T-helper type cytokines

Inflammation of the sinonasal mucosa is accompanied by characteristic profiles of increased cytokine expression.Reference Daines and Orlandi¹⁵ Cytokines are chemical messengers that direct the immune response by facilitating communication between diverse cell populations. They are proteins produced by structural and infiltrating immune cells, and they interact with specific cell-surface receptors. Cytokines are involved in the stimulation and regulation of the immune system, and have a wide variety of functions, ranging from cellular proliferation to chemotaxis.

Cytokines are divided into groups depending on the type of cluster of differentiation 4⁺ T-helper cell that produces them.Reference Daines and Orlandi^15–Reference Derycke, Eyerich, Van Crombruggen, Perez-Novo, Holtappels and Deruyck¹⁷ The best defined subsets of cluster of differentiation 4⁺ T-helper cells are the T-helper 1, 2 and 17 subsets (Figure 1). T-helper 1 cells produce interleukin (IL)-2 and interferon gamma. These cytokines activate macrophages and are predominantly involved with cellular immune response. T-helper 2 cells produce IL-4, IL-5 and IL-13. These cytokines target B cells and are involved with humoral immunity, stimulation of immunoglobulin synthesis, mast cell proliferation, and eosinophil production, recruitment and survival. T-helper 17 cells produce IL-17, IL-22 and chemokines. These cytokines recruit neutrophils and monocytes, and promote inflammation. T-helper 1, 2 and 17 cytokines can stimulate their own production through an autocrine mechanism, enhance their own effects and stimulate production of other cytokines. They can also inhibit other subsets of cells in the vicinity to produce polarisation of the immune response.

Fig. 1

Subsets of cluster of differentiation 4⁺ T-helper cells and their target roles in immunity. CD = cluster of differentiation; T_H = T-helper; IL = interleukin; IFN = interferon

Cytokines are readily detectable in sinonasal secretions. Molecular-based studies such as real-time quantitative polymerase chain reaction and DNA microassay analysis have revealed a characteristic profile for chronic rhinosinusitis. This profile includes tumour necrosis factor alpha, IL-3, granulocyte macrophage colony stimulating factor, IL-1 beta, IL-5, IL-8 and IL-13.Reference Ramanathan and Lane¹⁸

A study by Riechelmann et al. examined the expression of 15 cytokines from the T-helper 1, 2 and 17 subsets in the sinonasal secretions of patients with acute rhinosinusitis, chronic rhinosinusitis without nasal polyps, and chronic rhinosinusitis with nasal polyps.Reference Riechelmann, Deutschle, Rozsasi, Keck, Polzehl and Burner¹⁹ Results from that study showed that compared with controls, all 15 cytokines examined were significantly higher in expression in patients with chronic rhinosinusitis, irrespective of the clinical type.Reference Riechelmann, Deutschle, Rozsasi, Keck, Polzehl and Burner¹⁹ Interleukin 5 levels were also found to be significantly higher in patients with chronic rhinosinusitis with nasal polyps, compared with patients with acute rhinosinusitis and chronic rhinosinusitis without nasal polyps.Reference Ramanathan and Lane¹⁸ The most common type of inflammatory cell present in nasal polyps is eosinophils, and therefore these findings support the role of IL-5 in the activation and prolonged survival of eosinophils.Reference Aukema and Fokkens^1, Reference Akdis, Bachert, Cingi, Dykewicz, Hellings and Naclerio^13, Reference Riechelmann, Deutschle, Rozsasi, Keck, Polzehl and Burner^19, Reference Bachert, Wagenmann, Hauser and Rudack²⁰ These studies highlight the important role of cytokines in chronic rhinosinusitis, and the variation in cytokine expression between chronic rhinosinusitis patients with and without nasal polyps.

Nuclear factor kappa B activation

Chronic inflammation in chronic rhinosinusitis is induced and maintained by a complex interaction of various cytokines. This process is activated by several transcription factors, one of which is nuclear factor kappa B.Reference Lawrence²¹ Nuclear factor kappa B is considered to be a prototypical component of the proinflammatory signalling pathway, and has an important role in the modulation of pro- and anti-inflammatory cytokines at the gene expression level.

