The New Perspective of Pathophysiology and Immunology Profile in Chronic Rhinosinusitis.

The New Perspective of Pathophysiology and Immunology Profile in Chronic Rhinosinusitis.

Widodo Judarwanto. Children Allergy Online Clinic, Jakarta Indonesia

Chronic rhinosinusitis (CRS) is one of the most common long-term illnesses in the world. The etiology of CRS is unknown, and no effective treatment has been established. Chronic rhinosinusitis (CRS) is a heterogeneous disease characterized by local inflammation of the upper airways and sinuses and is frequently divided into polypoid CRS (CRSwNP) and nonpolypoid CRS (CRSsNP). However, the mechanism of inflammation in CRS has still not been fully elucidated.

Chronic sinusitis is one of the more prevalent chronic illnesses in the United States, affecting persons of all age groups (see Epidemiology). It is an inflammatory process that involves the paranasal sinuses and persists for 12 weeks or longer (see Pathophysiology). The literature has supported that chronic sinusitis is almost always accompanied by concurrent nasal airway inflammation and is often preceded by rhinitis symptoms; thus, the term chronic rhinosinusitis (CRS) has evolved to more accurately describe this condition.

Most cases of chronic sinusitis are continuations of unresolved acute sinusitis; however, chronic sinusitis usually manifests differently from acute sinusitis. Symptoms of chronic sinusitis include nasal stuffiness, postnasal drip, facial fullness, and malaise.  Chronic sinusitis may be noninfectious and related to allergy, cystic fibrosis, gastroesophageal reflux, or exposure to environmental pollutants.Allergic rhinitis, nonallergic rhinitis, anatomic obstruction in the ostiomeatal complex, and immunologic disorders are known risk factors for chronic sinusitis

Immunopathophysiology

Stasis of secretions inside the sinuses can be triggered by (1) mechanical obstruction at the ostiomeatal complex due to anatomic factors or (2) mucosal edema caused by various etiologies (eg, acute viral or allergic rhinitis).

Mucous stagnation in the sinus forms a rich medium for the growth of various pathogens. The early stage of sinusitis is often a viral infection that generally lasts up to 10 days and that completely resolves in 99% of cases. However, a small number of patients may develop a secondary acute bacterial infection that is generally caused by aerobic bacteria (ie, Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catarrhalis). Initially, the resulting acute sinusitis involves only one type of aerobic bacteria. With persistence of the infection, mixed flora, anaerobic organisms, and, occasionally, funguscontributes to the pathogenesis, with anaerobic bacteria of oral flora origin often eventually predominating. In one study, these bacterial changes were demonstrated with repeated endoscopic aspiration in patients with maxillary sinusitis.Most cases of chronic sinusitis are due to acute sinusitis that either is untreated or does not respond to treatment.

The role of bacteria in the pathogenesis of chronic sinusitis is currently being reassessed. Repeated and persistent sinus infections can develop in persons with severe acquired or congenital immunodeficiency states or cystic fibrosis.

Current thinking supports that chronic rhinosinusitis (CRS) is predominantly a multifactorial inflammatory disease. Confounding factors that may contribute to inflammation include the following:

  • Persistent infection (including biofilms and osteitis)
  • Allergy and other immunologic disorders
  • Intrinsic factors of the upper airway
  • Superantigens
  • Colonizing fungi that induce and sustain eosinophilic inflammation
  • Metabolic abnormalities such as aspirin sensitivity

All of these factors can play a role in disruption of the intrinsic mucociliary transport system. This is because an alteration in sinus ostia patency, ciliary function, or the quality of secretions leads to stagnation of secretions, decreased pH levels, and lowered oxygen tension within the sinus. These changes create a favorable environment for bacterial growth that, in turn, further contributes to increased mucosal inflammation.

Localization of inflammatory mediators

Microarray analyses of sinus mucosa in pediatric patients with chronic rhinosinusitis (CRS) have recently demonstrated increased messenger RNA expression of the inflammatory chemokines CXCL5 and CXCL13 and of the innate immune mediators β-defensin 1 (DEFB1), serum amyloid A2 (SAA2), and serpin B4. The objectives of this study were to determine whether these gene products were expressed at the protein level in pediatric sinus mucosa and to determine their localization. Immunohistochemical analysis was used to identify protein expression and cellular localization of CXCL5, CXCL13, DEFB1, SAA2, and serpin B4. Coimmunofluorescence staining of inflammatory cells was performed to further evaluate expression of CXCL5 and CXCL13

Fifteen children with CRS who underwent endoscopic sinus surgery and 8 children who underwent craniofacial or neurosurgical procedures for abnormalities other than sinusitis. Protein expression and cellular localization of CXCL5, CXCL13, DEFB1, SAA2, and serpin B4 in pediatric sinus mucosa. Ciliated and basal cells in the pseudostratified epithelium stained positively for the 5 mediators examined in both cohorts. Except for serpin B4, goblet cells did not stain for any mediators in either cohort. Glandular cells stained positively for all 5 mediators in both cohorts. Coimmunofluorescence staining of inflammatory cells showed that CXCL13 was expressed in macrophages, T and B cells but not in neutrophils. CXCL5 was detected only in T cells.

