The role of oral methotrexate as a steroid sparing agent in refractory eosinophilic asthma.

The use of oral methotrexate for refractory eosinophilic asthma in a tertiary asthma referral centre, Glenfield Hospital, Leicester, was evaluated between January 2006 and December 2014. The patients ( n = 61) were carefully phenotyped at baseline with markers of airway inflammation. In addition, a structured oral methotrexate proforma was utilized to evaluate response to therapy and adverse events. Oral steroid withdrawal was attempted 3 months after commencing treatment. Several outcomes were evaluated at 12 months, including both efficacy and adverse effects; 15% ( n = 9/61) responded by achieving a decrease in daily oral corticosteroid dose (mean 8.43 (±8.76) mg), although we were unable to identify factors that predicted a treatment response. There were no other significant changes in any other clinical outcome measures. There was a high rate of adverse events (19/61 (31%)), primarily gastrointestinal/hepatitis. Our findings support the use of biological agents in preference to using oral methotrexate as a steroid sparing agent at the first instance. In the event of failure of these agents, oral methotrexate remains a therapeutic option, which can be considered in highly specialist severe asthma centres.

The relationship between biomarkers of fungal allergy and lung damage in asthma.

BACKGROUND: Immunological biomarkers are the key to the diagnosis of allergic bronchopulmonary aspergillosis (ABPA) and fungal sensitisation, but how these relate to clinically relevant outcomes is unclear. OBJECTIVES: To assess how fungal immunological biomarkers are related to fixed airflow obstruction and radiological abnormalities in moderate to severe asthma. METHODS: Cross-sectional study of 431 asthmatics. Inflammatory biomarkers, lung function and an IgE fungal panel to colonising filamentous fungi, yeasts and fungal aeroallergens were measured. CT scans were scored for the presence of radiological abnormalities. Factor analysis informed the variables used in a k-means cluster analysis. Fixed airflow obstruction and radiological abnormalities were then mapped to these immunological variables in the cluster analysis. RESULTS: 329 (76.3%) subjects were sensitised to ≥ 1 fungi. Sensitisation to Aspergillus fumigatus and/or Penicillium chrysogenum was associated with a lower post-bronchodilator FEV1 compared with those not sensitised to fungi ((73.0 (95% CI 70.2-76) vs. 82.8 (95% CI 78.5-87.2)% predicted, P < 0.001), independent of atopic status (P = 0.005)), and an increased frequency of bronchiectasis (54.5%, P < 0.001), tree-in-bud (18.7%, P < 0.001) and collapse/consolidation (37.5%, P = 0.002). Cluster analysis identified three clusters: (i) hypereosinophilic (n = 71, 16.5%), (ii) high immunological biomarker load and high frequency of radiological abnormalities (n = 34, 7.9%) and (iii) low levels of fungal immunological biomarkers (n = 326, 75.6%). CONCLUSIONS AND CLINICAL RELEVANCE: IgE sensitisation to thermotolerant filamentous fungi, in particular A. fumigatus but not total IgE, is associated with fixed airflow obstruction and a number of radiological abnormalities in moderate to severe asthma. All patients with IgE sensitisation to A. fumigatus are at risk of lung damage irrespective of whether they meet the criteria for ABPA.

Responsiveness to oral prednisolone in severe asthma is related to the degree of eosinophilic airway inflammation.

