Eleanor M. Minshall

Inflammation of small airways in asthma

This study was designed to examine the inflammatory process in the central and peripheral airways of surgically resected lungs from asthmatic and nonasthmatic subjects. Lung specimens were inflated with cryoprotective, rapidly frozen, and systematically sampled. Cryosections prepared from frozen tissue blocks were fixed in acetone/methanol and immunostained with monoclonal antibodies by using the alkaline phosphatase-anti-alkaline phosphatase technique to detect CD3 (T cells), major basic protein (total eosinophils), EG2 (activated eosinophils), anti-tryptase (mast cells), anti-elastase (neutrophils), and CD68 (macrophages). All airways from patients with asthma demonstrated a significant increase in the numbers of T cells and total and activated eosinophils compared with airways from nonasthmatic subjects (p < 0.001). In the patients with asthma, the numbers of activated eosinophils but not T cells were significantly greater in airways with an internal perimeter less than 2 mm compared with those with an internal perimeter greater than 2 mm (p < 0.05). There were also significantly higher numbers of major basic protein-positive eosinophils, when expressed as a fraction of the alveolar wall tissue, in patients with asthma compared with control subjects (p < 0.05). In asthmatic airways with an internal perimeter of more than 2 mm, there was a greater number of activated eosinophils in the tissue between the epithelium and the smooth muscle compared with the tissue between the smooth muscle layer and lung parenchyma (p < 0.05). In contrast, there was a greater number of total eosinophils in the outer airway layer compared with the inner airway layer (p < 0.05). These results show that there is a similar but more severe inflammatory process present in the peripheral compared with the central airways of patients with asthma, which is consistent with the fact that the smaller airways are a major site of obstruction in asthma.

In vivo expression of IL-12 and IL-13 in atopic dermatitis

Previous studies in atopic dermatitis (AD) have shown that acute and chronic skin lesions are associated with a TH2-type profile of cytokine expression. IL-12 and IL-13 are recently described cytokines, which possess TH1-and TH2-like actions, respectively. We have used the technique of in situ hybridization to examine the expression of IL-12 and IL-13 messenger RNA in skin biopsy specimens of acute and chronic skin lesions and uninvolved skin from patients with AD. When compared with normal control skin, the acute and chronic skin lesions and unaffected skin from patients with AD had significantly greater numbers of cells that were positive for IL-13 mRNA (p < 0.05). Acute AD skin lesions expressed a higher number of positive cells than those observed in chronic AD skin lesions (p < 0.05) or psoriasis skin lesions (p < 0.05) There was a significant increase in the numbers of IL-12 mRNA-positive cells in chronic skin lesions compared with acute lesions and uninvolved skin from patients with AD (p < 0.05). These data demonstrate that acute AD skin lesions are associated with an increased expression of IL-13 mRNA. In contrast, the relative increase in IL-12 mRNA in chronic AD skin lesions suggests a possible role for IL-12-producing cells in modulating chronic inflammation.

Eotaxin and monocyte chemotactic protein-4 mRNA expression in small airways of asthmatic and nonasthmatic individuals

BACKGROUND Although an eosinophilic infiltrate has been observed in the small airways of asthmatic individuals, the mechanisms responsible for cellular recruitment in the lung periphery remain to be clarified. Eotaxin and monocyte chemotactic protein (MCP)-4 are 2 eosinophil-associated chemokines shown to be upregulated at sites of allergic inflammation. However, their expression within the small airways of asthmatic individuals remains to be elucidated. OBJECTIVE We sought to determine the expression of eotaxin and MCP-4 in the peripheral airways and parenchyma of lungs of subjects with asthma and to assess their relationship to the numbers of resident eosinophils. METHODS We examined surgically resected lung tissue from 6 asthmatic and 10 nonasthmatic subjects for the presence of eotaxin and MCP-4 mRNA by in situ hybridization. Chemokine mRNA expression was examined with respect to the numbers of eosinophils within the airways, as detected by immunocytochemistry for major basic protein. RESULTS Numbers of chemokine mRNA-positive cells were significantly increased in the large and small airways of asthmatic subjects compared with nonasthmatic subjects. Although eotaxin and MCP-4 mRNA were widely expressed in the lungs of subjects with asthma, their expression was particularly evident within the bronchial epithelium and inflammatory cells. In the airways of the asthmatic individuals, the expression of eotaxin mRNA was significantly correlated to the numbers of eosinophils present. CONCLUSION There is an increased expression of eotaxin and MCP-4 mRNA within the peripheral airways of lungs of asthmatic subjects, suggesting that these chemokines contribute to the small airways and peripheral lung inflammation in asthma.

