INTRODUCTION

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Aminopeptidase N is a membrane-bound metalloprotease, and it has been shown to be identical to CD13 (1), a 150-kD cell surface glycoprotein, which was originally used as a marker for subpopulations of hematopoietic cells (2). CD13/aminopeptidase N is widely distributed in a variety of mammalian cells such as monocytes/macrophages, fibroblasts, neutrophils, endothelial cells, and epithelial cells (3, 4). CD13/aminopeptidase N is a receptor for human coronavirus (5), and it has an important role in HIV entry (6). This peptidase was shown to be involved in the degradation of extracellular matrix in tumor invasions (7) and the processing of peptide for presentation by antigen-presenting cells (8). Although little information is available concerning the regulation of CD13/aminopeptidase N expression, recent reports have shown that lymphokines such as IFN- and IL-4 upregulate the expression of CD13/ aminopeptidase N in all cell types (3, 9).

Sarcoidosis is a chronic inflammatory disease in which there is a systemic granulomatous process, and the lungs are most commonly involved in this disorder. Lung parenchymal lesions in patients with sarcoidosis are characterized by alveolitis associated with mononuclear cell infiltration and noncaseating granuloma formation and, in a few patients, with pulmonary fibrosis (10). Although the triggering agent of sarcoidosis is still uncertain, lymphokines released by activated CD4+ T lymphocytes play a pivotal role in the inflammatory process of this disorder. CD4+ T lymphocytes can be subdivided into two distinct populations, Th1 and Th2, defined by the spectrum of cytokines produced by these cells (11). Th1 cells generate IL-2, IFN-, and tumor necrosis factor-, and they promote cell-mediated immunity, whereas Th2 cells generate IL-4, IL-5, IL-6, and IL-10, and play a role in humoral immunity and allergic diseases (12). There is evidence that a balance of different cytokine producers is crucial for an effective immune response and the outcome of infectious and autoimmune diseases (13, 14). In sarcoidosis, a Th-1 response was shown to be predominant because lung lymphocytes from patients with sarcoidosis produced excessive amounts of IFN- and IL-2 (15). The Th-1 cytokines could be involved in the activation of macrophages at the site of inflammation and granuloma formation (15). In this study, we found increased activity of aminopeptidase in bronchoalveolar lavage fluid (BALF) and increased expression of CD13/aminopeptidase N protein in alveolar macrophages (AM) from patients with sarcoidosis that correlated with the activity of alveolitis in this disorder. Moreover, we demonstrated that CD13/aminopeptidase N may have a significant role in the pathogenesis of sarcoidosis as a T-cell chemoattractant.

	METHODS

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Reagents

CD13/aminopeptidase N (E.C.3.4.11.2; specific activity, 34 U/mg protein) and bestatin were purchased from Sigma Chemical Co. (St. Louis, MO). L-leucine-7-amino-4-methyl-coumarine (L-leucine-AMC) was purchased from Peptide Institute (Osaka, Japan). Monoclonal antibody against human CD13 was purchased from Becton Dickinson (San Jose, CA). Macrophage inflammatory protein (MIP)-1  and  were purchased from Pepro Tech (Rocky Hill, NJ).

Study Population

Clinical data of patients participating in this study are shown in Table 1.

Patients with sarcoidosis. Studies were made on 30 patients with sarcoidosis. The diagnosis of sarcoidosis was based on previously defined clinical and histologic criteria (10). None had received corticosteroid therapy before bronchoalveolar lavage (BAL) was performed.

Control subjects. The control population consisted of 13 normal, nonsmoking, male volunteers (NV) and 10 control patients (CP) (six men and four women). None of the NV showed any abnormalities on physical examination, chest radiography, or lung function tests. All the CP were free of interstitial lung disease; eight had localized lung cancer and two had no detectable lesion in the lungs, although they complained of hemosputum.

Bronchoalveolar Lavage

BAL was performed as described previously (16). Briefly, a flexible fiberoptic bronchoscope (Model 1T20; Olympus Co., Tokyo, Japan) was wedged into a segmental or subsegmental bronchus of the middle lobe or lingula, and lavage was performed with a total volume of 150 ml of sterile 0.9% saline in three 50-ml aliquots. The lavage fluid was gently aspirated by syringe after deep inspiration. In CP, BAL was performed on the opposite side from lung lesions. The fluid recovered was passed through a sterile gauze and centrifuged at 250 × g for 10 min at 4° C to precipitate cells, and the supernatant was stored at 70° C until examination. The precipitated cells were analyzed. The total number of cells, suspended in an appropriate volume of saline, was counted in a hemocytometer. Differential counts on 500 cells were carried out on smears of sedimented cells stained with May-Giemsa stain, and the percentages of AM, lymphocytes, neutrophils, and eosinophils were calculated. The cells were examined for the presence of lymphocyte markers CD4 and CD8, using murine monoclonal antibodies as described previously (17).

