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

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

LOREN W. HUNT, M.D., Division ofAllergic Diseases and Internal Medicine; THOMAS V. COLBY, M.D., Department of Laboratory Medicine and Pathology; DEBORAH A. WEILER, M.S., SANJIV SUR, M.D., Allergic Diseases Research Laboratory; JOSEPH H. BUTTERFIELD, M.D., Division ofAllergic Diseases and Internal Medicine

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
The role of mast cell mediators in pulmonary disorders of immediate hypersensitivity has been studied extensively. Less attention has been focused on the possible contribution of the mast cell to chronic inflammatory and fibrotic disorders of the lung. 1 Investigators have noted increased numbers of mast cells in pulmonary diseases such as sarcoidosis.? bleomycin-induced pulmonary fibrosis in rats,' asbestos-induced fibrosis in rats,4,S chronic mitral stenosis with pulmonary fibrosis," chronic fibrosing alveolitis," and chronic radiation pneumonitis in rats.v'? Ruitenberg and associates," in a study of mast cells in the human duodenum, and Ghanem and colleagues," who studied mast cells in guinea pigs, showed that many mast cells lose their metachromatic staining properties after traditional formalin fixation. Mast cells in the mucosal areas seemed much more sensitive to

loss of staining after formalin fixation than mast cells located deeper in connective tissue. Shanahan and co-workers" found similar formalin sensitivity of mucosal and submucosal mast cells in the lung, and inhibition of mast cell staining has also been observed in formalin-fixed human skin." Because 90% of the mast cells within the lung seem to be of the mucosal type, 15 histologic identification of mast cells after traditional formalin fixation has been particularly difficult. Tryptase, present in all mast cells, is an ideal marker for mast cell staining with use of immunohistochemical methods. 15 Using indirect immunofluorescent staining with antiserum to purified mast cell tryptase, we quantified mast cells in the peribronchiolar regions of specimens from patients who had idiopathic pulmonary fibrosis (IPF).

METHODS 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. Address reprint requests to Dr. L. W, Hunt, Division of Allergic Diseases, Mayo Clinic, Rochester, MN 55905, Mayo Clin Proc 67:941-948,1992

An immature human mast cell line (HMC-l) 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." Tryptase was purified from this line, and 941

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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.'? Paraffin blocks from 15 cases of IPF with open-lung biopsy specimens were selected for study by one of us (T.V.C.). Each block was from a well-inflated specimen with a moderate degree of inflammation and fibrosis. Sixteen specimens from autopsies of patients who had no known lung disease and histologically normal lung tissue were used as controls. We used the indirect immunofluorescent staining technique of Filley and associates," with minor modification. All tissue was fixed in 10% formalin and embedded in paraffin. Tissue sections (6 urn) were affixed to glass microscope slides with dilute LePage's Fast Bond glue. All slides were deparaffinized with xylene and ethyl alcohol, digested mildly with 0.1% trypsin at 37°C for 1 hour, washed with distilled water, and incubated overnight in 10% normal goat serum at 4°C. The tissue slides, prepared in duplicate, were then incubated with either rabbit antihuman tryptase antiserum or normal rabbit serum at 37°C for 30 minutes. The tissue slides were washed with Dulbecco's phosphate-buffered saline (pH 7.2 to 7.5) and suspended in 1% chromotrope 2R for 30 minutes, washed again, and subsequently incubated with fluorescein isothiocyanate-labeled goat antirabbit IgG at 37°C for 30 minutes. All tissue slides were washed again with phosphate-buffered saline, and, to retard fading, a coverglass was affixed with mounting medium that consisted of 0.1% p-phenylenediamine in 90% glycerol: 10% phosphate-buffered saline (pH 9.0)19 and sealed with clear nail polish. The slides were examined with a Zeiss microscope (Carl Zeiss, Oberkochen, Germany) equipped with both standard light and fluorescent systems. Mast cells were counted per high-power field (x400) directly adjacent to the lumen of bronchioles (0.5 to 2 mm in diameter), including mucosa, submucosa, muscularis, and adventitia of the airways. Bronchioles of this size were selected for mast cell counting to provide a uniform area of comparison between IPF and normal lung. Most biopsy specimens in IPF contain parenchymal areas that are heavily fibrotic and other areas that are relatively uninvolved. We thought that the observer bias of counting mast cells in only heavily fibrotic areas could be eliminated by selecting a similar landmark of peribronchial tissue for mast cell counting in both IPF and control groups. From one to three airways of this size are easily identified in slides prepared from lung tissue, and pulmonary mast cells are normally distributed in these areas." Fifteen adjacent fields were counted per bronchiole, and the results were averaged. For assessment of consistency in mast cell counting by the first observer (L.W.H.), seven cases were also counted by a second observer (S.S.),

