INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Hypersensitivity pneumonitis (HP) represents a group of lung diseases resulting from repeated inhalational exposures to a variety of nonpathogenic antigens, including bacteria, fungi, or animal proteins that are present in the susceptible person's environment (1). The disease is characterized by an intense lymphocytic infiltrate that involves the parenchyma and the alveolar structures of the lungs (2). It is well established that only a minority of exposed persons develop the disease, but factors that trigger the onset of HP remain unknown. In particular, there is no evidence of any sex, age, or genetically related susceptibility in HP (3, 4).

HP occurs generally in winter and has "flulike" symptoms (5). It is unknown whether these symptoms could be related to a concomitant viral infection as a part of the disease process. Recent studies from this laboratory have clearly demonstrated that lung viral infections can enhance the pathology of experimental HP induced in mice by repeated exposures to causative HP antigens (6). However, it is still unknown whether common respiratory viruses are present in the lower airways, the likely site of hypersensitivity reaction, of the patients during acute HP.

Bronchoalveolar lavage (BAL) is a useful technique that allows for sampling of the lower airways to directly analyze the cellular and biochemical events that characterize the lymphocytic alveolitis in HP (7). This technique can also provide specimens for the detection of pathogens in the lower airways (8). The highly sensitive polymerase chain reaction (PCR) method has been used to document rhinovirus in BAL cells recovered from the lower airways of volunteer subjects that were experimentally infected with this virus (9). The findings of our recent PCR-based study suggest that the human lungs may serve as a reservoir of common respiratory viruses that may potentially enhance the underlying inflammatory process responsible for certain pathologic conditions such as asthma or other lung diseases (10).

The purpose of this study was to document the presence of respiratory viruses in the lower airways of patients presenting with acute HP in order to determine the type and number of viruses that could play a potential role in modulating the pathogenesis of the disease in humans. The presence of viruses was documented in BAL cells, obtained from patients with acute HP and from unexposed healthy volunteers, by using a reverse transcription (RT) and polymerase chain reaction (PCR) panel suitable for the detection of most common human respiratory viruses. Immunocytochemistry was subsequently performed on BAL cells to localize proteins from the most prevalent viruses detected by PCR.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Study Patients

The study included 13 dairy farmers in whom an acute or recurrent episode of farmer's lung, a classic form of HP, had been diagnosed between December 1994 and April 1997. All patients were studied during active episode of HP. The diagnosis of HP was based on previously reported criteria (11) and on the results of BAL and high resolution computed tomography (HRCT). Six healthy unexposed volunteer subjects who resided in urban areas were included as control subjects in the study. The patients and control subjects were all nonsmokers and none of them had apparent symptoms suggestive of acute viral upper respiratory tract infection at the time of the study. Informed written consent was obtained from each subject in the study, which received approval from the local institutional ethics committee.

Bronchoalveolar Lavage

BAL was performed in the right middle lobe as previously described (12). The recovered lavage fluids were placed in 50-ml centrifuge tubes, kept on ice during the lavage procedure, and immediately centrifuged at 1,500 rpm for 10 min at 4° C to sediment BAL cells. The collected cells were pooled and total and differential cell counts were made on cytospin preparations of cells stained with crystal violet and Diff-Quik solution (BaxterHealthcare, Miami, FL). One aliquot of cells (10⁶) was fixed at 4° C for 10 min with paraformaldehyde (4% in 10 mM phosphate-buffered saline [PBS] at pH 7.4), washed with PBS and dehydrated in graded ethanols and stored at 20° C in 95% ethanol until needed for immunocytochemistry. The remaining BAL cells were washed three times with cold PBS and stored as pellets at 70° C until needed for subsequent analyses.

Nucleic Acid Extraction

BAL cells were incubated with 100 µl of proteinase K digestion solution (200 µg/ml in 100 mM TRIS HCl (pH, 7.5), 0.15 M NaCl, 1% sodium dodecyl sulfate, 12.5 mM ethylene diamine tetraacetate) for 2 h at 50° C. Total RNA was extracted from 75 µl of digested cells using RNeasy kit (Qiagen Inc., Mississauga, ON, Canada) and DNA was extracted from the remaining volume using the QIA amp Tissue kit (Qiagen). The extracted amounts of RNA and DNA were estimated by reading of absorbance at 260 nm.

Reverse Transcription

Total RNA (2 µg) was reverse transcribed into cDNA by incubation at 37° C for 1 h in a 60-µl reaction volume containing 10 mM TRIS HCl at pH 8.3, 50 mM KCl, 5 mM MgCl₂, 50 µM each of deoxynucleotides (dNTPs), 50 units of RNase inhibitor (Pharmacia, Montréal, PQ, Canada), 6 µg of random hexamers (Pharmacia) and 400 U of cloned Moloney murine leukemia virus reverse transcriptase (GIBCO-BRL; Burlington, ON, Canada).