Determination of Protease Activity of Aminopeptidase

Protease activity of aminopeptidase was assayed fluorometrically as described previously (18). In brief, 80 µl of 0.1 M TRIS-HCl buffer (pH 8.0), 100 µl of 100 µM L-leucine-AMC diluted with the buffer, and 20 µl samples were added into the wells of 96-multiwell plates (C8 White Maxisorp; Nunc, Roskilde, Denmark). The standard contained various amounts of AMC. The plates were incubated at 37° C for 1 h. The fluorescence intensity was measured in MTP-32 (Corona Electric Co., Ibaragi, Japan) with 365 nm for excitation and 450 nm for emission wavelengths. The aminopeptidase activity is expressed in nanomoles of substrate cleaved per hour.

Western Blotting

BAL cells dissolved in RPMI-1640 (Nissui Pharmaceutical Co., Tokyo, Japan) were incubated in tissue culture dishes (Falcon 3003; Falcon Plastics, Oxnard, CA) at 37° C in a humidified atmosphere of 5% CO₂ in air for 60 min, and then the plates were washed three times with PBS to remove nonadherent cells. Adherent AM were scraped from the plates using a cell scraper-M (Sumitomo Bakelite, Osaka, Japan) and sonicated twice at 20 kHz for 5 s. The resulting cell lysates were mixed with an equal volume of loading buffer (125 mM TRIS-HCl at pH 6.8, 4% SDS, 2 mg/ml methyl green, 10% glycerol). Fifty micrograms of protein were subjected to 10% polyacrylamide gel electrophoresis in SDS running buffer [25 mM TRIS-HCl at pH 8.3, 192 mM glycine, 0.1% SDS). After electrophoresis, the protein bands were transferred to an Immobilon-P membrane (Millipore, Bedford, MA) in a buffer containing 25 mM TRIS at pH 8.3, 192 mM glycine, 0.1% SDS, 20% methanol. The blots were pretreated with 1% skim milk and then incubated with antihuman CD13 antibody as primary antibody and with biotinylated antimurine IgG as secondary antibody. The reacted proteins were visualized by the use of a Vectastain ABC kit (Vector Laboratories, Burlingame, CA) according to the manufacturer's instruction. PBS containing 0.05% Tween 20 was used as washing buffer throughout the blotting experiments.

Measurements of BALF Cytokines and Serum Angiotensin-converting Enzyme

Enzyme immunoassays for human IL-2 and IFN- were performed essentially as described in detail previously (19). The detection limits of IL-2 and IFN- were 20 pg/ml. Serum angiotensin-converting enzyme (ACE) activity was determined by the technique of Lieberman (20). Normal values in our laboratory range were from 7.7 to 29.4 U/ml.

Isolation of Human Lymphocytes and T Cells

Lymphocytes were separated from peripheral blood of healthy donors by centrifugal elutriation in a Beckman JE-5.0 elutriation system (Beckman Instruments, Fullerton, CA) (21). Fractions enriched in lymphocytes (> 99%) were obtained at 3,000 rpm and at flow rates of 26 ml/min. Human T-cell enrichment columns (R&D System, Minneapolis, MN) were then used to purify human CD4+ and CD8+ T-cell populations via high-affinity negative selection according to the manufacturer's instruction. This isolation procedure typically yielded more than 94% CD4+ T cells and 88% CD8+ T cells. The cells were resuspended in chemotaxis medium RPMI 1640, 1% BSA (Sigma Chemical), 25 mM Hepes. More than 97% of the cells were viable, as judged by the trypan blue dye exclusion test.