BACKGROUND: Patients with severe asthma appear relatively corticosteroid resistant. Corticosteroid responsiveness is closely related to the degree of eosinophilic airway inflammation. The extent to which eosinophilic airway inflammation in severe asthma responds to treatment with systemic corticosteroids is not clear. OBJECTIVE: To relate the physiological and inflammatory response to systemic corticosteroids in asthma to disease severity and the baseline extent of eosinophilic inflammation. METHODS: Patients with mild/moderate and severe asthma were investigated before and after 2 weeks of oral prednisolone (Clintrials.gov NCT00331058 and NCT00327197). We pooled the results from two studies with common protocols. The US study contained two independent centres and the UK one independent centre. The effect of oral corticosteroids on FEV1 , Pc20, airway inflammation and serum cytokines was investigated. Baseline measurements were compared with healthy subjects. RESULTS: Thirty-two mild/moderate asthmatics, 50 severe asthmatics and 35 healthy subjects took part. At baseline, both groups of asthmatics had a lower FEV1 and Pc20 and increased eosinophilic inflammation compared to healthy subjects. The severe group had a lower FEV1 and more eosinophilic inflammation compared to mild/moderate asthmatics. Oral prednisolone caused a similar degree of suppression of eosinophilic inflammation in all compartments in both groups of asthmatics. There were small improvements in FEV1 and Pc20 for both mild/ moderate and severe asthmatics with a correlation between the baseline eosinophilic inflammation and the change in FEV1 . There was a ~50% reduction in the serum concentration of CXCL10 (IP-10), CCL22 (MDC), CCL17 (TARC), CCL-2 (MCP-1) and CCL-13 (MCP-4) in both asthma groups after oral corticosteroids. CONCLUSIONS AND CLINICAL RELEVANCE: Disease severity does not influence the response to systemic corticosteroids. The study does not therefore support the concept that severe asthma is associated with corticosteroid resistance. Only baseline eosinophilic inflammation was associated with the physiological response to corticosteroids, confirming the importance of measuring eosinophilic inflammation to guide corticosteroid use.

Protein phosphatase 5 mediates corticosteroid insensitivity in airway smooth muscle in patients with severe asthma.

BACKGROUND: The mechanisms driving glucocorticoid (GC) insensitivity in patients with severe asthma are still unknown. Recent evidence suggests the existence of GC-insensitive pathways in airway smooth muscle (ASM) caused by a defect in GC receptor (GRα) function. We examined whether other mechanisms could potentially explain the reduced sensitivity of ASM cells to GC in severe asthmatics. METHODS: Airway smooth muscle cells from healthy and severe asthmatic subjects were treated with TNF-α and responses to corticosteroids in both cohorts were compared by ELISA, immunoblot, immunohistochemistry and real-time PCR. Immunohistochemistry and flow cytometry assays were used to assess the expression of the protein phosphatase PP5 in endobronchial biopsies and ASM cells. RESULTS: The production of CCL11 and CCL5 by TNF-α was insensitive to both fluticasone and dexamethasone in ASM cells from severe asthmatic compared to that in healthy subjects. Fluticasone-induced GRα nuclear translocation, phosphorylation at serine 211 and expression of GC-induced leucine zipper (GILZ) were significantly reduced in ASM cells from severe asthmatics compared to responses in healthy subjects. Levels of PP5 were increased in ASM cells from severe asthmatics and PP5 knockdown using siRNA restored fluticasone repressive action on chemokine production and its ability to induce GRα nuclear translocation and GRE-dependent GILZ expression. In vivo PP5 expression was also increased in the ASM bundles in endobronchial biopsies in severe asthmatics. CONCLUSIONS: PP5-dependent impairment of GRα function represents a novel mechanism driving GC insensitivity in ASM in severe asthma.

Allergic Fungal Airway Disease.

Fungi are ubiquitous and form their own kingdom. Up to 80 genera of fungi have been linked to type I allergic disease, and yet, commercial reagents to test for sensitization are available for relatively few species. In terms of asthma, it is important to distinguish between species unable to grow at body temperature and those that can (thermotolerant) and thereby have the potential to colonize the respiratory tract. The former, which include the commonly studied Alternaria and Cladosporium genera, can act as aeroallergens whose clinical effects are predictably related to exposure levels. In contrast, thermotolerant species, which include fungi from the Candida, Aspergillus, and Penicillium genera, can cause a persistent allergenic stimulus independent of their airborne concentrations. Moreover, their ability to germinate in the airways provides a more diverse allergenic stimulus, and may result in noninvasive infection, which enhances inflammation. The close association between IgE sensitization to thermotolerant filamentous fungi and fixed airflow obstruction, bronchiectasis, and lung fibrosis suggests a much more tissue-damaging process than that seen with aeroallergens. This review provides an overview of fungal allergens and the patterns of clinical disease associated with exposure. It clarifies the various terminologies associated with fungal allergy in asthma and makes the case for a new term (allergic fungal airway disease) to include all people with asthma at risk of developing lung damage as a result of their fungal allergy. Lastly, it discusses the management of fungirelated asthma.

Developments in the field of allergy in 2014 through the eyes of Clinical and Experimental Allergy.

