INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Hypersensitivity pneumonitis (HP) represents a lung disorder characterized by a diffuse inflammatory granulomatous response to a wide variety of inhaled organic particles (1). Pigeon breeder's disease (PBD) is a common form of HP provoked by the inhalation of avian antigens. Patients exposed to low concentrations of bird droppings and feathers, usually in the domestic environment, develop a subacute or chronic disease and a number of them evolve to diffuse pulmonary fibrosis (2). However, why some patients improve or heal whereas other progress to fibrosis is essentially unknown. The disease is characterized by a lymphocytic alveolitis with an impairment of T-cell subsets, mainly delineated by an increase of CD8⁺ lymphocytes with an inversion of the CD4⁺/CD8⁺ ratio (3, 4). Strong evidence supporting a pathogenic role of local T-cell activation has resulted from studies performed in experimental models as well as in the human disease (1, 3).

The possible participation of other inflammatory cells in the pathogenesis of HP remains unclear. An immediate and transient neutrophil alveolitis after antigen inhalation has been demonstrated in bronchoalveolar lavage (BAL) of patients with HP, and an increase in neutrophils has been also occasionally reported in subacute/chronic patients with HP (6). In addition, alveolar macrophages obtained from patients with HP produce high levels of interleukin-8 (IL-8), a potent chemotactic agent for neutrophils (9). However, evidence of increased numbers of neutrophils derives only from BAL analysis, and quantitative studies on lung tissues are scanty.

Neutrophils secrete a wide variety of molecules that could be implicated in the pathogenesis of diffuse lung inflammation that occurs during the development of HP. Among them, two matrix metalloproteinases (MMPs) involved in the remodeling of the extracellular matrix, collagenase-2 (MMP-8) and gelatinase B (MMP-9), may play a role. Both enzymes are synthesized as inactive zymogens, and in contrast to other cells, in which MMPs are produced by de novo synthesis and rapidly secreted, in neutrophils MMP-8 and MMP-9 are stored in specific intracellular granules, and released after chemotactic stimulation in vitro and during inflammatory conditions in vivo (10).

Collagenase-2 is one of the three members of the collagenase subfamily of human MMPs. It is a 75 to 80-kD enzyme that shares with the other collagenases the ability to cleave native triple helix of types I, II, and III fibrillar collagens, at a single site producing fragments approximately three-fourths and one-fourth the size of the original molecule (14). Collagenase-2 preferentially cleaves type I collagen (15), which is the most abundant collagen type in the lung parenchyma. MMP-9 degrades primarily type IV collagen, but also entactin, proteoglycans, and possibly laminin, and therefore, it has been implicated in basement membrane breakdown. More recently, it has been suggested that MMP-9 may play a role for neutrophil migration through basement membranes, into sites of inflammation (16).

In this context, the aims of this study were, first, to determine whether increased quantities of neutrophils are present in the lung tissue of patients with hypersensitivity pneumonitis; and second to localize immunoreactive gelatinase B and collagenase-2.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Study Population

Fifteen nonsmoking patients with hypersensitivity pneumonitis were included in this study (12 females and 3 males, 37 ± 9 yr [mean ± SD]). The ethical committee of the institute approved the protocol, and informed written consent was obtained from each subject. Diagnosis of PBD was obtained according to international criteria including: (1) pigeon's exposure preceding disease, and positive serum antibodies against avian antigens; (2) shortness of breath with partial improvement upon avoidance of the avian antigen exposure; (3) clinical, radiological, and functional features of an interstitial lung disease (ILD); (4) more than 40% of lymphocytes in BAL; (5) lung histology compatible with HP (1, 17). Briefly, the tissue samples showed diffuse interstitial inflammation of mononuclear predominance, mainly lymphocytes, and frequent multinucleated giant cells in terminal and respiratory bronchioles, as well as in the alveolar walls. Foamy macrophages were seen in the alveolar spaces, and small and loosely arranged granulomas were observed in the interstitium. Biopsy cultures were negative for bacteria, mycobacteria, and fungi, and no changes suggestive of any other ILD were found. The lung samples were taken by open lung biopsy or by video-assisted thoracoscopy, usually 1 wk after hospital admission. None of the patients had been treated with steroids or immunosuppressive drugs at the time of biopsy.

