INTRODUCTION

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Summer-type hypersensitivity pneumonitis (SHP) is one of the most prevalent types of hypersensitivity pneumonitis (HP) in Japan (1). It is characterized by cough, fever, and dyspnea, which appear when patients are at home during the summer and midautumn (1). We first reported the clinical and immunological features of 42 cases of HP of unknown origin positive for anti-Cryptococcus neoformans antibodies (anti-Cryptococcus antibodies) in sera in 1978, although at that time the disease was not called SHP (2). In 1982 we reported further clinical and immunological studies of 16 patients with HP referred to as Japanese summer-type hypersensitivity pneumonitis (3). As C. neoformans was not isolated from the home environments of these patients, we hypothesized that saprophytic Cryptococcus albidus, which was frequently found in the patient home environments, might be the causative agent. Later, in 1984, Shimazu and coworkers proposed that Trichosporon cutaneum may be a causative agent for SHP (5, 6). Mizobe and coworkers suggested that anti-Cryptococcus antibody is really an anti-T. cutaneum antibody and that approximately 65% of all SHP cases were likely to be caused by serotype II T. cutaneum (7). Many investigators currently believe that T. cutaneum is the etiologic antigen of SHP (1, 8, 9). We have tried for many years to isolate T. cutaneum from patient home environments; however, T. cutaneum has rarely been found in the home environments of these patients. We have obtained no supportive data to conclude that T. cutaneum is the causal agent in our SHP cases; rather, we have found that T. cutaneum is not likely to be a causative antigen in our cases.

Summer-type HP is associated with increased levels of T lymphocytes, and low levels of plasma cells in bronchoalveolar lavage fluid (BALF). Our previous studies demonstrated the presence of IgG, IgA, and IgM anti-Cryptococcus and/or anti- Trichosporon antibody in BALF (10). We also demonstrated the presence of specific antibody-secreting cells (ASCs) in BALF by enzyme-linked immunospot (ELISPOT) assay (12).

To clarify a causal antigen for SHP, we attempted to isolate Cryptococcus and Trichosporon species from patient home environments. To study local antibody production in the lung, we cultured BAL cells to measure anti-Cryptococcus and anti- Trichosporon antibodies in the culture supernatants.

	METHODS

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Subjects

Twenty-two patients with subacute-type SHP (5 males and 17 females; mean age, 44.5 yr; range, 11 to 69 yr) underwent BAL. Nineteen patients were nonsmokers and 3 patients were current or ex-smokers. Some patients received corticosteroid hormone therapy before admission to our hospital. They all fulfilled the diagnostic criteria of SHP (1). Serum samples were obtained shortly after admission and preserved at 20° C until use. Patients with other lung diseases (n = 13) were studied as controls. We did not use a healthy subject control group for BAL. This study was approved by the Ethics Committee of Habikino Hospital (Osaka, Japan) and informed consent was obtained from each patient.

Bronchoalveolar Lavage

Bronchoalveolar lavage was performed by standard techniques (13) within 17 d (mean, 6.9 d) after admission. Shortly after BAL, a transbronchial lung biopsy (TBLB) was done. A fibrobronchoscope (Olympus, Tokyo, Japan) was wedged into a segmental bronchus of the right middle lobe. Three to four 50-ml aliquots of sterile physiological saline were instilled and recovered immediately. The recovered fluid was filtered through two layers of sterile gauze and centrifuged at 1,500 rpm for 5 min. A supernatant fluid was preserved at 20° C until assays were performed. The cell pellet was washed three times with RPMI 1640 supplemented with Penicillin (100 U/ml) and streptomycin (100 µg/ml) and resuspended in medium with 10% fetal calf serum (FCS) (medium). A differential count was made from a cytocentrifuged cell preparation stained with Wright-Giemsa. Lymphocyte surface markers were examined by flow cytometric analysis (FACScan, Becton Dickinson, San Jose, CA), using monoclonal antibodies. Fluorescein isothiocyanate (FITC)- or phycoerythrin (PE)-conjugated anti-CD3, anti-CD19, anti-CD4, and anti-CD8 monoclonal antibodies (MAbs) were purchased from Becton Dickinson (Becton Dickinson Immunocytometry Systems, San Jose, CA).