Evidence for Local Eosinophil Differentiation Within Allergic Nasal Mucosa: Inhibition with Soluble IL-5 Receptor

Eosinophil differentiation occurs within the bone marrow in response to eosinopoietic cytokines, particularly IL-5. Recently, however, eosinophil precursors (CD34/IL-5Rα+ cells) and IL-5 mRNA+ cells have been identified within the lungs of asthmatics, indicating that a population of eosinophils may differentiate in situ. In this report, we examined the presence of eosinophil precursors within allergic nasal mucosa and examined whether they undergo local differentiation following ex vivo stimulation. We cultured human nasal mucosa obtained from individuals with seasonal allergic rhinitis with either specific allergen, recombinant human IL-5 (rhIL-5), or allergen + soluble IL-5Rα (sIL-5Rα), shown to antagonize IL-5 function. Simultaneous immunocytochemistry and in situ hybridization demonstrated that there were fewer cells coexpressing CD34 immunoreactivity and IL-5Rα mRNA following culture with allergen or rhIL-5, compared with medium alone. Immunostaining revealed that the number of major basic protein (MBP) immunoreactive cells (eosinophils) was higher within tissue stimulated with allergen or rhIL-5, compared with unstimulated tissue. In situ hybridization detected an increase in IL-5 mRNA+ cells in sections from tissue cultured with allergen, compared with medium alone. These effects were not observed in tissue cultured with a combination of allergen and sIL-5Rα. Colocalization analysis indicated this expression to be mainly, but not exclusively, T cell (44%) and eosinophil (10%) derived. Our findings suggest that a subset of eosinophils may differentiate locally within allergic nasal mucosa, in what appears to be a highly IL-5-dependent fashion, and imply that this process might be regulated in vivo by endogenous production of sIL-5Rα.

Cytokine mRNA expression in asthma is not restricted to the large airways

BACKGROUND Although previous studies have established the presence of an eosinophil-rich cellular infiltrate in the small airways of asthmatic lungs, the expression of cytokines within the peripheral airways has been largely unexplored. The purpose of our study was to test the hypothesis that TH2-type cytokines are increased in the peripheral airways and parenchyma of asthmatic lungs. METHODS The presence of messenger ribonucleic acid (mRNA) encoding both T-helper (TH1)-type (IL-2, interferon-gamma) and TH2-type (IL-4, IL-5) cytokines in surgically resected lungs from six asthmatic and 10 nonasthmatic subjects was determined by in situ hybridization. Colocalization of IL-5 mRNA within the large and small airways was performed by simultaneous in situ hybridization and immunocytochemistry. RESULTS Expression of IL-5 mRNA-positive cells was significantly increased in the large and small airways and in the lung parenchyma of asthmatic subjects compared with nonasthmatic subjects. In the asthmatic individuals, the expression of IL-5 mRNA was increased in the small airways compared with the large airways. There was also an increase in the number of cells expressing IL-4 mRNA in the large and small asthmatic airways compared with the nonasthmatic airways. In contrast, the numbers of IL-2 and interferon-gamma mRNA-positive cells did not differ between asthmatic and nonasthmatic individuals. CONCLUSIONS We conclude that there is an increased expression of TH2-type cytokines within the peripheral airways of asthmatic lungs and suggest that the small airways contribute to the pathophysiology of asthma.

