Histology-based blood leukocyte profiling reveals parallel Th2 and Th17 signatures in seasonal allergic rhinitis.

BACKGROUND: Allergic rhinitis (AR), a common condition in the westernized world, is suggested to be more immunologically complex than the archetypical 'Th2' inflammation. New approaches are needed to decode this complexity. AIMS/OBJECTIVES: In this study, we explored a novel histology-based analysis for circulating blood leukocyte profiling in 16 patients with seasonal AR outside and during the pollen season. MATERIAL AND METHODS: Leukocytes were purified with minimal ex-vivo artefacts, embedded into agarose-paraffin pellets for immunohistochemistry-based immune cell profiling. Blood leukocyte mapping was performed. RESULTS: Samples collected during the pollen season had statistically increased eosinophils, neutrophils, monocytes, and CD8+ T-lymphocytes compared to the off-season baseline. In contrast, no change was observed for CD20+ B-lymphocytes and CD3+ T-lymphocytes. Subclassification of CD4+ T-helper cells demonstrated a parallel and significant expansion of Th2 and Th17-cells during the pollen season, while Th1-cells remained unchanged. Whereas absolute basophils numbers were unaltered, the basophil markers GATA2 and CPA3 increased during the pollen season. CONCLUSIONS AND SIGNIFICANCE: This study introduces a novel and applicable method for systemic immune cell screening and provides further evidence of complex and parallel Th2 and Th17-immune signatures in seasonal AR. It also forwards GATA2 and CPA3 as potential biomarkers for ongoing allergic inflammation.

Basophil sensitivity reflects long-term clinical outcome of subcutaneous immunotherapy in grass pollen-allergic patients.

BACKGROUND: Allergic rhinoconjunctivitis is a public health problem. Allergen Immunotherapy is an effective and safe treatment, that modifies the natural course of allergic disease and induces long-term tolerance. OBJECTIVE: To correlate basophil and antibody biomarkers of subcutaneous immunotherapy to clinical outcomes and cellular changes in target tissue. METHODS: Adults suffering from allergic rhinoconjunctivitis due to grass pollen allergy were randomized to receive subcutaneous immunotherapy (n = 18) or to an open control group (n = 6). Patients reported daily symptom and medication scores and weekly rhinitis related quality of life scores during four pollen seasons. Biomarkers were measured every 3 months for three years treatment and every 6 months in the follow-up year. Nasal and cutaneous allergen challenge tests were performed annually. Leukocyte subsets were assessed in nasal mucosa biopsies at baseline and after treatment. RESULTS: Subcutaneous immunotherapy led to a 447-fold decrease in basophil sensitivity during the first treatment year. This remained 100-fold lower than baseline during the 3 year-treatment period and 10-fold lower during the follow-up year (n = 18, P = .03). Decrease in basophil sensitivity after three weeks of treatment predicted long-term improvement in seasonal combined symptom and medication scores (á¿¥=-0.69, P = .0027) during three years of treatment. AUC of IgE-blocking factor correlated to nasal allergen challenge (á¿¥ = 0.63, P = .0012) and SPT (á¿¥ = 0.45, P = .03). Plasma cell numbers in the nasal mucosa increased during treatment (P = .02). CONCLUSION: Decrease in basophil sensitivity after three weeks of subcutaneous allergen immunotherapy predicted the clinical outcome of this treatment.

Effectiveness of bronchial thermoplasty in patients with severe refractory asthma: Clinical and histopathologic correlations.

