Short-term subcutaneous grass pollen immunotherapy under the umbrella of anti-IL-4: A randomized controlled trial.

BACKGROUND: Allergen immunotherapy is currently the only disease-modifying treatment available for allergic rhinitis and allergic asthma. OBJECTIVES: We sought to evaluate the induction of sustained tolerance to allergen when anti-IL-4 was combined with a suboptimal course of grass pollen subcutaneous immunotherapy (SCIT) using the allergen-induced skin late-phase response (LPR) and exploratory immune monitoring as surrogate markers of therapeutic response. METHODS: In this randomized, double-blind, 3-group parallel design trial, 37 participants with seasonal allergic rhinitis received suboptimal SCIT (30,000 standardized quality units) in combination with anti-IL-4 (VAK694) and suboptimal SCIT (30,000 standardized quality units) plus placebo antibody or double placebo (placebo SCIT and placebo antibody) restricted to 13 weeks before the grass pollen season. The primary end point was the size of the LPR at 12 months. Exploratory end points included measures of the immunomodulatory activity of treatment by using IL-4 and IL-10 FluoroSpot assays, flow cytometry of T cells, and measurement of IgE, IgG4, and facilitated antigen binding. RESULTS: Both active treatment arms led to a substantial and sustained reduction of the LPR with no additional suppression with addition of anti-IL-4. Treatment with anti-IL-4 and SCIT compared with SCIT alone led to a sustained reduction in allergen-specific IL-4-producing cell counts (P < .01). Both active treatment arms led to induction of dual IL-4/IL-10-producing cells during the pollen season. CONCLUSION: The combination of anti-IL-4 with SCIT provided no additional benefit over SCIT alone in suppressing the allergen-induced skin LPR. A larger trial is needed to assess whether the observed ex vivo downregulation of TH2 responses might translate into clinical benefit.

Efficacy and safety of multiple doses of QGE031 (ligelizumab) versus omalizumab and placebo in inhibiting allergen-induced early asthmatic responses.

BACKGROUND: Omalizumab is an established anti-IgE therapy for the treatment of allergic diseases that prevents IgE from binding to its receptor. QGE031 is an investigational anti-IgE antibody that binds IgE with higher affinity than omalizumab. OBJECTIVE: This study compared the effects of QGE031 with those of omalizumab on clinical efficacy, IgE levels, and FcεRI expression in a clinical model of allergic asthma. METHODS: Thirty-seven patients with mild allergic asthma were randomized to subcutaneous omalizumab, placebo, or QGE031 at 24, 72, or 240 mg every 2 weeks for 10 weeks in a double-blind, parallel-group multicenter study. Inhaled allergen challenges and skin tests were conducted before dosing and at weeks 6, 12, and 18, and blood was collected until 24 weeks after the first dose. RESULTS: QGE031 elicited a concentration- and time-dependent change in the provocative concentration of allergen causing a 15% decrease in FEV1 (allergen PC15) that was maximal and approximately 3-fold greater than that of omalizumab (P = .10) and 16-fold greater than that of placebo (P = .0001) at week 12 in the 240-mg cohort. Skin responses reached 85% suppression at week 12 in the 240-mg cohort and were maximal at week 18. The top doses of QGE031 consistently suppressed skin test responses among subjects but had a variable effect on allergen PC15 (2-fold to 500-fold change). QGE031 was well tolerated. CONCLUSION: QGE031 has greater efficacy than omalizumab on inhaled and skin allergen responses in patients with mild allergic asthma. These data support the clinical development of QGE031 as a treatment of asthma.

Intravenous anti-IL-13 mAb QAX576 for the treatment of eosinophilic esophagitis.

