The aim of this study was to evaluate the cellular and biochemical characteristics of the bronchoalveolar lavage (BAL) fluid in patients with farmer's lung disease (FLD). Total cell numbers in BAL fluids from patients with FLD (n = 30) were significantly higher than in normal subjects (n = 7; p [less than] 0.01), and differential cell counts were significantly different. Lymphocytes were the most numerous cell type in BAL fluids from patients with FLD (65.4 [+ or -] 2.5 percent vs 6.8 [+ or -] 0.5 percent), and analysis of lymphocyte subsets revealed increased percentages of [CD3.sup.+] and [CD8.sup.+] cells (91.8 [+ or -] 0.9 percent vs 68.8 [+ or -] 3 percent, p [less than] 0.01, and 54.3 [+ or -] 3.1 percent vs 30.1 [+ or -] 3.2 percent, p [less than] 0.01, respectively). A marked increase in mast cell numbers, as revealed by the specific alcian blue/safranin staining, was observed in patients with FLD (4.2 [+ or -] 0.57 percent, n = 12, vs 0.18 [+ or -] 0.04 percent, n = 7, p [less than] 0.001). Histamine levels in BAL supernatants were increased in patients with FLD (mean = SEM, 4.4 [+ or -] 0.8 ng/ml vs 0.9 [+ or -] 0.1 ng/ml; median, 2.4 ng/ml vs 0.9 ng/ml, p [less than] 0.01), and correlated positively with mast cell numbers and percentages (r = + 0.63, p [less than] 0.03, and r = + 0.69, p [less than] 0.02, respectively); conversely, a negative correlation was found between histamine levels and [CD8.sup.+] lymphocyte percentages (r = 0.48, p [less than] 0.01). Raised neutrophil percentages (5.1 [+ or -] 0.8 vs 0.5 [+ or -] 0.18, p [less than] 0.05) and albumin concentrations (29.2 [+ or -] 3.9 mg/dl vs 3.4 [+ or -] 1.3 mg/dl, p [less than] 0.01) were also found in patients with FLD. These findings show that increased numbers of mast cells, lymphocytes, and neutrophils can be found in BAL fluids of patients with FLD. The increased histamine levels in the supernatants of BAL fluids indicate that mast cells are activated. These data allow us to postulate a role for mast cell accumulation and histamine release in the inflammatory process of FLD.
Farmer's lung disease (FLD) is an interstitial lung disease occurring in sensitized subjects following exposure to Micropolyspora faeni.[1-3] The histopathologic picture of the lung is characterized by a diffuse lymphocytic infiltrate with increased numbers of plasma cells and macrophages. Several clinical and experimental data suggest that FLD is an immune-mediated disease.[5,6] The underlying mechanism has been supposed to be an Arthus-type immune reaction:[7,8] antigen exposure would lead to the formation of immune complexes, and their deposition in the lung would be responsible for the inflammatory changes. However, some recent findings have led to a re-evaluation of the immunopathogenetic mechanism, which is supposed to be cell-mediated (type 4 immune reaction). Antigen challenge in patients with FLD does not cause any significant decrease of complement factors, and the histopathologic picture differs from the vasculitic changes observed in a typical Arthus reaction. Moreover, symptom-free subjects exposed to antigen can produce precipitating antibodies, without any evidence of lung disease. Conversely, the striking increase in T lymphocytes, which has been found in bronchoalveolar lavage (BAL) fluids from patients with FLD,[10,11] suggests that the disease could be related to lymphocyte activation and cytokine release.[11,12]
Among the soluble products of activated lymphocytes, there are some histamine-releasing factors, which in turn can activate basophils and mast cells;[13,14] therefore, it is reasonable to suppose that mast cells and basophils and their inflammatory mediators could be also involved in the pathogenesis of FLD.[15-19]
Haslam et al have found increased numbers of mast cells in BAL samples from 15 patients with extrinsic allergic alveolitis. According to electron microscopic examination, these mast cells resemble bronchial subepithelial tissue mast cells, and are often activated. The BAL cell lysates from patients with extrinsic allergic alveolitis contain higher concentrations of histamine than control subjects. In line with these data, Soler et al have observed an increase in mast cell numbers and histamine levels in BAL fluids from eight patients with extrinsic allergic alveolitis. In this study, we have evaluated differential cells counts and histamine concentrations in BAL fluids from 30 patients with FLD.
