Introduction

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Dipeptidyl peptidase I (DPPI) is a structurally and catalytically unique member of the papain family of cysteine proteases (1). Unlike its monomeric relatives cathepsins B, L, K, and S, DPPI is a tetramer of enzymatic subunits, each consisting of a propeptide, heavy chain, and light chain (2, 3). DPPI removes NH₂-terminal dipeptides from proteins or peptides lacking basic residues or proline in the NH₂- and CO₂H-terminal positions, respectively, of the dipeptide (4, 5), and also has endoproteolytic activity of unknown significance (6). Although the key in vivo roles of DPPI are not yet known, in vitro studies suggest several intracellular activities of DPPI. These include protein degradation and turnover and activation of neuraminidase (7), platelet factor XIII (8), and granule-associated serine proteases such as granzymes A (9) and B (9), elastase (9), cathepsin G (9), tryptase (10), and chymase (11). In addition, the discovery of DPPI secretion by cytotoxic T lymphocytes (12) and mast cells (3) suggests possible extracellular activities.

DPPI is expressed in several tissues and cells. DPPI messsenger RNA (mRNA) and protein are found in extracts of many tissues, with particularly high expression in spleen, kidney, liver, and lung (13, 14). Although the specific cell sources of DPPI expression in these tissues are unknown, among the cell types known to express DPPI are bone marrow-derived leukocytes that contain secretory granules, including myelomonocytes, cytotoxic T lymphocytes, and mast cells (3, 15). In lysates of these cells, DPPI activity has been localized to the cytoplasmic granular fraction, suggesting that DPPI is localized in the lysosomes and secretory granules of these cells (9). Functional evidence for a secretory granular location of DPPI is demonstrated by its secretion from cytotoxic T lymphocytes (12) and mast cells (3).

With a goal of clarifying possible roles for DPPI in lung diseases, we sought to determine the cells that express DPPI in lung tissue. We hypothesized that lung mast cells are major producers of DPPI and that DPPI cleaves extracellular matrix proteins after secretion by mast cells. To address these hypotheses we used immunohistochemical techniques to identify cells expressing DPPI in normal dog airways and lung. We also analyzed DPPI's ability to cleave the extracellular matrix proteins fibronectin, laminin, and type I, type III, and type IV collagen.

	Materials and Methods

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Materials

All chemicals were from Sigma (St. Louis, MO) unless otherwise noted. Dog DPPI was purified from cultured dog mastocytoma cells as previously described (3).

Cell Culture

Dog BR mastocytoma cells were cultured in Dulbecco's modified Eagle's medium-H16 medium containing 2% supplemented calf serum as previously described (16). Cells were maintained at 37°C in 5% CO₂ and 95% air, cultivated to a density of 1 × 10⁶ cells/ml, and were harvested by centrifugation.

DPPI Assay

DPPI activity was measured spectrophotometrically by monitoring hydrolysis of L-Ala-Ala-p-nitroanilide at 37°C (2). A total of 0.25 to 1 µg of DPPI was incubated for 5 min in 500 µl of activation buffer (100 mM Na₂HPO₄ buffer, 20 mM NaCl, 1 mM ethylenediaminetetraacetic acid [EDTA], and 4 mM cysteine [pH 6]), followed by the addition of 400 µl of substrate buffer (100 mM Na₂HPO₄, 20 mM NaCl, 1 mM EDTA, 125 µM L-Ala-Ala-p-nitroanilide, and 1% dimethylsulfoxide [pH 6]). Release of free nitroaniline was measured at 410 nm for 10 min.

Affinity Purification of Rabbit Antidog DPPI Antibody

To minimize background in immunohistochemistry experiments, polyclonal rabbit antisera raised against DPPI as previously described (3) were purified by affinity chromatography. Briefly, 7 mg of Affi-Gel 15 (Bio-Rad Laboratories, Hercules, CA) suspended in 50 mM sodium acetate (pH 5.5) were reacted with 600 µg of purified dog DPPI for 1.5 h at 18°C. The DPPI-conjugated resin was poured into a 3 × 20 mm column and equilibrated with phosphate-buffered saline (PBS) (pH 7.4). Rabbit antidog DPPI serum (2 ml) was then applied to the column. Unbound proteins were eluted with PBS and bound proteins eluted with 50 mM glycine-HCl (pH 2.5). Fractions of 0.5 ml were collected, immediately neutralized with 2 M Tris base, and tested by dot-blot for immunoreactivity with dog DPPI.

