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Background The glucocorticoid receptor (GR) is able to participate in regulation of transcription by a variety of mechanisms, one of which involves DNA binding and recruitment of regulatory cofactors. The best-studied forms of the receptor are the 777-amino-acid α and the 742-amino-acid β variants. The β isoform, which does not bind cortisol in human subjects, has been proposed to be a dominant-negative inhibitor of the transcriptional activation-competent GRα isoform. Objective GRα has roles in both transcriptional activation and repression. We wished to determine the influence of GRβ on genes that are normally transcriptionally repressed by glucocorticoids. We studied IL5 and IL13 , which both contribute to the asthmatic phenotype. Methods We used transient transfection systems and coimmunoprecipitation experiments to determine whether GRβ has repressive activity on the promoters of the human IL5 and IL13 genes. Results GRβ is able to act as a transcriptional repressor of cytokine genes and mediates its function through the recruitment of histone deacetylase complexes. Conclusion GRα and GRβ act in a similar manner on IL5 and IL13 promoters, serving to repress transcription. In this circumstance GRβ does not act as a dominant-negative inhibitor of GRα.

Background Inflammation is a key feature of asthma and allergic disease. The proinflammatory cytokines IL-4 , IL-5 , and IL-13 are clustered on chromosome 5q with GM-CSF in close proximity, and each of these cytokines has been implicated in the pathogenesis of inflammatory disease. Although the expression of IL-4, IL-5, and IL-13 is coordinately regulated, the T H 2-associated transcription factor c-Maf is thought to be involved only in the regulation of IL-4, the cytokine thought to be the main driver of T H 2 differentiation. Objective We sought to determine whether c-Maf influenced the expression of proinflammatory cytokines other than IL-4 in the Jurkat human T-cell line. Methods RT-PCR, ELISA, and promoter-driven CAT assays were used to determine the effect of c-Maf overexpression on cytokine genes. A biotinylated oligo pulldown assay was used to demonstrate recruitment of c-Maf to the GM-CSF promoter. Results We found that in addition to induction of IL-4, c-Maf could upregulate GM-CSF expression at both mRNA and protein levels, and that c-Maf could strongly activate the promoters of GM-CSF and IL-4 but not IL-5 . Recruitment of c-Maf to the -33 to -97 bp region of the GM-CSF promoter was demonstrated. Conclusion We propose a novel role for c-Maf in the transcriptional regulation of GM-CSF in human T cells. Clinical implications These data suggest that c-Maf may be a therapeutic target affecting both IL-4 and GM-CSF.

Background Overexpression of the transcriptional regulatory factor activator protein 1 might contribute to T-cell glucocorticoid (GC) refractoriness in GC-resistant asthma. Objective We sought to address the hypothesis that clinically GC-resistant asthma is accompanied by failure of systemic GCs to inhibit phosphorylation of c-jun and c-jun N-terminal kinase (JNK) in bronchial mucosal cells. Methods We performed enumeration of total (CD45 + ) leukocytes and cells expressing c-fos and total and phosphorylated c-jun and JNK in bronchial biopsy sections from 9 GC-sensitive and 17 GC-resistant asthmatic patients taken before and after oral prednisolone (40 mg/1.72 m 2 body surface area daily for 14 days) using specific antibodies, immunohistochemistry, and image analysis. Results At baseline, mean total (CD45 + ) mucosal leukocytes, total cells expressing phosphorylated c-jun and JNK, and mean percentages of cells in which these molecules were phosphorylated were similar in both groups, whereas mean total numbers of c-fos–immunoreactive cells were increased in the GC-resistant asthmatic subjects ( P = .04). After prednisolone, the mean total cells expressing phosphorylated c-jun and JNK and the mean percentages of cells in which these molecules were phosphorylated were significantly reduced in the GC-sensitive ( P ≤ .02) but not the GC-resistant asthmatic subjects. Mean total CD45 + leukocytes and c-fos–immunoreactive cells were not significantly altered in either group. Conclusion Clinical GC responsiveness in asthma is accompanied by reduced phosphorylation of bronchial mucosal c-jun and JNK, a phenomenon not seen in resistant patients. Clinical implications Dysregulation of activator protein 1 activation leading to clinical GC resistance might reflect identifiable environmental influences and is a target for future therapy.