Most of the knowledge of signalling in inflammation has been obtained by studying members of the interleukin (IL)-1 and tumour necrosis factor (TNF)-alpha receptor families, and the toll-like microbial pattern recognition receptors, which belong to the IL-1 receptor family.Reference Lawrence²¹ Interleukin 1 and TNF-alpha are archetypal proinflammatory cytokines that are rapidly released upon tissue injury or infection.Reference Bachert, Wagenmann, Hauser and Rudack²⁰ Toll-like receptors are part of a germline-encoded non-self-recognition system that is programmed to trigger inflammation.Reference Akira, Uematsu and Takeuchi²² These receptors, albeit structurally unrelated, use similar signal transduction mechanisms, such as the activation of IκB kinase and nuclear factor kappa B.Reference Ghosh and Karin²³ IκB kinase is an enzyme involved in the propagation of cellular responses to inflammation and forms part of the upstream nuclear factor kappa B signal transduction cascade.Reference Lawrence²¹

There are two main pathways for nuclear factor kappa B activation.Reference Lawrence^21, Reference Strnad and Burke²⁴ The canonical pathway is triggered by microbial products and proinflammatory cytokines such as TNF-alpha and IL-1, and leads to the activation of nuclear factor kappa B proteins, and RelA- or cRel-containing complexes.Reference Karin and Ben-Neriah²⁵ The non-canonical pathway is triggered by TNF-family cytokines such as lymphotoxin beta, cluster of differentiation 40 ligand, B cell activating factor and receptor activator of nuclear factor kappa B ligand, and leads to the activation of RelB/p52 complexes.Reference Senftleben, Cao, Xiao, Greten, Krahn and Bonizzi^26–Reference Novack, Yin, Hagen-Stapleton, Schreiber, Goeddel and Ross²⁸ These two pathways are differentiated by their requirement for the IκB kinase subunits. The IκB kinase complex consists of two kinase subunits, IκB kinase alpha and IκB kinase beta, and a regulatory subunit IκB kinase gamma. IκB kinase beta regulates activation of the canonical pathway through phosphorylation of IκB, and requires the IκB kinase gamma subunit but not IκB kinase alpha.Reference Zandi, Rothwarf, Delhase, Hayakawa and Karin²⁹ IκB kinase alpha regulates activation of the non-canonical pathway through phosphorylation and processing of p100, the precursor for p52.Reference Lawrence^21, Reference Ghosh and Karin^23, Reference Senftleben, Cao, Xiao, Greten, Krahn and Bonizzi²⁶ IκB kinase alpha is independent of both IκB kinase beta and IκB kinase gamma.Reference Lawrence^21, Reference Ghosh and Karin^23, Reference Senftleben, Cao, Xiao, Greten, Krahn and Bonizzi²⁶

Inflammation is a process of innate immunity in response to physical, physiological or oxidative stress, and is associated with activation of the canonical pathway.Reference Hoesel and Schmid³⁰ The canonical pathway primarily responds to TNF-alpha and IL-1 signalling, and prototypical proinflammatory cytokines, and therefore has an important role in the pathogenesis of chronic inflammatory diseases.Reference Lawrence²¹

In the canonical pathway, the inactive form of nuclear factor kappa B lies in the cytoplasm, and is constituted by a heteromeric complex of p50, p65 and an inhibitor of the nuclear factor kappa B family of proteins (IκB alpha).Reference Strnad and Burke^24, Reference Fraczek, Rostkowska-Nadolska, Kapral, Szota, Krecicki and Mazurek³¹ In response to a variety of proinflammatory signals, mediated through toll-like receptors, IL-1 receptors, TNF receptors and antigen receptors, the activation of nuclear factor kappa B leads to the phosphorylation and degradation of IκB alpha. The released p50/p65 heterodimer consequently translocates to the nucleus, where it binds to specific consensus sequences within the promoters of the nuclear factor kappa B target genes to enhance gene expression.

