Gabriele Holtappels

Differentiation of chronic sinus diseases by measurement of inflammatory mediators

Background:  Chronic rhinosinusitis (CRS) clinically is a heterogeneous group of sinus diseases, which may cover different disease entities, or may represent a disease continuum. Studying inflammatory cells and mediators in clearly defined disease subgroups may lead to a better differentiation of chronic sinus diseases.

Different types of T-effector cells orchestrate mucosal inflammation in chronic sinus disease

BACKGROUND Chronic rhinosinusitis with nasal polyps (CRSwNP) is characterized by the accumulation of inflammatory cells; however, an eosinophil predominance is seen in white (Belgian), but not Asian (south Chinese), patients with polyps. OBJECTIVE We sought to investigate the association of inflammatory cell predominance with regulatory T-cell and T-effector cell patterns. METHODS Nasal mucosal tissue was obtained from 26 consecutive Belgian patients with CRSwNP and 21 Belgian control subjects and 29 south Chinese patients with CRSwNP and 29 south Chinese control subjects, who all underwent phenotyping, including nasal endoscopy and computed tomographic scanning. Tissues were investigated for granulocytes and their products and T-effector/regulatory T cells and related cytokines. RESULTS Both CRSwNP groups were comparable in terms of symptoms, computed tomographic scan results, and nasal endoscopy results, but asthma comorbidity was significantly higher in white patients. Tissue from white patients with CRSwNP was characterized by eosinophilic inflammation (eosinophil cationic protein/myeloperoxidase ratio > 2), whereas samples from Asian patients were biased toward neutrophilic inflammation (eosinophil cationic protein/myeloperoxidase ratio = 0.25). Both CRSwNP groups demonstrated significant upregulation of the T-cell activation marker soluble IL-2 receptor alpha and significant downregulation of Foxp3 expression and TGF-beta1 protein content versus their respective control groups. However, whereas white patients displayed a significant increase in T(H)2 cytokine and related marker levels versus control subjects and versus Asian patients, the latter showed a T(H)1/T(H)17 cell pattern versus control tissue. CONCLUSION Nasal polyps (CRSwNP) from white and Asian patients are both characterized by T-cell activation and impaired regulatory T-cell function; however, T-effector cells in the samples from white patients were T(H)2-biased, whereas samples from their Asian counterparts demonstrated a T(H)1/T(H)17 polarization.

Nasal polyposis: from cytokines to growth.

Nasal polyposis (NP) is a chronic inflammatory condition that is mostly characterized by an infiltration of eosinophils. How this eosinophilic inflammation leads to polyp formation remains largely unclear. In order to identify the most important factors in polyp growth, first we report the histologic features of two early stage manifestations of eosinophilic nasal polyps compared to their surrounding normal mucosa and mature polyps from the same patients. Histomorphologic analysis of these early stage manifestations of NP showed the presence of eosinophils, forming a subepithelial cap over a pseudocyst area that was filled with albumin. In mature NP, a large pseudocyst area containing albumin was surrounded by subepithelial eosinophilia. Second, in an approach to quantify and to study possible relations between eosinophilic inflammation and changes in extracellular tissue components we measured interleukin-5 (IL-5), eotaxin, eosinophil cationic protein (ECP), leukotrienes (LTC4/D4/E4), transforming growth factor-β 1 (TGF-β1), fibronectin, hyaluronic acid, and albumin in nasal tissue homogenates of 31 subjects. Nasal polyp samples (n = 16) were obtained during routine endonasal sinus surgery, whereas control non-polyp samples (n = 15) from subjects with (6) and without (9) allergic rhinitis were obtained from the inferior turbinate during septum surgery. In the group of polyp patients 11 received no treatment, whereas 5 were treated with oral glucocorticoids (GCS) within 4 weeks before surgery. IL-5 was measurable in 8 of 11 untreated NP, whereas IL-5 could not be detected in all 15 controls nor in 4 of 5 oral corticoid-treated polyps. The comparison between the untreated polyp group and controls showed significantly higher concentrations of IL-5, eotaxin, ECP, and albumin in polyp supernatants, whereas TGF-β1 was significantly lower. In the oral GCS-treated group, ECP and albumin were significantly reduced compared to untreated nasal polyps. The same tendency, but not reaching significance, was seen for eotaxin and fibronectin, while no difference was found for LTC4/D4/E4 and hyaluronic acid between the groups. Our observations suggest a deposition of albumin (and possibly other plasma proteins) and extracellular matrix proteins, which may be regulated by the subepithelial eosinophilic inflammation, as a possible pathogenic principle of polyp formation and growth. IL-5 and eotaxin are found to be key factors for eosinophilic accumulation and activation in NP. Oral corticoid treatment may lead to the shrinkage of NP by downregulation of the eosinophilic inflammation and reduction of the extravasation and deposition of albumin in NP.

T-cell regulation in chronic paranasal sinus disease.