Immunoblotting

All lung tissues were obtained from the tissue sharing program at the University of California, San Francisco. Tissues were harvested from dogs at the time of death after experiments approved by the Committee on Animal Research. Lung tissue fragments containing airway and parenchyma were pulverized under liquid nitrogen with a mortar and pestle, resuspended in 100 mM sodium acetate containing 300 mM NaCl and 1 mM EDTA (pH 6.0), and then centrifuged at 14,000 × g for 10 min. The supernatant was adjusted to pH 4.2, heated at 37°C for 2 h, then centrifuged at 14,000 × g for 10 min. The resulting supernatant was subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions and then transferred to polyvinylidine difluoride (PVDF) membrane (Life Sciences Products, Boston, MA) in transfer buffer containing 25 mM Tris base, 200 mM glycine, and 15% methanol for 1 h at 4°C. The membrane was washed with 50 mM Tris-HCl, 0.5 M NaCl, and 0.01% Tween-20 (TBS; pH 7.5) and incubated in TBS for 1 h with a 1:1,000 dilution of affinity-purified rabbit antidog immunoglobulin. The membrane was then washed with TBS, incubated in TBS for 30 min with a 1:2,000 dilution of alkaline phosphatase-conjugated goat antirabbit antiserum, and washed again. Immunoreactivity was detected using the Fast Red TR/Naphthol AS-MX detection system.

Immunofluorescence Colocalization of DPPI and Chymase in Dog Airways

Lung and airway tissues were harvested from mixed-breed dogs, washed with PBS, fixed for 1 h in PBS containing 4% paraformaldehyde, then incubated in PBS containing 30% sucrose for 18 h at 4°C. Specimens were then washed in PBS before freezing in Tissue-Tek OCT compound (Miles, Elkhart, IN) at 70°C. Airway cryosections of 5 µm were equilibrated in PBS and then incubated for 15 min with blocking solution (PBS containing 5% dehydrated milk, 3% nonimmune goat serum, 0.1% Triton X-100, and 1% glycine) at 18°C. The blocking solution was removed and the tissues were probed with appropriate dilutions of primary and secondary antibodies. Primary antibodies were applied for 18 h at 4°C and secondary antibodies for 10 min at 18°C. Tissues were washed between steps with PBS containing 0.05% Tween (PBS-Tween) at 18°C. DPPI was detected by application of the following antibodies: (1) affinity-purified rabbit antidog DPPI, (2) biotin-conjugated goat antirabbit immunoglobulin (Ig)G (Zymed Laboratories, South San Francisco, CA) secondary antibody, then (3) streptavidin-Texas Red (Zymed Laboratories) applied simultaneously with fluorescein isothiocyanate (FITC)-conjugated goat antirabbit IgG (Zymed Laboratories). Chymase was detected by application of the following antibodies: (1) IgG fraction of rabbit antidog -chymase (17), and (2) FITC-conjugated goat antirabbit IgG applied simultaneously with streptavidin-Texas Red. To double-label cells for DPPI and -chymase, the following antibodies were applied sequentially: (1) affinity-purified rabbit antidog DPPI, (2) biotin-conjugated goat antirabbit IgG, (3) IgG fraction of rabbit antidog -chymase, and (4) FITC-conjugated goat antirabbit IgG applied simultaneously with streptavidin-Texas Red. Control experiments were performed by application of (1) IgG fraction of rabbit nonimmune serum, and (2) FITC-conjugated goat antirabbit IgG applied simultaneously with streptavidin-Texas Red. After washing with PBS-Tween, tissues were mounted in Vectashield (Vector Laboratories, Burlingame, CA) and observed on a Nikon E600 fluorescence microscope.

Immunolocalization of DPPI in Normal Dog Lung Parenchyma

Immunohistochemistry was performed using the histostain-DS kit (Zymed Laboratories). Tissue cryosections of 5 µm were equilibrated in 0.3% H₂O₂ and 90% methanol for 10 min and washed with PBS, then incubated for 15 min with blocking solution at 18°C. The blocking solution was removed and the tissues were probed with affinity-purified rabbit antidog DPPI overnight at 4°C. Tissues were washed with PBS-Tween, incubated with a biotin-conjugated goat antirabbit IgG for 10 min at 18°C, and washed with PBS-Tween, then incubated with streptavidin-alkaline phosphatase for 10 min at 18°C and washed again. Bound alkaline phosphatase was detected using 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium (BCIP/NBT) as a substrate. Note: an alkaline phosphatase detection system was used for these experiments because significant autofluorescence of the alveolar tissue precluded the use of fluorescent secondary antibodies.