Chronic inflammatory conditions such as chronic rhinosinusitis are influenced by nuclear factor kappa B modulation in diseased tissue. A study by Takeno et al. examined the relationship between the degree of nuclear factor kappa B activation and the levels of local cytokine gene expression in nasal polyp epithelium.Reference Takeno, Hirakawa, Ueda, Furukido, Osada and Yajin³² The results from that study showed a significant correlation between the degree of epithelial nuclear factor kappa B activation and the levels of IL-8, IL-16 and eotaxin messenger RNA (mRNA) expression. This highlights the important role of nuclear factor kappa B in the inflammatory process in chronic rhinosinusitis with nasal polyps. A study by Yang et al. has also shown that nuclear factor kappa B may act as anti-apoptotic molecules in eosinophils and are responsible for the recruitment-enhanced survival of inflammatory cells.Reference Yang, Cohn, Zhang, Homer, Ray and Ray³³

Previous studies have described the expression and localisation of cyclo-oxygenase 2 in chronic rhinosinusitis mucosa, and have shown that nuclear factor kappa B pathways are involved in the up-regulation of cyclo-oxygenase 2 expression.Reference Wang, Zhang, Li, Li, Zhang and Li^34–Reference Zhou, Shin, Guo, Youn, Bae and Kang³⁸ Cyclo-oxygenase is an enzyme that converts arachidonic acid to prostaglandins and thromboxane. As an inducible isoform, cyclo-oxygenase 2 has been shown to be induced by various inflammatory stimuli, such as cytokines and lipopolysaccharide, in cells in vitro and in inflamed sites in vivo.

Transforming growth factor beta and tissue remodelling

Chronic inflammation in chronic rhinosinusitis results in structural changes referred to as remodelling.Reference Akdis, Bachert, Cingi, Dykewicz, Hellings and Naclerio¹³ The remodelling pattern is not consistent in chronic rhinosinusitis, and there are different types of mucosal remodelling in chronic rhinosinusitis patients without nasal polyps and those with nasal polyps (Table I).Reference Tomassen, Van Zele, Zhang, Perez-Novo, Van Bruaene and Gevaert^39–Reference Spinozzi, de Benedictis and de Benedictis⁴³ Chronic rhinosinusitis without nasal polyps is characterised by fibrosis, basement membrane thickening, goblet cell hyperplasia, subepithelial oedema and mononuclear cell infiltration. In contrast, chronic rhinosinusitis with nasal polyps is characterised by severely oedematous stroma with albumin deposition, pseudocysts, and subepithelial and perivascular inflammatory cell infiltration.

Table I

Mucosal remodelling patterns in chronic rhinosinusitis with and without nasal polyps

The remodelling process aims to balance extracellular matrix production and degradation, and is regulated by several mediators, of which transforming growth factor beta has the main role. Transforming growth factor beta is involved in the attraction and induction of fibroblast proliferation, with an integral role in wound repair.Reference Leask and Abraham⁴⁴

In patients with chronic rhinosinusitis without nasal polyps, previous studies have shown that transforming growth factor beta 1 and 2 protein concentrations, and transforming growth factor beta receptor type I and III mRNA expressions, were significantly higher compared with controls.Reference Van Crombruggen, Zhang, Gevaert, Tomassen and Bachert^40–Reference Yang, Zhang, Van Crombruggen, Hu, Hong and Bachert⁴² In patients with chronic rhinosinusitis with nasal polyps, transforming growth factor beta 1 protein concentration, and transforming growth factor beta receptor type II and III mRNA expressions, were significantly lower compared with controls. In patients with chronic rhinosinusitis without nasal polyps, the up-regulation of the transforming growth factor beta signalling pathway results in excessive collagen deposition with fibrosis. In patients with chronic rhinosinusitis with nasal polyps, the down-regulation of the transforming growth factor beta signalling pathway results in oedema formation and lack of collagen production.

Potential therapeutic strategies

Most clinicians tend to classify chronic rhinosinusitis by clinically relevant phenotypes, such as the absence or presence of nasal polyps, severity, disease recurrence, and response to therapy.Reference Akdis, Bachert, Cingi, Dykewicz, Hellings and Naclerio¹³ However, there is increasing evidence to suggest that chronic rhinosinusitis should also be classified by endotypes. The presence of different inflammatory and remodelling patterns between chronic rhinosinusitis patients without nasal polyps and those with nasal polyps, as highlighted in this review article, reflects the different pathophysiological mechanisms involved, and these are associated with different responses to treatment.Reference Gevaert, Van Bruaene, Cattaert, Van Steen, Van Zele and Acke^45, Reference Van Bruaene and Bachert⁴⁶ Classifying chronic rhinosinusitis by endotypes therefore allows for targeted therapeutics, with improved effectiveness and better patient outcomes.