MATERIALS AND METHODS
Thirty patients (21 men and 9 women, ages ranging from 28 to to 62 years, mean age 45.4 [+ or -] 1.8), affected with FLD were included in the study (Table 1). The diagnosis was based on the following criteria: (1) documented exposure to moldy hay; (2) a symptomatic acute episode with chills, fever, cough, and breathlessness 4 to 8 h after farm exposure to a specific antigen; (3) radiologic features and/or functional pattern of interstitial lung disease; and (4) evidence of antibodies to Micropolyspora faeni. All patients had been exposed recently to the antigen and were still symptomatic at the time of bronchoscopy. None of them had been treated with corticosteroids before the initial investigation. Three of them were smokers and none had a personal or family history of allergic and respiratory disease. Skin prick tests for the most common allergens were negative in all the studied subjects. The control group was made of seven age-matched healthy nonatopic normal subjects (four men and three women, mean age 41.1 [+ or -] 4.2 years), who accepted to undergo fiberoptic bronchoscopy. All subjects were nonsmokers and each individual gave informed and signed consent prior to study.
Lung Function Tests
Lung function was evaluated in 18 patients at the time of hospitalization and before bronchoscopy (Table 2). A standard spirometer (Morgan, Chatham, United Kingdom) was used to evaluate lung volumes (total lung capacity [TLC]; vital capacity [VC]; forced expiratory volume in 1 s [FEV.sub.1]; maximal expiratory flow at 25 percent of the VC [MEF25]; [FEV.sub.1]/VC ratio). Diffusion capacity of carbon monoxide (transfer factor, Dco) was measured by the single-breath method. The results were expressed as percent of predicted values.
Bronchoscopy was performed between 8 and 9 AM on each occasion. Subjects abstained from food for at least 12 h before bronchoscopy. In all subjects, upper airways were anesthetized by 4 ml of 2 percent lidocaine. A flexible fiberoptic bronchoscope (Olympus BTI, Tokyo, Japan) was wedged into a segment of the right middle lobe, and three 50-ml aliquots of sterile prewarmed (37 [degrees] C) saline solution were infused. Fluid was gently aspirated immediately after each aliquot was introduced and collected in a sterile container. The mean percent fluid recovery was 75.3 [+ or -] 2.96 (mean [+ or -] SEM) in patients with FLD and 94 [+ or -] 5.5 in normal subjects, with a significant difference between the two types of subject (p[less than] 0.01).
Processing of BAL Fluid
After recovery, BAL fluid was strained through a monolayer of surgical gauze to remove mucus. A sample was reserved for total cell count. The total cell number was counted using a Nageotte's chamber and results were expressed as cells X[10.sup.3]/ml. The remaining fluid was immediately centrifuged at 800 rpm for 10 min at 4 [degrees] C. The cell pellet was washed twice with phosphate-buffered saline solution (without [Ca.sup.2+] and [Mg.sup.2+]) and resuspended in the same buffer. Aliquots of the cell suspensions ([4X10.sup.4] cells) were centrifuged at 500 rpm for 10 min in a cytocentrifuge (Labofuge AE, Heraeus Sepatech, Osterode, Germany). Cytocentrifuged slides were stained by May-Grunwald-Giemsa method. The differential count of macrophages, lymphocytes, neutrophils, and eosinophils was made under light microscope at X1,000, by counting approximately 300 cells in random fields. Due to the difficulty in detecting mast cells using May-Grunwald-Giemsa stain, they were counted in cell suspension after staining with alcian blue/safranin. Mast cells were quantified in 12 patients by counting at least 1,000 cells (BH-2 Olympus, Kontron, Milano, Italy). However, we cannot exclude that some basophils, which also stain with alcian blue/safranin, were present in BAL fluids. Photomicrographs were taken using a 35-mm microscope camera (PM-6, Olympus, Kontron), and film (Kodak Ektachrome 50 Professional) was used with semiautomatic controlled exposure times (EMM-7, Olympus, Kontron).
Immunophenotyping of Lymphocyte Subpopulations
The following monoclonal antibodies (MoAb) were used to characterize the phenotype of cells recovered from BAL. Leu-4 (CD3) MoAb (Becton Dickinson, Mountain View, Calif) defines T cells; it recognizes a component of the T cell antigen receptor. Leu-3a (CD4) (Becton Dickinson) positive cells include helper lymphocytes, whereas cells reactive with Leu-2a (CD8) (Becton Dickinson) MoAb include cytotoxic/suppressor cells. The frequency of BAL lymphocytes positive for the above reagents was determined by indirect immunofluorescence. Briefly, 50 ml of cell suspension ([10.sup.7]/ml) was incubated with the above MoAb for 30 min at 4 [degrees] C, washed three times in cold (4 [degrees] C) phosphate-buffered saline solution, and then incubated with an IgG fluorescein-conjugated [Fab.sub.2] goat antimouse immunoglobulin antiserum (Kallestad, Austin, Tex). After incubation for 30 min at 4 [degrees] C and further washing, 300 cells were examined using a microscope (Leitz Dialux) equipped with phase-contrast optics and an epi-illumination system (Leitz, Wetzlar, Germany).