Immunofluorescence of DPPI and Chymase in Cultured Mastocytoma Cells

Glass slides containing 2 × 10⁵ dog BR mastocytoma cells were prepared by cytocentrifugation of PBS-suspended mast cells at 600 rpm for 4 min. The slides were air-dried; fixed in Mota's solution (1% lead acetate, 47.5% ethanol, and 0.5% acetic acid); rinsed with PBS; dehydrated by immersion in 50, 70, and 100% ethanol; then air-dried again and frozen for storage. For antibody staining, the tissues were rehydrated by immersion in PBS, incubated for 15 min at 20°C with blocking solution, then incubated overnight at 4°C with appropriate dilutions of the affinity-purified rabbit antidog DPPI or rabbit antidog -chymase antibodies. Tissues were then washed in PBS-Tween, and the bound antibody detected with Texas Red-conjugated goat antirabbit IgG (Vector Laboratories) for DPPI detection, and with FITC-conjugated goat antirabbit IgG for -chymase detection.

DPPI Cleavage of Fibronectin and Laminin

Purified dog DPPI or bovine DPPI (Boehringer Mannheim, Mannheim, Germany) were activated for 5 min in 100 mM Na₂HPO₄ buffer (pH 5.5-7.5) containing 20 mM NaCl, 1 mM EDTA, and 4 mM cysteine. Bovine fibronectin or mouse laminin was added to activated DPPI at a 20:1 weight ratio and the resulting mixture incubated for 16 h at 37°C. Fibronectin and laminin were assayed for cleavage by SDS-PAGE. Control experiments were performed in the absence of DPPI or the presence of the DPPI inhibitor Gly-Phe-diazomethylketone (20 µM; Enzyme Systems Products, Dublin, CA).

Zymography

Zymography (18) was used to test DPPI cleavage of gelatin and types I, III, and IV collagen. Purified DPPI was electrophoresed in nonreducing conditions through 10% SDS-polyacrylamide gels copolymerized with 0.75 mg/ml gelatin, type I collagen (Collaborative Biomedical Products, Bedford, MA), type III collagen, or type IV collagen. After electrophoresis, gels were washed twice for 30 min in 2.5% Triton X-100, then incubated overnight at 37°C in 0.1 M Tris-HCl (pH 7.5) containing 2 mM dithiothreitol. Gels were stained for 12 min with 0.5% Coomassie Blue/ 10% acetic acid/30% methanol. Regions of proteolysis were visualized on gels destained in 10% acetic acid/30% methanol.

DPPI Cleavage of Soluble Gelatin

Purified dog DPPI was activated for 5 min in 100 mM Na₂HPO₄ buffer (pH 5.5-7.5) containing 20 mM NaCl, 1 mM EDTA, and 4 mM cysteine. Biotinylated gelatin (Boehringer Mannheim) was then added at a 1:1 to 1:1,000 weight ratio of gelatin:enzyme, and the resulting mixture incubated for 16 h at 37°C. Gelatinase activity was measured according to the instructions of the gelatinase activity assay kit (Boehringer Mannheim).

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

Immunoblotting of Lung Tissue for DPPI

The presence of DPPI immunoreactivity in normal dog lung, containing airway and alveolar tissue, was identified on immunoblots using our affinity-purified antidog DPPI antibody (Figure 1). A single band of immunoreactivity in lung tissue comigrating with adjacent purified dog DPPI confirmed the presence of DPPI in dog lung. Absence of other immunoreactive bands indicates that our affinity- purified antidog DPPI antibody is specific for DPPI and does not bind to other lung proteins.

View larger version (46K): [in this window] [in a new window]   Figure 1.   Immunoblot of normal dog lung for DPPI. Quantities of 250 ng of purified dog DPPI (lane 1) and 200 µg of an extract of dog lung, containing airway and lung parenchyma (lane 2) were separated under reducing conditions by SDS-PAGE, blotted to PVDF membrane, and probed with polyclonal rabbit antidog DPPI antibodies. Immunoreactivity was identified using a goat antirabbit IgG alkaline phosphatase detection system. Numbers to the left indicate the elution position of marker proteins (not shown).