Background Prostaglandin (PG) E 2 binds to 4 G-protein–coupled receptors designated EP 1 through EP 4 . Although PGE 2 plays an immunomodulatory role in asthma, there is little information on the expression of PGE 2 receptors in this disease. Objective We hypothesized that profiles of E-prostanoid (EP) receptor expression are altered on asthmatic bronchial inflammatory cells in vivo and further altered by allergen challenge in vivo and proinflammatory mediators in vitro . Methods The numbers and phenotypes of EP 1-4 immunoreactive induced sputum cells from atopic asthmatics (n=13; before and 24 hours after allergen inhalational challenge) and normal controls (n=9; 3 after saline challenge) and EP 1-4 expression on purified blood eosinophils from both groups (n=4 for each) before and after stimulation with LPS and/or IL-5 in vitro were measured by using single and double immunocytochemistry. Results Subsets of sputum cells of all phenotypes expressed all 4 EP receptors in both patients with asthma and controls. There were significantly greater numbers of macrophages expressing all 4 EP receptors and increased percentages of macrophages expressing EP 2 and EP 4 in patients with asthma compared with controls. Allergen bronchial challenge of patients with asthma was associated with a selective influx of eosinophils, but the percentages of these and other leukocytes expressing all 4 EP receptors were unchanged. Compared with sputum, only small percentages of peripheral blood eosinophils expressed each receptor, but this was increased by culture with exogenous IL-5 or LPS. Conclusion E-prostanoid receptor expression is increased on airway macrophages of patients with asthma at baseline and may be altered on eosinophils after allergen challenge in vivo in response to inflammatory stimuli.

Background: The genes encoding the cytokines IL-4, IL-5, IL-13, and GM-CSF are located in close proximity on human chromosome 5. We have previously described a motif in the promoters of these genes with a common palindromic sequence: CCAAG…CTTGG. These half sites are variably spaced and, within GMCSF , flank a second internal palindromic site. The GMCSF palindrome was shown to act as a strong enhancer of transcription when linked to a heterologous promoter. Objective: We sought to determine whether the related palindromic elements from IL4 , IL5 , and IL13 also act as enhancers of gene transcription. Methods: Reporter plasmids driven by palindromic elements were transfected into Jurkat T and HeLa cells to determine enhancer activity, and T-cell extracts were used in electrophoretic mobility shift and methylation interference assays to determine the DNA-binding characteristics of palindrome-binding proteins. Results: Enhancer activity was observed in unactivated T cells and HeLa cells, whereas in T cells the IL4 palindrome mediated an activation-specific response. Mutational analysis of this element revealed that both halves of the CCAAG…CTTGG palindrome and part of the intervening sequence were essential for mediating interaction with protein complexes. Electrophoretic mobility shift assays suggested that the 4 palindromes bound similar factors because complexes formed between Jurkat nuclear extracts and each palindromic element showed identical mobility, and these elements were able to cross-compete for binding. Conclusions: These data suggest that constitutively expressed factors are involved in mediating the enhancer function of these elements in T cells and that these factors might either be present or have closely related homologues in other cell types. Also, an activation-dependent factor might be recruited to modulate the function of the IL4 element. (J Allergy Clin Immunol 2003;111:826-32.)