The pathogenesis of asthma continues to be a major topic of interest to our authors with reviews and original papers on the role of viruses, mechanisms of inflammation, biomarkers, and phenotypes of asthma being major topics. A number of papers described new treatments for asthma focusing on blocking the Th2 response reflecting the fact that two decades of work in this area is finally bearing fruit. The pathogenesis of chronic rhinosinusitis is a growing area of interest, but there has been less on the genetics of airways disease than in previous years possibly reflecting the degree of rigour (and therefore a smaller body of work), with which these sorts of studies are now being undertaken. There continues to be a wide range of papers dealing with mechanisms of allergic disease ranging from clinical-based studies to basic research and the use of in vivo animal models especially mice. As before, mechanisms and new approaches to immunotherapy are common themes. Several were published in the allergens section investigating modification of allergens to increase their effectiveness and reduce the risk of adverse events. Risk factors for allergic disease was a common theme in the epidemiology section and food allergy a common theme in clinical allergy with papers on the development of protocols to induce tolerance and attempts to find biomarkers to distinguish sensitization from allergic disease. This was another exciting year for the editors, and we hope the readers of the journal.

Developments in the field of allergy in 2013 through the eyes of Clinical and Experimental Allergy.

2013 was another exciting year for allergy in general and Clinical and Experimental Allergy in particular. In the field of asthma and rhinitis, there continued to be a focus on heterogeneity and phenotypes with increasing use of biostatistical techniques to determine clusters of similar populations. Obesity- and aspirin-associated disease are intriguing associations with asthma which were explored in a number of papers. We published a number of excellent papers on mechanisms of airway inflammation and how this relates to physiology, pathology, genetics and biomarkers in both human and experimental model systems. In terms of mechanisms, there is less on individual cell types in allergic disease at the moment, but the immunology of allergic disease continued to fascinate our authors. Another area that was popular both in the mechanisms and in the epidemiology sections was early life events and how these lead to allergic disease, with an increasing focus on the role of the microbiome and how this influences immune tolerance. In the clinical allergy section, oral immunotherapy for food allergy is clearly a major topic of interest at the moment as was in vitro testing to distinguish between sensitization and allergic disease. There was less on inhalant allergy this year, but a good representation from the drug allergy community including some interesting work on non-IgE-mediated mechanisms. In the allergen section, important new allergens continue to be discovered, but the major focus as in the last couple of years was on working out how component-resolved approaches can improve diagnosis and management of food and venom allergy.

Developments in the field of allergy in 2012 through the eyes of Clinical & Experimental Allergy.

In 2012, we received 683 submissions and published 20 editorials, 38 reviews, 11 letters and 128 original articles. This represents an acceptance rate for original papers in the range of 20%. About 30% of original papers were triaged not to go out to review, either because the editors did not feel they had sufficient priority for publication or because the topic did not feel right for the readers of the journal. We place great emphasis on obtaining sufficient high-quality reviews to make our decisions on publication fair and consistent. Inevitably, however, there is a degree of luck about what gets published and which papers miss out, and we are always happy to receive an appeal on our decisions either at the triage stage or after review. This gives us the opportunity to revisit the decision and revise it or explain in more detail to the authors the basis for the decision. Once again in 2012, we were delighted by the quality of the papers submitted and the breadth and depth of research into allergic disease that it revealed. The pattern of papers submitted was similar in previous years with considerable emphasis on all aspects of asthma and rhinitis. We were particularly pleased with our special issue on severe asthma. Elucidating mechanisms using either animal models or patients has always been a major theme of the journal, and the excellent work in these areas has been summarized by Harissios Vliagoftis with a particularly interesting section on early-life events guiding the development of allergic disease, which understandably continue to be a major theme of research. Magnus Wickman summarized the papers looking at the epidemiology of allergic disease including work from birth cohorts, which are an increasingly rich source of data on risk factors for allergic disease, and two papers on the epidemiology of anaphylaxis. Giovanni Passalacqua discussed the papers in the clinical allergy section of the journal, and Adriano Mari who runs the excellent Allergome website discussed the papers looking at allergens including characterization and the relative usefulness of allergen arrays versus single extracts in diagnosis and management.

Isolation of Aspergillus fumigatus from sputum is associated with elevated airborne levels in homes of patients with asthma.

