Loss of B Cells in Patients with Heterozygous Mutations in IKAROS.

BACKGROUND: Common variable immunodeficiency (CVID) is characterized by late-onset hypogammaglobulinemia in the absence of predisposing factors. The genetic cause is unknown in the majority of cases, and less than 10% of patients have a family history of the disease. Most patients have normal numbers of B cells but lack plasma cells. METHODS: We used whole-exome sequencing and array-based comparative genomic hybridization to evaluate a subset of patients with CVID and low B-cell numbers. Mutant proteins were analyzed for DNA binding with the use of an electrophoretic mobility-shift assay (EMSA) and confocal microscopy. Flow cytometry was used to analyze peripheral-blood lymphocytes and bone marrow aspirates. RESULTS: Six different heterozygous mutations in IKZF1, the gene encoding the transcription factor IKAROS, were identified in 29 persons from six families. In two families, the mutation was a de novo event in the proband. All the mutations, four amino acid substitutions, an intragenic deletion, and a 4.7-Mb multigene deletion involved the DNA-binding domain of IKAROS. The proteins bearing missense mutations failed to bind target DNA sequences on EMSA and confocal microscopy; however, they did not inhibit the binding of wild-type IKAROS. Studies in family members showed progressive loss of B cells and serum immunoglobulins. Bone marrow aspirates in two patients had markedly decreased early B-cell precursors, but plasma cells were present. Acute lymphoblastic leukemia developed in 2 of the 29 patients. CONCLUSIONS: Heterozygous mutations in the transcription factor IKAROS caused an autosomal dominant form of CVID that is associated with a striking decrease in B-cell numbers. (Funded by the National Institutes of Health and others.).

Dysregulation of metabolic pathways in a mouse model of allergic asthma.

BACKGROUND: Asthma is a complex lung disease resulting from the interplay of genetic and environmental factors. To understand the molecular changes that occur during the development of allergic asthma without genetic and environmental confounders, an experimental model of allergic asthma in mice was used. Our goals were to (1) identify changes at the small molecule level due to allergen exposure, (2) determine perturbed pathways due to disease, and (3) determine whether small molecule changes correlate with lung function. METHODS: In this experimental model of allergic asthma, matched bronchoalveolar lavage (BAL) fluid and plasma were collected from three groups of C57BL6 mice (control vs sensitized and/or challenged with ovalbumin, n=3-5/group) 6 hour, 24 hour, and 48 hour after the last challenge. Samples were analyzed using liquid chromatography-mass spectrometry-based metabolomics. Airway hyper-responsiveness (AHR) measurements and differential cell counts were performed. RESULTS: In total, 398 and 368 dysregulated metabolites in the BAL fluid and plasma of sensitized and challenged mice were identified, respectively. These belonged to four, interconnected pathways relevant to asthma pathogenesis: sphingolipid metabolism (P=6.6×10-5 ), arginine and proline metabolism (P=1.12×10-7 ), glycerophospholipid metabolism (P=1.3×10-10 ), and the neurotrophin signaling pathway (P=7.0×10-6 ). Furthermore, within the arginine and proline metabolism pathway, a positive correlation between urea-1-carboxylate and AHR was observed in plasma metabolites, while ornithine revealed a reciprocal effect. In addition, agmatine positively correlated with lung eosinophilia. CONCLUSION: These findings point to potential targets and pathways that may be central to asthma pathogenesis and can serve as novel therapeutic targets.

Combined blockade of the histamine H1 and H4 receptor suppresses peanut-induced intestinal anaphylaxis by regulating dendritic cell function.