BACKGROUND: Eosinophilic esophagitis (EoE) is a chronic allergic disease with limited treatment options. OBJECTIVE: We evaluated QAX576, an mAb against IL-13, in the treatment of patients with EoE. METHODS: Patients (18-50 years) with proton pump inhibitor-resistant esophageal eosinophilia received intravenous QAX576 (6 mg/kg) or placebo (2:1) at weeks 0, 4, and 8 and were followed for 6 months. The primary end point was the responder rate for a greater than 75% decrease in peak eosinophil counts at week 12. Efficacy was to be declared if the lower 90% confidence limit for the proportion of responders on QAX576 was 35% or greater. Secondary end points included changes in esophageal eosinophil counts, symptoms assessed by questionnaire scores, and quantification of a series of biomarkers. RESULTS: Twenty-three patients completed the study up to week 12, and 18 continued to the end of the study. For the proximal and distal esophageal biopsies combined, the responder rate was 12.5% (90% confidence limit, 1% to 43%) with placebo, compared to 40.0% (90% confidence limit, 22% to 61%) with QAX576. Although the primary end point was not met, the mean esophageal eosinophil count decreased by 60% with QAX576 versus an increase of 23% with placebo (P = .004), and the decrease was sustained up to 6 months. There was a trend for improved symptoms, particularly dysphagia. QAX576 improved expression of EoE-relevant esophageal transcripts, including eotaxin-3, periostin, and markers of mast cells and barrier function, for up to 6 months after treatment. QAX576 was well tolerated. CONCLUSIONS: QAX576 significantly improved intraepithelial esophageal eosinophil counts and dysregulated esophageal disease-related transcripts in adults with EoE in a sustained manner.

Impact of botanical oils on polyunsaturated fatty acid metabolism and leukotriene generation in mild asthmatics.

BACKGROUND: Dietary supplementation with botanical oils that contain n-6 and n-3 eighteen carbon chain (18C)-PUFA such as γ linolenic acid (GLA, 18:3n-6), stearidonic acid (SDA, 18:4n-3) and α linolenic acid (ALA, 18:3n-3) have been shown to impact PUFA metabolism, alter inflammatory processes including arachidonic acid (AA) metabolism and improve inflammatory disorders. METHODS: The diet of mild asthmatics patients was supplemented for three weeks with varying doses of two botanical seed oils (borage oil [Borago officinalis, BO] and echium seed oil [Echium plantagineum; EO]) that contain SDA, ALA and GLA. A three week wash out period followed. The impact of these dietary manipulations was evaluated for several biochemical endpoints, including in vivo PUFA metabolism and ex vivo leukotriene generation from stimulated leukocytes. RESULTS: Supplementation with several EO/BO combinations increased circulating 20-22 carbon (20-22C) PUFAs, including eicosapentaenoic acid (EPA), docosapentaenoic acid (DPA), and dihommo-gammalinolenic acid (DGLA), which have been shown to inhibit AA metabolism and inflammation without impacting circulating AA levels. BO/EO combinations also inhibited ex vivo leukotriene generation with some combinations attenuating cysteinyl leukotriene generation in stimulated basophils by >50% and in stimulated neutrophils by >35%. CONCLUSIONS: This study shows that dietary supplementation with BO/EO alters 20-22C PUFA levels and attenuates leukotriene production in a manner consistent with a reduction in inflammation.

Group V phospholipase A2 in bone marrow-derived myeloid cells and bronchial epithelial cells promotes bacterial clearance after Escherichia coli pneumonia.

Group V-secreted phospholipase A(2) (GV sPLA(2)) hydrolyzes bacterial phospholipids and initiates eicosanoid biosynthesis. Here, we elucidate the role of GV sPLA(2) in the pathophysiology of Escherichia coli pneumonia. Inflammatory cells and bronchial epithelial cells both express GV sPLA(2) after pulmonary E. coli infection. GV(-/-) mice accumulate fewer polymorphonuclear leukocytes in alveoli, have higher levels of E. coli in bronchoalveolar lavage fluid and lung, and develop respiratory acidosis, more severe hypothermia, and higher IL-6, IL-10, and TNF-α levels than GV(+/+) mice after pulmonary E. coli infection. Eicosanoid levels in bronchoalveolar lavage are similar in GV(+/+) and GV(-/-) mice after lung E. coli infection. In contrast, GV(+/+) mice have higher levels of prostaglandin D(2) (PGD(2)), PGF(2α), and 15-keto-PGE(2) in lung and express higher levels of ICAM-1 and PECAM-1 on pulmonary endothelial cells than GV(-/-) mice after lung infection with E. coli. Selective deletion of GV sPLA(2) in non-myeloid cells impairs leukocyte accumulation after pulmonary E. coli infection, and lack of GV sPLA(2) in either bone marrow-derived myeloid cells or non-myeloid cells attenuates E. coli clearance from the alveolar space and the lung parenchyma. These observations show that GV sPLA(2) in bone marrow-derived myeloid cells as well as non-myeloid cells, which are likely bronchial epithelial cells, participate in the regulation of the innate immune response to pulmonary infection with E. coli.