BACKGROUND Chronic rhinosinusitis is an inflammatory disease with distinct cytokine and remodeling patterns. Chronic rhinosinusitis with nasal polyps (CRSwNP) is characterized by a T(H)2-skewed eosinophilic inflammation, whereas chronic rhinosinusitis without nasal polyps (CRSsNP) represents a predominant T(H)1 milieu. OBJECTIVE We aimed to study the direct tissue expression of transcription factors for T-cell subpopulations, including T regulatory cells, in relation to the cytokine expression patterns in the different disease subgroups. METHODS The expression of forkhead box P3 (FOXP3), T-box transcription factor (T-bet), GATA-3, retinoid acid-related orphan receptor C (RORc), the suppressive cytokines TGF-beta1 and IL-10, and T(H)1/ T(H)2/ T(H)17 cytokines (IFN-gamma, IL-4, IL-5, IL-13, IL-17) were analyzed by means of RT-PCR in 13 CRSsNP, 16 CRSwNP, and 10 control samples. Additional protein measurements were performed for TGF-beta1 and IFN-gamma. RESULTS In CRSwNP, we observed a significantly lower FOXP3 mRNA and TGF-beta1 protein expression, but a significantly higher T-bet, GATA-3, IL-5, and IL-13 mRNA expression compared with controls, whereas RORc was not significantly different compared with controls. In CRSsNP, FOXP3, T-bet, GATA-3, and RORc expression was not significantly different from controls, whereas TGF-beta1 mRNA, IFN-gamma mRNA, and protein were significantly higher in CRSsNP compared with controls. For IL-17, no significant differences were noted among all groups. CONCLUSION We demonstrate for the first time a decreased FOXP3 expression accompanied by an upregulation of T-bet and GATA-3 and a downregulation of TGF-beta1 in CRSwNP versus controls and CRSsNP.

Presence of IL-5 protein and IgE antibodies to staphylococcal enterotoxins in nasal polyps is associated with comorbid asthma

BACKGROUND Nasal polyps often are associated with asthma. The phenotype of these patients is unknown. OBJECTIVE To identify the mucosal factors associated with asthma comorbidity, we analyzed the inflammatory patterns of nasal polyps. METHODS Nasal polyps from 70 Belgian patients, 34% with asthma, were analyzed for type of inflammation, T-cell cytokines, and IgE antibodies to Staphylococcus aureus enterotoxins. The same investigations were repeated in 93 Chinese patients with polyps, a group with a low asthma comorbidity rate (8%). RESULTS In Belgian patients with polyps, 54% of samples showed eosinophilic inflammation. A classification tree evaluation identified IL-5 as the main positive determinant. Enterotoxin IgE in tissue (37%) was associated with significantly increased total IgE and eosinophil cationic protein concentrations. Expression of enterotoxin IgE, total IgE at greater than 1,442 kU/L, and eosinophil cationic protein at greater than 17,109 μg/L in samples with a total IgE concentration of greater than 246 kU/L significantly predicted asthma (odds ratio, 5.8-13). Only 7.5% of the samples from Chinese patients with polyps showed eosinophilic inflammation. IL-5 was confirmed as a positive determinant of eosinophilic inflammation, and enterotoxin IgE in tissue (17% of patients) was associated with significantly increased total IgE and eosinophil cationic protein concentrations. The expression of IL-5 or total IgE at greater than 790 kU/L in samples with an IL-5 concentration of greater than 194 pg/mL significantly predicted comorbid asthma (odds ratio, 17.2-96). CONCLUSION Mucosal inflammation in nasal polyps orchestrated by T(H)2 cytokines and amplified by S aureus enterotoxins is characterized by an increased eosinophilic inflammation and formation of IgE antibodies. This phenotype is associated with comorbid asthma in white and Asian patients with nasal polyps.

Oral steroids and doxycycline: Two different approaches to treat nasal polyps

BACKGROUND There is little scientific evidence to support the current practice of using oral glucocorticosteroids and antibiotics to treat patients with chronic rhinosinusitis and nasal polyps. OBJECTIVE We evaluated the effects of oral glucocorticoids and doxycycline on symptoms and objective clinical and biological parameters in patients with chronic rhinosinusitis and nasal polyps. METHODS In a double-blind, placebo-controlled, multicenter trial, we randomly assigned 47 participants with bilateral nasal polyps to receive either methylprednisolone in decreasing doses (32-8 mg once daily), doxycycline (200 mg on the first day, followed by 100 mg once daily), or placebo for 20 days. Participants were followed for 12 weeks. Patients were assessed for nasal peak inspiratory flow and symptoms and by nasal endoscopy. Markers of inflammation such as eosinophilic cationic protein (ECP), IL-5, myeloperoxidase, matrix metalloproteinase 9, and IgE were measured in nasal secretions. Concentrations of eosinophils, ECP, and soluble IL-5 receptor alpha were measured in peripheral blood samples. RESULTS Methylprednisolone and doxycycline each significantly decreased nasal polyp size compared with placebo. The effect of methylprednisolone was maximal at week 3 and lasted until week 8, whereas the effect of doxycycline was moderate but present for 12 weeks. Methylprednisolone significantly reduced levels of ECP, IL-5, and IgE in nasal secretions, whereas doxycycline significantly reduced levels of myeloperoxidase, ECP, and matrix metalloproteinase 9 in nasal secretions. CONCLUSION This is the first double-blind, placebo-controlled study to show a significant effect of oral methylprednisolone and doxycycline on size of nasal polyps, nasal symptoms, and mucosal and systemic markers of inflammation.