BACKGROUND: Signaling through histamine receptors on dendritic cells (DCs) may be involved in the effector phase of peanut-induced intestinal anaphylaxis. OBJECTIVES: The objective of this study was to determine the role of histamine H1 (H1R) and H4 receptors (H4R) in intestinal allergic responses in a model of peanut allergy. METHODS: Balb/c mice were sensitized and challenged with peanut. During the challenge phase, mice were treated orally with the H1R antagonist, loratadine, and/or the H4R antagonist, JNJ7777120. Bone marrow-derived DCs (BMDCs) were adoptively transferred to nonsensitized WT mice. Symptoms, intestinal inflammation, and mesenteric lymph node and intestine mucosal DCs were assessed. Effects of the drugs on DC chemotaxis, calcium mobilization, and antigen-presenting cell function were measured. RESULTS: Treatment with loratadine or JNJ7777120 individually partially suppressed the development of diarrhea and intestinal inflammation and decreased the numbers of DCs in the mesenteric lymph nodes and lamina propria. Combined treatment with both drugs prevented the development of diarrhea and intestinal inflammation. In vitro, the combination suppressed DC antigen-presenting cell function to T helper cells and DC calcium mobilization and chemotaxis to histamine. CONCLUSION: Blockade of both H1R and H4R in the challenge phase had additive effects in preventing the intestinal consequences of peanut sensitization and challenge. These effects were mediated through the limitation of mesenteric lymph node and intestinal DC accumulation and function. Identification of this histamine H1R/H4R-DC-CD4+ T-cell axis provides new insights into the development of peanut-induced intestinal allergic responses and for prevention and treatment of peanut allergy.

Role of prostaglandin D2 and CRTH2 blockade in early- and late-phase nasal responses.

BACKGROUND: Prostaglandin D2 (PGD2 ) plays an important role in allergic inflammation. The PGD2 receptor, CRTH2, is expressed on basophils, eosinophils, and Th2 lymphocytes and mediates chemotactic activity. OBJECTIVE: To define the role of CRTH2 in allergen-induced nasal responses in a mouse model of allergic rhinitis (AR), a potent, selective CRTH2 receptor antagonist, ARRY-063 was administered in a model of allergic rhinitis in mice. METHODS: ARRY-063 was administered orally to ovalbumin (OVA) sensitized and challenged mice. To assess nasal obstruction, respiratory frequency (RF) was monitored by whole-body plethysmography immediately after the 4th challenge (early-phase response, EPR) and 24 h after the 6th challenge (late-phase response, LPR). Nasal resistance (RNA ) was also measured in the LPR. PGD2 was administered with or without OVA to determine the effect of PGD2 on nasal responsiveness. Cytokine levels and histopathological changes in nasal tissue were analysed. RESULTS: Instillation of PGD2 in the nose of sensitized mice together with a low concentration of OVA induced both an EPR and LPR. Treatment with the CRTH2 receptor antagonist prevented the decreases in RF seen immediately following the 4th challenge of sensitized mice (EPR). In the LPR, decreases in RF and increases in RNA were also prevented by antagonist treatment associated with reduced cytokine levels and inflammation in nasal tissues. CONCLUSIONS: These data identify PGD2 as a mediator of both the EPR and LPR in this model of AR and suggest that antagonism of CRTH2 prevents the development of both the EPR and LPR as well as nasal inflammation.

IL-13-producing BLT1-positive CD8 cells are increased in asthma and are associated with airway obstruction.

BACKGROUND: The role of CD8 T lymphocytes in the pathogenesis of asthma is not well understood. We investigated whether a subset of IL-13-producing BLT1-positive CD8 T lymphocytes are present in asthmatic airways and are associated with impaired lung function. METHODS: Bronchoalveolar lavage (BAL) cells were obtained from asthmatic (n = 39) and healthy control (n = 28) subjects. Cells were stimulated with phorbol ester and ionomycin in the presence of brefeldin A and stained for CD8, BLT1, and intracellular IL-13. The frequency of IL-13-producing BLT1-positive CD8 T lymphocytes was compared between the two groups and related to lung function, serum IgE levels, and reticular basement membrane (RBM) thickness. RESULTS: A subset of CD8 T lymphocytes expressing BLT1 and producing IL-13 were detected in the airways of all asthmatic subjects. The frequency of this subset among recovered lymphocytes was significantly higher in the airways of asthmatic subjects compared with controls (mean ± SEM: 16.2 ± 1.4 vs 5.3 ± 0.5, respectively, P < 0.001) and correlated positively with serum IgE levels and RBM thickness. More importantly, the frequency of CD8 T lymphocytes co-expressing BLT1 and IL-13 was inversely related to FEV1 and FEF[25-75] percent predicted values (P < 0.001). CONCLUSIONS: A subset of CD8 T lymphocytes expressing BLT1 and producing IL-13 is present in the airways of asthmatics. The accumulation of these cells is associated with airway obstruction, suggesting that they may play a significant pathogenic role in bronchial asthma.