Cytokine mRNA gene expression in active and nonactive pulmonary sarcoidosis

Sarcoidosis is a multisystem granulomatous disease associated with the expansion and activation of CD4+ T-lymphocytes and macrophages. To investigate the immunopathology of active and nonactive pulmonary sarcoidosis, we have examined the expression of cytokine gene transcripts in bronchoalveolar lavage cells from 15 patients with active pulmonary sarcoidosis, eight patients with non-active pulmonary sarcoidosis, and nine normal controls. Using in situ hybridization, the percentage of cells expressing messenger ribonucleic acid (mRNA) for interleukin (IL)-1 beta, IL-2, IL-3, IL-4, IL-5, IL-6, IL-10, IL-12 and interferon-gamma (IFN-gamma) were compared in the groups studied. In individuals with active sarcoidosis, there were significantly greater proportions of cells expressing mRNA for IL-2, IL-10, IL-12 and IFN-gamma than in subjects with nonactive disease and normal controls (p < 0.01). There was no significant difference in the percentage of positive cells expressing IL-10 and IL-12 mRNA in the nonactive group compared to the normal controls (p > 0.05). No significant differences in the percentages of IL-3, IL-4 and IL-5 mRNA positive cells were observed between active and nonactive sarcoidosis patients and normal controls (p > 0.05). These results demonstrate that there is a preferential expression of T-helper type 1 cytokines in pulmonary sarcoidosis, and that cytokines related to macrophage activation are the most prominent. In addition, these data implicate an elevated expression of interleukin-2, -10 and -12 and interferon-gamma in active compared to nonactive sarcoidosis.

Expression of the IL-4 receptor α-subunit is increased in bronchial biopsy specimens from atopic and nonatopic asthmatic subjects

BACKGROUND Recent studies have provided evidence for increased IL-4 expression in the airways of atopic and nonatopic asthmatic subjects. IL-4 is believed to perform important regulatory roles in asthma; however, the expression of the IL-4 receptor has not been investigated. In this study we examined the mRNA and protein expression of the specific alpha-subunit of the IL-4 receptor (alphaIL-4R) in bronchial biopsy specimens obtained from atopic and nonatopic asthmatic subjects. METHODS Asthmatic subjects and nonasthmatic control subjects were recruited, and lung function measurements were performed before bronchoscopy. Endobronchial biopsy specimens were examined for the presence of alphaIL-4R mRNA and immunoreactivity by using in situ hybridization and immunocytochemistry, respectively. RESULTS alphaIL-4R mRNA-positive and immunoreactive cells were detected in the epithelium and subepithelium in biopsy specimens from all subjects. Expression of alphaIL-4R mRNA and protein was significantly increased in the epithelium and subepithelium of biopsy specimens from atopic asthmatic subjects compared with atopic control subjects (P <.05 and P <.001, respectively). Epithelial alphaIL-4R mRNA expression and immunoreactivity did not differ significantly between nonatopic asthmatic subjects and nonatopic control subjects. Although the numbers of alphaIL-4R mRNA-positive cells were augmented in the submucosa of intrinsic asthmatic subjects compared with nonatopic control subjects (P <.05), alphaIL-4R immunoreactivity did not differ significantly between these groups. Increased alphaIL-4R immunoreactive signals were also detected in the endothelial cell layer in both atopic and intrinsic asthmatic subjects compared with atopic and nonatopic control subjects, respectively (P <.05). Combined in situ hybridization immunocytochemistry performed on biopsy sections from asthmatic and control subjects demonstrated alphaIL-4R mRNA expression in CD3-positive T cells and tryptasepositive mast cells, with T cells comprising the larger proportion of alphaIL-4R mRNA-positive cells. Numbers of alphaIL-4R mRNA-positive or immunoreactive cells did not correlate with CD3-positive cell numbers, numbers of IL-4 mRNA-positive cells, or indices of pulmonary function. CONCLUSION These results demonstrate constitutive alphaIL-4R expression in normal airways and enhanced expression in airway tissue from asthmatic individuals.