Semiquantitative Histologic Assessment

The percentage of fibrosis present in the lung samples was analyzed as described elsewhere (2). Briefly, the assessment was done on the slide scanned completely in zigzag fashion, first at ×25 and then at ×100 magnification. In all cases, four slides, two of them stained with Masson's trichrome and two with hematoxylin-eosin were analyzed. We first determined the percentage of the lung biopsy with abnormal lung, either inflammation or fibrosis or both (extent of the lesion), and then the percentage of the abnormal lung with fibrosis. At ×25 a slide comprised approximately 8 to 10 fields, and at this magnification, the extent of the lesion was evaluated. At ×100, the percent of fibrosis was evaluated in an average of 40 fields. The percentage of fibrosis was expressed in multiples of 10. The assessment of fibrotic changes included young connective tissue rich in fibroblasts and relatively poor in mature collagen, as well as areas with well-developed "collagenization" (18). Twice the same pathologist (M.G.) evaluated the percent of fibrosis with a 6-mo difference. The intraclass correlation coefficient was 0.82 (p < 0.01). Concordance was additionally evaluated reducing the percent of fibrosis to five categories finding a weighted kappa coefficient of 0.6 (SD 0.23, p = 0.012) (19).

Lung tissue samples obtained from autopsies of patients who died from nonlung causes were used as controls (4 females and 2 males, 43 ± 9 yr old). Selected lung fragments, which appeared macroscopically and microscopically normal, were used.

Quantitative Analysis of Neutrophils

Tissue samples stained for immunoreactive myeloperoxidase and counterstained with hematoxylin were used to evaluate the neutrophils/total cells ratio. The analysis was made by using a digital procedure with the software Imagenia 5000 (Biocom, Paris, France) (20). The images were acquired in videotape with a charge-coupled device (CCD) camera connected to a standard Zeiss light microscope, using a ×63 planapochromatic objective, and the quantification was made in 30 different lung areas per slide. The estimation of the whole population of cells was carried out by counting the total number of nuclei stained with hematoxylin and the number of neutrophils by counting the positive myeloperoxidase positive stained cells.

Immunohistochemistry

Tissue sections were deparaffinized, rehydrated and then blocked with 3% H₂O₂ in methanol for 30 min followed by antigen retrieval performed with citrate buffer 10 mM pH 6.0 for 5 min in microwave. Tissue sections were then incubated with an antibody diluent with background-reducing components (Dako, Carpinteria, CA) diluted 1/100 in phosphate-buffered saline (PBS) for 45 min. Primary monoclonal anti MMP-8 (20 µg/ml) and MMP-9 (5 µg/ml) antibodies (Fuji Chemical Ind., Ltd., Toyama, Japan), and rabbit anti-human myeloperoxidase (Dako, Carpinteria, CA) were used. A secondary biotinylated anti- immunoglobulin followed by horseradish peroxidase-conjugated streptavidin (BioGenex, San Ramon, CA) was used according to the manufacturer's instructions. 3-amino-9-ethyl-carbazole (AEC; BioGenex) in acetate buffer containing 0.05% H₂O₂ was used as substrate (21). The sections were counterstained with hematoxylin. The primary antibody was replaced by nonimmune serum for negative control slides.

BAL

BAL was performed using a fiberoptic bronchoscope wedged in two separate segments of the right middle lobe or lingula. A volume of 300 ml of normal saline was instilled in 50-ml aliquots, with an average return of 70%. The recovered BAL fluid (BALF) was filtered through sterile gauze and measured, and then centrifuged at 250 g for 10 min at 4° C. The supernatant was kept frozen at 70° C until use. The cell pellet was resuspended in 1 ml PBS and an aliquot was used to evaluate the total number of cells. Other aliquots were fixed in carbowax, and three slides per sample were stained with hematoxylin-eosin, Giemsa, and toluidine blue, and used for differential cell count. Six nonsmoking healthy normal individuals were lavaged as control subjects (3 females and 3 males, 29 ± 9 yr old).

