Expression of chemotaxis in regulators of tissues of exudative lesions of Reinke’s space

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Exudative tumor-like neoplasms of the Reinke space in vocal folds are widespread in the population, more often among representatives of vocal professions, e.g., actors, singers, teachers, lecturers and represent a serious medical and social problem. In pathogenesis of such neoplasms, a key role is given to chronic phonotrauma, intoxication during smoking against the background of the nearly complete absence of lymphatic drainage in the Reinke space. Cellular factors of tissue and immune homeostasis are of great importance in morphogenesis of this disorder. Switching of immune responses aimed at maintaining tissue homeostasis is accompanied by increased production of pro-inflammatory cytokines, as well as chemotaxis regulators and growth factors. The aim of our work was to study the levels of chemokines (MIP-1α, MIP-1β, MIP-3α, fractalkine, IL-8, I-TAC) in the tissues of exudative lesions from the Reinke’s space (EPPR).

Forty tissue samples of exudative lesions from Reinke’s space, in particular, vocal fold polyps, vocal nodules and neoplasms presenting as Reinke’s edema were taken as biological material for the study. The samples were taken intraoperatively when the neoplasm was removed, using an Olympus TYPE 150 fiber bronchoscope (Germany) using an integrated Lumenis Acu Pulse CO2 laser (Israel). The content of chemokines was determined in the supernatants of the tissues homogenates, using multiplex analysis with MAGPIX-100 immunoanalyzer (Bio-Rad, USA). Statistical processing was carried out using Statistica 10.0 for Windows software package. The results are presented as medians (Q0.25-Q0.75).

Chronic phonotrauma and/or exposure to toxic factors leads to increased permeability of the vascular endothelium, tissue edema and activation of cells involved in tissue and immune homeostasis, e.g., fibroblasts, monocytes, endotheliocytes. The study of the chemokine expression in the tissues of various exudative lesions of Reinke’s space enabled us to reveal the following features: (1) predominance of the CXC-chemokine content produced mainly by fibroblasts in “dense” neoplastic tissues, thus reflecting participation of the latter in the genesis of this pathology. (2) In the tissues of “soft” neoplasms with large proportion of liquid component, we have revealed increased concentrations of SS-chemokines and fractalkine. The latters are produced mainly by macrophages and endotheliocytes, thus, probably, reflecting a predominant role of these cells in development of myxoid polyps and Reinke’s edema.

