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CD8 alveolitis in sarcoidosis: Incidence, phenotypic characteristics, and clinical features
CD8 Alveolitis in Sarcoidosis: Incidence, Phenotypic Characteristics, and Clinical Features CARLOAGOSTINI,M.D., LIVIOTRENTIN, M.D., RENATO~AMBELLO,M.D., PIETROBULIAN,M.D., FOSCASIVIERO,M.D., MARIAMASCIARELLI,B.S., GIULIANAFESTI,M.D., ANGIOLOCIPRIANI, M.D., GIANPIETROSEMENZATO,M.D., Padua, /ta/y PURPOSE: Although the accumulation of CD4 cells in the lung and other involved tissues is regarded as the distinctive immunologic feature of sarcoidosis, a few sarcoid patients can present with CD8 alveolitis. This study evaluates the incidence as well as the clinical and immunologic features of sarcoidosis presenting with CD8 alveolitis. PATIENTSANDMRTHODS: Atotalof2,214consecutive bronchoalveolar lavage (BAL) specimens obtained from 481 patients with sarcoidosis between January 1985 and December 1991 were retrospectively analyzed. Subjects who entered the study had the following characteristics: (1) lymphocyte alveolitis and (2) lung CD4/CD8 ratio less than 1.0. Only data obtained from patients with a first episode of pulmonary involvement were included in the analysis (394 patients). RESULTS: Fifteen of the 394 patients studied at the time of diagnosis showed CD8 alveolitis as the presenting manifestation; the incidence of this phenomenon was 3.8%. A follow-up study of BAL T-cell subsets demonstrated that patients who showed high-intensity CD8 alveolitis at the onset of the disease maintained the CD8 pattern of alveolitis during relapses. Phenotypic analysis of lung T cells revealed that the accumulation of CD8 lymphocytes was due to the discrete local increase of CD45RO+ “memory” cells equipped with a number of accessory structures, including adhesion molecules and class II major histocompatibility complex-related HLA-DR antigen. CONCLUSIONS: The accumulation of CD8 cells in the sarcoid lung is likely to reflect a homing of memory cells due to the ongoing immunologic
From the Padua University School of Medicine, Department of Clinical Medicine, First Medical Clinic and Clinical Immunology Branch (CA, LT, RZ, PB, MM, GS), Padua, Italy; the Department of Clinical Pathology (FS), Cittadella Hospital, Padua, Italy; and the Department of Pneumophthisiology (GF, AC), Padua Hospital, Padua, Italy. This work was supported by grants from Minister0 dell’Universit8 e della Ricerca Scientifica e Tecnologica (Rome). Requests for reprints should be-addressed to Gianpietro Semenzato. M.D., lstitutodi Medicina Clinica dell’llniversita di Padova, Clinica Medica 1 O, Via Giustiniani 2. 35128 Padova, Italy. Manuscript submitted September 16, 1992, and accepted in revised form February 8, 1993.
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response against the unknown antigen causing the disease. Although CD8 alveolitis can be considered a relatively rare event in sarcoidosis, the possibility that an increase of CD8 cells in the BAL fluid might be sustained ,by an underlying sarcoid inflammatory process should never be dismissed on clinical grounds in patients with interstitial lung disease.
arcoidosis is a granulomatous disorder of unknown etiology characterized by the accumulaS tion of activated CD4+ helper/inducer lymphocytes and monocyte-macrophages in the affected organs [l]. In particular, the accumulation of immunocompetent cells in the lower respiratory tract, which is referred to as alveolitis, represents a crucial step along the mechanisms leading to sarcoid granuloma formation and to the development of endstage fibrosis [2]. Studies performed using cells obtained from bronchoalveolar lavage (BAL) have demonstrated that CD4 alveolitis occurs early in patients with sarcoidosis [3,4]. As a consequence of the persistent stimulation by the unknown antigen causing the disease, pulmonary CD4 cells release cytokines that account for the granuloma formation. Inasmuch as CD4 alveolitis represents the distinctive immunologic feature of sarcoidosis [2,4], the number of BAL T cells, and in particular the number of BAL CD4 cells, has been proposed as a useful parameter to define different phases of this disorder [2,5,6]. In past years, there has been considerable interest in using the phenotypic profile of BAL lymphoid constituents to distinguish diseases characterized by CD4 alveolitis (i.e., sarcoidosis, tuberculosis, berylliosis, and asbestosis) and interstitial lung disorders with CD8 alveolitis. Nevertheless, recent data indicate that not all sarcoid patients show CD4 alveolitis. In fact, rare but well-documented cases demonstrate CD8 alveolitis from the onset of the disease [7-91. The real incidence of this phenomenon and its clinical significance, however, remain to be established because, until now, reports describing sarcoid patients with CD8 alveolitis have been sporadic. This study was designed to assess the frequency and the clinical characteristics of CD8 alveolitis as 95
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the presenting manifestation in sarcoidosis. To this aim, BAL data obtained over the course of 7 years from a large series of patients observed at the onset of the disease were retrospectively analyzed. The expression of accessory molecules by T cells accumulating in the lung of patients with CD8 alveolitis was also evaluated. The demonstration that a compartmentalization of activated lung CD8 cells may occur in a small but significant percentage of sarcoid patients suggests caution in using the BAL findings for diagnostic purposes.