Negative regulation of airway responsiveness that is dependent on gammadelta T cells and independent of alphabeta T cells.

The mechanisms regulating airway function are complex and still poorly understood. In diseases such as asthma, involvement of immune-dependent mechanisms has been suggested in causing changes in airway responsiveness to bronchoconstrictors. We now demonstrate that gammadelta T cells can regulate airway function in an alphabeta T cell-independent manner, identifying them as important cells in pulmonary homeostasis. This function of gammadelta T cells differs from previously described immune-dependent mechanisms and may reflect their interaction with innate systems of host defense.

The interaction between human monocytes and red cells. Specificity for IgG subclasses and IgG fragments.

Studies were carried out in order to characterize the specificity of IgG sub-classes and IgG fragments for rosette formation using red cells and human mononuclear cells. Rosette formation of red cells coated with anti-D was inhibited by free IgG(1) and IgG(3); less inhibition occurred with IgG(2) and IgG(4). Red cells specifically coated with IgG(1) and IgG(3) by chromic chloride were bound to monocytes. Rosette inhibition of anti-D-coated red cells occurred with free Fc fragment of IgG globulin, and only partly with F(ab')(2). Inhibitory capacity of Fc fragments of IgG and gamma(1) heavy chain from heavy chain disease was reversed by the cleavage of disulfide bonds. No inhibition was noted with Fab, or with pepsin components II, III, or IV. These studies indicated that the mononuclear receptor was specific for IgG(1) and IgG(3). The peptide portion of IgG globulin which attached to the mononuclear cell appeared to reside in the N-terminal portion of the Fc fragment and also appeared to require the integrity of the inter-heavy chain disulfide bond. A specific receptor for C3 was not confirmed.

Independent regulation of Ca2+ entry and release from internal stores in activated B cells.

Addition of crosslinking antibody to B lymphocytes results in a rapid rise in cytoplasmic-free Ca2+ ([Ca2+]i) due to release of Ca2+ from internal stores and uptake of Ca2+ across the plasma membrane. Inositol 1,4,5-trisphosphate is believed to mediate the release of internal Ca2+ stores and has also been proposed to mediate extracellular Ca2+ entry. We have compared the properties of these two pathways for Ca2+ mobilization by dissociating the [Ca2+]i changes in ligand-activated human B cells after loading of the cells with the Ca2+ chelator BAPTA. In the present paper we show that: (a) the sustained increase in [Ca2+]i is due to increased unidirectional influx of external [Ca2+]i; (b) entry of extracellular Ca2+, but not release of internal stores, is sensitive to the transmembrane potential; and (c) entry of extracellular Ca2+, but not release of internal stores, is inhibited by increasing [Ca2+]i. These findings suggest that the permeation pathways mediating the translocation of Ca2+ across the plasma membrane and endoplasmic reticulum membrane are not identical.

T cells expressing specific V beta elements regulate immunoglobulin E production and airways responsiveness in vivo.

The role of T cells expressing specific V beta elements was examined in the regulation of allergen-specific immunoglobulin (Ig)E production and airways responsiveness (AR). In BALB/c mice, inhalation of the allergen ovalbumin (OVA) induced an IgE anti-OVA response, immediate cutaneous reactivity, and increased AR. These results were associated with an expansion of V beta 8.1/8.2 T cells in local draining lymph nodes of the airways and the lung. Transfer of V beta 8.1/8.2 T cells from sensitized mice stimulated an IgE anti-OVA response, immediate cutaneous hypersensitivity, and increased AR in naive syngeneic recipients. In contrast, OVA-reactive V beta 2 T cells inhibited these effects. These data demonstrate for the first time that T cells with different V beta specificities play a critical role in the in vivo regulation of allergen-specific IgE production and AR.

Nerve growth factor triggers microfilament assembly and paxillin phosphorylation in human B lymphocytes.