CXCL5, CXCL13, DEFB1, SAA2, and serpin B4 were expressed at the protein level in the sinus mucosa of controls and pediatric patients with CRS and exhibited cell-specific localization. These mediators, not typically associated with pediatric CRS, may be involved in the inflammatory response and mucus hypersecretion seen in pediatric CRS.

Regulation and expression of IL-32

Activated T lymphocytes and their interaction with resident tissue cells, particularly epithelium, play important roles in inflammatory processes in chronic rhinosinusitis (CRS). The role of interleukin-32 (IL-32), a recently discovered proinflammatory cytokine, in CRS. IL-32 is a recently described cytokine, which is expressed in a variety of tissue cells and involved in the pathogenesis of several chronic inflammatory diseases.

Human sinus epithelial cells were isolated from biopsies and stimulated with different cytokines, which play a role in the pathogenesis of CRS. IL-32 mRNA expression was analyzed using real-time-PCR, IL-32 protein was determined by Western blot and flow cytometry as well as immunofluorescent staining in primary sinus epithelial cells and nasal biopsies from patients with CRS and healthy controls.

IL-32 mRNA was upregulated by TNF-α and IFN-γ in primary sinus epithelial cells, whereas IL-1 β, IL-4, IL-13, and IL-17 did not influence IL-32 expression. IL-32 mRNA expression was significantly higher in human primary sinonasal epithelial cells (HSECs) cocultured with Th1 cells compared with HSECs cocultured with Th0 or Th2 cells. IL-32 mRNA expression was significantly higher in biopsies from sinus epithelial tissue of CRS patients with nasal polyps compared with healthy subjects (P = 0.01). IL-32 was detected in biopsies from patients with CRS, whereas it was scarcely present in control tissues.

The induction of IL-32 by TNF-α, IFN-γ and Th1 cells as well as its increased expression in sinus tissues from CRS patients with nasal polyps demonstrated a potential role for IL-32 in the pathogenesis of CRS.

The expression of mRNA for IL-32 was elevated in epithelial cells from uncinate tissue from patients with CRSsNP compared with patients with CRSwNP, control subjects, and epithelial cells from nasal polyp (NP) tissue. Production of IL-32 was induced by IFN-γ, TNF, and dsRNA in primary airway epithelial cells. In whole-tissue extracts, the expression of IL-32 protein was significantly elevated in patients with CRSwNP compared with patients with CRSsNP and control subjects. Immunohistochemistry data showed that IL-32 was detected in mucosal epithelial cells and inflammatory cells in the lamina propria. Levels of IL-32 were correlated with the levels of CD3 and macrophage mannose receptor in NP tissue. Immunofluorescence data showed IL-32 co-localization with CD3-positive T cells and CD68-positive macrophages in NPs. Overproduction of IL-32 may be involved in the pathogenesis of CRS, although the role of IL-32 in the inflammation in CRSsNP and CRSwNP may be different.

Sinonasal epithelial cell expression of toll-like receptor 9

Innate immune recognition of pathogens by sinonasal epithelial cells may play an important role in the pathogenesis of chronic rhinosinusitis (CRS). Previous studies have indicated that toll-like receptor (TLR) mRNA is present in sinonasal mucosa, and levels of TLR9 expression are decreased in recalcitrant CRS with nasal polyps (CRSwNP). However, the cellular source and function of TLR9 in the sinonasal epithelium is not known. In this study, primary epithelial cell cultures were analyzed from control subjects and CRSwNP patients to determine the presence and function of TLR9 protein.

Primary epithelial cell cultures were established from 5 controls and 10 CRSwNP patients undergoing sinus surgery. Flow cytometry was used to confirm purity of epithelial cells and to assess expression of TLR9 protein. Epithelial cells were stimulated with TLR9 agonist, and mRNA was analyzed by real-time PCR for expression of human beta-defensin (HBD) 2 and interleukin (IL)-8.

Flow cytometry showed TLR9 protein in 100% of epithelial cells from controls and CRSwNP patients. The level of expression was 50% lower in CRS patients than in controls. Stimulation of epithelial cells with TLR9 agonist produced a 1.5- to 9-fold increase in HBD-2 and IL-8 mRNA expression.