Organization of secondary lymphoid tissue and local IgE formation to Staphylococcus aureus enterotoxins in nasal polyp tissue

Background:  Bilateral nasal polyposis (NP) is characterized by high concentrations of IgE in NP tissue, which show no relation to the atopic status. We aimed to study the relationship between systemic and local IgE formation, nasal carriage of Staphylococcus aureus and nasal polyposis.

TGF-β signaling and collagen deposition in chronic rhinosinusitis

BACKGROUND Chronic rhinosinusitis is an inflammatory disease with distinct cytokine and remodeling patterns. OBJECTIVE The objective was to analyze the presence of TGF-beta isoforms, receptors, intracellular signaling, and collagen deposition in chronic rhinosinusitis. METHODS Sinonasal mucosal samples obtained from chronic rhinosinusitis with nasal polyps (CRSwNP; n = 13), chronic rhinosinusitis without nasal polyps (CRSsNP; n = 13), and controls (n = 10) were analyzed for TGF-beta isoforms 1 and 2 by means of ELISA and IHC, and for TGF-beta R1, 2, and 3 by RT-PCR and IHC. As downstream proteins, phospho-Smad 2 (pSmad 2) and collagen were analyzed by performing immunostaining and picrosirius red staining, respectively. RESULTS TGF-beta 1 and 2 protein concentrations, TGF-beta receptor (R) I and TGF-beta RIII mRNA expression, the number of pSmad 2-positive cells, and total collagen amount were significantly higher in CRSsNP versus controls. In CRSwNP, TGF-beta 1 protein concentration, TGF-beta RII and TGF-beta RIII mRNA expression, the number of pSmad 2-positive cells, and total collagen amount were significantly lower versus controls. Only TGF-beta 2 protein was found higher in CRSwNP versus controls. CONCLUSION A high TGF-beta 1 protein expression, increased TGF-beta RI expression, and a high number of pSmad 2-positive cells all indicate an enhanced TGF-beta signaling in CRSsNP, whereas a low TGF-beta 1 protein concentration, a decreased expression of TGF-beta RII, and a low number of pSmad 2-positive cells in CRSwNP indicate a low level of TGF-beta signaling in CRSwNP. These findings are compatible with the remodeling patterns observed, reflected by a lack of collagen in CRSwNP, and excessive collagen production with thickening of the collagen fibers in the extracellular matrix in CRSsNP.

Human beta-defensins and toll-like receptors in the upper airway.

Background:  Measurement of innate markers in nasal mucosa, tonsils and adenoids might lead to new views about the role of innate immunity in the upper airway. In this study, the expression of human β‐defensins (HBD) 2 and 3 and toll‐like receptors (TLR) 2 and 4 in various upper airway diseases was investigated.

Specific IgE against Staphylococcus aureus enterotoxins: an independent risk factor for asthma.

BACKGROUND The role of IgE in patients with severe asthma is not fully understood. OBJECTIVE We sought to investigate whether IgE to Staphylococcus aureus enterotoxins might be relevant to disease severity in adult asthmatic patients. METHODS Specific IgE antibody concentrations in serum against enterotoxins, grass pollen (GP), and house dust mite allergens and total IgE levels were measured in adult cohorts of 69 control subjects, 152 patients with nonsevere asthma, and 166 patients with severe asthma. Severe asthma was defined as inadequately controlled disease despite high-dose inhaled corticosteroids plus at least 2 other controller therapies, including oral steroids. RESULTS Enterotoxin IgE positivity was significantly greater in patients with severe asthma (59.6%) than in healthy control subjects (13%, P< .001). Twenty-one percent of patients with severe asthma with enterotoxin IgE were considered nonatopic. Logistic regression analyses demonstrated significantly increased risks for enterotoxin IgE-positive subjects to have any asthma (OR, 7.25; 95% CI, 2.7-19.1) or severe asthma (OR, 11.09; 95% CI, 4.1-29.6) versus enterotoxin IgE-negative subjects. The presence of GP or house dust mite IgE antibodies was not associated with either significantly increased risk for asthma or severity. Oral steroid use and hospitalizations were significantly increased in patients with enterotoxin IgE and nonatopic asthma. GP IgE was associated with a higher FEV(1) percent predicted value, and enterotoxin IgE was associated with a lower FEV(1) percent predicted value. CONCLUSIONS Staphylococcal enterotoxin IgE antibodies, but not IgE against inhalant allergens, are risk factors for asthma severity. We hypothesize that the presence of enterotoxin IgE in serum indicates the involvement of staphylococcal superantigens in the pathophysiology of patients with severe asthma.