Increasing evidence suggests that the nervous system is involved in allergic inflammation. One of the potential regulatory molecules of the neuroimmune system is nerve growth factor (NGF). Recent studies from our group demonstrated the presence of a functional NGF receptor (NGFR) on human B lymphocytes. Moreover, we showed that gp140trk tyrosine kinase, which serves as an NGFR, was involved in transduction of early signaling events in human B lymphocytes. The mechanisms by which NGF initiates the signaling cascade and the link between the neuroimmune systems are unknown. We have focused on the role of the cytoskeleton as a possible mediator for transduction of signals induced by NGF. Polymerized actin (F-actin) content was determined by fluorescent staining and immunoblotting with antiactin antibody. Addition of NGF caused a time- and concentration-dependent increase in F-actin content, and maximum effects were noted after 1 min. These increases in F-actin content and NGF-induced thymidine incorporation could be blocked by incubating the cells with cytochalasin D and botulinum C2 toxin before the addition of NGF. Incubation of human B lymphocytes with 10 nM K252a, an inhibitor of Trk kinase, decreased NGF-induced microfilament assembly by 75%. In immunoprecipitation experiments, addition of NGF to B cells induced a rapid increase in the tyrosine phosphorylation of paxillin, one of a group of focal adhesion proteins involved in linking actin filaments to the plasma membrane. Coimmunoprecipitation studies demonstrated the association between gp140trk kinase and paxillin. Together, these observations suggest that actin assembly is involved in NGF signaling in human B cells, and that paxillin may be essential in this pathway after phosphorylation by gp140trk kinase.

Absence of ZAP-70 prevents signaling through the antigen receptor on peripheral blood T cells but not on thymocytes.

Recently, a severe combined immunodeficiency syndrome with a deficiency of CD8+ peripheral T cells and a TCR signal transduction defect in peripheral CD4+ T cells was associated with mutations in ZAP-70. Since TCR signaling is required in developmental decisions resulting in mature CD4 (and CD8) T cells, the presence of peripheral CD4+ T cells expressing TCRs incapable of signaling in these patients is paradoxical. Here, we show that the TCRs on thymocytes, but not peripheral T cells, from a ZAP-70-deficient patient are capable of signaling. Moreover, the TCR on a thymocyte line derived from this patient can signal, and the homologous kinase Syk is present at high levels and is tyrosine phosphorylated after TCR stimulation. Thus, Syk may compensate for the loss of ZAP-70 and account for the thymic selection of at least a subset of T cells (CD4+) in ZAP-70-deficient patients.

Development of eosinophilic airway inflammation and airway hyperresponsiveness in mast cell-deficient mice.

Mast cells are the main effector cells of immediate hypersensitivity and anaphylaxis. Their role in the development of allergen-induced airway hyperresponsiveness (AHR) is controversial and based on indirect evidence. To address these issues, mast cell-deficient mice (W/W v) and their congenic littermates were sensitized to ovalbumin (OVA) by intraperitoneal injection and subsequently challenged with OVA via the airways. Comparison of OVA-specific immunoglobulin E (IgE) levels in the serum and numbers of eosinophils in bronchoalveolar lavage fluid or lung digests showed no differences between the two groups of mice. Further, measurements of airway resistance and dynamic compliance at baseline and after inhalation of methacholine were similar. These data indicate that mast cells or IgE-mast cell activation is not required for the development of eosinophilic inflammation and AHR in mice sensitized to allergen via the intraperitoneal route and challenged via the airways.

Requirement for CD8+ T cells in the development of airway hyperresponsiveness in a marine model of airway sensitization.

To study the role of CD8+ T cells in allergic sensitization, we examined the effects of in vivo depletion of CD8+ T cells prior to sensitization on IgE production, immediate type cutaneous hypersensitivity and development of altered airway responsiveness. BALB/c mice were thymectomized and treated with anti-CD8 antibody resulting in depletion of CD8+ T cells (<1%) in spleen and lymphoid tissues. In these mice, sensitization to ovalbumin (OVA) via the airways still resulted in IgE anti-OVA responses and immediate cutaneous reactions to OVA, but the animals were unable to develop airway hyperresponsiveness, eosinophil infiltration of the lung parenchyma, or IL-5 production in the local lymph nodes of the airway. Transfer of CD8+ T cells from naive animals during sensitization (on day 8 of the 10-d protocol) fully restored the ability to develop airway hyperresponsiveness and this was accompanied by IL-5 production and eosinophil accumulation in the lung. These data indicate a critical role for CD8+ T cells in the production of IL-5 and the development of altered airway responsiveness after antigen sensitization through the airways.