PATIENTS AND METHODS Selection of Patients A total of 2,214 consecutive BAL specimens obtained from 481 patients with sarcoidosis between January 1985 and December 1991 were retrospectively analyzed. In all cases, the diagnosis was made by biopsy specimens obtained either from the lungs, lymph nodes, conjunctiva, or liver and showing noncaseating epithelioid cell granulomas, with no evidence of inorganic material known to cause granulomatous diseases. Only data obtained from patients presenting with a first episode of pulmonary involvement were included in the analysis (394 patients; 160 males and 234 females; mean age 35 f 8 years). Subjects who entered the study had the following characteristics: (1) lymphocytic alveolitis (greater than 30 X lo3 lymphocytes/ml) and (2) lung CD4KD8 ratio less than 1.0 (this cutoff limit corresponding to twice the standard deviation from the mean of the corresponding control values). Twelve BAL specimens obtained from as many patients with sarcoidosis at the onset of the disease were excluded from the study due to inadequate cell recovery or peripheral blood cell contamination of BAL specimens. Each patient evaluated at the onset of the disease underwent history and physical examination, routine blood studies, chest radiography, physiologic lung testing (vital capacity [VC]; total lung capacity; diffusing capacity [DLoo]), 67Ga lung scanning, and BAL. One hundred seventy-four of the 2,214 BAL specimens showed a pulmonary CD4KD8 ratio less than 1.0 (incidence 7.8%). A total of 145 BAL specimens with low lung CD4KD8 ratios were excluded from the statistical analysis because the absolute number of BAL cells was within the normal range (136 patients during steroid therapy or in the inactive phase of the disease following steroid therapy, and 9 patients at the onset of the disease). Another 12 BAL samples with high-intensity alveolitis and low CD4/CD8 ratios were obtained during the follow-up of patients with CD8 alveolitis. Two patients with low CD4/CD8 pulmonary ratios showed an anamnestic exposure to inhaled antigens that
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are known to favor a CD8 immune response in the lung (Micropolyspora faeni), and were also excluded from the study. Two hundred sixty-two of the 394 newly diagnosed sarcoid patients showed more than 30 X lo3 lymphocytes/ml of BAL fluid (cumulative incidence of the lymphocyte alveolitis at the onset of the disease: 66.6%). Fifteen of the 394 patients (11 males and 4 females; mean age 43 f 6 years) showed CD8 alveolitis (incidence of the CD8 alveolitis as the presenting manifestation: 3.8%), 207 patients showed CD4 alveolitis (CD4/CD8 ratio greater than 4, incidence 56.8%), and 40 patients showed a CD4KD8 ratio ranging between 1.0 and 4.0 (incidence 10.1%). One hundred thirty-two of the 394 newly diagnosed sarcoid patients showed an absolute number of BAL lymphocytes within the normal range (incidence 33.4%). The occurrence of concomitant interstitial lung diseases usually associated with an increase of pulmonary CD8+ cells was carefully excluded in patients with CD8 alveolitis enrolled for the study. In particular, none of the patients with CD8 alveolitis showed a history of exposure to hypersensitivity pneumonitis antigens or evidence of serum antibodies against M. faeni and all were HIV-1-seronegative.
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Follow-Up Study Among the 15 newly diagnosed sarcoid patients with high-intensity CD8 alveolitis, 13 cases were evaluated during a follow-up. In these patients, the differential count of BAL cell populations and the analysis of BAL T-cell subsets were performed at 6to 12-month intervals. Aside from the BAL analysis, each evaluation included chest radiography, lung function testing, and routine blood studies. At the time of the first evaluation, according to radiologic staging, five of these patients belonged to stage I and eight patients to stage II. Initial lung physiologic tests in these patients showed the following average values: VC, 72 i 5% predicted; DLoo (single-breath corrected for volume and hemoglobin), 80 f 4% predicted. 67Ga lung scan was positive in all patients with CD8 alveolitis. In the 207 patients who showed CD4 alveolitis at the onset of the disease, the means of VC and DLoo values were 80 f 11% and 85 f 13%, respectively. A total of 83.7% of this group of patients showed positive results on 67Ga lung scanning. At the initial evaluation, patients with CD8 alveolitis did not receive anti-inflammatory and/or immunosuppressive therapy. After the first 6 months, four patients (Patients 1 through 4) were given steroid therapy (prednisone, 1 mg/kg/d). Besides the presence of alveolitis, the reasons for treatment included: (1) extrathoracic sarcoidosis 1993
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(Patient 1) and (2) persistence of the high-intensity alveolitis and progressive impairment on airway function testing (Patients 2,3, and 4). The average period of follow-up for this group of patients was 45 f 19.1 months (range: 18 to 64 months). Nine patients were never given treatment during their follow-up period (Patients 5 through 13). The mean time that untreated patients were in the study group was 21 f 15.4 months (range: 10 to 54 months). Preparation of Cell Suspensions and Evaluation With Monoclonal Antibodies BAL was performed following local anesthesia according to the method previously described [lo]. Briefly, a total of 150 to 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. The percentage of BAL fluid recovered was 58.6 f 5.7% of the injected fluid. Cells recovered from the BAL were washed three times with phosphate-buffered saline, resuspended in RPM1 1640 (Grand Island Biological Co., Paisley, Scotland), and then counted. A differential count of macrophages, lymphocytes, neutrophils, and eosinophils (made from total counts of 300 cells) was accomplished by morphologic criteria in cytocentrifuged smears stained with Wright-Giemsa. BAL cell subpopulations were characterized by monoclonal antibodies (MAbs), most of which belonged to the OK (Ortho Pharmaceuticals, Raritan, NJ), Leu (Becton Dickinson, Sunnyvale, CA), Immunotech SA (Marseille, France), and Dako (Glostrup, Denmark) series, including those belonging to CD3 (Leu-4, OKT3), CD4 (Leu-3, OKT4), and CD8 (Leu-2, OKT8). In four patients, BAL CD8 cells were further characterized employing a panel of MAbs recognizing accessory molecules, including CDlla/LFA-1 (IOT16), CD18//3-chain of LFA-1 (MHM23), CD44 (J-173), CD45RO (UCHLl), CD45RA (Leu-18), CD54/ICAM-1 (84HlO), CD58/LFA-3 (AICD58), and LAM-l (Leu-8). The specificity of all these reagents has already been reported in detail [ll]. The HLA-DR MAb (Becton Dickinson), which recognizes a nonpolymorphic class II major histocompatibility complex (MHC)associated determinant, was also used. The frequency of BAL lymphocytes positive for the above reagents was determined by flow cytometry, as previously described in detail [12]. Briefly, cells were analyzed using a FACScan cytofluorograph (Becton Dickinson) using fluorescein isothiocyanate-conjugated and/or phycoerythrinconjugated MAbs, and data were processed using 468
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the Consort 30 program (Becton Dickinson). Ten thousand cells bearing the typical lymphocyte scatter were scored. Statistical Methods Data of groups were expressed as mean f standard error (SEM), and comparisons between mean values were made using nonparametric tests. Statistical analysis was performed by utilizing the Statistical Analysis System [13]. A p value of less than 0.05 was considered significant.