Immunofluorescence Colocalization of DPPI and Chymase in Dog Airway

To identify cells expressing DPPI in normal dog airway, we examined tissue sections by immunofluorescence microscopy using two polyclonal antibodies, antidog DPPI and antidog -chymase antibodies. The DPPI antibodies revealed immunoreactive cells in airway subepithelium (Figures 2A and 2C). The -chymase antibodies revealed a similar pattern of immunoreactive cells in the airway subepithelium, suggesting that the DPPI-immunoreactive cells may be mast cells (Figures 2D and 2F). Further pursuing this possibility by double-labeling the subepithelial cells for DPPI and chymase, we show that all of the cells in airway tissues strongly immunoreactive for DPPI are chymase-expressing mast cells (Figures 2G-2I). A number of controls demonstrate that the signals identified are specific for DPPI and chymase. First, treatment of tissues with nonimmune rabbit serum demonstrated no immunofluorescence of cells in the airway subepithelium, indicating that the DPPI- and chymase-immunoreactivity is not due to nonspecific binding of rabbit Ig or secondary antibodies to mast cells (Figures 2J-2L). Second, by labeling the bound rabbit antidog DPPI antibody with a biotin-conjugated goat antirabbit IgG antibody, we occupied the rabbit IgG epitopes and blocked binding of the subsequently administered FITC-conjugated goat antirabbit IgG antibody (Figures 2B and 2C), thus demonstrating that the FITC-conjugated antirabbit IgG antibody binds specifically to the antichymase antibodies in double-labeled sections. Third, by simultaneously applying streptavidin-Texas Red and FITC-conjugated goat antirabbit IgG to tissues probed with the rabbit antichymase antibody alone, we show that streptavidin-Texas Red does not bind nonspecifically to the rabbit antichymase antibody (Figures 2E and 2F). Nor does streptavidin-Texas Red (unlike avidin-Texas Red) bind nonspecifically to canine mast cells (19, 20). Finally, red fluorescence of airway epithelium is due to direct binding of the streptavidin-Texas Red to proteins in the airway epithelium.

View larger version (63K): [in this window] [in a new window]   Figure 2.   Immunofluorescent colocalization of DPPI and chymase in normal dog airways. Cryosections of 5 µm were immunostained with rabbit antidog DPPI (A-C), rabbit antidog -chymase (D-F), both antidog DPPI and antidog -chymase antibodies (G-I), or rabbit nonimmune IgG (J-L). DPPI and -chymase immunoreactivity were imaged using biotinylated goat antirabbit IgG/streptavidin-Texas Red and FITC-conjugated goat antirabbit IgG, respectively. Tissue sections were photographed in the same microscopic field using a filter specific for FITC (A, D, G, and J) or Texas Red (B, E, H, and K), or were double-exposed using both filters (C, F, I, and L). Cells immunoreactive for chymase, DPPI, or both proteins fluoresce green, red, or yellow, respectively. (Original magnification: ×40.)

Immunolocalization of DPPI in Dog Lung Parenchyma

Next, we used nonfluorescent immunohistochemical techniques to identify DPPI-expressing cells in normal dog lung. Our DPPI antibodies revealed immunoreactive cells within alveoli (Figure 3B). At high power, these cells exhibited the size, shape, nuclear morphology, and tissue location of alveolar macrophages. Alveolar tissue treated with rabbit nonimmune serum showed no immunoreactivity (Figure 3A).

View larger version (66K): [in this window] [in a new window]   Figure 3.   Immunolocalization of DPPI in normal dog lung parenchyma. Tissue sections were immunostained using either rabbit nonimmune IgG (A), or rabbit antidog DPPI antibodies (B). DPPI-immunoreactive cells (arrow) were stained blue using an alkaline phosphatase BCIP/NBT detection system. A high-power image of DPPI immunoreactivity in an alveolar macrophage is shown in the inset of B. (Original magnification: ×40, inset: ×100.)

Immunolocalization of DPPI and Chymase in Cultured Mastocytoma Cells

The report of secretion of DPPI by mast cells hypothesized that DPPI is packaged in the secretory granules of mast cells (3). To test this hypothesis, we used immunofluorescence to examine the intracellular location of DPPI in cultured dog BR mastocytoma cells, which express both DPPI and chymase. Our DPPI and -chymase antibodies (Figure 4B) both produced a granular pattern of cytosolic fluorescence. The similarity of the DPPI pattern of immunofluorescence to that of chymase, which is known to reside in mast-cell secretory granules, suggests that DPPI also resides in the same or similar population of granules.

