Effect of steroids on nasal inflammatory cells and cytokine profile.

OBJECTIVES: To assess the cellular and humoral effects of unilateral topical steroid application on the nasal mucosa of chronic sinusitis patients. STUDY DESIGN: Cohort study with randomized grouping. METHODS: Chronic sinusitis patients awaiting endonasal sinus surgery were randomly divided into a study group and a control group. All patients underwent allergy studies and categorized as allergic or nonallergic. Patients in the study group were exposed unilaterally to fluticasone propionate nasal spray, 100 microg/day for 4 to 6 weeks before the planned surgery. The control group was not exposed to steroids. At the time of surgery, anterior ethmoid mucosa specimens were obtained. The samples were immunostained for CD3, MBP, CD68, elastase, and tryptase. In situ hybridization was used for the detection of interleukin (IL)-4, IL-5, and interferon gamma (IFN-gamma) mRNA. The results were compared between the treated and untreated sides of exposed patients, as well as with nontreated controls. RESULTS: Twenty-one patients consented to participate in the study. The number of CD3-, MBP-, and tryptase-positive cells was significantly less in the treated and untreated sides of exposed patients, compared with unexposed controls. IL-4 and IL-5 mRNA were significantly downregulated in the treated side of allergic patients, as compared with nontreated patients. This effect could not be shown for IL-4 in nonallergic patients. CONCLUSIONS: Topical steroid therapy had an anti-inflammatory effect on both allergic and nonallergic chronic sinusitis patients. The unilateral application of the steroid spray affected the contralateral side as well.

Glucocorticoid receptor mRNA and protein in fetal rat lung in vivo: modulation by glucocorticoid and androgen.

Pulmonary glucocorticoid receptor (GR) is essential to timely preparation for the onset of breathing air at birth. We have previously used primary culture of late-gestation fetal rat lung cells to demonstrate differential regulation of GR by glucocorticoid depending on cell type. In this study, we hypothesized that the action of glucocorticoid on GR mRNA expression and protein elaboration in lung cells might be modulated by interactions present in vivo but not in primary culture. Given that male sex hormone (androgen) has an inhibitory effect on antenatal lung development, we also postulated that androgen would decrease antenatal lung GR. We report that antenatal maternal injection of the glucocorticoid dexamethasone (1 mg/kg) enhanced fetal lung cellular levels of GR mRNA and protein as assessed by in situ hybridization and immunocytochemistry (ICC), respectively. ICC was performed using polyclonal rabbit anti-human antibody that reacts with rat GR whether bound to ligand or not and does not interfere with GR binding to DNA. Levels of GR mRNA and protein were enhanced in cells throughout all areas of the lung tissue, suggesting that interactions occurring in intact tissue may override the previously reported direct inhibition by glucocorticoid of GR protein elaboration in isolated fetal rat lung epithelial cells. Furthermore, antenatal administration of the androgen 5alpha-dihydrotestosterone (0.2 mg/kg) reduced tissue levels of GR mRNA and protein, consistent with androgenic inhibition of antenatal lung development by decreasing GR. We conclude that glucocorticoids and androgens exert opposite effects on fetal lung GR.

Granulocyte/macrophage-colony stimulating factor in allergen-induced rhinitis: cellular localization, relation to tissue eosinophilia and influence of topical corticosteroid.

BACKGROUND: Allergen-induced late nasal responses are associated with recruitment of T lymphocytes and eosinophils, and preferential messenger RNA (mRNA) expression of 'TH2-type' cytokines. We previously showed that topical steroid inhibited the late response and associated tissue eosinophilia. In this study we tested the hypothesis that granulocyte/macrophage-colony stimulating factor (GM-CSF) may contribute to late-responses and tissue eosinophilia and is inhibitable by topical corticosteroid. METHODS: Nasal biopsies were taken before and 24 h after nasal allergen provocation following 6 weeks of treatment with either a nasal corticosteroid spray (fluticasone propionate) or a matched placebo nasal spray twice daily. Cryostat sections were processed by immunohistochemistry and in situ hybridization to assess cytokine mRNA expression for GM-CSF. RESULTS: Increases in T lymphocytes and eosinophils were seen in the nasal mucosa after allergen challenge (p = 0.01) which were accompanied by a 5-fold increase in cells expressing mRNA for GM-CSF (p = 0.01). Double immunohistochemistry/in situ hybridization demonstrated that the majority of GM-CSF mRNA+ cells were co-localized to CD68+ (40%), or T cells (40%) with a lesser contribution from eosinophils (<20%). Topical steroid treatment was accompanied by a decrease in both the CD3+ and major basic protein (MBP+) cells expressing GM-CSF mRNA (p = 0.01) with a corresponding proportionate increase in the % of macrophages expressing GM-CSF. CONCLUSIONS: The results indicate that after allergen provocation, eosinophils are recruited to the nasal mucosa and that, at least in part, this may be due to GM-CSF. Topical nasal corticosteroid inhibits late responses and the associated eosinophilia, possibly indirectly by decreasing GM-CSF from T lymphocytes or reducing autocrine production of GM-CSF from eosinophils.