RESULTS Retrospective analysis of BAL CD4 and CD8 cell subsets showed that the pulmonary CD4/CD8 ratio was less than 1 in 24 of the 394 patients evaluated at the onset of the disease. The cumulative incidence of the low CD4/CD8 ratio was 6.1%. High-intensity lymphocytic alveolitis (greater than 30 X lo3 lymphocytes/ml of BAL fluid) was present in 15 of the 24 patients with a low CD4/CD8 ratio; in the remaining 9 cases, the absolute number of pulmonary CD8 lymphocytes was in the normal range. Thus, in our series of patients, the estimated incidence of high-intensity CD8 alveolitis at the onset of the disease was 3.8%. By contrast, high-intensity CD4 alveolitis was present in 207 of the 394 newly diagnosed sarcoid patients (incidence 56.8%). Cell recovery, differential cell count, and phenotypic characteristics of BAL T-cell subpopulations of the 15 patients with CD8 alveolitis are indicated in Table I. Sarcoid patients with CD8 alveolitis showed a statistically significant increase in cell recovery and in percentage and absolute number of BAL lymphocytes with respect to the control subjects. Phenotypic analysis of BAL T-cell subpopulations revealed a significant increase in the absolute number of CD3+ cells. However, CD8+ lymphocytes were markedly increased both in percentage and in absolute number. Even if the percentage of CD4+ cells was lower in sarcoid patients as compared with that in control subjects, we observed a statistically significant increase in their absolute number. The pulmonary CD4/CD8 ratio was reversed in all patients. It is noteworthy that the number of CD3+ cells was superimposed on the sum of CD4 and CD8 cells, arguing against the hypothesis that a reduced staining of CD4 cells might account for the decrease of the CD4/CD8 ratio. The majority of patients with high-intensity CD8 alveolitis (13 of the 15 cases) entered a follow-up study. During the follow-up period, four patients were given steroid therapy (30.7%) whereas nine patients were never given treatment (69.3%). Figure 1 shows sequential BAL data and lung function testing results obtained during the study in four 95
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patients who were given steroid treatment for the reasons detailed above. Immunosuppressive therapy led to a progressive decline of the absolute number of CD8 cells and to an increase in the CD4KD8 ratio in Patients 1,2, and 3. Regression of alveolitis was associated with an improvement on lung function testing (Figure 1, Patients 1,2, and 3). Patient 4, who showed progressive involvement of the pulmonary parenchyma and never responded to the steroid therapy completely, maintained the CD8 phenotypic pattern of the alveolitis during the follow-up period and, in particular, during the exacerbation phases of the disease. At present, this patient’s chest radiograph shows stage III disease; the patient has recurrent episodes of pleurisy, and typical sarcoid granulomas are still present in lung tissue specimens obtained by transbronchial biopsy. Data related to BAL findings of three of the nine patients who never received steroid treatment during their follow-up period (Patients 56, and 7) are shown in Figure 2. The CD8 alveolitis abated even if after variable periods of time. Recently, Patient 5 had a reexacerbation of the CD8 alveolitis in the absence of symptoms or lung function abnormalities. The frequency of repeated BAL analysis was less in six other untreated patients during their follow-up period. BAL findings of these last patients overlapped those described in Figure 2. In particular, Patients 8 and 9 showed a spontaneous regression of the CD8 alveolitis after a 6-month follow-up period, whereas in Patients 10, 11, 12, and 13, the CD8 alveolitis spontaneously improved after 1 year of follow-up. The CD4/CD8 ratio remained persistently low (less than 1) in four patients (Patients 8, 9, 10, and 13 after 12, 15, 18, and 15 months of follow-up, respectively), whereas in Patients 11 and 12, the BAL CD4KD8 ratio returned to the normal range after 15 and 18 months of follow-up, respectively. BAL findings and the disease course of patients with CD4 and CD8 alveolitis were also compared. Patients with CD8 alveolitis showed an increased number of CD3 lymphocytes with respect to patients with CD4 alveolitis (Table I). According to the group definition, the percentage and absolute number of CD4 cells and the CD4/CD8 ratio were higher in patients with CD4 alveolitis, whereas CD8 cells predominated in patients with CD8 alveolitis. In regard to the clinical course of the disease, 89 of the 207 patients with high-intensity CD4 alveolitis received steroid therapy during their follow-up period. The incidence of steroid therapy was 42.9% in patients with CD4 alveolitis, in comparison to 30.7% in patients with CD8 alveolitis. Therapy led to a progressive improvement in the parameters under study in 80 patients with CD4 alveolitis (inciNovember
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Figure 1. Data of BAL findings and lung function testing from four patients with high-intensity CD8 cell alveolitis who were given steroid treatment during their follow-up. At entry, each patient was followed for 6 months without therapy. Individual data of CD4/CD8 ratio and absolute number of CD8+ and CD4+ cells are represented by different symbols (0, 0, and n , respectively). Vital capacity and DLco are represented by different bars (open bars and solid bars, respectively); data of lung function tests are expressed as percent of predicted values. The shaded area at the top of each panel represents the time period during which patients were treated with steroid therapy.