View larger version (11K): [in this window] [in a new window]   Figure 4.   Immunolocalization of DPPI and chymase in cultured dog mast cells. Dog BR mast cells cytospun onto glass slides were immunolabeled with affinity-purified rabbit antidog DPPI (A), or rabbit antidog -chymase (B), as in Figure 2. Each primary antibody reveals a punctate pattern of cytoplasmic fluorescence consistent with packaging of the proteases in mast-cell secretory granules. (Original magnification: ×100.)

Hydrolysis of Fibronectin but Not Laminin by DPPI

Because DPPI is synthesized and secreted by mast cells, we sought to determine whether DPPI cleaves extracellular matrix proteins, such as fibronectin and laminin, that are known to be associated with mast cells (21). Purified dog DPPI cleaves fibronectin (Figure 5A, lanes 2-4), but not laminin (Figure 5B), into multiple fragments at pH 5.5 to 7.5. The optimum pH for DPPI cleavage of fibronectin is pH 5.5. Degradation of fibronectin into multiple high molecular-weight fragments suggests that the cleavage proceeds by an endoproteolytic mechanism. Inhibition of fibronectin cleavage by the DPPI inhibitor Gly-Phe-diazomethylketone (Figure 5A, lanes 5-7) and cleavage of fibronectin by commercially available bovine DPPI (Figure 5A, lanes 8 and 9) show that DPPI, and not a contaminating protease, is responsible for this cleavage and that fibronectin hydrolysis is not a feature of dog DPPI alone. The absence of fibronectin cleavage by DPPI in the presence of Gly-Phe-diazomethylketone indicates that fibronectin cleavage involves active site-mediated hydrolysis rather than a nonenzymatic effect on fibronectin stability at a low pH.

View larger version (31K): [in this window] [in a new window]   Figure 5.   Cleavage of fibronectin but not laminin by DPPI. Bovine fibronectin (0.5 µM) was incubated with dog DPPI (0.3 µM) in a phosphate buffer at pH 5.5 (A, lanes 2 and 5 [counted from left to right]), 6.5 (lanes 3 and 6), and 7.5 (lanes 4 and 7) for 16 h at 37°C, in the absence (lanes 2-4) or presence (lanes 5-7) of the DPPI inhibitor Gly-Phe-diazomethylketone (20 µM). Lane 1 is native fibronectin. Fibronectin (0.5 µM) was also incubated with bovine DPPI (1.2 µM) at pH 5.5 in the absence (lane 8) or presence (lane 9) of Gly-Phe-diazomethylketone. The generation of fibronectin fragments was detected by SDS-PAGE on gradient gels of 4 to 20% acrylamide as indicated by the arrows. (B) An SDS-PAGE gel of mouse laminin (0.3 µM) that was incubated with dog DPPI (0.3 µM) at pH 5.5, 6.5, or 7.5 for 16 h at 37°C. The lack of new protein bands indicates that laminin was not hydrolyzed endoproteolytically by DPPI.

DPPI Hydrolysis of Gelatin and Collagen Types I, III, and IV

To determine whether DPPI cleaves gelatin or types I, III, or IV collagen, zymography was performed. Purified dog DPPI that was electrophoresed through gelatin-, and types I, III, and IV collagen-embedded gels and subjected to zymography revealed bands of activity at 175 and 80 kD (Figure 6). The band of activity at 175 kD corresponds to the DPPI tetramer. Faint activity at 80 kD seen on some zymograms most likely represents a DPPI dimer. Similar bands of activity were detected by zymography performed at pH 5.2 (not shown). DPPI is inactive by casein zymography (data not shown), indicating that DPPI is not active as an endoprotease against all potential protein targets.

View larger version (34K): [in this window] [in a new window]   Figure 6.   DPPI cleavage of gelatin and collagen types I, III, and IV. A total of 1 µg of purified dog DPPI was subjected to electrophoresis under nonreducing conditions in gels copolymerized with 0.75 mg/ml gelatin (A), type I collagen (B), type III collagen (C), or type IV collagen (D). Gels were incubated in 0.1 M Tris-HCl (pH 7.5) containing 2 mM dithiothreitol for 16 h at 37°C, then stained with Coomassie Blue. Proteolytic cleavage of the substrates is identified by a major band at 175 kD.