A study by Gevaert et al. investigated the therapeutic potential of inhibiting interleukin (IL)-5 with a humanised monoclonal antibody, mepolizumab, as a treatment option for chronic rhinosinusitis with nasal polyps.Reference Gevaert, Van Bruaene, Cattaert, Van Steen, Van Zele and Acke⁴⁵ This was a randomised, double-blind, placebo-controlled study, where patients with chronic rhinosinusitis with nasal polyps received two single intravenous injections of mepolizumab. The results from that study showed that mepolizumab significantly reduced the size of nasal polyps, based on endoscopic scoring and computed tomography scans. Further long-term studies are required at this stage to further assess the full effects of mepolizumab.

Interestingly, recent strategies have also included the use of anti-sense oligonucleotides, such as TPI ASM8, to target IL-5 receptors.Reference Amini-Vaughan, Martinez-Moczygemba and Huston⁴⁷ TPI ASM8 modulates the expression of the common beta chain of IL-5 receptors to inhibit T-helper 2 type cytokines. Initially developed for asthma, studies have shown that inhaled TPI ASM8, with increasing doses over 4 days, results in a 60 per cent and 68 per cent reduction in sputum eosinophils after 7 and 24 hours respectively. It is hoped that the broader therapeutic potential for anti-sense oligonucleotides will be demonstrated for other subsets of hypereosinophilic syndromes such as chronic rhinosinusitis in the future.

The inhibition of nuclear factor kappa B has also been explored as a novel target for therapeutics in chronic rhinosinusitis. A study by Valera et al. investigated dehydroxymethylepoxyquinomicin (‘DHMEQ’) as a potential nuclear factor kappa B inhibitor for the treatment of chronic rhinosinusitis with nasal polyps.Reference Valera, Umezawa, Brassesco, Castro-Gamero, Queiroz and Scrideli⁴⁸ Dehydroxymethylepoxyquinomicin is derived from the antibiotic epoxyquinomicin. In that study, dehydroxymethylepoxyquinomicin was shown to directly inhibit the nuclear translocation and transcription of nuclear factor kappa B in vitro. Further studies are required at this stage to determine the ideal doses and schedules of dehydroxymethylepoxyquinomicin, and its efficacy in controlled clinical trials.

Given its regulatory function in both inflammation and remodelling processes, transforming growth factor beta also represents a novel therapeutic target.Reference Yang, Zhang, Van Crombruggen, Hu, Hong and Bachert⁴² Corticosteroids are currently the first-line therapy for chronic rhinosinusitis with nasal polyps, as recommended by the European position paper on rhinosinusitis and nasal polyps and by American guidelines.Reference Fokkens, Lund, Mullol, Bachert, Alobid and Baroody^4, Reference Kou, Hu, Yao, Wang, Shen and Kang^49, Reference Meltzer, Hamilos, Hadley, Lanza, Marple and Nicklas⁵⁰In vitro corticosteroids have been shown to suppress transforming growth factor beta 1 induced fibroblast proliferation and differentiation in nasal polyps.Reference Serpero, Petecchia, Sabatini, Giuliani, Silvestri and Di Blasi⁵¹

Long-term, low-dose macrolide antibiotic therapy (e.g. clarithromycin) is effective in the treatment of chronic rhinosinusitis through anti-inflammatory effects.Reference Kou, Hu, Yao, Wang, Shen and Kang^49, Reference Wallwork, Coman, Mackay-Sim and Cervin⁵² A study by Wallwork et al. showed that when applied in vitro, clarithromycin significantly reduced the expression of transforming growth factor beta in cultured nasal mucosal specimens from chronic rhinosinusitis patients.Reference Wallwork, Coman, Mackay-Sim and Cervin⁵² However, when clarithromycin was given therapeutically to chronic rhinosinusitis patients for three months, nasal biopsies taken before and after treatment showed no differences in the cellular expression of transforming growth factor beta.