BACKGROUND: The effectiveness of bronchial thermoplasty (BT) has been reported in patients with severe asthma, yet its effect on different bronchial structures remains unknown. OBJECTIVE: We sought to examine the effect of BT on bronchial structures and to explore the association with clinical outcome in patients with severe refractory asthma. METHODS: Bronchial biopsy specimens (n = 300) were collected from 15 patients with severe uncontrolled asthma before and 3 months after BT. Immunostained sections were assessed for airway smooth muscle (ASM) area, subepithelial basement membrane thickness, nerve fibers, and epithelial neuroendocrine cells. Histopathologic findings were correlated with clinical parameters. RESULTS: BT significantly improved asthma control and quality of life at both 3 and 12 months and decreased the numbers of severe exacerbations and the dose of oral corticosteroids. At 3 months, this clinical benefit was accompanied by a reduction in ASM area (median values before and after BT, respectively: 19.7% [25th-75th interquartile range (IQR), 15.9% to 22.4%] and 5.3% [25th-75th IQR], 3.5% to 10.1%, P < .001), subepithelial basement membrane thickening (4.4 µm [25th-75th IQR, 4.0-4.7 µm] and 3.9 µm [25th-75th IQR, 3.7-4.6 µm], P = 0.02), submucosal nerves (1.0 ‰ [25th-75th IQR, 0.7-1.3 ‰] immunoreactivity and 0.3 ‰ [25th-75th IQR, 0.1-0.5 ‰] immunoreactivity, P < .001), ASM-associated nerves (452.6 [25th-75th IQR, 196.0-811.2] immunoreactive pixels per mm2 and 62.7 [25th-75th IQR, 0.0-230.3] immunoreactive pixels per mm2, P = .02), and epithelial neuroendocrine cells (4.9/mm2 [25th-75th IQR, 0-16.4/mm2] and 0.0/mm2 [25th-75th IQR, 0-0/mm2], P = .02). Histopathologic parameters were associated based on Asthma Control Test scores, numbers of exacerbations, and visits to the emergency department (all P ≤ .02) 3 and 12 months after BT. CONCLUSION: BT is a treatment option in patients with severe therapy-refractory asthma that downregulates selectively structural abnormalities involved in airway narrowing and bronchial reactivity, particularly ASM, neuroendocrine epithelial cells, and bronchial nerve endings.

Elevated exhaled nitric oxide in allergen-provoked asthma is associated with airway epithelial iNOS.

BACKGROUND: Fractional exhaled nitric oxide is elevated in allergen-provoked asthma. The cellular and molecular source of the elevated fractional exhaled nitric oxide is, however, uncertain. OBJECTIVE: To investigate whether fractional exhaled nitric oxide is associated with increased airway epithelial inducible nitric oxide synthase (iNOS) in allergen-provoked asthma. METHODS: Fractional exhaled nitric oxide was measured in healthy controls (n = 14) and allergic asthmatics (n = 12), before and after bronchial provocation to birch pollen out of season. Bronchoscopy was performed before and 24 hours after allergen provocation. Bronchial biopsies and brush biopsies were processed for nitric oxide synthase activity staining with nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d), iNOS immunostaining, or gene expression analysis of iNOS by real-time PCR. NADPH-d and iNOS staining were quantified using automated morphometric analysis. RESULTS: Fractional exhaled nitric oxide and expression of iNOS mRNA were significantly higher in un-provoked asthmatics, compared to healthy controls. Allergic asthmatics exhibited a significant elevation of fractional exhaled nitric oxide after allergen provocation, as well as an accumulation of airway eosinophils. Moreover, nitric oxide synthase activity and expression of iNOS was significantly increased in the bronchial epithelium of asthmatics following allergen provocation. Fractional exhaled nitric oxide correlated with eosinophils and iNOS expression. CONCLUSION: Higher fractional exhaled nitric oxide concentration among asthmatics is associated with elevated iNOS mRNA in the bronchial epithelium. Furthermore, our data demonstrates for the first time increased expression and activity of iNOS in the bronchial epithelium after allergen provocation, and thus provide a mechanistic explanation for elevated fractional exhaled nitric oxide in allergen-provoked asthma.

Early phase resolution of mucosal eosinophilic inflammation in allergic rhinitis.

BACKGROUND: It is widely assumed that apoptosis of eosinophils is a central component of resolution of allergic airway disease. However, this has not been demonstrated in human allergic airways in vivo. Based on animal in vivo observations we hypothesised that steroid-induced resolution of human airway eosinophilic inflammation involves inhibition of CCL5 (RANTES), a CC-chemokine regulating eosinophil and lymphocyte traffic, and elimination of eosinophils without evident occurrence of apoptotic eosinophils in the diseased tissue. OBJECTIVE: To determine mucosal eosinophilia, apoptotic eosinophils, general cell apoptosis and cell proliferation, and expression of CCL5 and CCL11 (eotaxin) in human allergic airway tissues in vivo at resolution of established symptomatic eosinophilic inflammation. METHODS: Twenty-one patients with intermittent (birch and/or grass) allergic rhinitis received daily nasal allergen challenges for two seven days' periods separated by more than two weeks washout. Five days into these "artificial pollen seasons", nasal treatment with budesonide was instituted and continued for six days in a double blinded, randomized, placebo-controlled, and crossover design. This report is a parallel group comparison of nasal biopsy histochemistry data obtained on the final day of the second treatment period. RESULTS: Treatments were instituted when clinical rhinitis symptoms had been established. Compared to placebo, budesonide reduced tissue eosinophilia, and subepithelial more than epithelial eosinophilia. Steroid treatment also attenuated tissue expression of CCL5, but CCL11 was not reduced. General tissue cell apoptosis and epithelial cell proliferation were reduced by budesonide. However, apoptotic eosinophils were not detected in any biopsies, irrespective of treatment. CONCLUSIONS: Inhibition of CCL5-dependent recruitment of cells to diseased airway tissue, and reduced cell proliferation, reduced general cell apoptosis, but not increased eosinophil apoptosis, are involved in early phase steroid-induced resolution of human allergic rhinitis.