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Exudative tumor-like neoplasms of the vocal folds are quite widespread in the population, more often among representatives of voice-speech professions: actors, singers, teachers, announcers and represent a serious medical and social problem. According to experts, the incidence of this pathology ranges from 55 to 68% among voice professionals [1]. The main clinical forms of pathology are vocal fold polyps, vocal nodules, Reinke's edema. The leading causes of the development of pathology are considered to be intense, prolonged overexertion of the vocal folds associated with forced voice formation, the so-called "hard attack" and the impact of aggressive tobacco smoke on the vocal folds [1]. In addition to the mechanical component, other etiological factors include inhalation of toxic aerosols (for example, gas aerosol during welding, chemicals, smoke), pharyngolaryngeal reflux [1]. The pathogenesis is based on microcirculatory disorders caused by insufficient lymphatic drainage and accompanied by the accumulation of edematous fluid in the Reinke's space. Intense vocal loads lead to overstrain of the vocal apparatus, damage to microvessels, an increase in the permeability of the latter and the release of plasma into the interstitial space. Features of the drainage system of the vocal folds prevent the timely outflow of fluid into the regional lymph collectors. The formation of ischemic-hypoxic disorders against this background leads to the activation of local factors of the immune system that are part of the larynx - associated lymphoid tissue - LALT (Larynx-Associated Lymphoid Tissue) [6]. growth, which contributes to the progression of pathology. Chemokines are involved not only in the coordination of leukocyte movement during inflammatory processes, but are also important in many physiological and pathological processes.
In this regard, the aim of this study was to study the level of chemokines MIP-1α, MIP-1β, MIP-3α, fractalkine, IL-8, I-TAC in the tissues of exudative lesions of Reinke's space.
The material for the study was 40 tissue samples of vocal fold neoplasms taken intraoperatively from patients with a diagnosis according to ICD-10 J38.1 Polyp of the vocal fold and larynx and admitted for surgical treatment. Neoplasms were removed using an Olympus TYPE150 fiber bronchoscope (Germany) under endotracheal anesthesia under the control of a Haag-Streit Surgical Möller Wedel MV-500 operating microscope (Germany) using an integrated CO2 laser Lumenis Acu Pulse (Israel) according to the Hirano method. Tissue samples were distributed as follows: group 1 - 10 tissue samples with angiomatous type of polyps, group 2 - 9 myxoid type polyps, group 3 - vocal cord nodules (n=7), group 4 - Reinke's edema (n=14).
Definition of chemokines
were carried out in the tissue supernatant EPPR. To obtain the supernatant, tissue homogenates were centrifuged at 10,000 g for 20 min; the obtained supernatant was stored at –20°C until the study. The quantitative content of chemokines in the obtained supernatants was assessed using multiplex analysis on a MAGPIX-100 immunoanalyzer using the Bio-Plex multiplex analysis system (Bio-Rad, USA) to determine fractalkine, IL-8, I-TAC, MIP-1α, MIP -1β, MIP-3α (reagents "Merk", Germany) in accordance with the instructions of the company. For the purpose of standardization, the amount of protein in the homogenate was determined by the biuret method using the TOTAL PROTEIN-OLVEKS reagent kit (St. Petersburg, Russia). Calculated concentrations of cytokines are presented in pg/mg of protein.
Statistical processing
The material was carried out using the Statistica 10.0 for Windows software package. The values ​​of the indicators are presented as Me (Q25; Q75), where Me is the median, Q25 and Q75 are the interquartile interval. The groups were compared using the non-parametric Mann-Whitney test, taking into account the Bonferroni correction, differences were considered significant at p≤0.02.

The presented results indicate a change in the ratio of various chemokines in the studied samples (table 1).
The tissues of the so-called "dense" polyps, which include the angiomatous type of polyps and vocal nodules, and morphogenesis is dominated by fibroblast proliferation, dilation of the capillary bed, stromal hyalinosis with a pronounced thickening of the subbasal membrane, diffuse deposition of fibronectin, neoangiogenesis phenomena, contain the highest concentration of chemokines, representatives α - chemokines (CXC-family), in particular, IL-8 and interferon - γ-induced T-cell alpha chemoattractant (I-TAC; CXCL11 family).

Table -1 The concentration of chemokines in the tissues of exudative lesions of Reinke's space (Me (Q25; Q75))