Background: APC 366, a selective inhibitor of mast cell tryptase, has been shown to inhibit antigen-induced early asthmatic response (EAR), late asthmatic response (LAR), and bronchial hyperresponsiveness (BHR) in a sheep model of allergic asthma. Objective: The purpose of this study was to investigate the effects of APC 366 on antigen-induced EAR, LAR, and BHR in mild atopic asthmatics not on any anti-inflammatory therapy. Methods: Sixteen mild atopic asthmatics, each with a demonstrable antigen-induced EAR, LAR, and BHR to histamine, were recruited into this randomized, double-blinded, crossover study. APC 366 (5 mg)/placebo was administered by aerosol inhalation 3 times per day on treatment days 1 through 4. Allergen challenge was carried out on day 4. Histamine challenge was performed the following morning, 1 hour after final dosing. Results: Subjects were shown to have a significantly smaller overall mean area under the curve for the LAR ( P = .012) and mean maximum fall in FEV 1 for the LAR ( P = .007) after pretreatment with APC 366 in comparison with placebo. No significant effects on BHR were demonstrable. Although the EAR was reduced by 18% after treatment with APC 366 in comparison with placebo, this was not statistically significant. Conclusion: Short-term repeated administration of APC 366 significantly reduced the magnitude of antigen-induced LAR in atopic asthmatics, which supports the role of mast cell tryptase in the pathophysiology of the LAR. (J Allergy Clin Immunol 2001;107:1039-45.)

Background: Glucocorticoid-resistant bronchial asthma is characterized by failure of corticosteroids to suppress key asthma-relevant, cell–mediated inflammatory responses in the airways. Objective: The mechanism of this phenomenon is not clear but may involve aberrant expression of the β-isoform of the glucocorticoid receptor. Methods: We have measured expression of the α- and β-glucocorticoid receptor isoforms in tuberculin-driven cutaneous cell–mediated inflammatory lesions in people with asthma who are glucocorticoid sensitive and resistant after 9 days of therapy with oral prednisolone (40 mg/day) or matching placebo in a random order, crossover design. Results: After placebo therapy, the mean numbers of cells expressing glucocorticoid receptor α immunoreactivity in the lesions evoked in glucocorticoid-sensitive and -resistant patients with asthma were statistically equivalent. The numbers of cells expressing glucocorticoid receptor β were significantly elevated in the patients who were glucocorticoid resistant, resulting in an 8-fold higher ratio of expression of glucocorticoid receptor α/glucocorticoid receptor β in the patients who were glucocorticoid sensitive. Glucocorticoid receptor α/glucocorticoid receptors β were colocalized to the same cells. Oral prednisolone therapy was associated with a significant decrease in the numbers of cells expressing glucocorticoid receptor α but not glucocorticoid receptor β in the subjects who were glucocorticoid sensitive. No significant change was found in the numbers of cells expressing glucocorticoid receptor α and glucocorticoid receptor β in the patients who were glucocorticoid resistant. Prednisolone therapy reduced the ratio of glucocorticoid receptor α/glucocorticoid receptor β expression for the patients who were glucocorticoid sensitive to a level seen in the patients who were glucocorticoid resistant before therapy. Conclusion: Because glucocorticoid receptor β inhibits α-glucocorticoid receptor–mediated transactivation of target genes, the increased expression of glucocorticoid receptor β in inflammatory cells might be a critical mechanism for conferring glucocorticoid resistance. (J Allergy Clin Immunol 2000;105:943-50.)

Background: Increased levels of histamine have been previously demonstrated in patients with chronic idiopathic urticaria. Objective: The purpose of the study was to determine whether increased numbers of mast cells are present in lesional skin from such patients. Methods: Mast cells have been quantified in lesional ( n = 11) and nonlesional ( n = 9) skin from patients with chronic idiopathic urticaria and compared with site-matched skin from healthy control subjects ( n = 10). Mast cells were identified by using a sensitive, double-labeling immunohistochemical technique with specific monoclonal antibodies to mast cell tryptase and chymase and quantified under light microscopy. Results: No significant differences in mast cell numbers from lesional, nonlesional, or control skin were observed ( p > 0.1, Student's t test). In both patients with urticaria and control subjects, more than 99% of cutaneous mast cells contained tryptase and chymase. Conclusions: These data indicate that increased skin histamine in chronic idiopathic urticaria is not caused by increased mast cells and may alternatively reflect an increase in histamine content per mast cell, enhanced mast cell activation, or recruitment of basophils into skin in patients with chronic idiopathic urticaria. (J ALLERGY CLIN IMMUNOL 1995;96:360-4.)

