Study population

12 HP patients were included in the study (9 males and 3 females; mean age 38.3 ± 6.4 yr). The majority of the patients had farmer's lung disease (10 patients); 1 patient had bird fancier's lung, 1 patient had mushroom worker's lung. The following criteria for HP diagnosis were used: a) history of exposure to HP antigens, b) a symptomatic acute episode with chills, fever, cough, breathlessness 4 to 8 hours after exposure to specific antigens, c) radiological features (mainly diffuse reticular pattern) and/or a functional pattern of interstitial lung disease, and d) evidence of antibodies against S. rectivirgula in all except one case (bird fancier's lung). Each patient underwent bronchoscopy for transbronchial biopsy (TBB) and BAL analysis. BAL was performed according to the technical recommendations and guidelines for the standardization of BAL procedures [12]. Briefly, a total of 200 ml of saline solution was injected in 25-ml aliquots via fiberoptic bronchoscopy, with immediate vacuum aspiration after each aliquot. Immediately after the BAL, the fluid was filtered through gauze and the volume measured. A volume of 100-200 ml of BAL recovery and a sample of 50% of the instilled volume with a minimum of 50 ml was considered acceptable. The percentage of BAL recovery was 54.9% ± 4.2. Cells recovered from the BAL were washed 3 times with PBS, resuspended in endotoxin tested RPMI 1640 (Sigma Chemical Co., St. Louis, MO) supplemented with 20 mM HEPES and L-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, and 10% FCS (ICN Flow, Costa Mesa, CA) and then counted. A standard morphological and immunologic analysis of BAL cellular components was performed and included cell recovery, differential count of macrophages, lymphocytes, neutrophils, and eosinophils, and flow cytometry analysis of the CD4/CD8 BAL T-cell ratio.

Five healthy controls were selected (3 men and 2 women; average age 37.3 ± 4.3 yr; 2 non-smoking healthy adults and 3 non-smoking subjects evaluated for complaints of cough without lung disease). They showed normal physical examinations, chest X-rays, lung function tests and BAL cell numbers.

Purification of alveolar macrophages and T cells

Alveolar macrophages (AMs) and T cells were enriched from the BAL cell suspensions by rosetting with neuraminidase-treated SRBC followed by F/H gradient separations and residual CD3⁺ lymphocytes were removed using high-gradient magnetic separation columns (Mini MACS, Miltenyi Biotec, Germany) [13]. Following this multistep selection procedure more than 95% of the above cells were viable, as judged by the trypan blue exclusion test. Staining with mAb showed that more than 99% of purified lymphocytes were CD3+ T cells.

Monoclonal antibodies and cytokines

The commercially available conjugated or unconjugated mAbs used belonged to the Becton Dickinson and PharMingen series and included: CD3, CD4, CD8, isotype matched controls. Anti-IL-4 and anti-IFNγ mAbs were purchased from PharMingen (San Diego, CA). Purified rabbit anti-human CXCL10 polyclonal antibody (R&D Systems Inc, Minneapolis, MN) and anti-hCXCR3 mAb (R&D Systems Inc) were also used.

Immunohistochemical analysis of CXCR3+ cells and CXCL10 producing cells

Open lung biopsies from 8 patients with clinical and histological diagnosis of hypersensitivity pneumonitis were studied by immunohistochemistry for the immunophenotype characterization of inflammatory cells and for CXCR3 and CXCL10 expression.

Immunohistochemistry for the characterization of inflammatory infiltrate was carried out using the following antibodies (Dako Glostrup, Denmark): CD45 (1:20), CD43 (1:40), CD45RO (1:100), CD20 (1:100), CD3 (1:50), CD68 (1:50), CD4 (1:100), and CD8 (1:100). The immunoreaction products were developed using the avidin-biotin-peroxidase complex method. Immunostaining for CXCR3 was performed as previously described. Briefly, after the microwave antigen retrieval procedure and neutralization of endogenous peroxidase activity, the slides were incubated with primary antibody for 1 hr in a humidified chamber at 37°C (anti-hCXCR3 mAb 1:100). Immunoreactivity was detected using biotinylated secondary antibodies incubated for 45 min followed by a 30 min incubation with avidin-peroxidase and visualized by a 7 min incubation with the use of 0.1% 3,3'-diaminobenzidene tetrahydrochloride as the chromogen. Parallel control slides were prepared either lacking primary antibody or lacking primary and secondary antibodies, or stained with normal sera to control for background reactivity. The intensity of antibody staining was classified in three groups: strong, weak, negative.