Expression of epsilon germ-line gene transcripts and mRNA for the epsilon heavy chain of IgE in nasal B cells and the effects of topical corticosteroid.

We have studied the expression of the gene encoding the epsilon heavy chain of IgE in nasal B cells of hayfever patients. We developed probes to detect transcripts of the epsilon germ-line gene and the rearranged gene by in situ hybridization of biopsy sections from the nasal mucosa. We compared tissue from hayfever patients out of season with that of normal controls, and also of hayfever patients treated with topical corticosteroid (fluticasone propionate) or placebo for 6 weeks and then challenged with antigen. epsilon chain mRNA was expressed in an unexpectedly high proportion of nasal B cells of both hayfever patients and normal subjects. However, although similar numbers of B cells were found in both groups, the proportion of cells that express epsilon chain mRNA was several times higher in the hayfever patients. No transcripts of the epsilon germ-line gene were detected in either group before allergen challenge. When hayfever patients were administered antigen locally, early (10-30 min) and late (1-24 h) symptoms ensued. After 24 h, coincident with an increase in the number of cells expressing mRNA for IL-4 in the tissue, epsilon germ-line gene transcripts appeared in the nasal B cells. The induction by allergen of IL-4 mRNA and epsilon germ-line gene transcripts was suppressed by fluticasone propionate treatment. Our results suggest that local IgE synthesis and cytokine regulation of heavy chain switching to IgE occur in the nasal mucosa.

Increases in IL-12 messenger RNA+ cells accompany inhibition of allergen-induced late skin responses after successful grass pollen immunotherapy.

IL-12, a novel cytokine produced by tissue macrophages and B lymphocytes, stimulates proliferation of TH1-type T lymphocytes. We recently showed that in patients with summer hay fever, immunotherapy was effective and was associated with inhibition of allergen-induced late skin responses and increases in local interferon-gamma messenger RNA-positive cells. In this study 10 patients were reassessed after 4 years of immunotherapy and compared with 10 untreated patients with hay fever. Intradermal grass pollen challenge was performed, the late response was measured, and biopsies were performed at 24 hours. In situ hybridization of biopsy sections was performed by using a riboprobe coding for IL-12 mRNA. When immunotherapy and control subjects were compared, there was a marked reduction in the size of the late skin response (p = 0.0001). Significant increases in allergen-induced IL-12 mRNA+ cells in cutaneous biopsy specimens occurred only in the immunotherapy-treated group (all 10 patients, p = 0.002). At allergen-challenged sites, IL-12+ cells correlated positively with interferon-gamma + cells (r = 0.64, p < 0.05) and inversely with IL-4+ cells (r = -0.67, p < 0.05). The principal cell source (55% to 80%) of IL-12 message was the tissue macrophage (CD68+ cells). We suggest that IL-12 may promote TH1 responses and inhibit late-phase responses after successful immunotherapy.

Evidence for major basic protein immunoreactivity and interleukin 5 gene activation during the late phase response in explanted airways.