Culture of BAL Lymphocytes

Bronchoalveolar lavage fluid cells were suspended in medium at a cell density of 1 × 10⁶ lymphocytes/ml. One milliliter of BAL cell suspension was cultured in duplicate in Falcon 2054 tubes (Becton Dickinson, Labware, Lincoln Park, NJ) without stimulation at 37° C in 5% CO₂-humidified air. On Day 7, the cultures were harvested for centrifugation at 1,500 rpm for 5 min. The resultant supernatants were stored at 20° C until use.

Preparation of Antigens

As previously reported (10), freeze-dried culture filtrates (MW > 10,000) of C. neoformans IFO 608 (serotype A) and CBS 132 (serotype A-D); T. cutaneum TIMM 1318, IFO 0174, Kubota strain (serotype II), and IFO 1198 (serotype I); and C. albidus (Iwata) strains were used as test antigens for enzyme-linked immunosorbent assay (ELISA) and precipitation reaction. IFO strains were previously obtained from the Institute of Fermentation (Osaka, Japan). The TIMM 1318 strain was purchased from the Research Center for Fungal Diseases (Teikyo University Medical School, Tokyo, Japan). The CBS strain was provided by Meiji Pharmaceutical College, Tokyo, Japan). The Kubota and Iwata strains were isolated from patient home environments. These fungi were cultured in Czapek-Dox or Sabouraud medium with neopeptone dialysate, with shaking, for 7 to 10 d. After heating to 60° C for 2 h to kill active fungi, cells were centrifuged to obtain culture supernatants. Culture supernatants were concentrated by DIAFRO ultrafiltration membrane PM-10 (Amicon, Danvers, MA), dialyzed in water, and freeze-dried. Between culture filtrates of C. neoformans (A and A-D) and rabbit antisera against C. neoformans (serotype A), one or two precipitating lines were observed by agar-gel double diffusion and crossed immunoelectrophoresis (11). Between rabbit antisera against T. cutaneum (TIMM 1318) and culture filtrates of serotype II and serotype I T. cutaneum, five to seven precipitation lines and two precipitating lines were formed in serotype II and serotype I, respectively.

Enzyme-linked Immunosorbent Assay

Culture filtrate antigens of C. neoformans (IFO 608) and T. cutaneum (TIMM 1318) were used for ELISA, because of their lower nonspecific binding and higher antigen specificity compared with other antigens (12). The optimal conditions for preparing microtiter plates and performing ELISA analyses were described previously (10, 12). Briefly, a polystyrene ELISA plate (Nunc Immunoplate; Nalge Nunc International, Rochester, NY) was coated with 100 µl of a 20-µg/ml antigen solution diluted in phosphate-buffered saline (PBS) and then blocked with 2% bovine serum albumin (BSA). After washing in PBS-0.05% Tween 20, 100-µl test samples (serum diluted 1:1,000, BAL fluid diluted 1:10, or culture supernatant) were applied to the plates and incubated at 37° C for 1 h. After washing three times, the plates were incubated at 37° C for 1 h with alkaline phosphatase-conjugated goat anti-human IgG, IgA, or IgM (Biosource International, Camarillo, CA; diluted 1:7,500 or 1:10,000). After further washings, the plates were developed with p-nitrophenyl phosphate substrate (Sigma, St. Louis, MO) at room temperature for 30 min. The reaction was stopped by 50 µl of 3 N NaOH and the optical density at 405 nm was determined by either Immunoreader NJ 2000 (Intermed) or Labsystems Multiskan Bichromatic (Labsystems, Helsinki, Finland). Culture supernatants and BAL fluid were measured simultaneously.

Double Diffusion in Agar Gel

Serum precipitating antibody was measured by the double diffusion method in agar gel.

Preparation of Rabbit Anti-Cryptococcus albidus Sera

Anti-C. albidus rabbit serum was prepared by intravenous injection of New Zealand White rabbits, weighing 3.0-3.5 kg, with 2-3 × 10⁸ heat-killed C. albidus (Iwata strain) three times. Serum samples were obtained before each immunization. Antibody activity to C. albidus, C. neoformans, and T. cutaneum was measured by agglutination test and agar-gel double diffusion methods.