Indoor bioaerosols, such as mold spores, have been associated with respiratory symptoms in patients with asthma; however, dose-response relationships and guidelines on acceptable levels are lacking. Furthermore, a causal link between mold exposure and respiratory infections or asthma remains to be established. The aim of this study was to determine indoor concentrations of Aspergillus fumigatus and a subset of clinically relevant fungi in homes of people with asthma, in relation to markers of airways colonization and sensitization. Air and dust samples were collected from the living room of 58 properties. Fungal concentrations were quantified using mold-specific quantitative PCR and compared with traditional microscopic analysis of air samples. Isolation of A. fumigatus from sputum was associated with higher airborne concentrations of the fungus in patient homes (P = 0.04), and a similar trend was shown with Aspergillus/Penicillium-type concentrations analyzed by microscopy (P = 0.058). No association was found between airborne levels of A. fumigatus and sensitization to this fungus, or dustborne levels of A. fumigatus and either isolation from sputum or sensitization. The results of this study suggest that the home environment should be considered as a potential source of fungal exposure, and elevated home levels may predispose people with asthma to airways colonization.

What is severe asthma?

Asthma is common, and some individuals are severely affected by it. Learned institutions have sought to provide a definition of 'severe asthma' to facilitate research and clinical care. This is a challenging undertaking given the difficulty in defining asthma and the lack of supportive evidence for a distinct severe asthma phenotype. In this review, we discuss the rationale for a definition of severe asthma and the relative merits of the sequential attempts that have been made to produce such a definition. The difficulty in disentangling control and severity is highlighted, as is the heterogeneity of phenotype in severe asthma, and potential for misclassification. We conclude that the search for a singular definition of severe asthma is problematic, though likely to continue. We suggest the alternative strategy of using classifiers with a specific aim related to symptoms, pathophysiology or service provision.

Phenotyping airways disease: an A to E approach.

The airway diseases asthma and chronic obstructive pulmonary disease (COPD) are heterogeneous conditions with overlapping pathophysiological and clinical features. It has previously been proposed that this heterogeneity may be characterized in terms of five relatively independent domains labelled from A to E, namely airway hyperresponsiveness (AHR), bronchitis, cough reflex hypersensitivity, damage to the airways and surrounding lung parenchyma, and extrapulmonary factors. Airway hyperresponsiveness occurs in both asthma and COPD, accounting for variable day to day symptoms, although the mechanisms most likely differ between the two conditions. Bronchitis, or airway inflammation, may be predominantly eosinophilic or neutrophilic, with different treatments required for each. Cough reflex hypersensitivity is thought to underlie the chronic dry cough out of proportion to other symptoms that can occur in association with airways disease. Structural changes associated with airway disease (damage) include bronchial wall thickening, airway smooth muscle hypertrophy, bronchiectasis and emphysema. Finally, a variety of extrapulmonary factors may impact upon airway disease, including rhinosinusitis, gastroesophageal reflux disease, obesity and dysfunctional breathing. This article discusses the A to E concept in detail and describes how this framework may be used to assess and treat patients with airway diseases in the clinic.

Developments in the field of allergy in 2011 through the eyes of Clinical and Experimental Allergy.

As in previous years, we felt it would be of value to our readership to summarize the new information provided by the authors who have published in Clinical and Experimental Allergy in 2011 and set this in the context of recent advances in our understanding of the pathogenesis and management of allergic disease in all its many manifestations. In 2011, about 210 articles were published in Clinical and Experimental Allergy including editorials, reviews, opinion articles, guidelines, letters, book reviews and of course at the heart of the journal, papers containing original data. As before, this review is divided into sections based on the way the journal is structured, although this year we have grouped together all the papers dealing with mechanisms of allergic disease, whether they involve patients (clinical mechanisms), pure in vitro studies (basic mechanisms) or animal models (experimental models), as we felt this was a more coherent way to deal with the subject. In the field of asthma and rhinitis, the relationship between airway inflammation and airway dysfunction was of perennial interest to investigators, as were phenotypes and biomarkers. Aspirin hypersensitivity appeared in studies in several papers and there was new interest in asthma in the elderly. The mechanisms involved in allergic disease describe advances in our understanding of T cell responses, the relationship between inflammation and disease, mast cell and basophil activation, steroid resistance and novel therapies. In the section dealing with epidemiology, studies seeking to identify risk factors for allergic disease including vitamin D are prominent, as once again are studies investigating gene-environment interactions. The clinical allergy section focuses on drug allergy, food allergy and immunotherapy. The area of oral immunotherapy for food allergy is well covered and we were grateful to Stephen Durham for guest editing an outstanding special issue on immunotherapy in the centenary year of Leonard Noon's pioneering work. Lastly, in the field of allergens, the interest in component-resolved diagnosis continues to grow and there are also articles describing important novel cultivars and the effect of food processing on the allergenic properties of foods. Another terrific year, full of important and high-quality work,which the journal has been proud to bring to the allergy community.