Current evidence suggests that the events subsequent to antigen challenge in allergic asthmatics involve eosinophil activation and the synthesis of proinflammatory cytokines, in particular interleukin 5 (IL-5). However, little is known about how local inflammatory cell infiltration and activation are related to the changes in lung function following allergen exposure. We have developed a novel technique to investigate the local inflammatory events during late-onset allergic bronchoconstriction in lung explants from sensitized Brown-Norway (BN) rats. In this study we tested the hypothesis that the in vitro late airway response involves IL-5 gene activation and recruitment and activation of eosinophils. Explants were prepared from excised lungs of BN rats (n = 9) sensitized 2 wk previously to ovalbumin (OVA). Lungs were inflated with liquid agarose solution (2% wt/vol, 48 ml/kg) following perfusion with cold Ca2+/Mg(2+)-free Hanks' solution, and refrigerated briefly to gel the agarose, and 0.5- to 1.0-mm slices were prepared and cultured overnight at 37 degrees C. Airways were identified and challenged by direct application of OVA (20 micrograms). Cryostat sections of explants were immunostained for major basic protein (MBP) and IL-5 mRNA was detected by a 35S-uridine triphosphate-labeled probe and in situ hybridization. Explants harvested immediately prior to challenge showed little evidence of MBP and IL-5 mRNA expression. Explants harvested at 6 h which exhibited evidence of bronchoconstriction showed strong cell-associated immunostaining for MBP and high expression of IL-5 mRNA in the bronchial mucosa. colocalization studies performed in lung explants demonstrating late-onset airway responses suggested that the majority of IL-5 mRNA expression was not found in MBP-positive cells. When compared with explants from sham-sensitized rats (n = 4), there was a significant increase in MBP-positive and IL-5 mRNA-positive cells per millimeter of basement membrane of the airway. The presence of MBP immunoreactivity and IL-5 gene expression was not observed in explants taken from sensitized BN rats which did not undergo late-onset airway responses, indicating an association between inflammatory cell activation and airway constriction. The increase in MBP-positive cells several hours after OVA suggests activation, local recruitment, and/or differentiation of eosinophils. This study provides direct evidence for a temporal association between IL-5 expression, eosinophil infiltration, and the late response in individual cultured airways.

Eosinophil infiltration in nonallergic chronic hyperplastic sinusitis with nasal polyposis (CHS/NP) is associated with endothelial VCAM-1 upregulation and expression of TNF-alpha.

We studied potential mechanisms of eosinophil accumulation in nonallergic chronic hyperplastic sinusitis with nasal polyposis (CHS/NP). We measured expression of endothelial vascular cell adhesion molecule-1 (VCAM-1), which mediates selective eosinophil transendothelial migration, the cytokines interleukin (IL)-1 beta, TNF-alpha and IL-13 which upregulate VCAM-1 expression, and the chemokine RANTES which mediates lymphocyte, monocyte, and eosinophil chemotaxis in chronic hyperplastic sinusitis with nasal polyposis (CHS/NP) nasal polyps (nonallergic versus allergic) and middle turbinate biopsies from normal controls. By immunohistochemical staining, the density of EG2+ eosinophils was increased in both the nonallergic and allergic CHS/NP subgroups compared to normal controls. VCAM-1 expression was significantly increased in CHS/NP subjects compared to normal controls (P = 0.0005), with the highest intensity seen in nonallergic CHS/NP. By in situ hybridization, the densities of IL-1 beta, TNF-alpha, IL-13, and RANTES mRNA+ cells were all increased in nonallergic CHS/NP compared to normal controls (P = 0.009, 0.0005, 0.0005, and 0.001, respectively). In comparison to allergic CHS/NP, nonallergic CHS/NP had a significantly higher tissue density of TNF-alpha (P = 0.04) and a lower density of IL-13 (P = 0.005) mRNA+ cells. In general, VCAM-1 expression correlated strongly in CHS/NP with the density of TNF-alpha (R = .91, P = 0.0005) but not the density of IL-1 beta, IL-13, or RANTES mRNA+ cells. We conclude that upregulation of VCAM-1 and elaboration of RANTES may contribute to the marked accumulation of eosinophils in nonallergic CHS/NP. TNF-alpha may play a critical role in VCAM-1 upregulation in this nonallergic eosinophilic disorder.

Prednisolone treatment in asthma. Reduction in the numbers of eosinophils, T cells, tryptase-only positive mast cells, and modulation of IL-4, IL-5, and interferon-gamma cytokine gene expression within the bronchial mucosa.