Selective expansion of T cells expressing V beta 2 in toxic shock syndrome.

Infection with Staphylococcus aureus and the production of toxic shock syndrome toxin-1 (TSST-1) have been implicated in the pathogenesis of toxic shock syndrome. Previous in vitro studies have demonstrated that TSST-1 is a powerful but selective stimulator of human T cells, and that the majority of activated cells express the TCR V beta 2 gene segment. We therefore studied patients with toxic shock syndrome using a modification of the PCR to determine if expansion of V beta 2+ T cells is a marker of the in vivo disease process. Five of eight patients studied demonstrated markedly elevated levels of circulating V beta 2+ T cells, whereas none showed significantly elevated levels of T cells expressing other V beta gene segments. The results suggest that toxin-mediated T cell activation, which involves a large fraction of the human T cell repertoire, may be critical in the pathogenesis of this disease.

Reconstitution of T cell receptor signaling in ZAP-70-deficient cells by retroviral transduction of the ZAP-70 gene.

A variant of severe combined immunodeficiency syndrome (SCID) with a selective inability to produce CD8 single positive T cells and a signal transduction defect in peripheral CD4+ cells has recently been shown to be the result of mutations in the ZAP-70 gene. T cell receptor (TCR) signaling requires the association of the ZAP-70 protein tyrosine kinase with the TCR complex. Human T cell leukemia virus type I-transformed CD4+ T cell lines were established from ZAP-70-deficient patients and normal controls. ZAP-70 was expressed and appropriately phosphorylated in normal T cell lines after TCR engagement, but was not detected in T cell lines from ZAP-70-deficient patients. To determine whether signaling could be reconstituted, wild-type ZAP-70 was introduced into deficient cells with a ZAP-70 retroviral vector. High titer producer clones expressing ZAP-70 were generated in the Gibbon ape leukemia virus packaging line PG13. After transduction, ZAP-70 was detected at levels equivalent to those observed in normal cells, and was appropriately phosphorylated on tyrosine after receptor engagement. The kinase activity of ZAP-70 in the reconstituted cells was also appropriately upregulated by receptor aggregation. Moreover, normal and transduced cells, but not ZAP-70-deficient cells, were able to mobilize calcium after receptor ligation, indicating that proximal TCR signaling was reconstituted. These results indicate that this form of SCID may be corrected by gene therapy.

Interleukin-5 expression in the lung epithelium of transgenic mice leads to pulmonary changes pathognomonic of asthma.

We have generated transgenic mice that constitutively express murine interleukin (IL)-5 in the lung epithelium. Airway expression of this cytokine resulted in a dramatic accumulation of peribronchial eosinophils and striking pathologic changes including the expansion of bronchus-associated lymphoid tissue (BALT), goblet cell hyperplasia, epithelial hypertrophy, and focal collagen deposition. These changes were also accompanied by eosinophil infiltration of the airway lumen. In addition, transgenic animals displayed airway hyperresponsiveness to methacholine in the absence of aerosolized antigen challenge. These findings demonstrate that lung-specific IL-5 expression can induce pathologic changes characteristic of asthma and may provide useful models to evaluate the efficacy of potential respiratory disease therapies or pharmaceuticals.

Impact of a novel nutritional formula on asthma control and biomarkers of allergic airway inflammation in children.

