimmunity-allergic disregulation syndrome.

X-linked autoimmunity-allergic disregulation syndrome (XLAAD) is an X-linked recessive immunological disorder characterized by multisystem autoimmunity, particularly early-onset type 1 diabetes mellitus, associated with manifestations of severe atopy including eczema, food allergy, and eosinophilic inflammation. Consistent with the allergic phenotype, analysis of two kindreds with XLAAD revealed marked skewing of patient T lymphocytes toward the Th2 phenotype. Using a positional-candidate approach, we have identified in both kindreds mutations in JM2, a gene on Xp11.23 that encodes a fork head domain-containing protein. One point mutation at a splice junction site results in transcripts that encode a truncated protein lacking the fork head homology domain. The other mutation involves an in-frame, 3-bp deletion that is predicted to impair the function of a leucine zipper dimerization domain. Our results point to a critical role for JM2 in self tolerance and Th cell differentiation.

Impaired synaptic plasticity and cAMP response element-binding protein activation in Ca2+/calmodulin-dependent protein kinase type IV/Gr-deficient mice.

The Ca(2+)/calmodulin-dependent protein kinase type IV/Gr (CaMKIV/Gr) is a key effector of neuronal Ca(2+) signaling; its function was analyzed by targeted gene disruption in mice. CaMKIV/Gr-deficient mice exhibited impaired neuronal cAMP-responsive element binding protein (CREB) phosphorylation and Ca(2+)/CREB-dependent gene expression. They were also deficient in two forms of synaptic plasticity: long-term potentiation (LTP) in hippocampal CA1 neurons and a late phase of long-term depression in cerebellar Purkinje neurons. However, despite impaired LTP and CREB activation, CaMKIV/Gr-deficient mice exhibited no obvious deficits in spatial learning and memory. These results support an important role for CaMKIV/Gr in Ca(2+)-regulated neuronal gene transcription and synaptic plasticity and suggest that the contribution of other signaling pathways may spare spatial memory of CaMKIV/Gr-deficient mice.

Integration of calcineurin and MEF2 signals by the coactivator p300 during T-cell apoptosis.

T-cell antigen receptor (TCR)-induced thymocyte apoptosis is mediated by calcium-dependent signal transduction pathways leading to the transcriptional activation of members of the Nur77 family. The major calcium- and calcineurin-responsive elements in the Nur77 promoter are binding sites for myocyte enhancer factor-2 (MEF2). It has been shown that nuclear factor of activated T cells (NFAT) interacts with MEF2D and enhances its transcriptional activity, offering a plausible mechanism of activation of MEF2D by calcineurin. We report here that NFATp synergizes with MEF2D to recruit the coactivator p300 for the transcription of Nur77. Surprisingly, the enhancement of transcriptional activity of MEF2D by NFATp does not require its DNA-binding activity, suggesting that NFATp acts as a coactivator for MEF2D. Transient co-expression of p300, MEF2D, NFATp and constitutively active calcineurin is sufficient to recapitulate TCR signaling for the selective induction of the endogenous Nur77 gene. These results implicate NFAT as an important mediator of T-cell apoptosis and suggest that NFAT is capable of integrating the calcineurin signaling pathway and other pathways through direct protein-protein interaction with other transcription factors.

Ca(2+)-dependent gene expression mediated by MEF2 transcription factors.

Ca(2+) induction of a subset of cellular and viral immediate-early activation genes in lymphocytes has been previously mapped to response elements recognized by the MEF2 family of transcription factors. Here, we demonstrate that Ca(2+) activation of MEF2 response elements in T lymphocytes is mediated in synergy by two Ca(2+)/calmodulin-dependent enzymes, the phosphatase calcineurin, and the kinase type IV/Gr (CaMKIV/Gr), which promote transcription by the MEF2 family members MEF2A and MEF2D. Calcineurin up-regulates the activity of both factors by an NFAT-dependent mechanism, while CaMKIV/Gr selectively and independently activates MEF2D. These results identify MEF2 proteins as effectors of a pathway of gene induction in T lymphocytes which integrates diverse Ca(2+) activation signals and may be broadly operative in several tissues.

Calcium-dependent activation of TNF family gene expression by Ca2+/calmodulin kinase type IV/Gr and calcineurin.