Isolation and Identification of Yeast Form Fungi

Indoor samplings were performed by openplate and house dust cultures.

Open-plate culture. The culture plates for sampling of atmospheric molds were sterile petri dishes (20 by 90 mm) containing Sabouraud agar medium. Five plates were exposed for 30 min on the floor of every room. Plates were cultured for 7 d at room temperature.

House dust culture. House dusts were obtained from a vacuum cleaner. Culture plates containing Sabouraud agar medium were exposed to house dust particles floated in nylon bags.

Yeast form colonies were picked for subculture and identification. Species of isolated yeast were identified by morphologic and cultural methods with slide culture, using a physiologic method with API 20C AUX (Eiken Chemical, Tokyo, Japan) (14). Isolated strains were tested using serum samples from SHP patients and rabbit immune sera by indirect immunofluorescence assay (IFA) and agglutination reaction.

Statistical Analysis

Statistical analysis was done with the paired Student t test by StatView program (Brain Power, Calabasas, CA).

	RESULTS

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Bronchoalveolar Lavage Findings

Bronchoalveolar lavage was performed in 22 patients with SHP within 17 d of admission (last exposure to their home environment). Bronchoalveolar lavage fluids from 13 patients with interstitial lung diseases, such as idiopathic interstitial pneumonitis and broncholitis obliterans with organizing pneumonia, were used as control. The results are shown in Table 1. The recovery of lavage fluid was 49.9 ± 11.5% (mean ± SD). The cell analysis was as follows: total cells, 9.3 (± 3.4) × 10⁵ cells/ml, alveolar macrophages 28.6 ± 7.9%, lymphocytes 61.6 ± 12.1%, neutrophils 5.7 ± 5.8%, eosinophils 2.1 ± 3.1% (range, 0-13%), basophils 1.2 ± 0.8% (range, 0-3.5%), plasma cells 0.9 ± 1.1% (range, 0.1-3.1%; 320-28,520/ml). A fluorescence-activated cell sorting (FACS) analysis of lymphocyte surface makers revealed 92.1 ± 4.5% for CD3, 26.1 ± 10.1% for CD4, 65.7 ± 12.6% for CD8, 0.45 ± 0.28 for CD4⁺/CD8⁺ cell ratio, and 0.8 ± 1.2% for CD19, respectively. In this study, no data from healthy subjects were available; however, increases in total cell numbers and in CD4⁺ and CD8⁺ T cells, reversed CD4⁺/CD8⁺ cell ratios, and the presence of plasma cells and basophils (mast cells) were compatible with characteristic findings of BAL from HP patients (15, 16).

Antibody Activity in Serum

Serum antibodies were measured by ELISA in 19 patients, whose serum IFA titers (2) for heat-killed C. neoformans (serotype A) and T. cutaneum (TIMM 1318) cells were between 128 and 2,048 and no significant difference was observed in IFA titers between these antigens. As shown in Figure 1, the mean IgG, IgA, and IgM antibody activity to C. neoformans and T. cutaneum antigens were 1.23 ± 0.53 and 1.09 ± 0.53, 0.45 ± 0.25 and 0.31 ± 0.24, and 0.75 ± 0.50 and 0.50 ± 0.43 in diluted sera (1:1,000) from SHP patients, respectively. A significant correlation was observed between antibody activity to both antigens (r = 0.78 [IgG], r = 0.73 [IgA], and r = 0.86 [IgM]). However, the amount of IgA and IgM bound to C. neoformans antigen was significantly more than the amount bound to T. cutaneum antigen: p = 0.002 (IgA) and P < 0.0001 (IgM). Antibody activity in sera from healthy subjects was 0.07 ± 0.03 and 0.17 ± 0.09 (IgG), 0.02 ± 0.00 and 0.03 ± 0.01 (IgA), and 0.03 ± 0.01 and 0.04 ± 0.01 (IgM) to C. neoformans and T. cutaneum antigens, respectively. Low levels of IgG were reactive to T. cutaneum. Sera from a few patients with other lung diseases showed low antibody activity to T. cutaneum; however, their IFA titers for T. cutaneum were less than 16 (data not shown).