Chemotaxis Assays

Lymphocyte and T-cell migration was assessed by a 48-well microchemotaxis chamber technique as previously described (22). A 26- to 28-µl aliquot of CD13/aminopeptidase N diluted in chemotaxis medium was placed in the lower compartment and 50 µl of cell suspension (5 × 10⁶ lymphocytes or purified T cells/ml) was placed in the upper compartment of the chamber. The two compartments were separated by a polycarbonate filter (5-µm pore size; Neuroprobe, Cabin John, MD) which had been incubated overnight at 4° C in a solution containing 10 µg/ml fibronectin (Sigma Chemical). The chamber was incubated at 37° C for 3 h. At the end of the incubation period, the filter was removed, fixed, and stained with Diff-Quik solution (International Reagents Co., Kobe, Japan). The number of migrated cells in three high power fields (HPF ×400) was counted by light microscopy after coding the samples. The migration was expressed as chemotaxis index (CI) calculated: CI = Migration to stimuli/Migration to medium.

Inhibition of CD13/aminopeptidase N by Bestatin

Bestatin has been shown to be a specific inhibitor of aminopeptidases (23). After incubation of CD13/aminopeptidase N with bestatin for 30 min at 37° C, solutions were tested for enzymatic and chemotactic activities as described above.

Inhibition of Activity of BALF by Bestatin

BALF from NV or patients with sarcoidosis was preincubated with bestatin for 60 min at 37° C, and solutions were then tested for enzymatic and chemotactic activities as described above.

Statistical Analysis

All results are expressed as mean ± SEM. Statistical analysis was performed using Student's two-tailed unpaired t test for comparisons between two groups. Correlations between two parameters were evaluated using Pearson's test. Differences were considered significant if p values were 0.05 or less. Data were analyzed on a Macintosh computer using Statview software.

	RESULTS

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Aminopeptidase Activity in BALF

Aminopeptidase activity in the BALF from NV, CP, and patients with sarcoidosis is shown in Figure 1. A very low activity of aminopeptidase was detected in the BALF from NV and CP (6.8 ± 2.0, 9.1 ± 5.9 nmol/h, respectively). The mean value of aminopeptidase activity in the BALF from patients with sarcoidosis was significantly higher (49.9 ± 10.8 nmol/h) than that of NV and CP, but individual values were ranged widely, from 6 to 221 nmol/h.

View larger version (14K): [in this window] [in a new window]   Figure 1.   Aminopeptidase activity in the BALF from NV, CP, and patients with sarcoidosis. Aminopeptidase activity was assayed fluorometrically with L-leucine-AMC as a substrate, and was expressed in nanomoles of substrate cleaved per hour. Results are expressed as mean ± SEM. * Significantly different from the values of NV and CP (p < 0.05).

Comparison of Aminopeptidase Activity in BALF with Other Parameters in Patients with Sarcoidosis

The disease activity of pulmonary sarcoidosis, which is evaluated by clinical, roentgenographic, and physiologic markers, is important in predicting disease progression and long-term outcome of this disorder. Previous reports have shown that the number of lymphocytes, especially CD4+ T lymphocytes, in BALF is useful in determining the activity of alveolitis in sarcoidosis, and serum ACE levels are also useful in determining the disease activity of this disorder (24). Therefore, to determine the significance of the aminopeptidase activity in sarcoidosis, the correlations of the aminopeptidase activity with lymphocyte percentages in BALF, the ratio of CD4+ to CD8+ T lymphocytes in BALF, and serum ACE were examined. As shown in Figure 2, the aminopeptidase activity in the BALF showed significantly positive correlations with lymphocyte percentages and the ratio of CD4+ to CD8+ T lymphocytes. However, there was no significant correlation between aminopeptidase activity and serum ACE level (r = +0.356, p > 0.05). The significant correlations between the aminopeptidase activity and percentages of lymphocytes (r = +0.428, p = 0.0407) or CD4+/CD8+ ratio (r = +0.432, p = 0.030) were also observed in the patient populations whose BALF contained high aminopeptidase activity ( 18 nmol/h, n = 17). There were no significant differences in the aminopeptidase activity between male and female groups and between smokers and nonsmokers in patients with sarcoidosis. There was no significant correlation between the aminopeptidase activity and age.

View larger version (16K): [in this window] [in a new window]   Figure 2.   Correlation between aminopeptidase activity and percentages of lymphocytes in BALF from patients with sarcoidosis (A) and between aminopeptidase activity and the ratio of CD4+ to CD8+ T cells in BALF from patients with sarcoidosis (B). Aminopeptidase activity was assayed fluorometrically with L-leucine-AMC as a substrate, and was expressed in nanomoles of substrate cleaved per hour.