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and the mast cell counts per high-power field from the two observers showed high correlation (r = 0.94; P
RESULTS Cytocentrifuge preparations of neutrophils, eosinophils, hypodense eosinophils, lymphocytes, and cultured human

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fibroblasts did not stain at any dilution of the antiserum (Table I). 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 I:200, no staining of basophils was noted. HMC-I cells, HMC-l mastocytomas, dispersed human scalp mast cells, and mast cells in human urticaria pigmentosa all stained brightly with granular cytoplasmic fluorescence at antiserum dilutions of I: 10, I: 100, and 1:200. All preparations oflPF and control lung tissue were studied with an antitryptase antiserum dilution of 1:100. In the preparations of normal lung (Fig. I), mast cells were distributed within connective tissue around airways and vascular structures. Occasionally, a few mast cells were noted in the alveolar septa. In the specimens of IPF (Fig. 2, 3, and 4), increased numbers of mast cells were noted in peribronchial areasparticularly in areas of dense fibrosis, where mast cells were detected in greatest numbers. Indirect immunofluorescence and hematoxylin-eosin staining of specimens of IPF showed dense fibrosis adjacent to a bronchovascular bundle, juxtaposed with relatively uninvolved lung parenchyma (Fig. 2 A and B). Dense mast cell staining was noted in subpleural areas of early honeycombing (Fig. 2 C and D). In submucosal areas (Fig. 3), mast cells were seen in numbers often as high as 30 to 50 per high-power field, and degranulation was frequently evident. In contrast to the immunofluorescence method, an adjacent section stained with the metachromatic dye toluidine blue demonstrated the prominent formalin sensitivity of these cells; few mast cells were identifiable. In areas involved with fibrosis, numerous mast cells with irregular surfaces and degranulation were evident (Fig. 4). Mast cells had this characteristic appearance in all cases of IPF studied. In a comparison of mast cell numbers in cases of IPF and in normal controls (Fig. 5), we found a uniform distribution Table I.-Tissue Preparations Stained by Indirect Immunofluorescence for Mast Cell Tryptase

Preparation HMC-I (immature human mast cell line) Dispersed human scalp mast cells HMC-l-derived mastocytoma Human urticaria pigmentosa Basophils Neutrophils Eosinophils Hypodense eosinophils «1.081 g/ml) Lymphocytes Cultured human skin fibroblasts

Dilution of rabbit antitryptase antiserum 1:10 1:100 1:200

+ + + + +

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Fig. I. Staining of mast cells in submucosa in preparations of normal lung with an indirect immunofluorescence stain to mast cell tryptase. (x400.) A, Mast cells beneath the mucosa of a small bronchus are of uniform size and are ovoid or round. No extracellular material is present. (Nonspecific autofluorescence is evident in mucosal cells at lower right in both A and B.) B, Section of tissue adjacent to A, stained as a control with normal rabbit serum, shows no mast cells. C, Section of normal lung shows mast cells in alveolar septum near a small blood vessel.