View larger version (12K): [in this window] [in a new window]   Figure 1.   (A) IgG, (B) IgA, and (C ) IgM antibodies bound to ELISA plates coated with C. neoformans and T. cutaneum culture filtrate antigen in the sera of patients with SHP. Values represent the OD at 405 nm. Amounts of IgA and IgM antibodies bound to C. neoformans antigen (a-Cr) (OD405nm) were significantly more than those to T. cutaneum antigen (a-Tr). p = 0.065 (IgG), p = 0.002 (IgA), p < 0.0001 (IgM).

Antibody Activity in BAL Fluid

Antibody activity in BALF is shown in Figure 2. Mean IgG, IgA, and IgM antibody activities to C. neoformans and to T. cutaneum in patients with SHP were 1.38 ± 0.62 and 1.32 ± 0.58 (IgG), 1.31 ± 0.47 and 1.12 ± 0.49 (IgA), and 1.37 ± 0.60 and 1.17 ± 0.60 (IgM) in diluted BALF (1:10), respectively. A significant correlation was observed between antibody activities to C. neoformans and T. cutaneum (r = 0.75 [IgG], r = 0.87 [IgA], and r = 0.895 [IgM]). However, the IgA and IgM antibody levels bound to C. neoformans were significantly higher than to T. cutaneum: p = 0.002 (IgA) and p = 0.002 (IgM). When BALF (1:80) was employed for antibody determinations, similar results were obtained. For control BALF (1:10) antibody activities to C. neoformans and to T. cutaneum were 0.07 ± 0.04 and 0.17 ± 0.10 (IgG), 0.05 ± 0.08 and 0.05 ± 0.03 (IgA), and 0.01 ± 0.01 and 0.01 ± 0.01 (IgM), respectively. Only low levels of IgG were reactive to T. cutaneum.

View larger version (13K): [in this window] [in a new window]   Figure 2.   (A) IgG, (B) IgA, and (C ) IgM anti-Cryptococcus and anti-Trichosporon antibodies bound to ELISA plates coated with C. neoformans and T. cutaneum culture filtrates antigen in the BALFs of patients with SHP. Values represent the OD at 405 nm. Amounts of IgA and IgM antibodies bound to C. neoformans antigen were significantly more than those to T. cutaneum. p = 0.53 (IgG), p < 0.002 (IgA), p < 0.002 (IgM).

Antibody Activities in BAL Cell and PBMC Culture Supernatants

Mean antibody activity (OD_405nm) to C. neoformans and to T. cutaneum antigen was 0.35 ± 0.43 (mean ± SD) and 0.27 ± 0.36 (IgG), 0.63 ± 0.47 and 0.44 ± 0.38 (IgA), and 0.69 ± 0.71 and 0.46 ± 0.60 (IgM) in BAL culture supernatants, respectively (Figure 3). A significant correlation was observed in antibody activity between antigens: IgG, r = 0.91; IgA, r = 0.94; and IgM, r = 0.97. Although antibody levels varied individually, antibody activity to C. neoformans was significantly higher than those to T. cutaneum: p = 0.03 (IgG), p < 0.0001 (IgA), p < 0.0001 (IgM). These results suggest that BALF plasma cells may be secreting specific antibodies and that most of these antibodies may react with antigens, probably polysaccharides, common to C. neoformans and T. cutaneum (17). In contrast, no antibody was detected in culture supernatants of SHP peripheral blood mononuclear cells (PBMCs) with or without pokeweed mitogen (PWM) stimulation. Addition of PWM to BAL cell cultures diminished antibody production because of impaired cell viability (data not shown).

View larger version (11K): [in this window] [in a new window]   Figure 3.   (A) IgG, (B) IgA, and (C ) IgM anti-Cryptococcus and anti-Trichosporon antibodies bound to ELISA plates coated with C. neoformans and T. cutaneum culture filtrates antigen in BAL cell culture supernatants of patients with SHP. Values represent the OD at 405 nm. Amounts of antibodies bound to C. neoformans antigen were significantly more than those to T. cutaneum antigen. p = 0.03 (IgG), p < 0.0001 (IgA), p < 0.0001 (IgM).