Impaired metabolism of histamine in the skin of patients with chronic idiopathic urticaria (CIU) might explain the observed enhanced and prolonged skin responses to intradermal histamine. Histamine metabolism was measured in homogenates from unaffected forearm skin in nine patients with CIU and in skin of age- and sex-matched control subjects with a radiochromatographic assay, and the results are expressed as nanograms of histamine metabolized per milligram of protein per hour. Endogenous histamine content was determined by RIA. There was a highly significant increase in endogenous histamine content in the skin of patients with urticaria (407.8 +/- 188.3 ng/mg of protein) compared with that in skin of control subjects (240.0 +/- 73.0 ng/mg of protein) (mean +/- 1 SD; p less than 0.02), which suggests either an increase in mast cell number or histamine concentration per cell. No significant difference was observed in the metabolism of histamine between patient and control group; therefore, an alternative mechanism may underlie differences in skin reactivity to histamine.

Previous studies have suggested that there is a genetic predisposition for the development of asthma and atopy. A recent study has also demonstrated that there is a striking link between chromosome 11q and the IgE response underlying asthma and rhinitis. To assess the linkage between chromosome 11q (region D11S97) and atopy or bronchial hyperresponsiveness (BH), we have studied nine families of two and, in many instances, three generations with the index case having asthma and/or atopy. With variable number of tandem repeat analysis with the probe, p lambda-MS.51, we have been unable to confirm a significant link between region D11S97 of chromosome 11q and either atopy or BH to methacholine. We have demonstrated that atopy and BH produce similar log of odds scores with linkage analysis at each recombination fraction from 0.001 to 0.5 with both Hinf1 and Taq1 restriction digests and that the use of either a positive skin prick test or positive RAST as a definition of atopy does not significantly alter the log of odds score.

This study is of the effect of the blockade of histamine H1 receptors by a long-acting antihistamine on the immediate and late clinical response to antigen (Ag) and on the recruitment of eosinophils in the late-phase cutaneous reaction. Ten adult volunteers with late-phase reactions to the intradermal injection of either Dermatophagoides pteronyssinus or Phleum pratense (timothy) pollen performed a double-blind, crossover study. Each volunteer took astemizole, 10 mg, or identical placebo, daily for 2 weeks. Ag in the concentration that induced a late reaction in the screening visit was injected intradermally at the end of each drug period. The early reaction was measured serially for 30 minutes and the late reaction at 4 and 6 hours. Biopsies of the Ag and control sites were also performed at 6 hours. After a 6-week washout period, subjects then took the opposite medication for 2 weeks and returned for skin testing and biopsy. Skin testing demonstrated that astemizole inhibited the immediate response to both histamine and allergen but had no effect on the late response at 4 hours and at 6 hours. Biopsy specimens revealed no significant effect on eosinophil recruitment at 6 hours. We conclude that histamine H1-receptor blockade has no effect on the late cutaneous reaction to Ag.

