Substance P and Hemokinin 1 in Nasal Lavage Fluid of Patients with Chronic Sinusitis and Nasal Polyposis

Abstract

This pilot study was undertaken to isolate and quantify substance P (SP) and hemokinin 1 (HK-1) in the nasal lavage fluid of patients with chronic rhinosinusitis with nasal polyps to better elucidate the pathophysiology underlying this inflammatory process, which remains poorly understood. Mucus samples were collected from this introductory cohort of 10 patients diagnosed with chronic rhinosinusitis with nasal polyps at Tufts Medical Center (Boston, Massachusetts). Relative levels of SP and HK-1 were measured with enzyme-linked immunosorbent assay methods. Both inflammatory neuropeptides were found in detectable and comparable amounts in patient samples and in concentrations up to 100-fold those established in past literature. The presence of SP and HK-1 necessitates further investigation into their role in nasal polyposis and the potentiation of the chronic inflammation inherent to chronic rhinosinusitis. Downregulating these peptides could therefore provide novel treatment targets to manage this disease process.

Nasal polyps are edematous outgrowths of degenerated paranasal sinus mucosa, which often coexist with chronic inflammatory processes, most notably allergy and chronic rhinosinusitis (CRS).^1,2 Polyps primarily consist of mast cells known to be involved in allergic and inflammatory processes.³ Common symptoms of nasal polyposis include nasal obstruction, anosmia, rhinorrhea, and subsequent reduced quality of life.⁴ Polyps can also obstruct the sinus drainage pathways and potentiate CRS, for which there is an established association. Chronic sinusitis predisposes patients to recurrent infections, headaches, and antibiotic resistance due to repetitive antibiotic use, making this disease process not only limiting to individuals suffering from it but also part of a larger public health concern.

The pathogenesis of nasal polyposis is thought to be multifactorial, though it remains poorly understood and an active area of research. Previously proposed mechanisms include compromised integrity of the airway epithelial barrier, altered expression of cytokines and chemokines, activation of innate and adaptive immunity, and tissue remodeling.⁵

Treatment modalities for polyposis are limited to intranasal and systemic corticosteroids as well as surgical resection for cases refractory to medical management, but neither has been successful in preventing recurrence—in fact, it is estimated that up to 60% to 70% of patients will have a recurrence of nasal polyps after definitive functional endoscopic sinus surgery by 18 months.^6-8 Thus, further knowledge of this disease process is necessary to create more lasting treatment strategies.⁹

Previous studies have affirmed the role of inflammatory cytokines, such as IL-6 and IL-33, in nasal polyposis.^10,11 It was our hypothesis that 2 particular neuropeptides—substance P (SP) and hemokinin 1 (HK-1)—could also contribute to the inflammation involved in CRS and nasal polyposis. SP and HK-1 act at the neurokinin 1 receptor on mast cells and lymphocytes, and both have been implicated in chronic inflammation as well as nasal polyp formation and regrowth after surgical and steroid treatment. SP and HK-1 have also been shown to potentiate inflammatory states, such as asthma and arthritic disease.^12-14 SP was previously isolated in the nasal polyp tissue of patients with chronic sinusitis, but relevant literature has lain dormant for >2 decades^15,16; the presence of HK-1 in nasal polyp tissue has not yet been recorded, though its shared receptor and function with SP makes this feasible.¹⁷ The following pilot study embodies a preliminary investigation into the hypothesized role of both these neuropeptides in CRS with nasal polyps (CRSwNP) as a means to inform future studies, which would thereby bolster the expanding knowledge base of this disease process.

Methods

In this pilot study, mucus samples were collected at Tufts Medical Center from patients diagnosed with CRSwNP. The study was approved by the Tufts Medical Center Institutional Review Board. The introductory cohort consisted of 3 women and 7 men with a mean ± SD age of 53.7 ± 18.92 years (range, 29-86 years) who were known to have nasal polyps and CRS. Samples were suctioned from the nasal cavity, collected in a Lukens trap collection device, and then stored in 1 mL of normal saline in a ‒80°C freezer until analysis. After thawing on ice, samples were centrifuged at 3000 rpm for 5 minutes. Supernatants were analyzed for the presence of SP and HK-1 with the ELISA protocol.

Results

SP and HK-1 were measured in all 10 mucus samples in comparable concentrations ( Table 1 ). The range of concentrations of SP was larger than that for HK-1, with the former present at a mean concentration of 3.649 ± 1.411 ng/mL and the latter found at a mean concentration of 3.152 ± 0.4266 ng/mL. Notably, previously established levels of SP in mucus samples of control patients were recorded on the order of mcg/mL; comparing these data with the mean concentration of the neuropeptide in our cohort, we found a 100-fold increase in concentration relative to baseline.^18,19

Table 1.

Mean Concentrations of SP and HK-1 in Nasal Lavage Fluid Samples of 10 Patients with CRSwNP as Measured by ELISA.

			Mean Concentration, ng/mL
Patient	Age, y	Sex	SP	HK-1
1	58	F	2.153	2.655
2	40	M	2.113	3.791
3	29	F	6.715	3.146
4	86	M	2.881	3.584
5	34	M	3.700	3.411
6	67	M	4.362	3.101
7	67	F	4.835	3.189
8	53	M	3.851	3.342
9	34	M	2.511	2.950
10	69	M	3.364	2.347

Abbreviations: CRSwNP, chronic rhinosinusitis with nasal polyps; ELISA, enzyme-linked immunosorbent assay; F, female; HK-1, hemokinin 1; M, male; SP, substance P.

Conclusion

Both neuropeptides SP and HK-1 were found in detectable amounts in the samples of patients with nasal polyps and CRS, suggesting their production and secretion by either the nasal polyp tissue itself or peripheral sensory nerve endings, lending credence to their role in potentiating CRS refractory to standard-of-care treatments. Combining knowledge of SP and HK-1 in systemic inflammation with past research efforts to categorize and typify the molecular basis of CRSwNP suggests that these endogenous neuropeptides stimulate nasal polyps to release inflammatory cytokines into the surrounding mucus and nasal mucosal tissue.

The results of our study, as compared with the relatively low amounts in the controls of previous studies, suggest an association among SP, HK-1, and the inflammatory processes involved in CRSwNP. Preliminarily isolating these neuropeptides in the nasal lavage fluid of patients with CRSwNP in this pilot study informs future studies investigating their role in the pathogenesis of nasal polyposis and the chronic inflammation inherent to CRS, while providing novel targets for treatment. The study is limited in that the results were not compared with a control population of patients with nasal polyps who did not have CRS. Further studies will include patients with allergic rhinitis and compare the levels of SP and HK-1 in the nasal lavage fluid of those patients with levels in patients with CRS but without nasal polyps as well as control subjects who have nasal polyps but do not have CRS.

Author Contributions

Ashley Lonergan, primary author, specimen collection, data collection and analysis, drafting, final approval, accountability for work; Theoharis Theoharides, coauthor, data collection and analysis, drafting, final approval, accountability for work; Eirini Tsilioni, coauthor, data collection and analysis, drafting, final approval, accountability for work; Elie Rebeiz, senior author, data collection and analysis, drafting, final approval and revisions, accountability for work.

Disclosures

Competing interests: None.

Sponsorships: None.

Funding source: None.