Polymerase Chain Reaction

Five microliters of the transcribed cDNA product, or 0.2 µg of extracted DNA in the case of adenovirus, were subsequently amplified by PCR in a 50-µl reaction mixture containing 10 mM TRIS HCl at pH 8.3, 50 mM KCl, 2.5 mM MgCl₂, 50 µM each of dNTPs, 1.5 U of Taq DNA polymerase (GIBCO-BRL), and 50 pmol each of forward and reverse specific oligonucleotide primers. The primers and probes were selected from previously published reports (Table 1), and they were custom synthesized using an automated DNA synthesizer at a commercial source (Medicorp, Montréal, PQ, Canada). The primers used for adenovirus detect all 47 serotypes of this virus, and those for rhinovirus also detect other picornaviruses.

PCR amplification consisted of 40 cycles of denaturation at 94° C for 45 s (3 min for the first cycle), annealing at 55° C for 30 s, and extension at 72° C for 1 min (7 min for the last cycle) in a 96-gradient temperature thermal Robocycler (Stratagene, La Jolla, CA). After amplification, the PCR products were resolved by electrophoresis on ethidium bromide (EtBr)-stained 1.5% agarose gels. For adenovirus, 2 µl of the first PCR reaction were reamplified with nested primers and the products obtained underwent electrophoresis on EtBr-stained 2% agarose gels. The EtBr-stained PCR products were visualized in the gels under illumination with ultraviolet light. All samples, including positive and negative controls, were analyzed twice (on different days) and randomly to account for potential pitfalls caused by possible cross-contamination.

Positive and Negative Controls

Positive and negative controls for the PCR reactions consisted of nucleic acids extracted from virus-infected and uninfected cell cultures, respectively. Both positive and negative controls were run with the samples on the same day for PCR analysis. The following human respiratory viruses were obtained from the American Type Culture Collection (ATCC, Rockville, MD): adenovirus type 5, coronavirus subtypes OC43 and 229E, influenza viruses A (Weiss/43) B (Lee/40) C (Taylor 1233/47), parainfluenza virus type-1 (PIV-1) and type-3 (PIV-3), rhinovirus type 1B (HRV), and respiratory syncytial virus (RSV long strain type A).

Adenovirus was propagated in monolayers of A549 cells (ATCC) and grown in minimal essential medium (MEM) supplemented with 5% fetal bovine serum (FBS). All the influenza viruses, coronaviruses, and parainfluenza viruses were propagated in LLC2-monkey kidney cells (ATCC) grown in medium 199 supplemented with 1% horse serum (Hyclone, Logan, UT). RSV was grown in HEp2 cells (ATCC) in MEM with 5% FBS. For influenza viruses, 0.01% of trypsin was added to the cultures. All culture media contained 50 µg/ ml of gentamicin.

Successful extraction and integrity of nucleic acids was assessed by amplification of the constitutively expressed human -actin mRNA gene (for RNA) and ₁-antitrypsin DNA gene (for DNA) in each sample (Table 1). To avoid amplification of intronless DNA retropseudogenes of human -actin gene (16), which interfere with RT-PCR (17), each RNA sample was subjected to enzymatic digestion with RNase-free DNase prior to RT-PCR.

Southern Blot Analysis

To further confirm the specificity of PCR amplification products, a southern blot analysis was performed as previously described (17). Briefly, after southern transfer and crosslinking onto Hybond-N nylon membranes (Amersham, Arlington Heights, IL), the PCR products were hybridized with the specific oligonucleotide probes (Table 1), which were end-labeled with [-³²P]ATP using the T4 polynucleotide kinase reaction following the manufacturer's instructions (GIBCO-BRL). After hybridization, the specific PCR products were visualized by autoradiography after exposure to Kodak X-ray films.

Immunocytochemistry

BAL cells, previously stored in 95% ethanol, were rehydrated and cytospin slides were prepared for immunocytochemistry. Influenza A virus was detected with a mouse monoclonal antibody (clone IA-52) (Biogenesis, Sandown, NH), which recognizes a group-specific nucleoprotein in major subtypes (H1N1, H2N2, H3N2) of influenza A, with a secondary antibody consisting of biotinylated-goat antimouse antibody (DAKO Diagnostics Inc., Mississauga, ON, Canada) followed by use of the avidin-biotin alkaline phosphatase complex. Adenovirus was detected by using a biotinylated goat antiadenovirus hexon antibody (Biogenesis) and the avidin-biotin alkaline phosphatase system (DAKO). Nonspecific binding of conjugated antibodies was prevented by a preliminary incubation of cells with normal goat serum (5% in 50 mM TRIS-buffered saline at pH 7.6). The immunoreaction product was revealed by incubation with One-Step alkaline phosphatase NBT/BCIP substrate (Pierce, Rockford, IL) to which levamisole (1 µM) was added to block endogenous alkaline phosphatase activity. Positive controls included cytospin preparations of adenovirus 5-infected A549 cells and influenza A-infected Mk2 cells similarly stained with antiadenovirus and antiinfluenza A antibodies, respectively. Negative controls consisted of uninfected A549 and Mk2 cells.