Isolation of filamentous fungi from sputum in asthma is associated with reduced post-bronchodilator FEV1.

BACKGROUND: Fungal sensitization is common in severe asthma, but the clinical relevance of this and the relationship with airway colonization by fungi remain unclear. The range of fungi that may colonize the airways in asthma is unknown. OBJECTIVE: To provide a comprehensive analysis on the range of filamentous fungi isolated in sputum from people with asthma and report the relationship with their clinico-immunological features of their disease. METHODS: We recruited 126 subjects with a diagnosis of asthma, 94% with moderate-severe disease, and 18 healthy volunteers. At a single stable visit, subjects underwent spirometry; sputum fungal culture and a sputum cell differential count; skin prick testing to both common aeroallergens and an extended fungal panel; specific IgE to Aspergillus fumigatus. Fungi were identified by morphology and species identity was confirmed by sequencing. Four patients had allergic bronchopulmonary aspergillosis. RESULTS: Forty-eight percent of asthma subjects were IgE-sensitized to one fungal allergen and 22% to ≥ 2. Twenty-seven different taxa of filamentous fungi were isolated from 54% of their sputa, more than one species being detected in 17%. This compared with 3 (17%) healthy controls culturing any fungus (P < 0.01). Aspergillus species were most frequently cultured in isolation followed by Penicillium species. Post-bronchodilator FEV (1) (% predicted) in the subjects with asthma was 71(± 25) in those with a positive fungal culture vs. 83 (± 25) in those culture-negative, (P < 0.01). CONCLUSION AND CLINICAL RELEVANCE: Numerous thermotolerant fungi other than A. fumigatus can be cultured from sputum of people with moderate-to-severe asthma; a positive culture is associated with an impaired post-bronchodilator FEV (1) , which might be partly responsible for the development of fixed airflow obstruction in asthma. Sensitization to these fungi is also common.

Developments in the field of allergy in 2010 through the eyes of Clinical and Experimental Allergy.

In 2010 over 200 articles were published in Clinical and Experimental Allergy including editorials, reviews, opinion articles, letters, book reviews and of course at the heart of the journal, papers containing original data which have moved the field of allergy forward on a number of fronts. For the third year running the editors felt it would be of value to summarize the key messages contained in these papers as a snapshot of where the cutting edge of research into allergic disease is leading. We have broadly followed the sections of the journal, although this year the mechanistic articles are grouped together and the studies involving experimental models of disease are discussed throughout the paper. In the field of asthma and rhinitis phenotypes and biomarkers continue to a major pre-occupation of our authors. There is continued interest in mechanisms of inflammation and disordered lung function with the mouse model of asthma continuing to offer new insights. There is also a steady flow of papers investigating new therapies, including those derived from plants and herbs, although many are mechanistic with too few high quality clinical trials. The mechanisms involved in allergic disease are well covered with many strong papers using clinical material to ask relevant questions. Pro-pre and snybiotics continue to be of major interest to our authors and this remains a controversial and complicated field. The discipline of epidemiology has retained its interest in risk factors for the development of allergic disease with a view to refining and debating the reasons for the allergy epidemic. There is continued interest in the relationship between helminthic disease and allergy with a new twist in 2010 involving studies using infection with helminths as a potential treatment. The genetics of allergic disease continues to be very productive, although the field has moved on from only investigating single nucleotide polymorphisms of candidate genes to Genome Wide Association Studies and an increasing and welcome emphasis on gene-environment interactions. In the field of clinical allergy there is steady flow of papers describing patterns of drug allergy with renewed interest in reactions to contrast media, but food allergy is the major area of interest in this section of the journal. Lastly in the field of allergens there is a growing interest in the role of component resolved diagnosis in improving the diagnosis and management of allergic disease. Another excellent year, full of fascinating and high quality work, which the journal has been proud to bring to the allergy community.

Adhesion to fibronectin prolongs eosinophil survival.