CD40 ligand (L), FasL, and TNF-alpha are members of the TNF family of cytokines. All are expressed by T lymphocytes shortly after activation but have distinct effector functions. Transcription of these genes can be induced by stimulation of T cells by calcium ionophore alone and requires the calcineurin-dependent transcription factor NF of activated T cells. We have examined a second calcium-dependent signaling pathway, mediated by calcium/calmodulin-dependent kinase IV (CaMKIV) in transcriptional activation of TNF family genes. In reporter gene assays using constructs driven by the promoters of human CD40L, FasL, or TNF-alpha along with vectors expressing constitutively active CaMKIV and calcineurin, we have demonstrated that each promoter is activated by calcineurin and CaMKIV in a synergistic fashion. Furthermore, specific inhibition of CaMKIV by chemical means and by a dominant negative mutant of CaMKIV impairs the ionomycin-induced activity of all three promoters as well as protein expression of CD40L and TNF-alpha. Our results indicate that activation of gene expression by calcineurin and CaMKIV is common to members of the TNF cytokine family.

Genetics of atopic diseases.

Atopic (allergic) disorders develop out of a close interaction between genetic predisposition and environmental triggers. A unifying attribute underlying these disorders is atopy, defined as the predisposition of those affected to generate IgE antibodies to environmental antigens and to respond with immediate-type hypersensitivity reactions upon subsequent exposure. Atopy is a heritable trait, and recent studies have identified several genes that engender atopy by increasing either the production of or the responsiveness to IgE. Other genes that contribute to the development of allergic disorders include leukocyte histocompatibility alleles, which specify responsiveness to individual environmental antigens, and disease-related genes, which promote distinctive aspects of an allergic disorder, such as tissue localization. A model is presented whereby the evolution of specific allergic disorders is predicated on the confluence of predisposing genetic elements, coupled with exposure to environmental triggers.

The association of atopy with a gain-of-function mutation in the alpha subunit of the interleukin-4 receptor.

BACKGROUND: Atopic diseases are very common, and atopy has a strong genetic predisposition. METHODS: Using single-strand conformation polymorphism analysis and DNA sequencing, we searched for mutations in the a subunit of the interleukin-4 receptor that would predispose persons to atopy. We examined the prevalence of the alleles among patients with allergic inflammatory disorders and among 50 prospectively recruited adults. Subjects with atopy were identified on the basis of an elevated serum IgE level (> or = 95 IU per milliliter) or a positive radioimmunosorbent test in response to standard inhalant allergens. The signaling function of mutant interleukin-4 receptor a was examined by flow cytometry, binding assays, and immunoblotting. RESULTS: A novel interleukin-4 receptor alpha allele was identified in which guanine was substituted for adenine at nucleotide 1902, causing a change from glutamine to arginine at position 576 (R576) in the cytoplasmic domain of the interleukin-4 receptor alpha protein. The R576 allele was common among patients with allergic inflammatory disorders (found in 3 of 3 patients with the hyper-IgE syndrome and 4 of 7 patients with severe atopic dermatitis) and among the 50 prospectively recruited adults (found in 13 of 20 subjects with atopy and 5 of 30 without atopy; P=0.001; relative risk of atopy among those with a mutant allele, 9.3). The R576 allele was associated with higher levels of expression of CD23 by interleukin-4 than the wild-type allele. This enhanced signaling was associated with a change in the binding specificity of the adjacent tyrosine residue at position 575 to signal-transducing molecules. CONCLUSIONS: The R576 allele of interleukin-4 receptor alpha is strongly associated with atopy. This mutation may predispose persons to allergic diseases by altering the signaling function of the receptor.

Regulation of microtubule dynamics by Ca2+/calmodulin-dependent kinase IV/Gr-dependent phosphorylation of oncoprotein 18.

Oncoprotein 18 (Op18; also termed p19, 19K, p18, prosolin, and stathmin) is a regulator of microtubule (MT) dynamics and is phosphorylated by multiple kinase systems on four Ser residues. In addition to cell cycle-regulated phosphorylation, external signals induce phosphorylation of Op18 on Ser-25 by the mitogen-activated protein kinase and on Ser-16 by the Ca2+/calmodulin-dependent kinase IV/Gr (CaMK IV/Gr). Here we show that induced expression of a constitutively active mutant of CaMK IV/Gr results in phosphorylation of Op18 on Ser-16. In parallel, we also observed partial degradation of Op18 and a rapid increase of total cellular MTs. These results suggest a link between CaMK IV/Gr, Op18, and MT dynamics. To explore such a putative link, we optimized a genetic system that allowed conditional coexpression of a series of CaMK IV/Gr and Op18 derivatives. The result shows that CaMK IV/Gr can suppress the MT-regulating activity of Op18 by phosphorylation on Ser-16. In line with these results, by employing a chemical cross-linking protocol, it was shown that phosphorylation of Ser-16 is involved in weakening of the interactions between Op18 and tubulin. Taken together, these data suggest that the mechanism of CaMK IV/Gr-mediated suppression of Op18 activity involves both partial degradation of Op18 and direct modulation of the MT-destabilizing activity of this protein. These results show that Op18 phosphorylation by CaMK IV/Gr may couple alterations of MT dynamics in response to external signals that involve Ca2+.

