Immunofluorescent Staining for Mast Cells in Idiopathic Pulmonary Fibrosis: Quantification and Evidence for Extracellular Release of Mast Cell Tryptase

doi:10.1016/S0025-6196(12)60924-0

Mayo Clinic Proceedings

Volume 67, Issue 10, October 1992, Pages 941-948

https://doi.org/10.1016/S0025-6196(12)60924-0 Get rights and content

In many diseases, retrospective analysis for determining the presence of mast cells has been difficult because of their loss of metachromatic staining properties once tissue has undergone formalin fixation. We quantified mast cells in peribronchiolar tissue of idiopathic pulmonary fibrosis (IPF) and in normal human lung by using rabbit antiserum to human mast cell tryptase. In lung biopsy specimens from 15 patients with IPF, the mean number of mast cells per high-power field in connective tissue directly adjacent to the lumen of small airways (0.5 to 2 mm in diameter) and other fibrotic foci was 29.9 ± 10.8 in comparison with 13.7 ± 3.5 in 16 normal controls (P<0.001). In addition, mast cells in cases of IPF had an altered appearance—irregularity of the plasma membrane and release of extracellular tryptase. We conclude that the number of mast cells is increased in IPF and that the altered appearance of the mast cells suggests that they are activated and undergoing degranulation.

Section snippets

METHODS

An immature human mast cell line (HMC-1) was established from a peripheral blood specimen of a patient with mast cell leukemia. The cell line was maintained in vitro as continuously proliferating clones and in vivo as solid mastocytomas in nude mice.¹⁶ Tryptase was purified from this line, and New Zealand white rabbits were injected with purified human tryptase for antitryptase antiserum production. Specificity of the antiserum to purified human tryptase was demonstrated by immunoblotting.¹⁷

RESULTS

Cytocentrifuge preparations of neutrophils, eosinophils, hypodense eosinophils, lymphocytes, and cultured human fibroblasts did not stain at any dilution of the antiserum (Table 1). An enriched preparation of basophils showed granular fluorescence at the highest antiserum concentration (1:10), but at dilutions of 1:50 (not tabulated), 1:100, and 1:200, no staining of basophils was noted. HMC-1 cells, HMC-1 mastocytomas, dispersed human scalp mast cells, and mast cells in human urticaria

DISCUSSION

In this study, we quantified mast cells in open-lung biopsy specimens from patients with IPF and compared the results with similar determinations in autopsy specimens of normal lung tissue. Mast cell numbers were assessed in the tissue adjacent to small airways to provide uniform areas of comparison between the two groups.

Immunofluorescent staining of formalin-fixed, paraffin-embedded tissue for mast cell tryptase showed increased mast cell numbers in specimens of IPF. We believe that the