Patients with sarcoidosis were classified into two groups by chest radiographs: Group I without parenchymal involvement (Stage I), and Group II with parenchymal involvement (Stage II/III). By this classification, 15 patients belonged to Group I and 15 to Group II (Table 2). There was no significant difference in the mean ages between the two groups. The level of aminopeptidase activity in BALF was significantly higher in Group II than in Group I. The percentage of lymphocytes and the ratio of CD4+ to CD8+ T lymphocytes were also higher in Group II than in Group I. There was no significant difference in serum ACE level between these two groups. We measured Th1-type cytokines; but neither IFN- nor IL-2 was detectable in the BALF from patients with sarcoidosis.

Aminopeptidase Activity in Sera

Serum level of aminopeptidase activity was measured to determine whether it reflected the aminopeptidase activity in the BALF. We obtained sera from 3 NV, 3 CP, and 14 patients with sarcoidosis at the same time that BAL was performed. There was no significant difference in aminopeptidase activity in sera from NV, CP, and patients with sarcoidosis (145.3 ± 21.3, 188.3 ± 40.1, 178.0 ± 18.3 nmol/h, respectively). There was no significant correlation between BALF and serum aminopeptidase activity in patients with sarcoidosis (r = 0.091, p > 0.05).

The Expression of CD13/aminopeptidase N Protein in AM

Because CD13/aminopeptidase N is known to be expressed on monocytes/macrophages (9), the expression of CD13/aminopeptidase N protein in AM was determined by Western blotting and is documented in Figure 3. When 50 µg of AM lysate protein were separated on a SDS-PAGE, CD13/aminopeptidase N protein, which has a molecular mass of approximately 150 kD, was detected in normal volunteers at very low levels. The AM lysate protein from six of seven patients with sarcoidosis contained higher amount of CD13. Increased aminopeptidase activity in BALF was detected in six patients with sarcoidosis in accordance with results of CD13/aminopeptidase N protein expression in AM.

View larger version (48K): [in this window] [in a new window]   Figure 3.   Western blot analysis of CD13/aminopeptidase N protein expression in AM. Fifty micrograms of AM lysate protein were separated on SDS polyacrylamide gel and blotted. CD13/aminopeptidase N was detected by a monoclonal antihuman CD13 antibody. Lanes 1 to 3 contain lysates of AM from NV, and lanes 4 to 10 contain those from patients with sarcoidosis.

Chemotactic Activity of CD13/aminopeptidase N for T Lymphocytes

CD13/aminopeptidase N induced chemotactic migration of human lymphocytes in a concentration range from 10⁵ to 10¹ U/ml (Figure 4). Chemotactic activity induced by CD13/ aminopeptidase N at a concentration range from 10⁴ to 10² U/ml was equivalent to that of 50 ng/ml of MIP-. Checkerboard analysis showed that the effect of CD13/aminopeptidase N was chemotactic rather than chemokinetic (data not shown). Because there was a correspondence between the chemotactic response and the enzymatic activity (Figure 4), we examined the relationship of the chemotactic activity of CD13/aminopeptidase N to its enzymatic activity by using bestatin, a specific inhibitor for aminopeptidases. The inhibition of enzymatic activity of CD13/aminopeptidase N by bestatin by 96% led to inactivation of its chemotactic activity for lymphocytes by 93% (Figure 5). Bestatin did not inhibit the chemotactic activity of MIP-1 (data not shown). CD13/aminopeptidase N also manifested chemotactic activity for purified human CD4+ and CD8+ T lymphocytes in a concentration range from 10⁴ to 10¹ U/ml and 10³ to 10¹ U/ml, respectively, but the response of CD4+ T cells was greater than that of CD8+ T cells (Figure 6).

View larger version (32K): [in this window] [in a new window]   Figure 4.   Chemotactic and enzymatic activities of CD13/aminopeptidase N for human lymphocytes. Lymphocytes were separated from peripheral blood of healthy donors by centrifugal elutriation in a Beckman JE-5.0 elutriation system. MIP-1 is included as a positive control. The results are expressed as the chemotaxis index (CI), which represents the ratio of the number of cells in HPF in the test to control samples (medium control). Columns show the enzymatic activity of CD13/ aminopeptidase N, which was assayed fluorometrically with L-leucine-AMC as a substrate and was expressed in nanomoles of substrate cleaved per hour. Experiments were repeated four times with cells from different donors. Results are expressed as mean ± SEM. * Indicates significantly different from the value of medium control (p < 0.05).