Gelatin Zymography

Sodium dodecyl sulfate (SDS) polyacrylamide gels containing gelatin (1 mg/ml) were used to identify proteins with gelatinolytic activity from the BALF as described elsewhere (22). BALF was ×10 concentrated with a Speed Vac SC 110 (Thermo Instruments Systems, Holbrook, NY) and adjusted to 4 µg of protein for gel loading. After electrophoresis, gels were immersed in a solution of 2.5% Triton X-100, washed extensively with water, and incubated overnight at 37° C in glycine 100 mM pH 8.0, containing 10 mM CaCl₂ and 50 nM ZnCl₂. Identical gels were incubated but in the presence of 20 mM ethylenediaminetetraacetic acid (EDTA). Gels were stained with Coomassie Brilliant Blue R250 and destained in a solution of 7.5% acetic acid and 5% methanol. Gelatinase A activity was estimated using lung fibroblasts serum-free conditioned medium as standard, and gelatinase B using serum-free conditioned medium from phorbol myristate acetate (PMA)-stimulated osteogenic sarcoma, bone primary, human (U2-OS) cells (American Type Culture Collection, Rockville, MD). Gelatinolytic activities were quantified using 1D image analysis software (Eastman Kodak, Rochester, NY) which quantifies the surface and intensity of lysis bands. Results were expressed as progelatinase A and B arbitrary units (AU): AU = net intensity × 10⁴ per 4 µg of BAL protein.

Statistical Analysis

Values were expressed as mean ± SD. Association between BAL and tissue neutrophils and between tissue or BAL neutrophils and lung fibrosis was done using the Pearson correlation coefficient.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Baseline characteristics of the patients with hypersensitivity pneumonitis are summarized in Table 1. All patients showed clinical and functional evidence of ILD, with variable degrees of dyspnea, decreased lung capacities, and hypoxemia at rest worsening during exercise. Differential cell count in BALF was characterized by a marked lymphocytosis, usually over 50% (Table 2).

Neutrophil Quantification

All HP tissue samples revealed neutrophils located inside small vessels, as well as in the interstitial and alveolar spaces as identified using myeloperoxidase-positive stained cells as a marker (Figures 1A and 1B). The percentage of lung neutrophils evaluated by densitometric analysis is shown in Table 3. Lung neutrophil/total cell ratio varied from 0.7% to 4.8% (2.1 ± 1.4%). Control lungs showed scattered neutrophils, always inside vessels (not shown).

View larger version (85K): [in this window] [in a new window]   Figure 1.   Myeloperoxidase staining of HP lungs. (Panel A) neutrophils are mainly located close to the endothelium inside vessels (arrowhead ). Interstitial and intra-alveolar neutrophils can also be noticed (arrow; original magnification, ×4). (Panel B) High-power magnification showing a cluster of neutrophils (double arrowhead ) and an isolated neutrophil (arrow; original magnification, ×10). Tissue samples were counterstained with hematoxylin.

HP tissues displayed variable degrees of lung fibrosis (Table 3), and a significant positive correlation between the percent of lung fibrosis and the percent of lung tissue neutrophils was found (r = 0.6, p < 0.02). As expected, a similar but negative correlation between lung inflammation and the percent of lung neutrophils was found (r = 0.6, p < 0.02). Additionally, the number of granulomas was also examined. Tissue samples showed between 4 to 15 usually small, poorly differentiated granulomas. A negative correlation between the number of granulomas and the degree of lung fibrosis was found (r = 0.7; p < 0.01).

Regarding BAL cellular profile, an increase in neutrophils was noticed in 12 of 15 patients. However, the percentages were usually lower than those observed in lung tissues (1.0 ± 1.4%; Table 3). No relationship was found between BAL and lung tissue neutrophils (r = 0.21) or between BAL neutrophils and lung fibrosis (r = 0.03).

Immunohistochemical Localization of MMP-8 and MMP-9

Neutrophils were strongly positive for collagenase-2 (MMP-8) and gelatinase B (MMP-9) in all HP lungs. In Figure 2 are illustrated intravascular neutrophils in close relationship to endothelial cells of large vessels (Figure 2A), and peribronchiolar neutrophils (Figure 2B) showing immunoreactive MMP-8. No MMP-8-positive signal was observed in other than neutrophils. Gelatinase B signal was also strongly detected in neutrophils adherent to the endothelium of large and small vessels, and in the inflammatory infiltrate (Figures 2C and 2D). Endothelial cells were negative, and in some areas weakly reactive alveolar macrophages were observed (Figure 2C, inset).