We have investigated the effect of adhesion to fibronectin (Fn) on the survival of eosinophils in culture. Peripheral blood eosinophils from normal human donors were separated by immunomagnetic selection and cultured in RPMI on Fn- (100 micrograms/ml) coated microtiter plates for up to 96 h. Survival was measured by trypan blue exclusion. There was a significant enhancement of eosinophil survival with Fn as compared with both bovine serum albumin-coated and uncoated wells (p < 0.05-0.01). Fn-induced eosinophil survival was comparable to that obtained with exogenous interleukin 3 (IL-3) or granulocyte/macrophage colony-stimulating factor (GM-CSF) and was inhibitable by antibodies against Fn, very late antigen 4 (VLA-4), IL-3, and GM-CSF. Supernatants from Fn-, but not BSA-coated wells contained picogram amounts of IL-3 and GM-CSF, and eosinophils cultured on Fn for 24 h expressed mRNA for GM-CSF as determined by in situ hybridization. Therefore, Fn prolongs eosinophil survival in culture by triggering autocrine generation of cytokines by eosinophils. Since neutrophils lack VLA-4, this could provide a partial explanation for the preferential accumulation of eosinophils at sites of allergic inflammation, as well as the predominant tissue localization of eosinophils in healthy individuals.

Eosinophil adhesion to nasal polyp endothelium is P-selectin-dependent.

Tissue eosinophilia is a characteristic feature of a number of inflammatory diseases including asthma and nasal polyposis. Eosinophil migration into tissues is controlled in part by interactions between eosinophil adhesion receptors and counter-structures on the vascular endothelium. To determine the receptors used by eosinophils to adhere to vascular endothelium in allergic inflammation we have adapted the Stamper-Woodruff frozen section assay (FSA) to study eosinophil adhesion to nasal polyp endothelium. Immunohistology indicated that intercellular adhesion molecule 1 (ICAM-1), E-selectin and P-selectin were well expressed by nasal polyp endothelium, whereas expression of vascular cell adhesion molecule 1 (VCAM-1) was weak or absent. Unstimulated human peripheral blood eosinophils adhered specifically to nasal polyp endothelium. Adherence was temperature and divalent cation-dependent and saturable at cell densities > 5 x 10(6) cells/ml. Eosinophil adhesion was almost completely inhibited by a monoclonal antibody (mAb) against P-selectin and by a chimeric molecule consisting of the Fc portion of human IgG and the lectin binding domain of P-selectin, which binds to the P-selectin ligand on leucocytes. Anti-Mac-1 mAb partially inhibited eosinophil adhesion whereas mAb against E-selectin, L-selectin, ICAM-1, VCAM-1, very late activation antigen 4, and lymphocyte function-associated antigen 1 had no effect. P-selectin is stored in intracellular granules within the endothelial cell and in vitro is only transiently expressed. To determine if P-selectin was expressed on the membrane of the nasal polyp endothelium we compared P-selectin expression in normal skin and nasal polyps after acetone fixation, which permeabilizes cells, and paraformaldehyde, which only allows staining of membrane expressed receptors. In the skin, good expression was seen with acetone fixation but no expression was seen after paraformaldehyde treatment, whereas in nasal polyps, similar expression was observed with both fixatives. In addition immunofluorescence with confocal microscopy demonstrated lumenal staining of nasal polyp endothelium indicating that P-selectin was located on the surface of endothelial cells while in skin only an intracellular granular distribution was apparent. Lastly, whereas eosinophils bound consistently to nasal polyp endothelium, no binding was observed to blood vessels in normal skin further supporting the idea that eosinophils were binding to membrane expressed and not intracellular P-selectin. The importance of P-selectin in eosinophil adhesion to nasal polyp endothelium suggests that P-selectin antagonists may be effective at inhibiting eosinophil accumulation at sites of allergic inflammation.

Distinct mutations in two patients with leukocyte adhesion deficiency and their functional correlates.