View larger version (21K): [in this window] [in a new window]   Figure 5.   Inhibition of CD13/aminopeptidase N activity by bestatin. CD13/aminopeptidase N solution (2 mU/ml) was preincubated with an equal volume of bestatin solution (200 µg/ml) or medium alone at 37° C for 30 min. The solution was then tested for enzymatic and chemotactic activities as described in METHODS. Experiments were repeated three times with cells from different donors. All results are expressed as mean ± SEM. * Indicates significantly different from the value of untreated CD13/aminopeptidase N (p < 0.05).

View larger version (18K): [in this window] [in a new window]   Figure 6.   Chemotactic activity of CD13/aminopeptidase N for purified human CD4+ (closed circles) and CD8+ (open circles) T lymphocytes. Human T-cell enrichment columns were used to purify human CD4+ and CD8+ T cell population via high-affinity negative selection as described in METHODS. MIP-1  is included as a positive control. The results are expressed as the chemotaxis index (CI), which represents the ratio of the number of cells in HPF in the test to control samples (medium control). All experiments were repeated three times with cells from different donors. Results are expressed as mean ± SEM. * Indicates significantly different from the value of medium control (p < 0.05).

Effect of Bestatin on the Enzymatic and Chemotactic Activities in the BALF

BALF from six patients with sarcoidosis containing high aminopeptidase activity and from three NV was treated with bestatin or medium alone, and then tested for aminopeptidase and chemotactic activities. The treatment of BALF from patients with sarcoidosis with bestatin resulted in inhibition of the aminopeptidase activity of BALF (85 to 93% inhibition) (Table 3). Chemotactic activity for lymphocytes was detected in the BALF from patients with sarcoidosis, but not in that from NV, and the treatment of the BALF from patients with sarcoidosis with bestatin resulted in inhibition of the chemotactic activity in BALF (43 to 74% inhibition). The lymphocyte chemotactic activity of MIP-1, which was shown to be produced in the lung with sarcoidosis (25), was not inhibited by the treatment with bestatin (Table 3), indicating that bestatin might specifically inhibit the chemotactic activity of aminopeptidase in BALF from patients with sarcoidosis. Bestatin itself did not show any inhibitory effects in lymphocyte chemotaxis at the concentration used (data not shown).

	DISCUSSION

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We have shown here significantly higher aminopeptidase activity in BALF from patients with sarcoidosis than that from NV and CP. The activity closely correlated with the percentage of lymphocytes and the ratio of CD4+ to CD8+ T lymphocytes in BALF, and, moreover, was significantly higher in patients with sarcoidosis and lung parenchymal involvement than in those without the involvement. Although considerable difficulty exists in the classification of active versus nonactive pulmonary sarcoidosis, these results suggest that the aminopeptidase activity in BALF corresponds with the activity of alveolitis observed in this disorder.

This report describes for the first time that CD13/aminopeptidase N is a chemoattractant for T lymphocytes. Interestingly, the chemotactic activity was greater for CD4+ T cells than for CD8+ T cells. We were unable to detect any monocyte or neutrophil chemotaxis with CD13/aminopeptidase N (data not shown). Inhibition of the enzymatic activity of CD13/aminopeptidase N by bestatin resulted in abolishing the chemotactic activity, suggesting that the enzymatic activity of CD13/aminopeptidase N is required for the T-cell chemotactic activity. We recently reported that cathepsin G and CAP37/ azurocidin, which are neutrophil-derived proteases, were chemotactic for mononuclear cells and neutrophils, and their enzymatic activities were required for the chemotactic acitivity (26, 27). Thrombin, a trypsinlike serine protease, was also reported to have chemotactic activity for monocytes, and complexes of thrombin with thrombin inhibitors such as antithrombin III and hirudin showed less chemotactic activity (28).

We have shown that CD13/aminopeptidase N is chemotactic for total lymphocytes at a concentration greater than 10⁵ U/ml, and for CD4+ and CD8+ populations at a concentration greater than 10⁴ U/ml and greater than 10³ U/ml, respectively. Although the procedure to purify CD4+ and CD8+ T cells may partially decrease the responsiveness of cells to the chemoattractant, we cannot explain the inconsistencies about required doses at present.