Figure 2. Data of BAL findings and lung function testing from three representative patients with highintensity CD8 cell alveolitis who never received steroid treatment during their follow-up. Individual data of CD4/CD8 ratio and absolute number of CD8+ and CD4+ cells are represented by different symbols (0, 0, and n , respectively). Vital capacity and DLco are represented by different bars (open bars and solid bars, respectively); data of lung function tests are expressed as percent of predicted values.
dence 89.8%) and 3 of 4 patients with CD8 alveolitis. Nine patients with CD4 alveolitis (10.2%) and one of the four patients with CD8 alveolitis never completely responded to steroids. A spontaneous regression of the alveolitis was observed in 118 of the 207 patients with high-intensity CD4 alveolitis (incidence 57.0%) and in 69.3% of patients with CD8 alveolitis. Taken together, these data suggest that 470
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in both groups of patients, there are patients who improve, either spontaneously or with therapy, or who progressively worsen despite immunosuppressive therapy. This finding indicates that the phenotypic pattern of both forms of alveolitis is likely to be not relevant to the outcome of the sarcoid process. BAL CD8 cells from four patients were further 95
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(Figure 1, Patient 4) represents an additional indirect confirmation of the causal relationship between the local expansion of CD8 cells and the disease. An obvious implication of these findings is that the presence of CD8 alveolitis does not reliably help to discriminate between sarcoidosis and conditions usually associated with an increase of CD8 cells in the BAL. On the other hand, CD4 alveolitis is more discriminatory, although not absolutely, because, aside from sarcoidosis, other interstitial lung diseases are associated with an increase in pulmonary CD4 cells [3,14]. In other words, immunologic analysis of BAL cell constituents may contribute to a presumptive diagnosis by enhancing or decreasing the probability of sarcoidosis in the context of interstitial lung disease, but definitive conclusions cannot be drawn. As previously hypothesized for CD4 cells accumulating in the lung of patients characterized by CD4 alveolitis [2-41, CD8 lymphocytes are likely to participate in the ongoing immune response occurring in this disease. These cells could represent a long-term activated and homing population activeCOMMENTS ly proliferating in the lower respiratory tract as the result of persistent antigenic exposure to the etioIn this study, we demonstrated that CD8 alveolitis may occur in patients with sarcoidosis both at logic agent causing sarcoidosis. As a confirmation of the onset and during the relapsing phases of the this interpretation, we demonstrated that they are disease. The overall incidence of this phenomenon equipped with molecules that are usually expressed as the presenting manifestation was 3.8%; patients on activated T cells, including CD45R0, class II showing high-intensity CD8 alveolitis at the onset MHC determinants, and a number of adhesion molecules. It is worth noting that strong similarities in of the disease maintain the CD8 pattern of alveolitis during the active phases of the disease. Phenotypic the surface expression of accessory molecules have study of lymphocytes isolated from the BAL of pa- been observed between T cells recovered from patients with CD4 [15-181 and CD8 alveolitis (Results tients with CD8 alveolitis revealed that accumulating cells were CD8tKD45ROt memory T lymphosection). This finding supports the concept that faccytes bearing class II MHC determinants and a tors that usually induce the proliferation of CD4 number of adhesion molecules, including CDlla, cells may, in selected patients, stimulate an imCD18, CD44, CD54, and CD58. The observation mune response ultimately leading to CD8 alveolitis. that patients with sarcoidosis may present with Further, in vitro functional studies are needed to properties and the CD8 alveolitis has a relevant impact not only on the compare the immunoregulatory management of patients with interstitial lung dis- molecular characteristics of pulmonary CD8 cells of ease but also in terms of the actual concepts con- patients with CD8 and CD4 alveolitis. These studcerning the pathogenesis of the sarcoid inflammaies could provide insights into the events that regutory process. late the CD8 immune response in the lung of paDifferent interstitial lung disorders are associ- tients with sarcoidosis. ated with an increase of cytotoxic/suppressor CDs+ The mechanisms that favor the occurrence of cells in the BAL, including hypersensitivity pneuCD8 alveolitis rather than the more common CD4 monitis, acquired immunodeficiency syndrome, sil- alveolitis in sarcoid lung are presently unknown. Since the absolute number of pulmonary CD4 cells icosis, hiitiocytosis X, amiodarone pneumonitis, blastomycosis, and pulmonary complications dur- was also increased in our series of patients (Table I) with respect to that in healthy control subjects, a ing collagen vascular disorders, graft-versus-host disease, and HTLV-I-related myelopathy. All the possible cause could be that the in situ proliferation above conditions were ruled out in our cases, indiof CD8t cells in this selected group of cases reflects cating that the accumulation of CD8 cells was relatattempts of the host immune system to down-moded to the sarcoid inflammatory process. The evi- ulate the exaggerated function of pulmonary CD4t dence that the absolute number of CD8 cells lymphocytes. The evidence that the functional increased during the relapse of the sarcoid process properties of pulmonary CD8 suppressor cells are
characterized by a panel of MAbs recognizing accessory molecules. Double-color flow-cytometry studies performed on CD8t lymphocytes revealed that CD8 alveolitis was due to the accumulation of T cells bearing CD8, CD45RO antigens (greater than 95%) and lacking CD45RA and LAM-l determinants (less than 3%), thus indicating that proliferating CD8 cells were “memory” cells. CD8 memory cells also expressed a number of adhesion molecules (greater than 95%), including CDlla, CD18, CD44, CD54, CD58, and class II MHC-related HLA-DR determinants (values ranging between 18% and 41%, mean 32.5 f 7%). For a control group, we evaluated the expression of accessory molecules by T cells recovered from five patients with CD4 alveolitis, all of them showing high-intensity lymphocyte alveolitis (121,000 f 34,000 lymphocytes/ml) and a high CD4/CD8 ratio (9.7 f 1.2). There were no significant differences in the expression of CD45RA, CD45R0, HLA-DR, and adhesion molecules between T cells isolated from patients with CD4 and CD8 alveolitis (p = NS for all compared values).
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maintained in sarcoidosis [19] suggests that the accumulation of activated CD4 cells is not secondary to a generalized defect of CD8 suppressor cells. Alternatively, it is possible that CD4 effecters might be constitutionally defective in these patients [20,21]. The normal expression of the T4A-F antigens by CD4 cells isolated from the BAL and peripheral blood of two patients with CD8 alveolitis (data not shown) argues against this possibility. In conclusion, results presented here are consistent with the idea that a compartmentalization of activated CD8 cells may occasionally occur in sarcoidosis. This accumulation of CD8 cells is detectable from the early phases of the disease and maintains its peculiar phenotypic pattern during the subsequent phases of the disease. Although the incidence of the phenomenon is limited, the possibility that CD8 alveolitis might be sustained by a sarcoid inflammatory process should always be taken into account by clinicians in the management of patients with interstitial lung disorders.