Confocal microscopy for the identification of CXCR3+ cells

Paraffin sections were prepared for immunofluorescent labelling. Briefly, primary antibodies against CD3 and CXCR3 (1:100 diluted and 1:100 diluted in phosphate-buffered saline with 5 g/L bovine serum albumin and 1 g/L gelatine, respectively) and secondary antibodies (goat anti-mouse IgG and donkey anti-goat IgG) conjugated with TEXAS red or ALEXA 488 (Sigma) were used. Double labelling using both antibodies on the same section was performed. Primary antibodies and secondary antibodies were incubated for 1 h at room temperature. Nuclear staining was carried out with DAPI (Sigma) in PBS. Slides were stored at 4°C and analysed within 24 h. As a control, the primary antibody was omitted.

Immunofluorescence was evaluated with a confocal microscopy (Biorad 2100 Multiphoton; Hercules, CA), We used an argon laser at 488 nm in combination with a helium neon laser at 543 nm to excite the green (CD3) and red (CXCR3) fluorochromes simultaneously. Emitted fluorescence was detected with a 505–530 nm band pass filter for the green signal and a 560 nm long pass filter for the red signal. Images were analyzed using the Adobe Photoshop 7.0 program.

Phenotypic evaluation of BAL cells

The frequency of BAL cells positive for the above reagents was determined by overlaying the flow cytometry histograms of the samples stained with the different reagents as previously reported [12]. Cells were scored using a FACScan^® analyzer (Becton Dickinson), and data were processed using the Macintosh CELLQuest software program (Becton Dickinson). The expression of cytoplasmic cytokine was evaluated following permeabilization of cell membranes using 1:2 diluted PermeaFix (Ortho, Raritan, NJ) for 40 min. After permeabilization procedures anti-IL-4, anti-IFN-γ and anti-CXCL10 antibodies were added. Since pulmonary cells bear cytoplasmic cytokines in a unimodal expression pattern, indicating that the entire cell population exhibits relatively homogeneous fluorescence, the percentage of positive cells does not represent the most accurate way of enumerating positive cells. Mean fluorescence intensity (MFI) was used to compare the positivity of these specific antigens on different cell populations. To evaluate whether the shift of the positive cell peak was statistically significant, the Kolmogorov-Smirnov test for analysis of histograms was used according to the Macintosh CELLQuest software user's guide (Becton Dickinson).

For immunofluorescence analysis, control IgG1 and IgG2a and IgG2b were obtained from Becton-Dickinson; control rat antiserum consisted of ascites containing an irrelevant rat IgG2b; control rabbit antiserum consisted of rabbit IgG (purified protein) purchased from Serotec (Serotec, U.K.); goat-anti-rabbit IgG and goat F(ab')2 anti-rat IgG were obtained from Immunotech (Marseille, France).

Determination of IP-10/CXCL10 and Mig/CXCL9 mRNA levels

Each PCR product was analysed and quantitated by Bio-Rad's Image Analysis System Gel Doc using Quantity One software (Bio-Rad, Hercules, CA). Briefly, the images of the gels were acquired from the Gel Doc system densitometer and saved in digitised forms to perform volume analysis. The intensity of each band was differentiated by the intensity of the background, whose value was subtracted from each individual band and the resulting PCR product value was expressed in mm*mm*intensity of the pixels of the specific band in the gel.

Generation of macrophage supernatants

To verify the ability of AMs to release CXCL10, AMs (1 × 10⁶/ml) were isolated from the BAL of HP patients, resuspended in RPMI medium and cultured for 24 hr in 24-well plates at 37°C in 5% CO₂. In separate experiments AMs were stimulated with IFN-γ (100 U/ml), PMA (10 ng/ml) and LPS (10 μg/ml; Difco Lab., Detroit, MI). Following the incubation period, supernatants were harvested, filtered through a 0.45 μm Millipore filter and immediately stored at -80°C. At the end of the culture time AM viability was always greater than 95%. Chemotactic activity of supernatants was determined as reported below.