Two patients with leukocyte adhesion deficiency (LAD), one with a moderate phenotype (patient 14) and one with a severe phenotype (patient 2) who had been shown to have a normal sized beta subunit protein precursor, were analyzed in an attempt to determine the molecular basis for their disease. RNase mapping located possible mutations to two distinct but adjacent regions of the beta subunit cDNA. Sequencing of patient-derived cDNA clones in this region revealed a C for T difference at amino acid 149 in patient 14 which resulted in the substitution of a leucine for a proline, and an A for G substitution at amino acid 169 in patient 2 which mutated a glycine to an arginine. The mutated amino acids are in a region of the cDNA that is highly conserved between the beta subunits of the integrin family and are identical in all known integrin beta subunits. Co-transfection of the beta subunit cDNA containing the patient 2 mutation with the wild-type alpha subunit of LFA-1 in a mammalian expression system resulted in no expression of LFA-1. In the case of the mutation in patient 14 there was markedly diminished expression of LFA-1 with loss of function and loss of the epitope for a number of anti-beta mAbs. Normal half-life of the mutant beta subunits, and previous demonstration of a lack of alpha/beta complex formation during biosynthesis in patient cells, suggest a defect in association with the alpha subunit. Association with beta is required for expression of the alpha subunit of LFA-1. Loss of functional expression with both of these beta subunit mutations suggests that they lie in a site critical for association with the alpha subunit.

Messenger RNA expression of the cytokine gene cluster, interleukin 3 (IL-3), IL-4, IL-5, and granulocyte/macrophage colony-stimulating factor, in allergen-induced late-phase cutaneous reactions in atopic subjects.

Cryostat sections from skin biopsies from 24-h allergen-induced late-phase cutaneous reactions (LPR) in 14 human atopic subjects were hybridized with 35S-labeled RNA probes for a number of cytokines. mRNA was detected for interleukin 3 (IL-3) (8/14), IL-4 (10/14), IL-5 (11/14), and granulocyte/macrophage colony-stimulating factor (GM-CSF) (13/14). Only 5 of 14 gave hybridization signals for IL-2, and 0 of 14 for interferon gamma. Biopsies from diluent controls gave only occasional weak signals. These results suggest that cells infiltrating the site of the 24-h LPR transcribe mRNA for the IL-3, IL-4, IL-5, and GM-CSF gene cluster and support the hypothesis that atopy is associated with preferential activation of cells having a similar cytokine profile to the murine T helper type 2 subset.

Expression of mRNA and immunoreactivity for the granulocyte/macrophage colony-stimulating factor in activated human eosinophils.

Using in situ hybridization, we have shown that activated human peripheral blood eosinophils express mRNA for granulocyte/macrophage colony-stimulating factor (GM-CSF). Between 15 and 27% of eosinophils gave positive hybridization signals for GM-CSF mRNA after stimulation with the calcium ionophore A23187 or interferon gamma, and 4 and 6% after incubation with interleukin 3 (IL-3) or IL-5. Activated eosinophils also gave specific immunoreactivity with an anti-GM-CSF polyclonal antibody, suggesting translation of the mRNA. These data indicate that eosinophils may be an important source of GM-CSF at sites of allergic inflammation. Furthermore, the identification of GM-CSF production by human eosinophils suggests that the pro-inflammatory potential of this cell type may be substantially greater than hitherto recognized.

Lymphocyte CC chemokine receptor 9 and epithelial thymus-expressed chemokine (TECK) expression distinguish the small intestinal immune compartment: Epithelial expression of tissue-specific chemokines as an organizing principle in regional immunity.

The immune system has evolved specialized cellular and molecular mechanisms for targeting and regulating immune responses at epithelial surfaces. Here we show that small intestinal intraepithelial lymphocytes and lamina propria lymphocytes migrate to thymus-expressed chemokine (TECK). This attraction is mediated by CC chemokine receptor (CCR)9, a chemoattractant receptor expressed at high levels by essentially all CD4(+) and CD8(+) T lymphocytes in the small intestine. Only a small subset of lymphocytes in the colon are CCR9(+), and lymphocytes from other tissues including tonsils, lung, inflamed liver, normal or inflamed skin, inflamed synovium and synovial fluid, breast milk, and seminal fluid are universally CCR9(-). TECK expression is also restricted to the small intestine: immunohistochemistry reveals that intense anti-TECK reactivity characterizes crypt epithelium in the jejunum and ileum, but not in other epithelia of the digestive tract (including stomach and colon), skin, lung, or salivary gland. These results imply a restricted role for lymphocyte CCR9 and its ligand TECK in the small intestine, and provide the first evidence for distinctive mechanisms of lymphocyte recruitment that may permit functional specialization of immune responses in different segments of the gastrointestinal tract. Selective expression of chemokines by differentiated epithelium may represent an important mechanism for targeting and specialization of immune responses.