The proinflammatory CXC-chemokines GRO-alpha/CXCL1 and MIG/CXCL9 are concomitantly expressed in ulcerative colitis and decrease during treatment with topical corticosteroids.

BACKGROUND: Ulcerative colitis is characterized by relapsing mucosal inflammation where the lesions include tissue-damaging granulocytes. In addition, T cells and natural killer (NK) cells play important pathophysiologic roles. Chemokines are a large family of peptides that play key roles in the regulation of inflammation. The CXC-chemokines, growth-related oncogene (GRO)-alpha/CXCL1 and interleukin (IL)-8/CXCL8, both recruit neutrophils and possess mitogenic properties, whereas the interferon-dependent CXC-chemokines monokine induced by gamma-interferon (MIG)/CXCL9, interferon-gamma inducible protein of 10 kD/CXCL10, and IFN-inducible T cell alpha chemoattractant/CXCL11 recruit and activate T cells and NK cells. MATERIALS AND METHODS: The expression of CXC-chemokines was studied in eight controls and in 11 patients suffering from ulcerative colitis in the distal part of the colon, before and during topical treatment with corticosteroids. Perfusates (obtained before, after 7 days, and after 28 days of treatment) and pinch biopsies (obtained before and after 28 days of treatment) were collected by colonoscopy. The rectal release of GRO-alpha and MIG was determined by enzyme-linked immunosorbent assay (ELISA), and tissue expression of the chemokines was detected in colonic tissue by immunohistochemistry. RESULTS: In perfusates, high levels of GRO-alpha, IL-8, and MIG were detected compared with controls (p=0.02, 0.005, and p=0.03, respectively). During treatment with corticosteroids, both GRO-alpha and MIG decreased. In clinical nonresponders, characterized by sustained inflammation, the levels of GRO-alpha and MIG remained elevated. Both epithelial cells and granulocytes, present in the submucosa, expressed GRO-alpha and MIG as detected by immunohistochemistry. CONCLUSIONS: CXC-chemokines are likely to be important in the pathophysiology of ulcerative colitis and may become targets for novel treatment strategies. In addition, GRO-alpha may serve as a marker of disease activity.

Neural expression and increased lavage fluid levels of secretoneurin in seasonal allergic rhinitis.

Secretoneurin is a neuropeptide potentially involved in migration of eosinophils, monocytes, and dendritic cells. Whether secretoneurin is present in the human airway mucosa and whether it is released at ongoing allergic airway inflammation is currently unknown. In patients with allergic rhinitis, we have explored the occurrence of secretoneurin in nasal mucosal biopsies and lavage fluids before and during natural allergen exposure. Immunohistochemical analysis revealed an abundance of nerves displaying secretoneurin immunoreactivity, which were distributed predominantly around blood vessels and submucosal glands. A majority of nerve fibers containing vesicular acetylcholine transporter, tyrosine hydroxylase, calcitonin gene-related peptide, and vasoactive intestinal peptide were also secretoneurin-immunoreactive, indicating a localization of secretoneurin in cholinergic, adrenergic, and sensory nerves. Lavage fluid levels of secretoneurin were increased at allergen exposure (p < 0.01-0.05). Levels of secretoneurin did not correlate with eosinophil cationic protein (rho = 0.1, p = 0.7). We conclude that secretoneurin has a widespread occurrence in nasal mucosal nerves of patients with seasonal allergic rhinitis and that increased nasal lavage fluid levels of secretoneurin may characterize ongoing allergen exposure. These data favor a role of secretoneurin in the local traffic of immune cells in human airway mucosa.