We wish to thank Dr. Antonella Milani for her expert technical
assistance and Mr.
Martin Donach for his help in the preparation of the manuscript. Dr. R. Zambello and Dr. L. Trentin are the recipients of a fellowship of the Minister0 della Sanita, Superiore
di SanitB (Rome).
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lymphocytaire a lymphocytes T CD8+ au tours de la sarcoidose. tions. Presse Med 1991; 20: 593-5. 9. Oda H, Matsutake T, Sakito 0, et al. A case of lung sarcoidosis clinically difficult to distinguish from hypersensitivity Kyobu Shikkan Gakkai Zasshi 1991; 29: 501-6. 10. Agostini C. Garbisa S, Trentin L, efa/. Pulmonary
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4. Thomas PD, Hunninghake GW. Current concepts coidosis. Am Rev Respir Dis 1987; 135: 74760.
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lack of T4(+) inducer/helper T cell subsets in peripheral blood lymphocytes. J lmmunol 1984; 132: 1071-3. 21.Aozasa M, Amino N, lwatani Y. et al. Familial OKT4 epitope deficiency: studies on antigen density pathol 1985; 37: 48-55.
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From the Padua University School of Medicine, Department of Clinical Medicine, First Medical Clinic and Clinical Immunology Branch (CA, LT, RZ, PB, MM, GS), Padua, Italy; the Department of Clinical Pathology (FS), Cittadella Hospital, Padua, Italy; and the Department of Pneumophthisiology (GF, AC), Padua Hospital, Padua, Italy. This work was supported by grants from Minister0 dell’Universit8 e della Ricerca Scientifica e Tecnologica (Rome). Requests for reprints should be-addressed to Gianpietro Semenzato. M.D., lstitutodi Medicina Clinica dell’llniversita di Padova, Clinica Medica 1 O, Via Giustiniani 2. 35128 Padova, Italy. Manuscript submitted September 16, 1992, and accepted in revised form February 8, 1993.
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response against the unknown antigen causing the disease. Although CD8 alveolitis can be considered a relatively rare event in sarcoidosis, the possibility that an increase of CD8 cells in the BAL fluid might be sustained ,by an underlying sarcoid inflammatory process should never be dismissed on clinical grounds in patients with interstitial lung disease.
arcoidosis is a granulomatous disorder of unknown etiology characterized by the accumulaS tion of activated CD4+ helper/inducer lymphocytes and monocyte-macrophages in the affected organs [l]. In particular, the accumulation of immunocompetent cells in the lower respiratory tract, which is referred to as alveolitis, represents a crucial step along the mechanisms leading to sarcoid granuloma formation and to the development of endstage fibrosis [2]. Studies performed using cells obtained from bronchoalveolar lavage (BAL) have demonstrated that CD4 alveolitis occurs early in patients with sarcoidosis [3,4]. As a consequence of the persistent stimulation by the unknown antigen causing the disease, pulmonary CD4 cells release cytokines that account for the granuloma formation. Inasmuch as CD4 alveolitis represents the distinctive immunologic feature of sarcoidosis [2,4], the number of BAL T cells, and in particular the number of BAL CD4 cells, has been proposed as a useful parameter to define different phases of this disorder [2,5,6]. In past years, there has been considerable interest in using the phenotypic profile of BAL lymphoid constituents to distinguish diseases characterized by CD4 alveolitis (i.e., sarcoidosis, tuberculosis, berylliosis, and asbestosis) and interstitial lung disorders with CD8 alveolitis. Nevertheless, recent data indicate that not all sarcoid patients show CD4 alveolitis. In fact, rare but well-documented cases demonstrate CD8 alveolitis from the onset of the disease [7-91. The real incidence of this phenomenon and its clinical significance, however, remain to be established because, until now, reports describing sarcoid patients with CD8 alveolitis have been sporadic. This study was designed to assess the frequency and the clinical characteristics of CD8 alveolitis as 95
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the presenting manifestation in sarcoidosis. To this aim, BAL data obtained over the course of 7 years from a large series of patients observed at the onset of the disease were retrospectively analyzed. The expression of accessory molecules by T cells accumulating in the lung of patients with CD8 alveolitis was also evaluated. The demonstration that a compartmentalization of activated lung CD8 cells may occur in a small but significant percentage of sarcoid patients suggests caution in using the BAL findings for diagnostic purposes.