Migration activity of pulmonary T cells in response to CXCLIO

T-cell migration was measured in a 48-well modified Boyden chamber (AC48 Neuro Probe Inc., USA). The chamber is made of two sections: different chemotactic stimuli were loaded in the bottom section while cells were added in the top compartment. Polyvinylpyrrolidone-free polycarbonate membranes with 3 to 5 μm pores (for lung T cells obtained from HP patients and the CXCR3+ and CXCR3- T-cell lines, respectively) (Osmonics, Livermore, CA) and coated with fibronectin were placed between the two chamber parts. Only the bottom face of filters was pretreated with fibronectin; the fibronectin pretreatment maximizes attachment of migrating cells to filters, avoiding the possibility that they may not adhere. Using this procedure in preliminary experiments we demonstrated that only a trivial number of cells may be recovered in the bottoms of the wells. To avoid the shedding of fibronectin, fibronectin-treated filters were extensively washed. In preliminary experiments, fibronectin-treated filters did not induce spontaneous chemotaxis in absence of chemokines.

To evaluate the migratory properties of pulmonary T lymphocytes rhIP-10/CXCL10 (200 ng/ml) were used. The CXCR3- and CXCR3+ cell lines (300-19, kindly provided by Dr. B. Moser, Theodor-Kocher Institute, University of Bern, Switzerland) were used as negative and positive controls. 30 μl of chemokines or control medium were added to the bottom wells, and 50 μl of 5.0 × 10⁶ cells/ml T cells or CXCR3-/+ cells resuspended in RPMI 1640 were added to the top wells. The chamber was incubated at 37°C with 5% CO₂ for 2 hrs. The membranes were then removed, washed with PBS on the upper side, fixed and stained with DiffQuik (Dade AG, Düdingen, Switzerland). Cells were counted in three fields per well at 800× magnification. All assays were performed in triplicate. In blocking experiments, cell suspensions were preincubated before chemotaxis assay for 30 min at 4°C with anti-human CXCR3 mAb at a concentration of 20 μg/ml.

Chemotactic activity of the fluid component of BAL and macrophage supernatants

The CXCR3(-) and CXCR3(+) cell lines were also used to evaluate both the chemotactic activities of macrophage supernatants and the fluid component of BAL samples. Supernatants from cell cultures and the fluid components of BAL were obtained as reported above and used undiluted; different concentrations of CXCL10 were utilized as a positive control. Chemotactic assays were performed as reported above. In blocking experiments, anti-CXCL10 was added to the cell supernatants before chemotaxis assay at a concentration of 20 μg/ml.

Statistical analysis

Data were analysed with the assistance of the Statistical Analysis System. Data are expressed as mean ± SD. Mean values were compared using the ANOVA test. A P value <0.05 was considered as significant.

Immunohistochemical analysis of CXCR3 expression in lung biopsies

In all cases typical pathological examination showed features with poorly formed non-necrotizing granulomas and widespread thickening of the alveolar walls by a diffuse lymphocytic infiltrate. Pleural lymphoid aggregates were seen in a few cases and pleural lymphoid aggregates were seen in a few samples.

Diffuse interstitial lymphocytic infiltrates were characterized by an accumulation of T cells and a few B-lymphocytes. Sub-pleural and peri-bronchiolar nodules consisted mostly of T lymphocytes mainly represented by CD8 cytotoxic T lymphocytes which strongly stained for CXCR3 in all cases (Figure 1A and 1B). Marked CXCR3 immunostaining was also seen in peribronchial lymphocytic infiltrate and in the interstitial non-necrotising granuloma (Figure 2A and 2B) Both interstitial and intra-alveolar macrophages (CD 68 positive) showed weak or negative CXCR3 staining and multinucleated giant cells always stained negatively (Fig. 2C inset). Endothelial and epithelial cells close to more intense lymphocytic infiltrate were sometimes positively marked.

Confocal microscopy analysis of lung biopsies confirmed that lymphocyte infiltrates were formed by T cells coexpressing CXCR3 (Figure 3 panels A, B and C).

Morphological and phenotypical features of cells obtained from the BAL

Morphological and phenotypical features of cells obtained from the BAL of 12 patients with HP and 5 controls are reported in Table 1. All HP subjects showed a high intensity lymphocytic alveolitis sustained by CD8(+) Tc1 cells (Table 1 and Figure 4A and 4B). These cells were CXCR3(+) and bore IFN-γ but not IL-4 receptor (Figure 4C). Furthermore, pulmonary T cells expressed activatory molecules such as CD103 and IL-12β2 receptor (Figure 4C). The percentage and absolute number of BAL CXCR3(+) was significantly higher in HP patients with respect to control subjects (Table 2).