Albumin and Histamine Determinations
Albumin was assayed by radial immune diffusion, according to the method of Mancini et al (LC-Partigen albumin Behringwerke AG, Marburg, Germany). The method has a sensitivity of 2.4 mg/dl. Histamine concentration in BAL supernatants was measured by an automated flurometric method, according to the technique of Ruff et al.
Results were expressed as means [+ or -] SEM or medians and ranges, as needed. Student's t test, Mann-Whitney U test, and least squares linear regression coefficient were used for statistical analysis.
Lung Function Tests
Lung function tests showed a mild impairment (mixed restrictive and obstructive defect) in patients with FLD (Table 2).
Differential Cell Counts in BAL Fluids From Patients With FLD
An increase in total cell number was found in BAL fluids from patients with FLD (460.2X [10.sup.3] [+ or -] 36.5X[10.sup.3]/ml vs 175X[10.sup.3] [+ or -] 14X[10.sup.3]/ml in controls, p [less than] 0.01), and significant changes were also observed in the differential cell counts. Macrophage percentage, but not absolute counts, were significantly reduced in BAL fluids from subjects with FLD (28.6 [+ or -] 2.4 percent vs 92.6 [+ or -] 0.6 percent, p [less than] 0.01). Lymphocytes were the predominant cell type in BAL fluids from patients with FLD (65.38 [+ or -] 2.52 percent vs 6.8 [+ or -] 0.5 percent, p [less than] 0.01), and analysis of lymphocyte subsets revealed a significant increase in [CD3.sup.+] cells (91.8 [+ or -] 0.9 percent vs 68.8 [+ or -] 3 percent; p [less than] 0.01) and [CD8.sup.+] cells (54.5 [+ or -] 3.1 percent vs 30.1 [+ or -] 3.2 percent; p [less than] 0.01) (Table 1). Furthermore, raised neutrophil percentages were found in patients with FLD (p [less than] 0.05). Mast cell counts after alcian blue/safranin staining were available in 12 patients with FLD. In these subjects, a significant increase in BAL mast cell concentration was observed (p [less than] 0.001). Mast cells from patients with FLD were often degranulated, whereas mast cells from normal subjects were well granulated.
Histamine and Albumin Concentrations
Histamine concentrations in BAL fluids were higher in patients with FLD than in normal control subjects (means [+ or -] SEM, 4.44 [+ or -] 0.85 ng/ml vs 0.9 [+ or -] 0.13 ng/ml; median, 2.4 ng/ml vs 0.9 ng/ml; range, 0.5 to 24 ng/ml and 0.5 to 1.5 ng/ml, respectively; p [less than] 0.001) (Fig 1). A significant difference was also found when total histamine contents in BAL supernatants from patients with FLD and normal control subjects were compared (median, 270 ng vs 110 ng; range, 163 to 3,128 ng vs 57 to 258 ng; p [less than] 0.04). Albumin levels in BAL fluids were higher in patients with FLD than in normal subjects mean [+ or -] SEM, 29.2 [+ or -] 3.9 mg/dl vs 3.4 [+ or -] 1.3 mg/dl, p [less than] 0.01). No significant correlation was found between albumin levels and histamine concentrations in BAL fluids from patients with FLD (r = -0.06, p = NS). Mast cell numbers and percentages correlated with histamine levels in the supernatants of BAL fluids from patients with FLD (r = +0.63, p [less than] 0.03 and r = +0.69, p [less than] 0.02, respectively) (Fig 2). By contrast, no correlation was found among total lymphocytes, macrophage or neutrophil percentages, and histamine levels. CD8+ lymphocyte percentages correlated negatively with histamine concentrations (r = -0.48, p [less than] 0.01).
Several cytologic changes were found in BAL fluids from patients with FLD: total cell numbers were increased, lymphocytes represented the pre-dominant cell type, and mast cell and neutrophil percentages were significantly raised. Conversely, macrophage percentages, but not absolute counts, were significantly reduced. Histamine concentrations were high in BAL fluids from patients with FLD and correlated positively with mast cell numbers and percentages, and negatively with CD8+ lymphocyte percentages.