Statistical Analysis

The proportions of subjects with positive virus detections were compared between the groups by using Fisher's exact test. When necessary, the data were transformed for homogeneity of variance and the group means were compared using a multivariate analysis of variance. The Bonferroni procedure was used to correct for multiple comparisons between the groups. Correlation between variables was performed by simple regression analysis. A p value < 0.05 was considered to be statistically significant.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

The clinical histories, pulmonary function tests, and BAL cytology of the study patients are summarized in Table 2. Patients with HP had significantly higher numbers of total cells and more lymphocytes and neutrophils in their BAL than did control subjects (p < 0.01). Although the percentage of alveolar macrophages was lower in the HP group, the absolute number of these cells was significantly higher with respect to the total cells recovered in the BAL (mean ± SD × 10³/ml = 44 ± 26 in the HP group versus 9 ± 3 in the control group, p < 0.01). The mean age was greater in the HP group than in the control group.

Representative agarose gels and southern blots showing specific PCR products for common respiratory viruses and housekeeping genes detected in the BAL cells recovered by lavage from the study subjects are illustrated in Figure 1. All specimens analyzed in the study showed positive housekeeping gene products, indicating that negative viral detections were not a result of unsuccessful nucleic acid extraction, nucleic acid degradation, or presence of PCR inhibitors. In addition, complete agreement between duplicate sample analyses was achieved, indicating that no cross-contamination between samples occurred in this study to produce false-positive results.

View larger version (45K): [in this window] [in a new window]   Figure 1.   Representative agarose gels and southern blots showing specific PCR products of common respiratory viruses and housekeeping genes detected in BAL cells obtained from patients with HP (Subjects 1 to 13) and unexposed healthy volunteers (Subjects 14-19).

The results of PCR analysis show that influenza A was detected in the BAL in six of 13 patients and in two of six control subjects. Adenovirus, coronavirus subtype OC43, and parainfluenza type 3 virus were detected in one of 13 patients but not in any control subjects. None of the other six viruses, including picornaviruses (rhinovirus) and respiratory syncytial virus, were detected by PCR in any sample of the study subjects.

Representative results of immunostaining for influenza A and adenovirus proteins in BAL cells and appropriate virus-infected and uninfected controls from cell culture are shown in Figure 2. Both infected cell lines showed positive virus-specific immunostaining (Figures 2A and 2B), whereas the uninfected cell lines showed no positive staining (Figures 2C and 2D). Influenza A proteins were detected, principally in alveolar macrophages (Figure 2E), in 69% (nine of 13) of patients and in 33% (two of six) of control subjects. Adenovirus was not detected by immunocytochemistry in BAL cells (Figure 2F).

View larger version (76K): [in this window] [in a new window]   Figure 2.   Immunocytochemical detection of influenza A nucleoproteins and adenovirus hexon proteins. Both influenza A-infected MDCK cells (A) and adenovirus-infected A549 cells (B) showed virus-specific immunostaining products. No immunoreactive viral proteins were detected in uninfected control MDCK cells (C ) or A549 cells (D). Influenza A proteins were detected in alveolar macrophages (E ), whereas adenovirus proteins were not detected in BAL cells (F ). (Indirect alkaline phosphatase method. Nuclei were counterstained with methyl green. Scale bar represents 50 µm).

Patients with documented influenza A proteins had significantly higher numbers of total cells, but similar proportion of lymphocytes, in their BAL than did patients with negative influenza A proteins (Figure 3). In addition, the proportion of influenza-A-antigen-containing alveolar macrophages correlated with the number of total BAL cells but not with the percentage of lymphocytes in this group (Figure 4).

View larger version (15K): [in this window] [in a new window]   Figure 3.   Number of total cells (A) and percentage of lymphocytes (B) recovered in the BAL of patients and control subjects with or without influenza A proteins. Patients with documented influenza A proteins had higher numbers of total cell counts in the BAL than did patients without influenza A proteins. Open circles represent individual values, closed circles are mean values, and bars represent standard deviations. POS = positive influenza A proteins; NEG = negative influenza A proteins.