Functional TLR9 protein is expressed by normal and diseased sinonasal epithelial cells. The level of TLR9 expression is decreased in CRSwNP patients, consistent with the previous finding of decreased TLR9 mRNA in whole sinonasal tissue. These findings suggest that impaired innate immune responses to pathogens via TLR9 on sinonasal epithelial cells may represent a critical mechanism in chronic inflammatory sinus disease.

Dual nature of T cell-epithelium interaction

T-cell infiltration of submucosa, release of proinflammatory cytokines leading to epithelial activation, and contributions to inflammation are observed in chronic rhinosinusitis (CRS). Molecular mechanisms and kinetics of T-cell interaction with sinus epithelium leading to activation followed by subsequent apoptosis of epithelial cells were the focus of the current study.

Primary human sinus epithelial cells and T cells generated from sinus tissues of healthy individuals and patients with CRS with or without allergy and sinus tissue biopsies were characterized in terms of activation (surface marker expression, cytokine production via real-time PCR, confocal microscopy, ELISA) and apoptosis (annexin V/7-amino-actinomycin D staining, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay, receptor expression by flow cytometry, confocal microscopy) of epithelial cells.

Primary human sinus epithelial cells isolated from patients with CRS were at an activated state with upregulated expression of HLA-DR, IFN-gamma-inducible protein 10, monokine induced by IFN-gamma, and TNF-related apoptosis-inducing ligand (TRAIL) compared with healthy individuals. The expressions of these chemokines, HLA-DR, TRAIL, and TNF receptor 2 were significantly induced by IFN-gamma, whereas TRAIL receptor 4 was downregulated. Epithelial cells started to undergo apoptosis 48 hours after IFN-gamma stimulation when the transcription of proinflammatory cytokines and chemokines decreased to initial levels. The essential factors for sinus epithelial apoptosis were T(H)1 cells and IFN-gamma. Epithelial apoptosis was enhanced by Fas-Fas-ligand and TRAIL-TRAIL receptor 2 interactions. Remarkable apoptosis of epithelial cells and shedding was observed in CRS in situ. Epithelial cell interaction with activated T cells is a biphasic phenomenon in CRS. Initially activated T cells lead to activation and induction of proinflammatory functions of epithelial cells, and thereafter their apoptotic death, resulting in no more contribution to inflammation, takes place.

Association of the -14C/G MET and the -765G/C COX-2 Gene Polymorphisms

Chronic rhinosinusitis with nasal polyps is strongly associated with other diseases, including asthma and allergy. The following study tested the association of the -765G/C polymorphism of cyclooxygenase-2 (COX-2) encoding gene and the -14C/G polymorphism of protooncogen MET (MET) encoding gene with a risk of chronic rhinosinusitis with nasal polyps in a Polish population. Sitarek et al reported observed of one hundred ninety-five patients of chronic rhinosinusitis with nasal polyps as well as 200 sex-, age-, and ethnicity-matched control subjects without chronic sinusitis and nasal polyps were enrolled in this study. Among the group of patients, 63 subjects were diagnosed with allergy and 65 subjects with asthma, respectively.

DNA was isolated from peripheral blood lymphocytes of patients as well as controls, and gene polymorphisms were analyzed by restriction fragment length polymorphism-polymerase chain reaction (RFLP-PCR). Ten percent of the samples have been confirmed by a second method single-strand conformation polymorphism (SSCP)-PCR. We reported that the -765G/C COX-2  and the -14C/G MET  were associated with an increased risk of chronic rhinosinusitis with nasal polyps among analyzed group of patients. Moreover, the group of patients without allergy or asthma indicated the association of the -765C/G  genotype of the COX-2 as wells as the -14C/G  genotype of MET with an increased risk of chronic rhinosinusitis with nasal polyps. Finally, it was also found that the selected group of patients with allergy or asthma indicated a very strong association of the -765G/C genotype of the COX-2 with an increased risk of chronic rhinosinusitis with nasal polyps.

These study suggest that COX-2 and MET gene polymorphisms may have deep impact on the risk of rhinosinusitis nasal polyp formation, which may also depend on asthma or allergy. Our results showed that the -765G/C polymorphism of COX-2 gene and the -14C/G polymorphism of the MET gene may be associated with the risk of chronic rhinosinusitis with nasal polyps in a Polish population.

Matrix metalloproteinase 2 and 9 expression and severity of chronic rhinosinusitis

Matrix metalloproteinase (MMP) is involved in the remodeling process of inflammatory airway diseases and is correlated with the severity of asthma. MMP was associated with the severity of chronic rhinosinusitis with nasal polyps (CRSwNPs).  Wang et al reported that investigated the effect of allergy on the expression of MMP in the polyp.