BALF from patients with sarcoidosis contained high chemotactic activity for lymphocytes when compared with that from NV, and the treatment of the BALF with bestatin partially decreased its chemotactic activity. These results indicate that CD13/aminopeptidase N is responsible, at least in part, for the chemotactic activity for lymphocytes in BALF from patients with sarcoidosis. Although various chemotactic factors, including IL-8, IL-15, and MIP-1 and , have been shown to be chemoattractants for lymphocytes (22), this study suggests that enzymatically active CD13/aminopeptidase N may have a significant role in T-lymphocyte involvement as a chemoattractant in pulmonary sarcoidosis.

Activated macrophages have an important role in initiating and amplifying immunologic and inflammatory responses in various inflammatory lung diseases, including sarcoidosis (10, 29). In sarcoidosis, T lymphocytes and macrophages play a pivotal role in orchestrating the inflammatory process. CD13/ aminopeptidase N expressed in macrophages was shown to be an ectoenzyme marker that increases as macrophages mature or become activated (4). In this study, increased CD13/aminopeptidase N protein was detected in AM from six of seven patients with sarcoidosis whose BALF contained high aminopeptidase activity, indicating that AM may be responsible, at least in part, for the aminopeptidase activity detected in BALF from patients with sarcoidosis. However, further study is necessary to clarify the mechanism of the shedding of CD13/aminopeptidase N from cell membrane in pulmonary sarcoidosis.

The level of serum aminopeptidase activity of patients with sarcoidosis was not elevated when compared with that of NV or CP. The family of aminopeptidases mainly consists of leucine aminopeptidase (E.C.3.4.11.1), CD13/aminopeptidase N (E.C.3.4.11.2), and cystyl aminopeptidase (E.C.3.4.11.3) (30). L-leucine-AMC, which was used in this study as a substrate to detect aminopeptidase activity, could be cleaved by all these aminopeptidases (9). Recent reports have shown that serum aminopeptidase activity is mainly ascribed to CD13/aminopeptidase N (31). However, the amount of permeability in the sarcoid lung is known to be minimal (32), and this study has shown that increased expression of CD13 protein is detected in AM from patients with sarcoidosis. These findings suggest that BALF aminopeptidase may be produced locally in the lung.

Both aminopeptidase protein and mRNA were shown to be induced by IFN- (3). IFN- was not detectable in the BALF from patients with sarcoidosis in this study (detection limit; 20 pg/ml). A recent report showed that IFN- is detectable in 5 or 10 times-concentrated BALF from 12 of 21 patients with sarcoidosis tested (the detection limit; 0.4 pg/ml) (33), suggesting that the level of IFN- may be lower than the detection limit in our assay. Thus, it is still possible that Th1-type cytokine(s) are responsible for increased CD13/aminopeptidase N in BALF and AM from patients with sarcoidosis. We found that BALF obtained from patients with active pulmonary sarcoidosis upregulated CD13/aminopeptidase N activity in human peripheral monocytes (unpublished observation), indicating that factor(s) that can contribute to CD13/aminopeptidase N expression may be present in the sarcoid lung.

ACE is known to be produced by epithelioid cells of granulomas, and serum ACE levels are higher in clinically active than in inactive disease (34). However, in this study, there was no significant increase in serum ACE levels of patients with lung parenchymal involvement when compared with those without the involvement, corresponding with previous reports which have shown that serum ACE levels reflect the total-body granuloma burden and not just the degree of lung involvement, and the changing levels of serum ACE are more useful in monitoring disease activity (35). CD13/aminopeptidase N in the BALF may be more specific as a marker of the extent of lung involvement in sarcoidosis than serum ACE.

The data presented here raise the possibility that CD13/ aminopeptidase N activity may serve as a marker of the activity of alveolitis in sarcoidosis, and suggest that CD13/aminopeptidase N may participate in the mechanism of T-cell involvement in this disorder. Increased BALF aminopeptidase activity was found not only in patients with sarcoidosis but also in those with other interstitial lung diseases such as idiopathic pulmonary fibrosis, bronchiolitis obliterance organizing pneumonia, and collagen vascular diseases with interstitial pneumonia (unpublished observation). Therefore, it is possible that CD13/aminopeptidase N plays a role in recruiting lymphocytes to disease sites in various interstitial lung diseases. A greater understanding of the regulation of the production and action of this enzyme may lead to new insights for the control and treatment of interstitial lung diseases.