PATIENTS AND METHODS Selection of Patients A total of 2,214 consecutive BAL specimens obtained from 481 patients with sarcoidosis between January 1985 and December 1991 were retrospectively analyzed. In all cases, the diagnosis was made by biopsy specimens obtained either from the lungs, lymph nodes, conjunctiva, or liver and showing noncaseating epithelioid cell granulomas, with no evidence of inorganic material known to cause granulomatous diseases. Only data obtained from patients presenting with a first episode of pulmonary involvement were included in the analysis (394 patients; 160 males and 234 females; mean age 35 f 8 years). Subjects who entered the study had the following characteristics: (1) lymphocytic alveolitis (greater than 30 X lo3 lymphocytes/ml) and (2) lung CD4KD8 ratio less than 1.0 (this cutoff limit corresponding to twice the standard deviation from the mean of the corresponding control values). Twelve BAL specimens obtained from as many patients with sarcoidosis at the onset of the disease were excluded from the study due to inadequate cell recovery or peripheral blood cell contamination of BAL specimens. Each patient evaluated at the onset of the disease underwent history and physical examination, routine blood studies, chest radiography, physiologic lung testing (vital capacity [VC]; total lung capacity; diffusing capacity [DLoo]), 67Ga lung scanning, and BAL. One hundred seventy-four of the 2,214 BAL specimens showed a pulmonary CD4KD8 ratio less than 1.0 (incidence 7.8%). A total of 145 BAL specimens with low lung CD4KD8 ratios were excluded from the statistical analysis because the absolute number of BAL cells was within the normal range (136 patients during steroid therapy or in the inactive phase of the disease following steroid therapy, and 9 patients at the onset of the disease). Another 12 BAL samples with high-intensity alveolitis and low CD4/CD8 ratios were obtained during the follow-up of patients with CD8 alveolitis. Two patients with low CD4/CD8 pulmonary ratios showed an anamnestic exposure to inhaled antigens that
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are known to favor a CD8 immune response in the lung (Micropolyspora faeni), and were also excluded from the study. Two hundred sixty-two of the 394 newly diagnosed sarcoid patients showed more than 30 X lo3 lymphocytes/ml of BAL fluid (cumulative incidence of the lymphocyte alveolitis at the onset of the disease: 66.6%). Fifteen of the 394 patients (11 males and 4 females; mean age 43 f 6 years) showed CD8 alveolitis (incidence of the CD8 alveolitis as the presenting manifestation: 3.8%), 207 patients showed CD4 alveolitis (CD4/CD8 ratio greater than 4, incidence 56.8%), and 40 patients showed a CD4KD8 ratio ranging between 1.0 and 4.0 (incidence 10.1%). One hundred thirty-two of the 394 newly diagnosed sarcoid patients showed an absolute number of BAL lymphocytes within the normal range (incidence 33.4%). The occurrence of concomitant interstitial lung diseases usually associated with an increase of pulmonary CD8+ cells was carefully excluded in patients with CD8 alveolitis enrolled for the study. In particular, none of the patients with CD8 alveolitis showed a history of exposure to hypersensitivity pneumonitis antigens or evidence of serum antibodies against M. faeni and all were HIV-1-seronegative.
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Follow-Up Study Among the 15 newly diagnosed sarcoid patients with high-intensity CD8 alveolitis, 13 cases were evaluated during a follow-up. In these patients, the differential count of BAL cell populations and the analysis of BAL T-cell subsets were performed at 6to 12-month intervals. Aside from the BAL analysis, each evaluation included chest radiography, lung function testing, and routine blood studies. At the time of the first evaluation, according to radiologic staging, five of these patients belonged to stage I and eight patients to stage II. Initial lung physiologic tests in these patients showed the following average values: VC, 72 i 5% predicted; DLoo (single-breath corrected for volume and hemoglobin), 80 f 4% predicted. 67Ga lung scan was positive in all patients with CD8 alveolitis. In the 207 patients who showed CD4 alveolitis at the onset of the disease, the means of VC and DLoo values were 80 f 11% and 85 f 13%, respectively. A total of 83.7% of this group of patients showed positive results on 67Ga lung scanning. At the initial evaluation, patients with CD8 alveolitis did not receive anti-inflammatory and/or immunosuppressive therapy. After the first 6 months, four patients (Patients 1 through 4) were given steroid therapy (prednisone, 1 mg/kg/d). Besides the presence of alveolitis, the reasons for treatment included: (1) extrathoracic sarcoidosis 1993
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(Patient 1) and (2) persistence of the high-intensity alveolitis and progressive impairment on airway function testing (Patients 2,3, and 4). The average period of follow-up for this group of patients was 45 f 19.1 months (range: 18 to 64 months). Nine patients were never given treatment during their follow-up period (Patients 5 through 13). The mean time that untreated patients were in the study group was 21 f 15.4 months (range: 10 to 54 months). Preparation of Cell Suspensions and Evaluation With Monoclonal Antibodies BAL was performed following local anesthesia according to the method previously described [lo]. Briefly, a total of 150 to 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. The percentage of BAL fluid recovered was 58.6 f 5.7% of the injected fluid. Cells recovered from the BAL were washed three times with phosphate-buffered saline, resuspended in RPM1 1640 (Grand Island Biological Co., Paisley, Scotland), and then counted. A differential count of macrophages, lymphocytes, neutrophils, and eosinophils (made from total counts of 300 cells) was accomplished by morphologic criteria in cytocentrifuged smears stained with Wright-Giemsa. BAL cell subpopulations were characterized by monoclonal antibodies (MAbs), most of which belonged to the OK (Ortho Pharmaceuticals, Raritan, NJ), Leu (Becton Dickinson, Sunnyvale, CA), Immunotech SA (Marseille, France), and Dako (Glostrup, Denmark) series, including those belonging to CD3 (Leu-4, OKT3), CD4 (Leu-3, OKT4), and CD8 (Leu-2, OKT8). In four patients, BAL CD8 cells were further characterized employing a panel of MAbs recognizing accessory molecules, including CDlla/LFA-1 (IOT16), CD18//3-chain of LFA-1 (MHM23), CD44 (J-173), CD45RO (UCHLl), CD45RA (Leu-18), CD54/ICAM-1 (84HlO), CD58/LFA-3 (AICD58), and LAM-l (Leu-8). The specificity of all these reagents has already been reported in detail [ll]. The HLA-DR MAb (Becton Dickinson), which recognizes a nonpolymorphic class II major histocompatibility complex (MHC)associated determinant, was also used. The frequency of BAL lymphocytes positive for the above reagents was determined by flow cytometry, as previously described in detail [12]. Briefly, cells were analyzed using a FACScan cytofluorograph (Becton Dickinson) using fluorescein isothiocyanate-conjugated and/or phycoerythrinconjugated MAbs, and data were processed using 468
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the Consort 30 program (Becton Dickinson). Ten thousand cells bearing the typical lymphocyte scatter were scored. Statistical Methods Data of groups were expressed as mean f standard error (SEM), and comparisons between mean values were made using nonparametric tests. Statistical analysis was performed by utilizing the Statistical Analysis System [13]. A p value of less than 0.05 was considered significant.