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Fig. 2. Mast cell staining in idiopathic pulmonary fibrosis (IPF). (xlOO.) A, Section of IPF stained by indirect immunofluorescence for mast cell tryptase shows numerous mast cells, predominantly in fibrotic region adjacent to bronchovascular bundle. B, Section of IPF adjacent to A, stained with hematoxylin-eosin, demonstrates prominent involvement of IPF around a bronchovascular area; juxtaposed is a relatively uninvolved area (at right) with a few normal alveolar structures. C, Dense mast cell infiltration is evident in region of early subpleural scarring. D, Section of tissue adjacent to C, stained with hematoxylin-eosin, demonstrates early honeycomb formation at pleural surface.

of mast cell numbers in peribronchial tissue in the normal lung (mean, 13.7 ± 3.5 cells per high-power field). The number of mast cells in the peribronchial tissue of patients with IPF showed a broader distribution, three cases having mast cell counts of more than 40 per high-power field. The mean mast cell count per high-power field was 29.9 ± 10.8 for cases of IPF, which was significantly different from controls (P
Immunofluorescent staining of formalin-fixed, paraffinembedded tissue for mast cell tryptase showed increased mast cell numbers in specimens ofIPF. We believe that the presence of increased numbers of lung mast cells in IPF has generally not been appreciated because of loss of the metachromatic staining properties of mast cells when tissue is fixed in formalin and traditional metachromatic stains are used. In the current study, immunofluorescent staining for tryptase was helpful because tryptase is present in all mast cells and minimal or no loss of mast cell staining occurs with formalin fixation. The additional findings of considerable irregularity of the mast cell margins and extracellular deposition of tryptase in IPF are of particular interest. The granular appearance of extracellular tryptase is compatible with findings in other studies that have shown that tryptase is secreted with and stabilized by heparin proteoglycan." The lack of

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Fig. 3. Mast cell staining in bronchial submucosal tissue of idiopathic pulmonary fibrosis. (x400.) A, High-power magnification of submucosal area shows dense infiltration with mast cells that have irregular surfaces and degranulation (arrows). B, Serial section adjacent to A, stainedwith hematoxylin-eosin, reveals prominent dense fibrosis in submucosal area. C, Anotheradjacent serial section, overlaidwith normal rabbit serumas a control, demonstrates no cellularstaining. D, Serial sectionadjacentto C, stainedwith toluidine blue, fails to depict identifiable mastcells;this specimen demonstrates profoundformalin sensitivity of mast cells.

similar evidence for degranulation in the control slides leads us to believe that this finding is not an artifact. To our knowledge, the finding of extracellular tryptase in IPF has not previously been reported. Human mast cell tryptase, originally purified from human lung mast cells, is a neutral protease that is present in all mast cells and is released on mast cell activation by antigenic and nonantigenic stimuli. Increased plasma levels of tryptase are a reliable indicator of mast cell activation in systemic anaphylaxis and mastocytosis." Tryptase levels in specimens obtained by bronchoalveolar lavage increase after challenge with a bronchopulmonary allergen." Little is known, however, of the function of mast cell tryptase in vivo--particularly in chronic diseases such as pulmonary fibrosis. The role of mast cell hyperplasia and degranulation in pulmonary fibrosis is also unclear. Cocultivation of mast cells and fibroblasts enhances the release of mast cell media-

tors," stimulates mast cell growth and development," and increases the longevity of mast cells in vitro." In addition, human pleural macrophages cultured with fibroblast supernatants differentiate into "mast cell-like" cells with substantial histamine content and increased granularity." Collectively, these data suggest that the increased numbers of mast cells noted in fibrotic lung disorders could be attributable to enhancement of mast cell growth by proliferation of activated fibroblasts in the chronic inflammatory reaction. 3D Other investigators suggested that mast cells enhance fibrotic responses through stimulation of growth and development of fibroblasts." Mast cell mediators cause mutagenesis of fibroblasts and mesothelioid cells in situ." In addition, fibroblasts have been shown to phagocytose mast cell granules," and both rat and human fibroblasts ingest and degrade 35S-labeled heparin proteoglycan." In tight-skinned mice, fibrosis in skin lesions can be inhibited by blocking