These results are in line with those obtained by Haslam et al and Soler et al who described an increase in mast cell numbers and histamine concentrations in BAL fluids from patients with extrinsic allergic alveolitis. The raised histamine levels in BAL fluids are likely to be due to mast cell/basophil activation, since this mediator is contained almost exclusively in these cells. According to Haslam et al, electron microscopic examination of BAL mast cells from patients with extrinsic allergic alveolitis indicates that they have features suggestive of activation. The main mechanism of activation of mast cells and basophils in vivo is represented by antigen binding to surface IgE. This event may lead to the release of histamine and other potent inflammatory mediators. However, no evidence of an IgE-mediated histamine release in patients with FLD is available so far.
Another important stimulus for basophil histamine release is represented by complement anaphylatoxins, which could be generated following immune complex formation and complement activation. However, in vitro, these anaphylatoxins can induce histamine release from human basophils, but not from human lung mast cells. The increase in lymphocyte numbers, in particular in CD3+ and CD8+ cells, suggests the possible activation and participation of these cells in the pathogenesis of FLD.
Activated lymphocytes can release soluble factors able to evoke histamine release from human basophils and tissue mast cells (histamine-releasing factors).[13,14] Moreover, interleukin 3 (IL-3), a hematopoietic factor that can stimulate the differentiation and the growth of basophils and mast cells, and can be produced by activated lymphocytes, can induce histamine release.[27,28] Other cytokines (IL-5, IL-6, granulocyte-macrophage colony-stimulating factor and low-molecular-weight B-cell growth factor) produced by activated lymphocytes can enhance basophil histamine release induced by IgE-dependent and IgE-independent stimuli.[29-31] Therefore, it is tempting to speculate that activated lymphocytes in patients with FLD could produce cytokines able to influence the activity of histamine-releasing cells. The mast cell increase in BAL fluids from patients with FLD could be the result of recruitment, proliferation, and/or changes in the distribution between the interstitium and the alveoli induced by lymphocyte-derived interleukins as IL-3 and IL-4, that have been recognized as growth factors for mast cells.[27,32] A mast cell hyperplasia has been described in experimental hypersensitivity pneumonitis in guinea pigs exposed to repeated intratracheal challenge with M faeni. These observations suggest a possible involvement of mast cells/basophils in the pathogenesis of FLD.
The high histamine levels in BAL fluids from patients with FLD are not accompanied by bronchoconstriction, although a bronchial hyperreactivity to both inhaled histamine and methacholine occurs in 20 to 50 percent of patients with FLD. The clinical picture of FLD is characterized by dyspnea, cough, fever, and chills, but the breathlesness reflects an impairment of diffusion lung capacity rather than a narrowing of respiratory airways. A mild impairment of lung function with a mixed restrictive and obstructive defect was found in our patients with FLD. By contrast, the increased histamine levels in BAL fluids from asthmatic patients are associated with bronchoconstriction and hypersecretion. Histamine concentrations in BAL fluids from patients with FLD are lower than those found in postchallenge BAL fluids from asthmatic patients; probably, histamine secretion in patients with FLD is a less acute event due to a prolonged mast cell activation, whereas the early antigen-induced reaction in asthmatic patients reflects a rapid and massive IgE-mediated mast cell degranulation. Measurement of inflammatory mediators other than histamine (peptide leukotrienes, prostaglandin [D.sub.2], paf-acether) and interleukins in BAL fluids from patients with FLD will contribute to elucidate the pathogenetic mechanism of the disease and the relationship between lung inflammation and clinical picture.
The elevation in albumin levels could be due to the increased vascular permeability, which in turn may be related to histamine release. Histamine is a proinflammatory mediator, but it has also been recognized as moderator of different immune functions; in particular, it can exert an inhibitory effect on T lymphocyte (CD8+ cells) proliferation, following specific interaction with membrane receptors. Therefore, it can be hypothesized that released histamine may have a damping effect on T lymphocyte activation, an activity that may be relevant to the evolution of inflammation in FLD. In our study, histamine levels correlated negatively with CD8+ counts, and this is in line with a possible damping effect of histamine on the proliferation of these cells. It is also interesting to note that neutrophil percentage was increased in BAL fluids from patients with FLD. Neutrophils can release inflammatory mediators and histamine-releasing factors, which in turn could participate to the pathogenesis of FLD.
In conclusion, our results showed increased numbers of mast cells, lymphocytes, and neutrophils in BAL fluids from patients with FLD. Histamine levels were increased, probably because of mast cell activation. A role for mast cell accumulation and histamine release in hypersensitivity pneumonitis can be hypothesized.
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