Study population	Cell Recovery	Lymphocytes	CD4 T cells		CD8 T cells
	cells × 103/ml	%	%	cells × 103/ml	%	cells × 103/ml
HP (n. 12)	351.9* ± 62.3	44.4** ± 8.1	25.6** ± 5.3	38.6*** ± 9.3	53.7** ± 6.3	83.4*** ± 8.5
Controls (n. 5)	138.6 ± 12.7	8.2 ± 2.2	48.3 ± 3.2	5.4 ± 0.9	23.7 ± 2.2	2.5 ± 0.3

Significance as follows: *p < 0.05; **p < 0.01; ***p < 0.001

CXCR3 mediates pulmonary T cell chemotaxis

To define the biological activities of CXCR3, highly purified T cells obtained from the BALs of patients with HP were assessed for their migratory capabilities in response to different concentrations of CXCL10. The evaluation of the migratory potential of T lymphocytes obtained from the BAL of the controls was prevented by the low number of cells recovered. For this reason, the 300-19 T-cell lines expressing high levels of CXCR3 or not expressing CXCR3 were used as positive and negative controls respectively for the in vitro chemotaxis assay (Figure 5, panel B and C respectively).

As shown in panel A of Figure 4, the migratory capability of T lymphocytes of patients with HP is regulated by CXCR3. In fact, CXCR3+ lung T cells exhibited a strong, definite migration in response to CXCL10. To further verify the functional role of the CXCL10 receptor, CXCR3+ pulmonary T cells were preincubated with anti-CXCR3 neutralizing antibody: the blocking of the receptor determined a marked inhibition of CXCL10-induced chemotaxis (panel A). These data suggest that pulmonary T lymphocytes that sustain T-cell alveolitis in patients with HP express a functional CXCR3 receptor and actively migrate in response to CXCR3 ligands.

Lung macrophages express CXCR3 ligands and release ligands showing chemotactic activity on CXCR3(+) cells

In order to analyse whether CXCL10 is expressed in vivo by lung cells of patients with hypersensitivity pneumonitis, BAL cells were stained with a anti-CXCL10 antibody as described above. Flow cytrometric analysis (Figure 6, panels A and B) revealed that AMs of patients with HP express CXCL10; macrophages retrieved from control subjects lacked the CXCR3 ligand (panel C).

Measurement of mRNA levels of the CXCR3 ligands demonstrated that unstimulated alveolar macrophages isolated from the BAL of HP subjects expressed increased mRNA levels of CXCL9 and CXCL10 with respect to macrophages obtained from control subjects (Table 2 and figure 7). Spearman Rank correlation coefficients between BAL T CD8(+)/CXCR3(+) T cell number and levels of CXCR3 ligands were also calculated. Interestingly, a positive correlation was demonstrated between mRNA levels of CXCL10 and CXCL9 and the absolute numbers of lung CD8(+)/CXCR3(+) T cells (r 0.815, p < 0.001 and r 0.825, p < 0.001, respectively).

Cell-free supernatants were obtained from 24-hour cultured AMs in the presence of IFN-γ and tested for their ability to induce T-cell migration. Supernatants obtained from AMs of patients with HP exerted chemotactic activity on the CXCR3(+) cell line; the CXCR3(-) cell line did not migrate in the presence of supernatants (data not shown). The addition of an anti-CXCL10 neutralizing antibody inhibited chemotactic activities of supernatants. The inhibitory activity shown by the neutralizing antibody was not complete, suggesting that other CXCR3 ligands (CXCL9 and CXCL11) are likely to be present in supernatants.

CXCR3 ligands may be demonstrated in the fluid component of BAL

To assess whether CXCR3 ligands are released in vivo in the lung microenvironment, the fluid component of BAL obtained from 10 HP patients was evaluated for chemotactic activity on CXCR3(+) cell lines (Figure 8). Measurable biological activity was demonstrated in 7 out of 10 patients with HP; this migration was partially abrogated by an anti-CXCL10 neutralizing antibody.