Interleukin 8 and I-TAC, belonging to the family of α-chemokines (CXC-chemokines) and produced mainly by fibroblasts, monocytes, endothelial cells, are actively involved in the activation, migration and chemotaxis of cells, are able to determine the type of cells penetrating into the tissue from the bloodstream through endothelium, as well as the direction of migration [2, 3]. The highest density of IL-8 receptors was found on neutrophils, monocytes/macrophages, and activated T-lymphocytes, after interaction with which CXCR1 binds to G-protein, leading to phosphoinositide hydrolysis, intracellular Ca2+ mobilization, and cell chemotaxis [3].
I-TAC receptors are predominantly expressed on activated Th1 lymphocytes and endotheliocytes. The activator and main regulator of I-TAC production is IFN-γ, negative control is carried out by anti-inflammatory cytokines IL-4 and IL-10.
I-TAC promotes the recruitment of activated Th1-lymphocytes to the zone of pathology formation. There is evidence of an angiostatic effect of I-TAC, which exhibits an angiostatic effect by secreting CXCR3 on endothelial cells [3, 5].
At the same time, in tissue samples of myxoid polyps and in Reinke's edema, characterized by the presence of an excess fluid component in the interstitium, thickening and expansion of the walls of blood vessels, thinning and fenestration of the endothelial lining, an increase in the level of β-chemokines (CC-chemokines) was observed: MIP- 1α (CCL3), MIP-1β (CCL4) and MIP-3α (CCL20) and δ-chemokines (CX 3 C-chemokines) - fractalkine (CX3CL1) [7, 12].
It is known that all representatives of β-CA are chemoattractants and activators of both innate (monocytes, dendritic cells, NK cells) and adaptive (T-lymphocytes) immunity cells. At the same time, the profile of receptors for CC - chemokines on cells can selectively change depending on the degree of cell differentiation. Thus, all T lymphocytes express CCR1, Th1 lymphocytes express CCR5 and CXCR3, and Th2 lymphocytes preferentially express CCR3 [12].
Macrophage inflammatory proteins - MIP (lα; 1β; 3α; 3β), being expressed mainly on activated monocytes / macrophages, less often on fibroblasts, epithelial cells, vascular smooth muscle cells, manifest their effect by binding to extracellular proteoglycans and ligating chemokine receptors CCR1 (ligand CCL3) and CCR5 (CCL3 and CCL4 ligands). Receptors for proteins of the MIP family (lα; 1β; 3α; 3β) are also present on dendritic cells and platelets [7, 11].
Fractalkin is the only member of the CX 3 C chemokine family present on monocytes and endotheliocytes both in soluble and mucin-bound form and plays an important role in the chemotaxis of innate (monocytes, NK cells) and adaptive (T-lymphocytes) immunity cells [ 4].
Chronic overstrain of the vocal folds or exposure to toxic factors, components of bile and hydrochloric acid during reflux leads not only to an increase in the permeability of the vascular endothelium, the accumulation of a fluid component in the interstitium and stroma of the vocal folds, but also to the activation of cells involved in tissue and immune homeostasis, for example, fibroblasts, monocytes, endotheliocytes. In the stroma of the vocal folds, the population of fibroblasts, which are factors of innate immunity, is quite numerous and, upon activation, can change its phenotype to an inflammatory one and increase secretory activity [8]. There is evidence that fibroblasts are able to modulate macrophage paracrine signaling during early expression of pro-inflammatory cytokines (TNF-α, IL-6, IL-12) and subsequent expression of chemokines and growth factors (IL-8, I-TAC, MCP-1, TGF -β) [10]. It is fibroblasts that can determine the degree and duration of the response of macrophages. Morphometric studies performed by us earlier showed that the representation of fibroblasts in the tissues of "dense" neoplasms, in particular, in angiomatous types of polyps and vocal nodules, is the largest among all forms of exudative lesions of Reinke's space, which is probably why, in these neoplasms, the highest concentration of these above chemokines. At the same time, a certain role in the stimulation of fibroblasts of the interaction of TLRs with the molecular patterns of the bacterial wall cannot be excluded, since the larynx is located at the intersection of the respiratory system and the gastrointestinal tract, and the microflora on the luminal surface of the vocal fold can penetrate into the epithelium and activate innate defense mechanisms in your own plate. Fibroblasts are among the first to encounter infectious agents, since they constitutively express TLRs 1, 2, 3, 4, 5, 6, and 9 [10]. The literature describes the hypothesis of a change in the fibroblast phenotype to inflammatory when microvessels are damaged and exposed to primary inflammatory mediators, such as cyclooxygenase-2, HIF-1α, proteases (MMP-2, 9), proteins (fibronectin, decorin), capable of changing signaling and activating


About the authors

Michael A. Kovalev

A. Vishnevsky Central Military Clinical Hospital (Branch 3)


Head, Department of Otorhinolaryngology

Russian Federation, Odintsovo, Moscow Region

Evgeniya V. Davydovа

Chelyabinsk Regional Clinical Hospital

Author for correspondence.

PhD, MD (Medicine), Head, Rehabilitation Department

Russian Federation, Chelyabinsk


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Copyright (c) 2022 Kovalev M.A., Davydovа E.V.

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