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disclosed a similar close interaction between mast cells and connective tissue cells in vivo." Norrby," who induced mast cell degranulation and release of histamine by injecting compound 48/80 into rat skin or peritoneal cavity, found changes in the proliferative rate of adjacent fibroblasts and alterations in connective tissue ground substance. Histamine levels in bronchoalveolar fluid from patients with IPF are increased in comparison with those of normal subjects. This observation was thought to be evidence of ongoing mast cell degranulation during the active disease process.Pr'? These findings are of interest because both histamine and heparin induce proliferation of and collagen synthesis by human lung fibroblasts derived from normal and fibrotic lung tissue." This effect could be mediated in part by binding of heparin proteoglycan to basic fibroblast growth factor, a process that would enhance binding of this growth factor to its receptor and result in increased fibroblast

growth." Electron microscopic studies also lend support to a role for mast cells in fibrotic disorders. Kawanami and colleagues? showed not only increased numbers of pulmonary mast cells in patients with pulmonary fibrosis but also ultrastructural evidence that the mast cells, with irregular membranes and the appearance of membrane exocytotic vesicles, may have been activated. In their study, however,no extracellular material was noted. In contrast to our data, these investigators found a 20-fold increase in mast cells when the areas of severe fibrosis were compared with lung parenchymal tissue and alveolar walls where few mast cells are normally found. Our study compared numbers of bronchiolar and peribronchiolar mast cells in patients with IPF and normal specimens by examining a uniform landmark of airways of comparable size. Because we selected patients with mild to moderate IPF, our smaller (twofold) increase in mast cells may represent milder disease or may reflect differences in methods, with electron microscopy being more sensitive than immunofluorescent staining. For many of our patients, we had no data available on pulmonary function that could be correlated with mast cell numbers, and we are not aware Fig. 4. Mast cells in area of dense idiopathic pulmonary fibrosis. of studies that have compared mast cell counts in IPF within (x400.) A, Note irregular cellular margins and extracellular gran- a uniform landmark with a variable of disease severity such ules (arrows). Mast cells in this area were larger than those as the extent of reduction in lung volume. elsewhere. B, Serial section adjacent to A, stainedwith hematoxyLykke and associates" found increased numbers of mast lin-eosin, shows densefibrosis. C, Another adjacent serialsection, cells in five cases of pulmonary fibrosis and also ultrastrucoverlaid with normal rabbit serum as a control, demonstrates no tural evidence of atypical degranulation of mast cells, charcellularfluorescence. acterized by scroll-like granules and granules with a tubular structure. Moreover, Haslam and colleagues" found that mast cell degranulation." Mast cells and fibroblasts, when mast cells were significantly more common in fibrotic areas cocultured, undergo a phenomenon of "transgranulation," of pulmonary fibrosis than in the alveolar septa. In their whereby mast cell granules appear to be taken up by fibro- report, particulate forms of granules were illustrated; thus, blasts when the cell surfaces are apposed." Light micro- degranulation in involved tissue was suggested. The apprescopic studies of rat intestinal mesenteric tissue have also ciable loss of toluidine blue metachromatic staining of the

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Fig. 5. Mean mast cell counts per high-power field (hpj) in normal lung (13.7 ± 3.5) in comparison with counts in idiopathic pulmonary fibrosis (IPF) (29.9 ± 10.8) (P
mast cells from the cases in our study ("formalin sensitiv-

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ity"!") suggests that the mast cells are either predominantly

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of the mucosal type or in an activated state, compatible with degranulation. We interpret our findings as evidence of increased numbers of mast cells and ongoing active mast cell degranulation in IPF.

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