Group V secretory phospholipase A2 modulates phagosome maturation and regulates the innate immune response against Candida albicans.

Phospholipase A(2) (PLA(2)) hydrolyzes the sn-2 position of cell membrane phospholipids to release fatty acids and lysophospholipids. We have previously reported that group V secretory PLA(2) (sPLA(2)) translocates from the Golgi and recycling endosomes of mouse peritoneal macrophages to newly formed phagosomes and regulates the phagocytosis of zymosan, suggesting a role in innate immunity. Here we report that in macrophages lacking group V sPLA(2), phagosome maturation was reduced 50-60% at early time points while the binding of zymosan was unimpaired. The ability of group V sPLA(2) to regulate phagocytosis extended to phagocytosis of IgG- and complement-opsonized sheep RBC. Moreover, macrophages lacking group V sPLA(2) had delays in phagocytosis, phagosome maturation, and killing of Candida albicans. Cytokine production and eicosanoid generation were not impaired by the lack of group V sPLA(2). Furthermore, in a model of systemic candidiasis, mice lacking group V sPLA(2) had an increased fungal burden in the kidney, liver, and spleen at day 7 postinfection and increased mortality. Thus, group V sPLA(2) regulates phagocytosis through major phagocytic receptors and contributes to the innate immune response against C. albicans by regulating phagocytosis and killing through a mechanism that is likely dependent on phagolysosome fusion.

Group V secreted phospholipase A2 contributes to LPS-induced leukocyte recruitment.

Secreted phospholipases A(2) (sPLA(2)s) are well known for their contribution in the biosynthesis of inflammatory eicosanoids. These enzymes also participate in the inflammatory process by regulating chemokine production and protein expression of adhesion molecules. The majority of sPLA(2) isoforms are up-regulated by proinflammatory stimuli such as bacterial lipopolysaccharide (LPS), which predominantly increases the expression of group V sPLA(2) (sPLA(2)-V). Furthermore, it has recently been shown that sPLA(2)-V is a critical messenger in the regulation of cell migration during allergic airway responsiveness. Herein, we investigated the effect of sPLA(2)-V on LPS-mediated leukocyte recruitment and its capacity to modulate adhesion molecule expression. We conducted our study in the murine air pouch model, using sPLA(2)-V null mice (sPLA(2)-V(-/-)) and control wild-type (WT) littermates. We observed that LPS (1 microg/ml)-mediated leukocyte emigration in sPLA(2)-V(-/-) was attenuated by 52% and 86% upon 6 and 12 h of treatment respectively, as compared to WT mice. In WT mice, treatment with the cell-permeable sPLA(2) inhibitor (12-epi-scalaradial; SLD) reduced LPS-mediated leukocyte recruitment by 67%, but had no additional inhibitory effect in sPLA(2)-V(-/-) mice. Protein analyses from the air pouch skin were carried out upon LPS-challenge, and the expression of intercellular adhesion molecule (ICAM)-1 and vascular cell adhesion molecule (VCAM)-1 were both significantly reduced in sPLA(2)-V(-/-) mice as compared to control WT mice. Together, our data demonstrate the role of sPLA(2)-V in LPS-induced ICAM-1 and VCAM-1 protein overexpression and leukocyte recruitment, supporting the contribution of sPLA(2)-V in the development of inflammatory innate immune responses.

Differential expression of leukocyte immunoglobulin-like receptors on cord-blood-derived human mast cell progenitors and mature mast cells.