The expression of MMP-2 and -9 was investigated in recurrent nasal polyps of 30 patients and in nonrecurrent nasal polyps of 31 patients undergoing endoscopic sinus surgery. These  expressions were then compared with those in control nasal mucosal samples obtained from 32 patients with chronic hypertrophic rhinitis. Demographic data, Lund-Mackay (LM) score, polyp grade, and allergy status were obtained for all patients. Tissue samples were assessed via immunohistochemistry.

MMP-2 and -9 were constantly expressed in recurrent NPs, primary NPs, and control nasal mucosa. The expression of MMP-9 was significantly enhanced in glands and MMP-2 positivity was significantly increased in surface epithelium for patients with NPs when compared with  control nasal mucosa. The expression of MMP-9 and -2 was not correlated with polyp grade and LM score. Allergic status is an independent factor in the expression of MMP-2 and -9.

These results suggested up-regulation of MMP-9, and MMP-2 in gland and surface  epithelium, respectively, were characteristic of NPs. Therefore, patients with allergy will exhibit greater MMP-2 and -9 positivity. However, the MMP-2 and -9 expression intensity was not correlated with the severity of CRS with nasal polyposis.

Acquired cilia dysfunction

Cilia are complex and powerful cellular structures of the respiratory mucosa that play a critical role in airway defense. Respiratory epithelium is lined with cilia that perform an integrated and coordinated mechanism called mucociliary clearance. Mucociliary  clearance is the process by which cilia transport the mucus blanket overlying respiratory mucosa to the gastrointestinal tract for ingestion. It is the primary means by which the airway clears pathogens, allergens, debris, and toxins. The complex structure and regulatory mechanisms that dictate  the form and function of normal cilia are not entirely understood, but it is clear that ciliary dysfunction results in impaired respiratory defense. The current knowledge of cilia dysfunction in chronic rhinosinsusitis was conducted.

Ciliary dysfunction may be primary, the result of genetic mutations resulting in abnormal cilia structure, or, more commonly, secondary, the result of environmental, infectious, or inflammatory stimuli that disrupt normal motility or coordination. Patients with chronicrhinosinusitis (CRS) have been found to have impaired mucociliary clearance. Many biochemical, environmental, and mechanical stimuli have been shown to influence ciliary beat frequency, and common microbial pathogens of respiratory mucosa such as Pseudomonas aeruginosa and Haemophilus influenzae  have developed toxins that appear to interrupt normal mucociliary function. Furthermore, inflammatory mediators known to be present in patients with CRS appear to impair secondarily mucociliary clearance.

References:

  • Soyka MB, et al.Regulation and expression of IL-32 in chronic rhinosinusitis. Allergy. 2012 Apr 9.
  • Wu X, Mimms R, Lima R, Peters-Hall J, Rose MC, Peña MT. Localization of inflammatory mediators in pediatric sinus mucosa.  Arch Otolaryngol Head Neck Surg. 2012 Apr;138(4):389-97.
  • Keswani A, et al. Differential expression of interleukin-32 in chronic rhinosinusitis with and without nasal polyps. Allergy. 2012 Jan;67(1):25-32.
  • Basinski TM, et al. Dual nature of T cell-epithelium interaction in chronic rhinosinusitis. J Allergy Clin Immunol. 2009 Jul;124(1):74-80.e1-8.
  • Ramanathan M Jr, et al. Sinonasal epithelial cell expression of toll-like receptor 9 is decreased in chronic rhinosinusitis with polyps. Am J Rhinol. 2007 Jan-Feb;21(1):110-6.
  • Basinski TM, et al. Dual nature of T cell-epithelium interaction in chronic rhinosinusitis. J Allergy Clin Immunol. 2009 Jul;124(1):74-80.e1-8.
  • Sitarek P, Zielinska-Blizniewska H, Dziki L, Milonski J, Przybylowska K, Mucha B, Olszewski J, Majsterek I.  Association of the -14C/G MET and the -765G/C COX-2 Gene Polymorphisms with the Risk of Chronic Rhinosinusitis with Nasal Polyps in a Polish Population. DNA Cell Biol. 2012 Mar 14.
  • Am J Rhinol Allergy. 2012 Jan-Feb;26(1):1-6. Acquired cilia dysfunction in chronic rhinosinusitis. Gudis D, Zhao KQ, Cohen NA.
  • Wang LF, Chien CY, Chiang FY, Chai CY, Tai CF. Corelationship between matrix metalloproteinase 2 and 9 expression and severity of chronic rhinosinusitis with nasal polyposis. Am J Rhinol Allergy. 2012 Jan-Feb;26(1):e1-4.

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