Induction of interleukin-6 after stimulation of human B-cell CD21 by Epstein-Barr virus glycoproteins gp350 and gp220.

The cellular receptor for Epstein-Barr virus (EBV) is the type 2 complement receptor, CD21. At initial infection, EBV virion glycoproteins gp350 and gp220 bind to CD21. We report here that the cross-linking of CD21 by gp350/220 results in increased amounts of interleukin 6 (IL-6) RNA and IL-6 protein. This effect could be blocked with anti-gp350/220 and anti-CD21 monoclonal antibodies. Induction of IL-6 in B cells by EBV could be mimicked by treatment with the protein kinase C (PKC) activator phorbol 12,13-dibutyrate but not with the calcium ionophore ionomycin. IL-6 induction by EBV was inhibited with the PKC-specific inhibitor bisindolylmaleimide or the protein tyrosine kinase inhibitors methyl 2,5-dihydroxycinnamate and herbimycin A, indicating that the induction of IL-6 following CD21 cross-linking is mediated through PKC- and protein tyrosine kinase-dependent pathways.

Serine 16 of oncoprotein 18 is a major cytosolic target for the Ca2+/calmodulin-dependent kinase-Gr.

Oncoprotein 18 (Op18) is a cytosolic protein that was initially identified due to its up-regulated expression in acute leukemia and its complex pattern of phosphorylation in response to diverse extracellular signals. We have previously identified in vivo phosphorylation sites and some of the protein kinase systems involved. Two distinct proline-directed kinase families phosphorylate Ser25 and Ser38 of Op18 with overlapping but distinct site preference. These two kinase families, mitogen-activated protein (MAP) kinases and cyclin-dependent cdc2 kinases, are involved in receptor-regulated and cell-cycle-regulated phosphorylation events, respectively. During analysis of Op18 phosphorylation in the Jurkat T-cell line, we also found that Ser16 of Op18 is phosphorylated in response to a Ca2+ signal generated by T-cell receptor stimulation or the Ca2+ ionophore ionomycin. As suggested by a previous study, T-cell-receptor-induced phosphorylation events may be mediated by the Ca2+/CaM-dependent protein kinase type Gr (CaM kinase-Gr). The present study shows that activation of this protein kinase correlates with phosphorylation of Ser16 of Op18, and in vitro experiments reveal efficient and selective phosphorylation of this residue. The CaM kinase-Gr is only expressed in certain lymphoid cell lines, and the present study shows that ionomycin-induced phosphorylation of Op18 Ser16 is restricted to cells expressing this protein kinase. Finally, CaM kinase-Gr-dependent in vitro phosphorylation of a crude cellular extract reveals a striking preference of this protein kinase for Op18 compared to other cellular substrates. In conclusion, the results suggest that Ser16 of Op18 is a major cytosolic target for activated CaM kinase-Gr.

A Ca2+/calmodulin-dependent protein kinase, CaM kinase-Gr, expressed after transformation of primary human B lymphocytes by Epstein-Barr virus (EBV) is induced by the EBV oncogene LMP1.

CaM kinase-Gr is a multifunctional Ca2+/calmodulin-dependent protein kinase which is enriched in neurons and T lymphocytes. The kinase is absent from primary human B lymphocytes but is expressed in Epstein-Barr virus (EBV)-transformed B-lymphoblastoid cell lines, suggesting that expression of the kinase can be upregulated by an EBV gene product(s). We investigated the basis of CaM kinase-Gr expression in EBV-transformed cells and the mechanisms that regulate its activity therein by using an EBV-negative Burkitt lymphoma cell line, BJAB, and BJAB cells converted to expression of individual EBV proteins by single-gene transfer. CaM kinase-Gr expression was upregulated in BJAB cells by EBV latent-infection membrane protein 1 (LMP1) but not by LMP2A or by nuclear proteins EBNA1, EBNA2, EBNA3A, and EBNA3C. In LMP1-converted BJAB cells, the kinase was functional and was dramatically activated upon cross-linking of surface immunoglobulin M. Overlapping cDNA clones that encode human CaM kinase-Gr were sequenced, revealing 81% amino acid identity between the rat and human proteins. Transfection of BJAB cells with an expression construct for the human enzyme resulted in a functional kinase which was shown by epitope tagging to localize primarily to cytoplasmic and perinuclear structures. Induction of CaM kinase-Gr expression by LMP1 provides the first example of a Ca2+/calmodulin-dependent protein kinase upregulated by a viral protein. In view of the key role played by LMP1 in B-lymphocyte immortalization by EBV, these findings implicate CaM kinase-Gr as a potential mediator of B-lymphocyte growth transformation.