The leukocyte Ig-like receptors (LILRs) comprise a family of cell-surface immunoregulatory receptors with activating and inhibitory members. The inhibitory LILRs possess cytoplasmic ITIMs that down-regulate signaling by nonreceptor tyrosine kinase cascades. The activating members have a truncated cytoplasmic domain and signal through the FcR gamma chain. We examined the expression of LILRs on human mast cells during their development in vitro. Progenitor mast cells expressed cell surface inhibitory LILRB1, -B2, -B3, and -B4 and activating LILRA1. However, although mature cord blood-derived mast cells (hMCs) had detectable mRNA encoding multiple LILRs, none were expressed on the cell surface. Culture of progenitor mast cells or hMCs with various cytokine combinations failed to retain or induce cell surface expression of the LILRs. It is interesting that hMCs expressed LILRB5 in cytoplasmic granules and upon cross-linking of the high-affinity IgE receptor, released LILRB5 into the culture medium. Our results demonstrate that LILRs are developmentally regulated in human mast cells and that LILRB5 is expressed in mast cell granules and the release of soluble LILRB5 following IgE FcR-dependent stimulation, which has potential for amplification of mast cell-dependent, inflammatory responses.

Mechanisms by which botanical lipids affect inflammatory disorders.

Changes in diet over the past century have markedly altered the consumption of fatty acids. The dramatic increase in the ingestion of saturated and n-6 fatty acids and concomitant decrease in n-3 fatty acids are thought to be a major driver of the increase in the incidence of inflammatory diseases such as asthma, allergy, and atherosclerosis. The central objective of the Center for Botanical Lipids at Wake Forest University School of Medicine and the Brigham and Women's Hospital is to delineate the mechanisms by which fatty acid-based dietary supplements inhibit inflammation leading to chronic human diseases such as cardiovascular disease and asthma. The key question that this center addresses is whether botanical n-6 and n-3 fatty acids directly block recognized biochemical pathways or the expression of critical genes that lead to asthma and atherosclerosis. Dietary supplementation with flaxseed oil, borage oil, and echium oil affects the biochemistry of fatty acid metabolism and thus the balance of proinflammatory mediators and atherogenic lipids. Supplementation studies have begun to identify key molecular and genetic mechanisms that regulate the production of lipid mediators involved in inflammatory and hyperlipidemic diseases. Echium oil and other oils containing stearidonic acid as well as botanical oil combinations (such as echium and borage oils) hold great promise for modulating inflammatory diseases.

Group V sPLA2: classical and novel functions.

Group V sPLA(2) is unique among the family of secretory sPLA(2) enzymes in being able to bind to cell membranes through both interfacial-binding and through binding to proteoglycan. The function of group V sPLA(2) as an enzyme and its cross-talk with cPLA(2)alpha in initiating eicosanoid generation is well documented. Evidence, though, is emerging on the ability of this molecule to act as a regulator of several intracellular and extracellular pathways independently of its ability to provide arachidonic acid for eicosanoid generation, acting within the cell or as a secreted enzyme. In this article we will provide an overview of the properties of the enzyme and how they relate to our current understanding of its function.

Group V secretory phospholipase A2 amplifies the induction of cyclooxygenase 2 and delayed prostaglandin D2 generation in mouse bone marrow culture-derived mast cells in a strain-dependent manner.

Activation of mouse bone marrow-derived mast cells (BMMC) with stem cell factor (SCF) or IgE and antigen elicits exocytosis and an immediate phase of prostaglandin (PG) D(2) and leukotriene (LT) C(4) generation. Activation of BMMC by SCF, IL-1beta and IL-10 elicits a delayed phase of PGD(2) generation dependent on cyclooxygenase (COX) 2 induction. Cytosolic phospholipase A(2) alpha provides arachidonic acid in both phases and amplifies COX-2 induction. Pharmacological experiments implicate an amplifying role for secretory (s) PLA(2). We used mice lacking the gene encoding group V sPLA(2) (Pla2g5-/-) to definitively test its role in eicosanoid generation by BMMC. Pla2g5-/- BMMC on a C57BL/6 genetic background showed a modest reduction in exocytosis and immediate PGD(2) generation after activation with SCF or with IgE and antigen, while LTC(4) generation was not modified. Delayed-phase PGD(2) generation and COX-2 induction were reduced approximately 35% in C57BL/6 Pla2g5-/- BMMC and were restored by exogenous PGE(2). There was no deficit in either phase of eicosanoid generation by Pla2g5-/- BMMC on a BALB/c background. Thus, group V sPLA(2) amplifies COX-2 expression and delayed phase PGD(2) generation in a strain-dependent manner; it has at best a limited role in immediate eicosanoid generation by BMMC.