Serum Precipitating Antibody

Serum precipitating antibody to culture filtrate antigens of C. neoformans serotypes A and A-D, and to C. albidus were found in 81, 81, and 78% of patients with SHP, respectively, whereas antibody to serotype II T. cutaneum TIMM 1318, IFO 0174, Kubota, and serotype I IFO 1198 was found in 62, 57, 67, and 33% of patients with SHP, respectively. The percentages of positive reactions were higher for Cryptoccocus antigens than for Trichosporon antigens (Table 2).

Double Diffusion and Agglutination Reaction with Anti-Cryptococcus albidus Rabbit Sera

In agglutination reactions (Table 3) and double diffusion in agar gels (Figure 4), anti-C. albidus sera raised in rabbits showed strong reactivity not only to homologous C. albidus but also to C. neoformans and T. cutaneum. One, two, and three precipitating lines were formed between antisera and T. cutaneum, C. albidus, and C. neoformans antigens, respectively. As shown in Figure 4, one of them completely fused. These results suggest the presence of a common antigen among these fungi, probably glucuronoxylomannan (GXM) (7, 18, 19). Culture filtrates of C. albidus and C. neoformans contain more antigens, which may be absent from T. cutaneum, and vice versa (11).

View larger version (65K): [in this window] [in a new window]   Figure 4.   Double diffusion in agar gel. Central hole, anti-C. albidus rabbit serum. Outer holes, culture filtrate antigens of (1) C. albidus, (2) C. neoformans serotype A, (3) C. neoformans serotype A-D, (4) type II T. cutaneum (TIMM 1318), (5) C. laurentii, (6 ) type I T. cutaneum (IFO 1198), and (7 ) type II T. cutaneum (IFO 0174). One of the precipitating lines completely fused.

Isolation and Identification of Home Environment Fungi

As shown in Table 4, from 1990 to 1996, saprophytic Cryptococcus species such as C. albidus and C. laurentii were isolated from 15 and 2 of 24 patient home environments (62.5 and 8.3%; total, 70.8%), respectively. Cryptococcus albidus was isolated from 1 of 10 control homes examined. Trichosporon cutaneum was not found in our cases, except for one case reported previously. No C. neoformans was found.

	DISCUSSION

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Hypersensitivity pneumonitis, or extrinsic allergic alveolitis (EAA), consists of a group of immunologically mediated pulmonary diseases associated with repeated exposure to a wide variety of finely dispersed organic dusts that affect the periphery of the lung. In HP, IgG, IgA, and IgM and specific antibodies are elevated in BALF when values were expressed in terms of albumin (12, 20, 21). Increasing numbers of lymphocytes and plasma cells infiltrate alveolar walls (alveolitis) (8, 9, 22). High levels of lymphocytes, particularly CD8⁺ T cells, and a low level of plasma cells are found in the BALF obtained from patients with active alveolitis (12, 16, 20). These data suggest that cell-mediated and humoral immunity may participate in the immunopathogenesis. Drent and co-workers have reported that patients with EAA who underwent BAL within 2 to 7 d of their last exposure to antigens showed a higher percentage of plasma cells in BALF and that the increase in plasma cells in lavage fluid might be a feature of recent antigen exposure, suggesting active alveolitis (20). We obtained similar results in our patients with SHP. These findings suggest that plasma cells may be secreting antibodies to inhaled antigen in the lung.

In this study we investigated specific antibody production in the lungs of patients with SHP. We cultured BAL cells in vitro and measured anti-Cryptococcus and anti-Trichosporon antibodies in culture supernatants. We clearly demonstrated IgG, IgA, and IgM anti-Cryptococcus and anti-Trichosporon antibodies in all culture supernatants (Figure 3). A significant correlation was observed in antibody binding between Cryptococcus antigen and Trichosporon antigen (IgG, r = 0.91; IgA, r = 0.94, IgM, r = 0.97); however, the levels of IgG, IgA, and IgM antibody bound to C. neoformans antigen was significantly higher than to T. cutaneum antigen: p = 0.03 (IgG), p < 0.0001 (IgA), and p < 0.0001 (IgM). These results indicate that BALF lymphocytes, probably plasma cells, are secreting antibodies in the lung and that most of the antibodies may be cross-reactive to C. neoformans and T. cutaneum. We also performed antibody determinations in BALF and serum by ELISA. The results have shown that IgG antibody activity was similar in BALF (Figure 2A) and in serum (Figure 1A); however, IgA antibody activity was much higher in BALF (Figure 2B) than in serum (Figure 1B) .These results agree with our previous investigations, which showed that the level of IgA anti-Cryptococcus antibody-secreting cells (ASCs) were higher than levels of IgG and IgM in BALF (IgA > IgM > IgG) (12). Most BALF IgA antibody may be produced by the local plasma cells, while BALF IgG antibody may be in part originated systemically and in part locally produced, since serum proteins (albumin and IgG) leak from blood vessels into the alveolar spaces owing to increased permeability in inflammatory responses (21, 23). According to our previous study, only a low percentage of total BALF plasma cells were antigen-specific ASCs (12). The recruitment of antibody-forming cells to the lung may be nonspecific, but IgA predominant (24).