BACKGROUND: A novel nutritional formula (NNF) enriched in eicosapentaenoic (EPA) and gamma-linolenic fatty acids and antioxidants reduces airway inflammation and improves clinical outcomes in critically ill patients, but NNF has not been evaluated in chronic inflammatory diseases such as persistent asthma. OBJECTIVE: To evaluate the efficacy, compliance, and safety of NNF in asthmatic children. METHODS: Children, 6-14 years of age, with mild to moderate persistent asthma, on as needed albuterol alone, were randomized to receive daily NNF (n=23) or control formula (n=20) for 12 weeks, with multiple assessments of asthma control, spirometry, measures of airway inflammation, formula tolerance, and adverse events. RESULTS: Daily consumption of either NNF or a control formula showed improvement in asthma-free days over time (P=0.04) but there was no difference between groups. However, the NNF group had lower exhaled nitric oxide levels compared with the control group at weeks 4, 8, and 12 (P<0.05). An overall group difference in log FEV1 PC20 (P=0.05) was found in favour of the NNF group as well. Significantly higher levels of EPA in plasma (P<0.01) and peripheral blood mononuclear cell (PBMC) (P<0.01) phospholipids in the NNF group compared with control group within 2 weeks indicated good adherence with daily NNF intake. There were no differences in adverse events for NNF vs. control after 12 weeks. CONCLUSIONS: Both NNF and control groups demonstrated improvement in asthma-free days. NNF-treated group had reduced biomarkers of disease activity. Rapid PBMC fatty acid composition changes reflected an anti-inflammatory profile. Dietary supplementation with NNF was safe and well tolerated (ClinicalTrials.gov number NCT01087710).

A prostacyclin agonist with thromboxane inhibitory activity for airway allergic inflammation in mice.

BACKGROUND: ONO-1301 is a novel drug that acts as a prostacyclin agonist with thromboxane A(2) (TxA(2)) synthase inhibitory activity. We investigated the effect of ONO-1301 on development of airway allergic inflammation. METHODS: Mice sensitized and challenged to ovalbumin (OVA) received ONO-1301, OKY-046 (TxA(2) synthase inhibitor), beraprost, a prostacyclin receptor (IP) agonist, ONO-1301 plus CAY10449 (selective IP antagonist) or vehicle during the challenge period. Twenty-four hours after the OVA challenge, airway hyperresponsiveness (AHR) to methacholine was assessed and bronchoalveolar lavage was performed. Lung specimens were excised for goblet cell staining and analysis of lung dendritic cells (DCs). Bone marrow-derived dendritic cells (BMDCs) were generated, in the presence or absence of drugs, for analysis of DC function. RESULTS: Mice that received ONO-1301 showed significantly lower AHR, airway eosinophilia, T-helper type 2 cytokine levels, mucus production and lung DCs numbers than vehicle-treated mice. These effects of ONO-1301 were mostly reversed by CAY10449. BMDCs treated with ONO-1301 alone showed lower DC functions, such as expression of costimulatory factors or stimulation to spleen T cells. CONCLUSIONS: These data suggest that ONO-1301 may suppress AHR and airway allergic inflammation through modulation of DCs, mainly mediated through the IP receptor. This agent may be effective as an anti-inflammatory drug in the treatment of asthma.

Lithium: a modulator of cyclic AMP-dependent events in lymphocytes?

Theophylline, salbutamol, isoproterenol, and dibutyryl cyclic AMP inhibited E-rosette formation by human T lymphocytes and immunoglobulin M secretion from human plaque-forming B cells and augmented T-suppressor cell activity in three patients with agammaglobulinemia. Lithium chloride increased mitogen-induced lymphocyte proliferation and inhibited suppressor cell activity. In the presence of lithium, the effects of all the drugs except dibutyryl cyclic AMP could be prevented. The data suggest a role for lithium in the modulation of cyclic AMP-dependent events in lymphocytes. Its potential role as an inhibitor of suppressor cell activity warrants further attention.

Purine nucleoside phosphorylase deficiency: altered kinetic properties of a mutant enzyme.

Erythrocyte purine nucleoside phosphorylase from two brothers had 0.5% of normal activity. It differed from the normal enzyme by a tenfold increase in the Michaelis constant for inosine, an inability of inosine to protect against thermal lability, and a more positive net charge. The altered kinetic properties may account for the milder disease in the patients compared to the previously described cases. The data provide evidence for a structural gene mutation and genetic heterogeneity in the new disease of purine nucleoside phosphorylase deficiency and T cell dysfunction.