Human eosinophils (EOSs) stimulated under optimal conditions with 5 × 10 8 unopsonized zymosan particles at 37 °C for 30 minutes produced an average total immunoreactive leukotriene (LT) C 4 of 1.6 ng per 10 6 EOSs, and 30% to 60% of the generated product remained cell associated. The dose-response characteristics of zymosan-induced LTC 4 generation were different from those of phagocytosis, suggesting that the two events were independent. Pretreatment of EOSs with 10 −8 mol/L of formyl-methionyl-leucyl-phenylalanine for 30 minutes led to a twofold to fivefold augmentation of LTC 4 generation by cells subsequently activated by unopsonized zymosan. Optimal EOS activation with 1 μmol/L of the calcium ionophore A23187 at 37 °C for 15 minutes produced more than 100 times greater quantities of LTC 4 than with zymosan. The amount of immunoreactive LTC 4 that remained cell associated after calcium ionophore A23187 stimulation reached a maximum after 5 minutes and then declined. Of the relatively small amount generated in the first minute, 71% was cell associated, but this figure declined to 9% after 15 minutes, by which time there had been a redistribution of the LTC 4 to the supernatant. Inflammatory leukocytes may respond to zymosan because the cells recognize either one or both of its major polysaccharide components, glucan and mannan. Glucan, but not mannan, stimulated EOSs to generate LTC 4 in a dose- and time-dependent manner. Under optimal conditions, there was no significant difference in the total quantities of LTC 4 elaborated by EOSs stimulated by glucan and by unopsonized zymosan. This suggests that zymosan may induce leukotriene generation in the human EOS through a glucan recognition mechanism.

We have measured plasma histamine, serum neutrophil chemotactic activity (NCA) and complement (C3 and C4) over a 24-hour period in patients experiencing either early- and late-phase (dual) or single early asthmatic reactions to inhaled allergens. There was a significant biphasic elevation in plasma histamine, which paralleled the fall in forced expiratory volume in 1 sec in 10 patients with dual responses, whereas in seven subjects with single early reactions, only a single early increase in histamine concentrations was observed. In general, in the individual subjects, the changes in plasma histamine paralleled both the elevations in serum NCA and the decreases in forced expiratory volume in 1 sec. By gel filtration on Sephacryl S-400, anion exchange chromatography on DEAE Sephacel, and chromatofocusing with Polybuffer Exchanger 94, the major NCA of both the early and the late reactions was associated with proteins having an estimated molecular size of 600,000 daltons, an elution from DEAE Sephacel at 0.15M to 0.30M of NaCl (pH 8.1), and a pI of approximately 6.5. There were no appreciable changes in serum C3 and C4 up to 24 hr after challenge in subjects with late-hase responses. The patterns of asthmatic response were not related to either the total or allergen-specific serum IgE or IgG 4 concentrations. These results support the view that mediators of hypersensitivity participate in late-phase as well as early asthmatic reactions.

The release of heat-stable neutrophil chemotactic activity (NCA) has been detected after challenge of isolated human lung tissue with anti-IgE. The major NCA released (NCA L ) had similar physicochemical properties to the NCA detected in the circulation of asthmatic subjects after bronchial challenge with specific antigen (NCA AG ). NCA L and NCA AG (1) had molecular weights of approximately 600,000 daltons as estimated by Sephacryl S-300 gel-filtration chromatography; (2) both eluted from DEAE-Sephacel (pH 7.8) between 0.1 and 0.2 molar NaCI; (3) had isoelectric points of between 6.5 and 6.8 as determined by chromatofocusing on Polybuffer Exchanger 94. In contrast to NCA AG , lung-derived neutrophil chemotactic activity appeared to be more heterogeneous after gel-filtration and anion-exchange chromatography. The release of NCA was complete by 15 min and there was no evidence of further release up to 12 hr. These observations indicate that high-molecular-weight NCA released from human lung tissue has similar properties to NCA AG and would support the view that NCA AG originates from lung tissue after antigen bronchial challenge in asthmatic subjects.