View larger version (91K): [in this window] [in a new window]   Figure 2.   Immunohistochemical localization of MMP-8 and MMP-9 in HP lungs. Immunoreactive neutrophil collagenase and gelatinase B were revealed by 3-amino-9-ethylcarbazole. Tissue samples were counterstained with hematoxylin. (Panel A) Numerous MMP-8-labeled intravascular neutrophils are adhered to the endothelium (arrowhead ); interstitial positive neutrophils in the lung interstitium are indicated by an arrow (original magnification, ×40). (Panel B) High-power magnification showing peribronchiolar MMP-8-stained neutrophils (original magnification, ×100). (Panel C ) Immunoreactive gelatinase B was seen in neutrophils adherent to the endothelium as signaled by arrowheads, and in the inflammatory infiltrate by arrows (original magnification, ×40). (Panel D) High-power magnification exhibiting positive staining for gelatinase B in a neutrophil inside a small vessel (arrowhead ), and in the interstitium (arrow). (Panel E ) MMP-8 staining in normal lung (original magnification, ×40). (Panel F ) MMP-9 staining in normal lung (original magnification, ×40). (Panel G) HP specimens incubated with nonimmune serum showed no staining (original magnification, ×40).

Normal lungs were negative or showed scattered labeled MMP-8 or MMP-9 neutrophils (Figures 2E and 2F). Control samples incubated with nonimmune sera were negative (2G).

Gelatin Zymography of BALF

BAL samples obtained from patients with HP and control subjects were 10-fold concentrated and aliquots were adjusted to 4 µg protein concentration. Zymograms revealed a significant increase of gelatinases A and B as compared with BAL controls samples from normal nonsmoker subjects, which exhibited faint bands of 72 kD corresponding to progelatinase A activity or of 92 kD representing gelatinase B activity. (Figure 3A, lanes C1 and C2). The levels of enzyme activity were variable in the BAL derived from different patients, and no correlation with the percentage of the various BAL inflammatory cells was found. In some samples (lanes 3, 4, 6, and 7), intense bands of approximately 92 and 84 kD corresponding to progelatinase B and its activated form were observed. Additionally, a 130-kD band usually identified as lipocalin-associated gelatinase B latent form, specific for neutrophils was also noticed. Progelatinase B activity showed an approximately 3- to 20-fold increase over controls (Figure 3B); gelatinase B activated form (approximately 84 kD) represented about 10% of proenzyme activity. Progelatinase A activity also showed approximately 3- to 20-fold increase in band intensity over controls; however, there was no correlation in the increase of both gelatinases in the same patient (Figure 3B). The activated form of progelatinase A (~ 62 kD) represented also approximately 10% of proenzyme form. All gelatinolytic bands were fully inhibited by EDTA (not shown).

View larger version (46K): [in this window] [in a new window]   View larger version (20K): [in this window] [in a new window]   Figure 3.   (A) Identification of gelatinolytic enzymes in BALF from patients with HP by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gelatin zymography. BAL samples (4 µg of protein in 8 µl) were mixed with an equal volume of Laemmli sample buffer containing 3% SDS. Lanes C1 and C2: Normal control samples. Lanes 1-9: HP samples from nine patients. GA: Conditioned medium derived from human lung fibroblasts as marker for progelatinase A. GB: Conditioned medium derived from PMA-stimulated U2-OS cells as marker for progelatinase B. Zones of enzymatic activity appear as clear bands over a dark background. Gelatinolytic activity bands were inhibited by EDTA (not shown). (B) Quantitative image analysis of the net intensity of the gelatinolytic band's mobility corresponding to progelatinases A and B displayed in A. Results are expressed as progelatinase A (dark bars) and progelatinase B (white bars) activity arbitrary units over the average of six nonsmoking healthy control subjects.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

In this study, we defined as having subacute/chronic HP those patients with at least 3 mo of persistent symptomatology, mainly characterized by progressive dyspnea on exercise. The patients displayed bilateral reticulonodular shadowing and ground-glass attenuation on computed tomography, and a predominantly restrictive functional pattern with hypoxemia at rest worsening during exercise. Likewise, diffuse lung inflammation and a noteworthy increase in BAL lymphocytes were observed in all the patients, substantiating the presence of active disease. Moreover, previous work has demonstrated that these subacute/chronic patients exhibit an acute response when challenged with avian antigens (23). In addition, all lung tissues revealed some degree of fibrosis.

Although lymphocytic accumulation characterizes the alveolitis of HP, our results demonstrated that, in the subacute/ chronic forms, there is a persistent presence of neutrophils in the alveolar septa and alveolar spaces, which may contribute to the pathogenesis of ongoing lung damage. Several chemokines may participate increasing the lung neutrophil traffic, and there is some evidence suggesting that BAL macrophages from patients with HP synthesize high levels of IL-8, a potent neutrophil chemoattractant (9). Moreover, in vitro studies have shown that neutrophils stimulated with IL-8 induce cell degranulation and maximal exocytosis of gelatinase B (24). Neutrophil accumulation in the lung parenchyma may amplify tissue damage by releasing a variety of putative injurious substances including oxygen free radicals, and proteolytic enzymes.