IL-4 and IL-5 mRNA expression in induced sputum of asthmatic subjects: Comparison with bronchial wash

BACKGROUND The local production of TH2 -type cytokines is thought to orchestrate the ongoing eosinophilic inflammation and contribute to the pathophysiologic features of allergic asthma. Previous studies investigating cytokine expression in asthmatic individuals have used invasive fiberoptic bronchoscopy techniques. To date, there have been no reports of cytokine mRNA expression in induced sputum as a means of quantifying local inflammatory events. OBJECTIVES We examined whether IL-4, IL-5, and IFN-gamma mRNA expression could be detected in cells from induced sputum in subjects with mild asthma and normal control subjects. In addition, we compared the profile of inflammatory cells and cytokine mRNA in sputum and bronchial wash fluid. METHODS Cells positive for IL-4, IL-5, and IFN-gamma mRNA were determined by using in situ hybridization on cytospun aliquots of sputum induced by successive inhalations of hypertonic saline. Inflammatory cells were quantified by using immunologic cell surface markers and immunocytochemistry. RESULTS IL-4 and IL-5 mRNA were detected in the sputum of all asthmatic subjects, and the number of cells expressing these cytokines was significantly higher than that found in control subjects. Colocalization studies showed CD3-positive T cells were the major sources of IL-4 and IL-5 mRNA. CONCLUSIONS This study demonstrates that induced sputum can be used to detect mRNA for TH2 -type cytokines in bronchial asthma and that the increase in IL-4 and IL-5 mRNA expression is similar to that seen with more invasive techniques. The qualitative differences in inflammatory cell numbers between sputum induction and bronchial wash are consistent with their sampling of different airway compartments.

Upregulation of αGM-CSF-receptor in nonatopic asthma but not in atopic asthma ☆ ☆☆ ★ ★★

Abstract Background: Intrinsic asthma is characterized by an increased number of activated eosinophils and macrophages and an increased expression of the hematopoietic growth factor granulocyte-macrophage colony-stimulating factor (GM-CSF) in the bronchial mucosa. Objective: This study was carried out to investigate the expression of αGM-CSF receptor (αGM-CSFr) messenger RNA and protein in the bronchial mucosa of patients with intrinsic or atopic asthma and of control subjects and to correlate the expression of αGM-CSFr to the number of EG2 + cells (eosinophils) and CD68 + cells (macrophages) and pulmonary function. Methods: Nineteen patients with stable asthma (9 with atopic and 10 with intrinsic asthma) and 22 normal control subjects (12 atopic and 10 nonatopic subjects) were recruited, and FEV 1 (percent predicted) and PC 20 were measured before bronchoscopy. Endobronchial biopsy specimens were obtained and examined for membrane-bound αGM-CSFr by using in situ hybridization and immunocytochemistry. Results: αGM-CSFr mRNA- and protein-positive cells were identified in biopsy specimens from all four groups studied. There was no significant difference in the number of cells expressing αGM-CSFr mRNA and protein in patients with atopic asthma compared with atopic and nonatopic control subjects. However, the numbers of αGM-CSFr mRNA- and protein-positive cells were significantly higher in nonatopic patients with asthma compared with atopic patients with asthma and atopic and nonatopic control subjects ( p + cells ( r 2 = 0.87, p + cells, and colocalization studies demonstrated that 80% of the cells expressing αGMCSFr mRNA were CD68 + . The expression of GM-CSF was also significantly increased in patients with intrinsic asthma compared with those with atopic asthma and control subjects ( p 1 ( r 2 = 0.61, p Conclusion: These results demonstrate that elevated numbers of cells expressing αGM-CSFr can be detected in nonatopic asthma but not in atopic asthma and suggest that this increased expression is predominantly macrophage-associated and may play an important pathophysiologic role in intrinsic asthma. (J Allergy Clin Immunol 1997;99:666-72.)

THE PATHOLOGY OF CHRONIC ASTHMA

Our understanding of the pathophysiology of asthma has undergone great advances in the past decade, particularly with the recognition of cytokines and the roles they may take in orchestrating the local immune response. With this information, it has been possible to target new therapeutic entities such as cytokine or chemokine receptors. Eosinophils and T lymphocytes have a special place in the inflammatory and structural alterations contributing to the asthmatic diathesis. It is possible that phenotype subsets of these cells exist and they hold the key to perpetuation of immunologic and physiologic abnormalities in asthma.