Monocytes and Macrophages in Asthma.

There is increasing evidence implicating the central role of cells of monocyte/macrophage lineage in the pathogenesis of bronchial asthma. This evidence comes from studies on peripheral blood monocytes. BAL fluid and cells and, more recently, airway immunohistochemistry. Elucidation of the mechanisms of macrophage interactions may eventually lead to novel approaches in anti-asthma therapy.

Phospholipase A(2).

Considerable progress has been made in characterizing the individual participant enzymes and their relative contributions in the generation of eicosanoids, lipid mediators derived from arachidonic acid, such as prostaglandins and leukotrienes. However, the role of individual phospholipase (PL) A(2) enzymes in providing arachidonic acid to the downstream enzymes for eicosanoid generation in biologic processes has not been fully elucidated. In this review, we will provide an overview of the classification of the families of PLA(2) enzymes, their putative mechanisms of action, and their role(s) in eicosanoid generation and inflammation.

LTC4 synthase polymorphism modifies efficacy of botanical seed oil combination in asthma.

UNLABELLED: Botanical seed oils reduce the generation of leukotrienes in patients with asthma. Our objective was to determine the efficacy of a botanical seed oil combination against airflow obstruction in asthma, and to determine the pharmacogenomic effect of the leukotriene C4 synthase (LTC4S) polymorphism A-444C. We conducted a randomized, double-blind, placebo-controlled, cross-over clinical trial in mild to moderate asthmatics to determine the change in FEV1 after 6 weeks of therapy with borage and echium seed oils versus corn oil placebo. We also examined the effect of the variant LTC4S -444C allele on the change in lung function. We did not identify a difference in FEV1 in the study cohort as a whole (n = 28), nor in the group of A homozygotes. In the C allele carriers (n = 9), FEV1 improved by 3% after treatment with borage and echium seed oils and declined by 4% after placebo corn oil (p = 0.02). All 9 C allele carriers demonstrated an improvement in their FEV1 on active treatment compared to placebo as compared to only 7 out of 19 A allele homozygotes (p = 0.007). We observed transient differences in ex vivo leukotriene generation from circulating basophils and granulocytes. We did not observe significant differences in urinary LTE4 levels. We conclude that compared to corn oil, a combination of borage and echium seed oils improves airflow obstruction in mild to moderate asthmatics who carry the variant allele in the LTC4S gene (A-444C). Botanical oil supplementation may have therapeutic potential in asthma if used in a personalized manner. TRIAL REGISTRATION: This trial was registered at http://www.clinicaltrials.gov as NCT00806442.

A novel anti-inflammatory role for secretory phospholipase A2 in immune complex-mediated arthritis.

Phospholipase A2 (PLA2) catalyses the release of arachidonic acid for generation of lipid mediators of inflammation and is crucial in diverse inflammatory processes. The functions of the secretory PLA2 enzymes (sPLA2), numbering nine members in humans, are poorly understood, though they have been shown to participate in lipid mediator generation and the associated inflammation. To further understand the roles of sPLA2 in disease, we quantified the expression of these enzymes in the synovial fluid in rheumatoid arthritis and used gene-deleted mice to examine their contribution in a mouse model of autoimmune erosive inflammatory arthritis. Contrary to expectation, we find that the group V sPLA2 isoform plays a novel anti-inflammatory role that opposes the pro-inflammatory activity of group IIA sPLA2. Mechanistically, group V sPLA2 counter-regulation includes promotion of immune complex clearance by regulating cysteinyl leukotriene synthesis. These observations identify a novel anti-inflammatory function for a PLA2 and identify group V sPLA2 as a potential biotherapeutic for treatment of immune-complex-mediated inflammation.

Effect of dietary fatty acids on inflammatory gene expression in healthy humans.