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In addition to secreting pro-fibrotic growth factors, mast cells also produce and secrete enzymes that activate these factors. Mast cell-derived enzymes chymase and tryptase are upregulated in human idiopathic interstitial pneumonia and IPF (Hirata et al., 2007; Hunt et al., 1992). Mechanical stress induces mast cells to secret chymase and tryptase in mice exposed to bleomycin (Shimbori et al., 2019).
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MC numbers are markedly increased in the fibrotic areas of lungs from patients with idiopathic pulmonary fibrosis (IPF) and correlate with the severity of fibrosis.4 As in asthma, the MCs in lungs with IPF demonstrate features of ongoing degranulation,5-7 histamine concentrations are increased in IPF BAL compared with those in control lungs,5,6 and tissue tryptase levels are elevated.8 There is extensive literature dating back decades describing the profibrotic interactions of MCs and fibroblasts (reviewed by Cruse and Bradding9).
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Lung tissue of patients with IPF displays increased mast cell numbers and these correlate with the severity of fibrosis.6 Mast cells in IPF lung tissue have signs of ongoing degranulation.116,117 Consistent with this the bronchiolar lavage of patients with IPF contains higher concentrations of histamine than controls.118,119
Mast cells and their activation contribute to lung health via innate and adaptive immune responses to respiratory pathogens. They are also involved in the normal response to tissue injury. However, mast cells are involved in disease processes characterized by inflammation and remodeling of tissue structure. In these diseases mast cells are often inappropriately and chronically activated. There is evidence for activation of mast cells contributing to the pathophysiology of asthma, pulmonary fibrosis, and pulmonary hypertension. They may also play a role in chronic obstructive pulmonary disease, acute respiratory distress syndrome, and lung cancer. The diverse mechanisms through which mast cells sense and interact with the external and internal microenvironment account for their role in these diseases. Newly discovered mechanisms of redistribution and interaction between mast cells, airway structural cells, and other inflammatory cells may offer novel therapeutic targets in these disease processes.
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In addition, histamine concentrations in the BAL fluid of patients with IPF are elevated about 10-fold that of control subjects (Casale et al., 1988; Rankin et al., 1987), and tryptase levels are increased in the lung tissue of IPF patients (Wygrecka et al., 2013). Mast cells present in pulmonary fibrosis show signs of on-going degranulation (Hunt et al., 1992; Kawanami et al., 1979) and coculture of human lung mast cells with human lung fibroblasts from IPF patients activates the mast cells to release tryptase (Wygrecka et al., 2013). Furthermore, infiltrating bFGF expressing cells are abundant in IPF and these cells have been identified as mast cells (Inoue et al., 2002, 1996; Qu et al., 1995), which are surrounded by collagen, elastic fibres and smooth muscle cell/myofibroblast-like cells (Inoue et al., 2002; Wygrecka et al., 2013).
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The level of statistical significance was set at P ≤ 0.05. Previous studies have demonstrated increased numbers of MCs in lungs of IPF patients.17–19 In accord with these reports, we also observed elevated number of MCs in IPF lungs (Figure 1A); moreover, the majority of MCs in IPF lungs were activated, as evinced by an increased index of degranulation (Figure 1B).
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Mast cells are increased in number in many fibrotic diseases and may play a crucial role in the development of fibrosis.44,45 The percentage of human mast cells in BAL fluid from patients with sarcoidosis or interstitial fibrosis is greater than in BAL fluid from healthy individuals,44,46 and patients with idiopathic interstitial pulmonary fibrosis show evidence of mast cell degranulation and elevated mast cell numbers.47 Concluding in a previous study show that Mast cells are located in connective tissue, including the lung, skin, the linings of the stomach and intestine, and other sites.
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: This study was supported in part by Grants AI 15231 and AI 09728 from the National Institutes of Health, Public Health Service, and Mayo Foundation.

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Immunofluorescent Staining for Mast Cells in Idiopathic Pulmonary Fibrosis: Quantification and Evidence for Extracellular Release of Mast Cell Tryptase

Section snippets

METHODS

RESULTS

DISCUSSION

Leuk Res

Lancet

J Immunol Methods

J Ultrastruct Res

J Biol Chem

Exp Cell Res

J Allergy Clin Immunol

Mast cell involvement in various inflammatory processes

Am Rev Respir Dis

The mast cell and signs of pulmonary fibroblast activation in sarcoidosis

Int Arch Allergy Appl Immunol

Mast cell heterogeneity and hyperplasia in bleomycin-induced pulmonary fibrosis of rats

Am Rev Respir Dis

Mast cells and inhalation of asbestos in rats

Thorax

Asbestos-induced fibrosis in rats: increase in lung mast cells and autacoid contents

Exp Lung Res

Pulmonary mast cells in mitral stenosis

Cardiovasc Res

Ultrastructure of pulmonary mast cells in patients with fibrotic lung disorders

Lab Invest

Early cellular events in pulmonary fibrosis

Exp Lung Res

Mast cells in the rat alveolar septa undergoing fibrosis after ionizing irradiation: ultrastructural and histochemical studies

Lab Invest

The ultrastructure of 60Co radiation pneumonitis in rats

Lab Invest

Effect of fixation on the light microscopical visualization of mast cells in the mucosa and connective tissue of the human duodenum

Int Arch Allergy Appl Immunol

Guinea pig mast cells: comparative study on morphology, fixation and staining properties

Int Arch Allergy Appl Immunol

Human lung mast cells: distribution and abundance of histochemically distinct subpopulations

Int Arch Allergy Appl Immunol

Formalin sensitivity and differential staining of mast cells in human dermis

Br J Dermatol

The ultrastructure of ⁶⁰Co radiation pneumonitis in rats