A main constituent of culture filtrate antigen of C. neoformans serotype A is capsular polysaccharide (CPS). Approximately 90% of CPS is glucuronoxylomannan (GXM) (18, 19, 25, 26) and about 10% of CPS is galactosylomannan-mannoprotein complex (GalXM-MP) (27). Extracellular and cell wall polysaccharides produced by serotype II T. cutaneum also consist of glucose, mannose, xylose, and D-glucuronic acid, which is similar in composition and structure to the CPS of C. neoformans (7, 28). Cryptococcus albidus strains also have polysaccharide GXM, consisting of a backbone of (1-3)- linked D-mannopyranosyl residues with a single branch of (1-2)-xylose or glucuronic acid, with an O-acetyl group, which is similar to that of C. neoformans serotype A (19). Cell surface antigenic factors of C. albidus (1, 2, 3, 7) are identical to those of C. neoformans (serotype A), with no more specific antigen in addition to antigens shared with C. neoformans serotype A (19, 26, 27). We have previously observed that most of the anti-C. neoformans IFA antibody in serum was absorbed with C. albidus, and vice versa. In this study we absorbed four SHP serum samples with heat-killed C. albidus to measured remaining antibody by the ELISA method. We observed that most anti-Cryptococcus and T. cutaneum antibodies could be absorbed by C. albidus. Even when a low level of antibody remained, the level was within the range of serum antibody levels from control subjects (data not shown). In light of this evidence, we examined antigenic cross-reactivity among C. albidus, C. neoformans, and T. cutaneum, using anti- C. albidus sera raised in rabbits. We observed that antisera showed similar agglutination titers against these fungi (Table 3). Antisera formed one, two, and three precipitating lines between culture filtrate antigens of T. cutaneum, C. albidus, and C. neoformans, respectively, by precipitation reaction. One of these precipitation lines completely fused (Figure 4). These data suggest that the antigen determinant of GXM may be completely identical among these fungi. However, we could not confirm whether small precipitating lines formed between C. neoformans and C. albidus antigen are against GalXM-MP or whether the GalXM-MP of C. albidus is similar to that of C. neoformans. We think that the anti-Cryptococcus antibody assayed in this study may be anti-GXM antibody.

Cryptococcus albidus is a saprophytic fungus that is often found in the air (29). We isolated one strain of C. albidus from 1 of 10 control subject homes examined; however, C. albidus strains were found in approximately 62.5% of the home environments of our patients with SHP. The frequency was significantly higher compared with control. Yoshida and co-workers found T. cutaneum in approximately 70% of their patients with SHP; however, they also found C. albidus strains with a significantly higher incidence in the homes of patients with SHP, compared with control homes (6).

An inhalation provocation test may be definite proof in determining a causative antigen in HP. In our previous study an inhalation provocation test with C. neoformans antigen was successful in three patients (3). In this study we had no chance to test an inhalation provocation with C. albidus antigen. Although an inhalation provocation test with C. albidus antigen still remains to be performed, we still claim that Cryptococcus species might be the etiologic antigen in our cases, because we have observed no evidence to conclude that T. cutaneum is the causal agent. It is probable that etiologic agents of SHP differ by area or individual case, because most SHP cases induced by T. cutaneum were reported in a southwestern area of Japan (5, 6), while our cases reside in mid-mainland Japan. These two areas are approximately 500 km apart.

In conclusion, both Cryptococcus and Trichosporon species may induce SHP. IgA and IgM antibody in BALF may be secreted locally by plasma cells in the lung.