Engagement of MHC class II molecules by staphylococcal superantigens activates src-type protein tyrosine kinases.

Staphylococcal exotoxins (SE) are superantigens that bind to monomorphic determinants on major histocompatibility complex (MHC) class II molecules and stimulate human peripheral blood T lymphocytes in a V beta-specific manner. SE also deliver activation signals via MHC class II molecules that initiate cell adhesion and cytokine gene transcription. These events are preceded by tyrosine phosphorylation and are antagonized by inhibitors of tyrosine kinases, indicating an essential role for these kinases in signaling via class II molecules. We report that stimulation of human peripheral blood monocytes with SE induced rapid and selective activation of the src-related protein tyrosine kinases (PTK) fgr and hck. SE also induced the activation of fgr and lyn in B cells. PTK activation by SE required MHC class II expression, and was greatly potentiated in the presence of T cells bearing toxin-specific V beta chains. These results indicate that in addition to their antigen and superantigen-presenting function, MHC class II molecules act as signal-transducing receptors that are coupled to src-type PTK.

Expression of a Ca2+/calmodulin-dependent protein kinase, CaM kinase-Gr, in human T lymphocytes. Regulation of kinase activity by T cell receptor signaling.

Ca2+/calmodulin-dependent protein kinase type Gr (CaM kinase-Gr) is a Ca2+/calmodulin-dependent protein kinase which is enriched in the brain and thymus. In this study, we examined the expression of CaM kinase-Gr in human lymphocytes and the regulation of its catalytic activity by antigen receptor signaling. CaM kinase-Gr was found selectively expressed in T lymphocytes in a developmentally regulated manner. It was present at severalfold higher levels in immature thymocytes (CD3low, CD4+CD8+) relative to mature thymocytes (CD3high, CD4+CD8-/CD8+CD4-) or to circulating T lymphocytes. The kinase was preferentially expressed in CD4+ T lymphocytes, but was not detected in B lymphocytes or in monocytes. The impact of T cell antigen receptor-CD3 complex (TCR.CD3) signaling on kinase activity was examined using Jurkat human leukemic T lymphocytes as a model. Treatment of Jurkat cells with anti TCR.CD3 monoclonal antibody induced rapid autophosphorylation of the kinase on serine residues and a dramatic, autophosphorylation-dependent enhancement of both Ca2+/calmodulin-dependent and autonomous kinase activity. Enzyme autophosphorylation and activation were dependent on the influx of extracellular Ca2+ following receptor signaling but could not be induced by an influx of extra-cellular Ca2+ triggered by ionophores, indicating that additional signals delivered via TCR.CD3 contribute to the activation of CaM kinase-Gr. These findings suggest a role for CaM kinase-Gr in T lymphocyte development and activation and indicate the presence of stringent regulatory mechanisms governing the activity of this kinase in situ.

Molecular basis of a multiple lymphokine deficiency in a patient with severe combined immunodeficiency.

We have previously reported that the T lymphocytes of a child with severe combined immunodeficiency are defective in the transcription of several lymphokine genes that include IL2, IL3, IL4, and IL5, which encode interleukins 2, 3, 4, and 5 (IL-2, -3, -4, and -5). To determine whether the defect in the patient's T lymphocytes involved a trans-acting factor common to the affected lymphokine genes, we examined the ability of nuclear factors from the patient's T lymphocytes to bind response elements present in the regulatory region of IL2. Nuclear factor NF-kB, activation protein 1 (AP-1), OCT-1, and NF-IL-2B binding activity were normal. In contrast, the binding of the nuclear factor of activated T cells (NF-AT) to its response element in the IL2 enhancer and to an NF-AT-like response element present in the IL4 enhancer was abnormal. To ascertain whether the abnormal NF-AT binding activity was related to an impaired function, we transfected patient and control T lymphocytes with constructs containing the reporter gene encoding chloramphenicol acetyl transferase (CAT) under the control of the entire IL2 regulatory region or of multimers of individual enhancer sequences. CAT expression directed by the IL2 regulatory region or by a multimer of the NF-AT-binding site was markedly lower in the patient relative to controls. In contrast, CAT gene expression directed by a multimer of the OCT-1 proximal (OCT-1p)-binding site was equivalent in patient and controls. These results indicate that an abnormality of/or influencing NF-AT may underlie the multiple lymphokine deficiency in this patient.