RESULTS Retrospective analysis of BAL CD4 and CD8 cell subsets showed that the pulmonary CD4/CD8 ratio was less than 1 in 24 of the 394 patients evaluated at the onset of the disease. The cumulative incidence of the low CD4/CD8 ratio was 6.1%. High-intensity lymphocytic alveolitis (greater than 30 X lo3 lymphocytes/ml of BAL fluid) was present in 15 of the 24 patients with a low CD4/CD8 ratio; in the remaining 9 cases, the absolute number of pulmonary CD8 lymphocytes was in the normal range. Thus, in our series of patients, the estimated incidence of high-intensity CD8 alveolitis at the onset of the disease was 3.8%. By contrast, high-intensity CD4 alveolitis was present in 207 of the 394 newly diagnosed sarcoid patients (incidence 56.8%). Cell recovery, differential cell count, and phenotypic characteristics of BAL T-cell subpopulations of the 15 patients with CD8 alveolitis are indicated in Table I. Sarcoid patients with CD8 alveolitis showed a statistically significant increase in cell recovery and in percentage and absolute number of BAL lymphocytes with respect to the control subjects. Phenotypic analysis of BAL T-cell subpopulations revealed a significant increase in the absolute number of CD3+ cells. However, CD8+ lymphocytes were markedly increased both in percentage and in absolute number. Even if the percentage of CD4+ cells was lower in sarcoid patients as compared with that in control subjects, we observed a statistically significant increase in their absolute number. The pulmonary CD4/CD8 ratio was reversed in all patients. It is noteworthy that the number of CD3+ cells was superimposed on the sum of CD4 and CD8 cells, arguing against the hypothesis that a reduced staining of CD4 cells might account for the decrease of the CD4/CD8 ratio. The majority of patients with high-intensity CD8 alveolitis (13 of the 15 cases) entered a follow-up study. During the follow-up period, four patients were given steroid therapy (30.7%) whereas nine patients were never given treatment (69.3%). Figure 1 shows sequential BAL data and lung function testing results obtained during the study in four 95
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patients who were given steroid treatment for the reasons detailed above. Immunosuppressive therapy led to a progressive decline of the absolute number of CD8 cells and to an increase in the CD4KD8 ratio in Patients 1,2, and 3. Regression of alveolitis was associated with an improvement on lung function testing (Figure 1, Patients 1,2, and 3). Patient 4, who showed progressive involvement of the pulmonary parenchyma and never responded to the steroid therapy completely, maintained the CD8 phenotypic pattern of the alveolitis during the follow-up period and, in particular, during the exacerbation phases of the disease. At present, this patient’s chest radiograph shows stage III disease; the patient has recurrent episodes of pleurisy, and typical sarcoid granulomas are still present in lung tissue specimens obtained by transbronchial biopsy. Data related to BAL findings of three of the nine patients who never received steroid treatment during their follow-up period (Patients 56, and 7) are shown in Figure 2. The CD8 alveolitis abated even if after variable periods of time. Recently, Patient 5 had a reexacerbation of the CD8 alveolitis in the absence of symptoms or lung function abnormalities. The frequency of repeated BAL analysis was less in six other untreated patients during their follow-up period. BAL findings of these last patients overlapped those described in Figure 2. In particular, Patients 8 and 9 showed a spontaneous regression of the CD8 alveolitis after a 6-month follow-up period, whereas in Patients 10, 11, 12, and 13, the CD8 alveolitis spontaneously improved after 1 year of follow-up. The CD4/CD8 ratio remained persistently low (less than 1) in four patients (Patients 8, 9, 10, and 13 after 12, 15, 18, and 15 months of follow-up, respectively), whereas in Patients 11 and 12, the BAL CD4KD8 ratio returned to the normal range after 15 and 18 months of follow-up, respectively. BAL findings and the disease course of patients with CD4 and CD8 alveolitis were also compared. Patients with CD8 alveolitis showed an increased number of CD3 lymphocytes with respect to patients with CD4 alveolitis (Table I). According to the group definition, the percentage and absolute number of CD4 cells and the CD4/CD8 ratio were higher in patients with CD4 alveolitis, whereas CD8 cells predominated in patients with CD8 alveolitis. In regard to the clinical course of the disease, 89 of the 207 patients with high-intensity CD4 alveolitis received steroid therapy during their follow-up period. The incidence of steroid therapy was 42.9% in patients with CD4 alveolitis, in comparison to 30.7% in patients with CD8 alveolitis. Therapy led to a progressive improvement in the parameters under study in 80 patients with CD4 alveolitis (inciNovember
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Figure 1. Data of BAL findings and lung function testing from four patients with high-intensity CD8 cell alveolitis who were given steroid treatment during their follow-up. At entry, each patient was followed for 6 months without therapy. Individual data of CD4/CD8 ratio and absolute number of CD8+ and CD4+ cells are represented by different symbols (0, 0, and n , respectively). Vital capacity and DLco are represented by different bars (open bars and solid bars, respectively); data of lung function tests are expressed as percent of predicted values. The shaded area at the top of each panel represents the time period during which patients were treated with steroid therapy.
Figure 2. Data of BAL findings and lung function testing from three representative patients with highintensity CD8 cell alveolitis who never received steroid treatment during their follow-up. Individual data of CD4/CD8 ratio and absolute number of CD8+ and CD4+ cells are represented by different symbols (0, 0, and n , respectively). Vital capacity and DLco are represented by different bars (open bars and solid bars, respectively); data of lung function tests are expressed as percent of predicted values.