In this study, a strong immunostaining for gelatinase B and collagenase-2 was found, and actually most of the neutrophils in the HP lung tissues showed intracellular stores of these metalloproteinases. The heterogeneity of neutrophil granules is much wider than previously thought and a highly mobilizable organelle, termed the secretory vesicle, may play a key role in modulating the neutrophil's surface protein profile in response to chemotactic factors (25). It has been shown that 50% of total cell gelatinase B is located in gelatinase granules with the remaining residing in specific granules (25). Exocytosis of gelatinase B from gelatinase granules may induce the degradation of type IV collagen in basement membranes and consequently, enhance neutrophil transmigration from capillaries to interstitium (16). However, recent evidence obtained in mice with a null mutation of the gelatinase B gene suggests that this enzyme is not required for polymorphonuclear cells' emigration (26). Interestingly, we also noticed gelatinase B-positive neutrophils in the lung interstitium, outside the vessels, confirming that the content of the different granules is released in different times and places.

Collagenase-2 has been identified as a protease associated with inflammatory processes and considering its substrate specificity for fibrillar collagens it is more likely that collagenase-2 is important for the ability of the neutrophil to make its way through tissues (27, 28). In general, both matrix metalloproteinases may have a profound effect on the inflammatory response and on the extracellular connective tissue remodeling. Overexpression of gelatinase B has been reported in a number of chronic inflammatory diseases including rheumatoid arthritis, chronic synovitis, pulmonary emphysema, and atherosclerosis (29). In the lung parenchyma, because gelatinase B substrates include several components of the basement membranes, primarily type IV collagen (33), the exaggerated activity of this enzyme in the lung microenvironment may result in disruption of the basement membranes. This pathological process may contribute to a variety of potential abnormalities, including the loss of normal reepithelialization, and the development of fibrosis (34, 35). By contrast, an intact basal lamina is essential for alveolar repair and correct reepithelialization following injury (36).

Equally collagenase-2, once released from specific granules and activated through proteolytic or oxidative mechanisms, appears to play a role in the connective tissue turnover occurring in inflammatory processes (27). The excessive production and activity of neutrophil collagenase may provoke a disorganized remodeling of the extracellular matrix. Moreover, degradation of some connective tissue macromolecules could also enhance a fibrotic response through the release of some matrix-bound growth factors, i.e., the transforming growth factor beta (37).

In the context of the potential capability of MMP-8 and MMP-9 to enhance a fibrotic response, it is interesting to note that a significant correlation between lung neutrophils and the percentage of pulmonary fibrosis was observed. Although the processes underlying the fibrotic response are varied and of enormous complexity, the persistence of tissue neutrophil activation has been suggested as one of them.

Neutrophils are rarely present in the lower respiratory tract of healthy individuals. In the early 1980s with the introduction of the BAL, it was observed that patients with a variety of ILDs, including idiopathic pulmonary fibrosis (IPF), ILD associated with collagen vascular disorders, and asbestosis, revealed increased numbers of neutrophils in their lungs (38, 39). In addition, several studies reported a positive correlation between the number of neutrophils in BAL and the clinical severity and/or the response to therapy in IPF (40, 41). However, these findings have not been confirmed by all investigators (42, 43). Unfortunately, evidence of increased neutrophils obtained directly in human lung tissue is scanty. Recent work has also suggested that neutrophil migration and activation is usually higher in patients with more aggressive and worse prognosis fibrotic lung disorder (44), and a similar situation may occur in HP where a number of patients evolve to interstitial fibrosis (2). In addition, in experimental fibrosis induced by bleomycin or silica, the persistent increase of neutrophil metabolic activity has been correlated with chronic inflammatory responses that resulted in scarring (45).

An additional interesting finding of our work was the lack of correlation between BAL and lung neutrophils, suggesting that the former may not reflect the distribution of neutrophils in the lung. Similar results have been reported in IPF (46).

In summary, our results demonstrate that in subacute/chronic hypersensitivity pneumonitis there is an increase of lung neutrophils, which contain gelatinase B and interstitial collagenase. Furthermore, increased neutrophil infiltration showed some correlation with the development of lung fibrosis and eventually might be a predictor of poor outcome. However, the continuing neutrophil activation may be a reflection of tissue damage and fibrosis, and not necessarily its cause. Further studies are required to unravel this concept.