We have tested the hypothesis that the beneficial effects of corticosteroids in asthma may result from reduction in the number of inflammatory cells infiltrating the bronchial mucosa with inhibition of cytokine gene expression. A randomized parallel group study was performed in 18 moderately severe asthmatic patients in whom an elective trial of corticosteroid treatment was indicated. Fiberoptic bronchoscopy was performed and bronchial biopsies taken from segmental carinae before and after 2 wk treatment with prednisolone (0.6 mg/kg/d) or matched placebo tablets. Immunohistology was performed on 6-microns cryostat sections using monoclonal antibodies. The number of cells expressing cytokine messenger RNA (mRNA) was assessed by in situ hybridization using S35-labeled riboprobes. When prednisolone- and placebo-treated groups were compared there was a decrease in airway methacholine responsiveness (p < 0.01) and an increase in FEV1 (p < 0.05) after prednisolone. This was accompanied by a reduction in CD3+ T lymphocytes (p < 0.05), "activated" EG2+ eosinophils (p < 0.02), and tryptase-only (mucosal-type) MCT cells (p < 0.02) but not MCTC (tryptase+chymase positive) cells in prednisolone-treated patients. In prednisolone-treated patients there was also a reduction in the number of cells expressing mRNA for interleukin-4 (IL-4, p < 0.01), and interleukin-5 (IL-5, p < 0.03) and an increase in cells expressing mRNA for interferon-gamma (IFN-gamma) (p < 0.01). These results support the view that corticosteroid treatment in asthma may act by modulation of cytokine expression with consequent inhibition of the local bronchial inflammatory infiltrate and tissue eosinophilia.

Evidence for distinct cytokine expression in allergic versus nonallergic chronic sinusitis.

BACKGROUND: The purpose of this study was to characterize the relationship between tissue cytokine expression and the cellular infiltrate present in chronic hyperplastic sinusitis with nasal polyposis (CHS/NP) and to compare the immunopathology and cytokine profile of patients with allergy versus patients without allergy. METHODS: Nasal polyp tissue samples from 12 patients with CHS/NP and nasal turbinate biopsy specimens from 10 normal control patients were examined for the expression of interleukin (IL)-4, IL-2, and interferon (IFN)-gamma cytokine messenger RNA (mRNA) species by in situ hybridization. These data were analyzed in conjunction with data previously reported for the cytokine mRNA species granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-3, and IL-5 and the immunocytochemical profile of the inflammatory cell infiltrate. Patients with allergy were distinguished from those without allergy on the basis of allergy skin tests. RESULTS: Tissue eosinophilia was a prominent feature of both allergic and nonallergic CHS/NP and correlated in both subgroups with the density of GM-CSF and IL-3 mRNA+ cells. In comparison with normal controls, patients with allergic CHS/NP had significantly higher CHS/NP had significantly higher tissue densities of GM-CSF, IL-3, IL-4, and IL-5 (p < or = 0.025). In contrast, patients with nonallergic CHS/NP had significantly higher tissue densities of GM-CSF, IL-3, and IFN-gamma (p < or = 0.001). The allergic and nonallergic subgroups showed distinct cytokine profiles with the most distinguishing cytokines of the allergic subgroup being IL-4 (p = 0.001) and IL-5 (p = 0.017) and of the nonallergic subgroup being IFN-gamma (p = 0.004). Furthermore, patients with allergic CHS/NP showed an increased density of CD3+ T lymphocytes compared with either controls or patients with nonallergic CHS/NP (p = 0.03). The density of CD3+ T lymphocytes was the only significant difference between patients with allergic and nonallergic CHS/NP. A clinical history of aspirin sensitivity was strongly correlated with nonallergic CHS/NP, as well as the nonallergic CHS/NP profile of cytokines, including IFN-gamma. CONCLUSION: We conclude that distinct mechanisms of eosinophilia exist in patients with allergic versus nonallergic CHS/NP. The allergic mechanism involves production of TH2-type cytokines, including GM-CSF, IL-3, IL-4, and IL-5, by infiltrating T lymphocytes. The nonallergic mechanism remains unknown but does involve production of GM-CSF, IL-3, and IFN-gamma. However, nonallergic eosinophilia is independent of IL-4 and IL-5, cytokines that contribute to tissue eosinophilia in allergic inflammation. Aspirin sensitivity is strongly correlated with nonallergic CHS/NP and production of the nonallergic CHS/NP profile of cytokines, including IFN-gamma.

The repertoire of anti-TNP antibodies in mice neonatally suppressed with anti-IgM.

Previous studies have shown that mice immunized with TNP-Ficoll when young, adult, or aged expressed different repertoires of anti-TNP antibodies. The aim of the present study was to find out whether this age-related nonrandom progression was driven by antigen, and whether it was regulated by the immune network through surface-Ig receptors on B lymphocytes. The approach utilized was to block receptor expression on B lymphocytes of mice by the chronic administration of anti-IgM from birth for approximately 1 year, and then compare their subsequent antibody response to that of age-matched control animals. The results obtained have shown that the age-dependent shift in the anti-TNP repertoire expressed could take place in animals whose B lymphocytes were blind to antigen and anti-id for the greater part of their lives and thus suggest that the regulatory events responsible for this shift may be (surface Ig) receptor independent.