dence 89.8%) and 3 of 4 patients with CD8 alveolitis. Nine patients with CD4 alveolitis (10.2%) and one of the four patients with CD8 alveolitis never completely responded to steroids. A spontaneous regression of the alveolitis was observed in 118 of the 207 patients with high-intensity CD4 alveolitis (incidence 57.0%) and in 69.3% of patients with CD8 alveolitis. Taken together, these data suggest that 470
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in both groups of patients, there are patients who improve, either spontaneously or with therapy, or who progressively worsen despite immunosuppressive therapy. This finding indicates that the phenotypic pattern of both forms of alveolitis is likely to be not relevant to the outcome of the sarcoid process. BAL CD8 cells from four patients were further 95
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(Figure 1, Patient 4) represents an additional indirect confirmation of the causal relationship between the local expansion of CD8 cells and the disease. An obvious implication of these findings is that the presence of CD8 alveolitis does not reliably help to discriminate between sarcoidosis and conditions usually associated with an increase of CD8 cells in the BAL. On the other hand, CD4 alveolitis is more discriminatory, although not absolutely, because, aside from sarcoidosis, other interstitial lung diseases are associated with an increase in pulmonary CD4 cells [3,14]. In other words, immunologic analysis of BAL cell constituents may contribute to a presumptive diagnosis by enhancing or decreasing the probability of sarcoidosis in the context of interstitial lung disease, but definitive conclusions cannot be drawn. As previously hypothesized for CD4 cells accumulating in the lung of patients characterized by CD4 alveolitis [2-41, CD8 lymphocytes are likely to participate in the ongoing immune response occurring in this disease. These cells could represent a long-term activated and homing population activeCOMMENTS ly proliferating in the lower respiratory tract as the result of persistent antigenic exposure to the etioIn this study, we demonstrated that CD8 alveolitis may occur in patients with sarcoidosis both at logic agent causing sarcoidosis. As a confirmation of the onset and during the relapsing phases of the this interpretation, we demonstrated that they are disease. The overall incidence of this phenomenon equipped with molecules that are usually expressed as the presenting manifestation was 3.8%; patients on activated T cells, including CD45R0, class II showing high-intensity CD8 alveolitis at the onset MHC determinants, and a number of adhesion molecules. It is worth noting that strong similarities in of the disease maintain the CD8 pattern of alveolitis during the active phases of the disease. Phenotypic the surface expression of accessory molecules have study of lymphocytes isolated from the BAL of pa- been observed between T cells recovered from patients with CD4 [15-181 and CD8 alveolitis (Results tients with CD8 alveolitis revealed that accumulating cells were CD8tKD45ROt memory T lymphosection). This finding supports the concept that faccytes bearing class II MHC determinants and a tors that usually induce the proliferation of CD4 number of adhesion molecules, including CDlla, cells may, in selected patients, stimulate an imCD18, CD44, CD54, and CD58. The observation mune response ultimately leading to CD8 alveolitis. that patients with sarcoidosis may present with Further, in vitro functional studies are needed to properties and the CD8 alveolitis has a relevant impact not only on the compare the immunoregulatory management of patients with interstitial lung dis- molecular characteristics of pulmonary CD8 cells of ease but also in terms of the actual concepts con- patients with CD8 and CD4 alveolitis. These studcerning the pathogenesis of the sarcoid inflammaies could provide insights into the events that regutory process. late the CD8 immune response in the lung of paDifferent interstitial lung disorders are associ- tients with sarcoidosis. ated with an increase of cytotoxic/suppressor CDs+ The mechanisms that favor the occurrence of cells in the BAL, including hypersensitivity pneuCD8 alveolitis rather than the more common CD4 monitis, acquired immunodeficiency syndrome, sil- alveolitis in sarcoid lung are presently unknown. Since the absolute number of pulmonary CD4 cells icosis, hiitiocytosis X, amiodarone pneumonitis, blastomycosis, and pulmonary complications dur- was also increased in our series of patients (Table I) with respect to that in healthy control subjects, a ing collagen vascular disorders, graft-versus-host disease, and HTLV-I-related myelopathy. All the possible cause could be that the in situ proliferation above conditions were ruled out in our cases, indiof CD8t cells in this selected group of cases reflects cating that the accumulation of CD8 cells was relatattempts of the host immune system to down-moded to the sarcoid inflammatory process. The evi- ulate the exaggerated function of pulmonary CD4t dence that the absolute number of CD8 cells lymphocytes. The evidence that the functional increased during the relapse of the sarcoid process properties of pulmonary CD8 suppressor cells are
characterized by a panel of MAbs recognizing accessory molecules. Double-color flow-cytometry studies performed on CD8t lymphocytes revealed that CD8 alveolitis was due to the accumulation of T cells bearing CD8, CD45RO antigens (greater than 95%) and lacking CD45RA and LAM-l determinants (less than 3%), thus indicating that proliferating CD8 cells were “memory” cells. CD8 memory cells also expressed a number of adhesion molecules (greater than 95%), including CDlla, CD18, CD44, CD54, CD58, and class II MHC-related HLA-DR determinants (values ranging between 18% and 41%, mean 32.5 f 7%). For a control group, we evaluated the expression of accessory molecules by T cells recovered from five patients with CD4 alveolitis, all of them showing high-intensity lymphocyte alveolitis (121,000 f 34,000 lymphocytes/ml) and a high CD4/CD8 ratio (9.7 f 1.2). There were no significant differences in the expression of CD45RA, CD45R0, HLA-DR, and adhesion molecules between T cells isolated from patients with CD4 and CD8 alveolitis (p = NS for all compared values).
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maintained in sarcoidosis [19] suggests that the accumulation of activated CD4 cells is not secondary to a generalized defect of CD8 suppressor cells. Alternatively, it is possible that CD4 effecters might be constitutionally defective in these patients [20,21]. The normal expression of the T4A-F antigens by CD4 cells isolated from the BAL and peripheral blood of two patients with CD8 alveolitis (data not shown) argues against this possibility. In conclusion, results presented here are consistent with the idea that a compartmentalization of activated CD8 cells may occasionally occur in sarcoidosis. This accumulation of CD8 cells is detectable from the early phases of the disease and maintains its peculiar phenotypic pattern during the subsequent phases of the disease. Although the incidence of the phenomenon is limited, the possibility that CD8 alveolitis might be sustained by a sarcoid inflammatory process should always be taken into account by clinicians in the management of patients with interstitial lung disorders.
We wish to thank Dr. Antonella Milani for her expert technical
assistance and Mr.
Martin Donach for his help in the preparation of the manuscript. Dr. R. Zambello and Dr. L. Trentin are the recipients of a fellowship of the Minister0 della Sanita, Superiore
di SanitB (Rome).
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