Over the past 100 years, changes in the food supply in Western nations have resulted in alterations in dietary fatty acid consumption, leading to a dramatic increase in the ratio of omega-6 (omega6) to omega3 polyunsaturated fatty acids (PUFA) in circulation and in tissues. Increased omega6/omega3 ratios are hypothesized to increase inflammatory mediator production, leading to higher incidence of inflammatory diseases, and may impact inflammatory gene expression. To determine the effect of reducing the omega6/omega3 ratio on expression of inflammatory pathway genes in mononuclear cells, healthy humans were placed on a controlled diet for 1 week, then given fish oil and borage oil for an additional 4 weeks. Serum and neutrophil fatty acid composition and ex vivo leukotriene B(4) production from stimulated neutrophils were measured at the start and end of the supplementation period and after a 2-week washout. RNA was isolated from mononuclear cells and expression of PI3K, Akt, NFkappaB, and inflammatory cytokines was measured by real-time PCR. A marked increase was seen in serum and neutrophil levels of long-chain omega3 PUFA concomitant with a reduction in the omega6/omega3 PUFA ratio (40%). The ex vivo capacity of stimulated neutrophils to produce leukotriene B(4) was decreased by 31%. Expression of PI3Kalpha and PI3Kgamma and the quantity of PI3Kalpha protein in mononuclear cells was reduced after supplementation, as was the expression of several proinflammatory cytokines. These data reveal that PUFA may exert their clinical effects via their capacity to regulate the expression of signal transduction genes and genes for proinflammatory cytokines.

Induced loss of Syk in human basophils by non-IgE-dependent stimuli.

In the general population, Syk expression in human basophils is highly variable and correlates well with the IgE-mediated responsiveness of these cells. Previous studies established that IgE-mediated stimulation results in loss of Syk expression. The current studies investigated whether stimulation through other receptors results in loss of Syk. Two classes of stimulation were examined, those that operate through the kinase Syk and those that operate through a GTP-binding protein. These studies demonstrated that aggregation of leukocyte Ig-like receptor LILRA-2 resulted in phosphorylation of Syk and c-Cbl, was inhibited by a third generation Syk inhibitor with an expected IC(50), and induced histamine release in strict proportion to release induced by anti-IgE Ab. Stimulation of LILRA-2 for 18 h resulted in modest loss of Syk that correlated with the more profound loss of Syk induced by anti-IgE Ab. Human recombinant histamine-releasing factor has also recently been shown to induce Syk phosphorylation and in the current studies has also been shown to induce loss of Syk in 18-h cultures. fMLP stimulation for 18 h was also found to induce modest loss of Syk. fMLP induced phosphorylation of c-Cbl that was sustained for at least 45 min. Phosphorylation of c-Cbl was inhibited by a Syk kinase inhibitor but with an IC(50) that was not consistent with Syk activity, suggesting another kinase was responsible for Cbl phosphorylation following fMLP. These studies demonstrate that it is possible to induce the loss of Syk expression in human basophils by a non-IgE-dependent mechanism and even by a mechanism that does directly involve Syk in the reaction complex.

Group V secretory PLA2 regulates TLR2-dependent eicosanoid generation in mouse mast cells through amplification of ERK and cPLA2alpha activation.

Mast cells may be activated through Toll-like receptors (TLRs) for the dose- and time-dependent release of eicosanoids. However, the signaling mechanisms of TLR-dependent rapid eicosanoid generation are not known. We previously reported a role for group V secretory phospholipase A(2) (PLA(2)) in regulating phagocytosis of zymosan and the ensuing eicosanoid generation in mouse resident peritoneal macrophages, suggesting a role for the enzyme in innate immunity. In the present study, we have used gene knockout mice to define an essential role for MyD88 and cytosolic PLA(2)alpha in TLR2-dependent eicosanoid generation. Furthermore, in mast cells lacking group V secretory PLA(2), the time course of phosphorylation of ERK1/2 and of cPLA(2)alpha was markedly truncated, leading to attenuation of eicosanoid generation in response to stimulation through TLR2, but not through c-kit or FcepsilonRI. These findings provide the first dissection of the mechanisms of TLR-dependent rapid eicosanoid generation, which is MyD88-dependent, requires cPLA(2)alpha, and is amplified by group V sPLA(2) through its regulation of the sequential phosphorylation and activation of ERK1/2 and cPLA(2)alpha. The findings support the suggestion that group V sPLA(2) regulates innate immune responses.