View larger version (13K): [in this window] [in a new window]   Figure 4.   Correlation between the proportion of influenza-A-positive alveolar macrophages and total cell counts (A) or percentage of lymphocytes (B) recovered in the BAL of patients with documented influenza A proteins. The number of total BAL cells were increased in correlation with the proportion of influenza-A-positive alveolar macrophages. Open circles denote individual values.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

A number of similarities exist between HP and an acute viral disease that suggest a role for respiratory viruses in the pathogenesis of HP. HP occurs in winter, and patients often complain of flulike symptoms (5). Patients with HP have increased bronchial responsiveness that is unrelated to past sensitization to causative antigens (18). The lymphocytic alveolitis that develops during acute HP is characterized by a predominant CD8+ T-cell infiltrate (19), consistent with an antiviral immune response. Respiratory viral infections are highly prevalent in winter and can induce significant increases in bronchial responsiveness (20). Recent animal studies from this laboratory have shown that Sendai virus infection considerably enhanced the pathology of experimental HP induced in mice by repeated exposures to the actinomycete Saccharopolyspora rectivirgula, the most common causative antigen of farmer's lung disease, a classic form of HP (6).

The present study has demonstrated that common respiratory viruses are present in the lower airways, the likely site of hypersensitivity reaction, of patients during acute HP. Because nasal aspirates samples were not taken at the same time as BAL, it was not possible to determine if these viruses were concomitantly present in the upper respiratory tract in the study subjects. The results show that Influenza A was the most frequently detected virus in the lower airways of our study patients and our unexposed healthy control subjects. Other important viruses, namely, respiratory syncytial virus (the most common cause of acute bronchiolitis in children [21]) and picornaviruses (which include rhinovirus, a major cause of the common cold) (22) were not detected by the highly sensitive PCR method used in this study.

Although the potential exists that use of bronchoscopy may cause artifactual contamination of the lower airways with viruses present in upper airways, such a possibility is unlikely in our study because: (1) none of the study subjects presented symptoms of acute upper respiratory infection; in particular, there was no evidence of runny nose or postnasal secretions at the time of bronchoscopy; (2) rhinovirus, which is a known ubiquitous pathogen of the upper respiratory tract (22), was not detected in any sample from any subject of the study; (3) the immediate treatment of the specimens at cold temperature did not permit optimal adsorption of contaminating viral particles, if any, and subsequent infection of BAL cells. Although inadvertant contamination of BAL specimens can be ruled out, it cannot be established directly whether these viruses resulted from a recent, active infection or if they represent inactive viral particles that could result from an abortive infection (23).

Unlike adenovirus, which can establish latent infection of the lung (24) and where viral DNA can potentially integrate into the host genome (25), influenza A is not known to produce a persistent infection in the human lung. The results of immunocytochemistry for influenza A and adenovirus, showing positive immunostaining for only influenza A proteins within alveolar macrophages, suggest a recent infection by the influenza virus. In addition, the significant increases in numbers of total BAL cells observed in the influenza-A-positive patients is consistent with recent infection, where an influx of phagocytes would facilitate clearance of virus-infected cells from the lungs. Alternatively, there is also a possibility of direct infection of alveolar macrophages, since these cells are susceptible to infection by various viruses, including influenza A (26).

Alveolar macrophages play an important role in protecting the lower airways against pathogens and inhaled particulate antigens (27). Studies have established that inhaled particulate antigens of 5 µm or less can reach the lower airways and be deposited by gravitational sedimentation in the alveoli (28), where causative HP antigens of similar size are likely to induce the reaction seen in HP. Previous studies have demonstrated that direct stimulation of alveolar macrophages with causative HP antigens can result in release of copious amounts of the proinflammatory cytokines, interleukin-1, and tumor necrosis factor- (29). We have shown that alveolar macrophages from patients with acute HP have a decreased capacity to suppress the proliferation of lymphocytes (30). Further investigations are required to determine whether a viral infection may enhance the proinflammatory cytokine response of alveolar macrophages to causative HP antigens and downregulate their immunosuppressive activity, thereby contributing to the development of a lymphocytic alveolitis, a hallmark of HP.

Other potential mechanisms whereby viral infections could enhance the pathology of HP may include virus-induced mucociliary dysfunction (31) and alteration of alveolar macrophage phagocytic functions (32), potentially leading to a defective clearance of inhaled particulate antigens. Viral infections could also increase secretion of particular chemokines (e.g., MIP-1, lymphotactin and RANTES) known to affect the recruitment of lymphocytes and enhance their effector immune functions (33).

In summary, this study has established that common respiratory viruses are present in the lower airways, the likely site of hypersensitivity reaction, of patients with an acute episode of HP. The positive correlation between proportion of influenza-A-positive cells and total number of BAL cells in patients with HP is consistent with the hypothesis that viral infections could modulate pulmonary immune responses to offending antigens during acute HP. Prospective studies are warranted to clarify the nature of the relationship that might exist between respiratory viral infections and the onset of HP, and to determine whether intervention with specific antiviral therapy such as protective immunization against the pertinent virus might help to modify the outcome of antigen exposure in this disease.