Four subjects with clinical histories of milk-induced asthma were studied (three allergic to cow's milk; one to soya milk). In each instance, skin prick tests, RAST (IgE and IgG4), the basophil histamine release, and serum precipitins, using appropriate milk extracts, were negative. After the ingestion of milk all the subjects developed a reproducible and dose-dependent increase in airflow limitation. Three patients (two allergic to cow's milk; one to soya milk) gave a characteristic immediate-type reaction, which was maximal at 30 min after challenge. The fourth individual developed an isolated late-phase response, with maximal airways obstruction 3 hr after ingesting milk. In the three subjects who gave an early reaction, wheezing was accompanied by an elevation in circulating neutrophil chemotactic activity (NCA). This was not observed in the individual with the isolated late reaction. By Sephacryl S-400 gel-filtration chromatography it was shown that NCA of the early reactions eluted with proteins having an estimated molecular weight of 600,000 daltons. The immediate asthmatic response in peak expiratory flow rate and the elevation in NCA were inhibited by the prior oral administration of either disodium cromoglycate (DSCG) or oral beclomethasone dipropionate (BDP). In contrast, DSCG had no effect on airways obstruction in the subject with the isolated late asthmatic response, although inhibition was achieved by BDP.

Neutrophils demonstrate increased complement receptor activity, measured by rosetting of C3b-coated erythrocytes, after asthma that was provoked experimentally. However, it is not clear whether the increased rosetting is due simply to increase in receptor numbers or whether other factors, such as cell adhesiveness, are involved. We have therefore enumerated granulocyte complement receptors, after asthma provoked experimentally, with monoclonal antibodies against the receptors and flow cytometry. There was a maximal 28.2 +/- 7.5% and 33.4 +/- 9.5% (mean +/- SEM; n = 15) increase in granulocyte CR1 and CR3, respectively, at 3 hours after asthma induced by antigen. There was a maximal 32.0 +/- 7.3% (mean +/- SEM; n = 7) increase in granulocyte CR1, but no change in granulocyte CR3, at 1 hour after exercise-induced asthma. No significant changes in granulocyte CR1 or CR3 were observed up to 6 hours after methacholine challenge, or after exercise in subjects who did not develop exercise-induced asthma. There was a maximal 33 +/- 9% (mean +/- SEM; n = 8) increase in granulocyte CR1 at 30 minutes, but no increase in granulocyte CR3, after histamine challenge of subjects with asthma. Incubation of whole blood with histamine in vitro did not lead to any enhancement in expression of granulocyte CR1. This suggests that antigen- and exercise-induced release of histamine may augment granulocyte CR1 expression through an indirect mechanism. These data indicate that there is increase in the numerical expression of CR1 on granulocytes, after asthma provoked experimentally, which is accompanied by increases in granulocyte CR3 after bronchoprovocation with antigen, but not histamine or exercise.

Eight subjects with asthma inhaled on separate occasions leukotriene E4 (LTE4) (6.1 nmol, geometric mean), methacholine, and diluent, which produced an average 41.0%, 37.0%, and 3.3% decrease in specific airway conductance (SGaw), respectively. When the SGaw had recovered to baseline levels at 60 minutes after challenge, the provocative dose of inhaled histamine that produced a 35% decrease in SGaw (PD35) was determined. The histamine PD35 observed after inhalation of LTE4 was 0.46 mumol, and this was significantly less than the histamine PD35 observed after inhalation of methacholine (0.88 mumol; p less than 10(-4) and diluent (0.97 mumol; p less than 10(-5). Histamine responsiveness was also enhanced by a fiftyfold lower dose of LTE4 (p = 0.005), and the enhancement was less than that elicited by the higher dose of LTE4 in the same individuals (p = 0.02). The changes in histamine PD35 during a 1-week period after LTE4 and methacholine challenges were compared in four subjects with asthma. There was a time-dependent enhancement in histamine responsiveness that reached a maximal of 3.5-fold at 7 hours after LTE4. The enhancement had disappeared by 1 week. Similar changes were not observed after methacholine challenge, which elicited the same degree of bronchoconstriction as LTE4. Inhalation of LTE4 in five normal subjects that produced a mean 37.6% decrease in SGaw did not change histamine responsiveness for up to 7 hours. These findings suggest that LTE4 may play a role in the perpetuation of nonspecific airway hyperresponsiveness in bronchial asthma.