DPPI Hydrolysis of Gelatin in Solution

By subjecting DPPI to SDS-PAGE we may have opened a previously blocked active site and converted DPPI from a dipeptidyl peptidase to a protease with endoproteolytic activity that is not present in solution. To test this possibility, we determined whether DPPI cleaves gelatin in solution. Figure 7 shows the concentration-dependent cleavage of biotinylated gelatin by DPPI. The specific activity of DPPI cleavage of gelatin is 7.3 ng gelatin cleaved/nmol DPPI/h. For comparison, chymase and gelatinase B cleaved biotinylated gelatin with specific activities of 33 ng gelatin cleaved/nmol chymase/h and 3,680 ng gelatin cleaved/nmol gelatinase B/h, respectively, in buffers that optimized the proteolytic activity of the enzyme tested. Thus, the optimal specific activities for DPPI and chymase cleavage of gelatin are 0.2% and 1%, respectively, that of gelatinase.

View larger version (18K): [in this window] [in a new window]   Figure 7.   Concentration-dependent cleavage of biotinylated gelatin by DPPI in solution. Biotinylated gelatin (2.5 ng) was incubated with increasing concentrations of dog DPPI in a phosphate buffer (pH 6.5) at 37°C for 16 h, then analyzed for cleavage using a gelatinase activity assay kit. Data are reported as means ± standard error for three separate experiments.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

In this study, we report that mast cells are the major source of DPPI in dog airways and alveolar macrophages in dog lung. We also show that DPPI is stored in mast-cell secretory granules, and that DPPI cleaves the extracellular matrix proteins fibronectin and types I, III, and IV collagen. Cells expressing DPPI in airway and lung were identified by immunohistochemistry using an affinity-purified rabbit antidog DPPI antibody. Resident airway mast cells were identified by double-labeling tissues for DPPI and chymase, a mast cell-specific protease. DPPI was found to cleave fibronectin and gelatin endoproteolytically in solution, as well as gelatin and types I, III, and IV collagen in SDS-PAGE gels. The finding of DPPI in airway mast cells suggests the possibility that DPPI plays a role in the turnover of extracellular matrix proteins in airway diseases, such as asthma, that are associated with mast-cell activation (22, 23). DPPI expression in alveolar macrophages suggests that it may be important for macrophage-specific functions such as antigen presentation.

Our observations support our hypothesis that DPPI is important in mast-cell biology. First is our detection of DPPI in resident airway mast cells. Previously, DPPI was shown to be found in transformed mast cells in culture (3, 9). Our current findings establish that normal mast cells synthesize DPPI. However, its roles in these cells have yet to be fully established. One identified intracellular role for DPPI is activation of the mast-cell serine proteases tryptase and chymase (10, 11). A second possible role is in mast-cell growth and differentiation, similar to its proposed involvement in cytotoxic T-cell growth and differentiation (24). Finally, DPPI immunoreactivity in resident airway mast cells suggests that these cells either synthesize DPPI continuously or store it in secretory granules. DPPI stored in secretory granules would be subject to regulated release outside the cell where it can interact with potential extracellular targets. Our previous studies have reported the secretion of DPPI by cultured mast cells (3). Whether this secretion is direct or due to regulated release from mast-cell secretory granules was not known. Earlier evidence supporting the packaging of DPPI from mast-cell secretory granules is the finding of DPPI activity in the granular fraction of mastocytoma cell extracts (9) and its corelease with tryptase, a known secretory granule-associated protease (3). Our current finding of DPPI immunoreactivity in granules of cultured mast cells provides further evidence that DPPI is stored in mast-cell secretory granules. From these granules, DPPI may then be released following mast-cell activation by mediators such as substance P or IgE-bound antigen, which crosslinks the IgE receptor Fc RI (25).

Secretion of DPPI from cells is not limited to mast cells. Cytotoxic T lymphocytes, neutrophils, and macrophages may also secrete DPPI. For example, DPPI is secreted by cytotoxic T lymphocytes stimulated with ionomycin (12). The possibility of neutrophil secretion of DPPI is supported indirectly by observations that DPPI and elastase activities colocalize to the granular fraction of myelomonocytic cells (9) and that elastase is secreted from activated neutrophils (26, 27). Macrophage secretion of DPPI is also indirectly supported by the observation that macrophages secrete cathepsins B, L, and S (28), three lysosomal cysteine proteases related to DPPI (1). Further, DPPI has been shown to be secreted by rat peritoneal macrophages treated with an anti-mannose 6-phosphate receptor antibody (29). Thus, DPPI is likely to be secreted by most of the major cell types known to synthesize it, suggesting that its extracellular activities are not limited to mast cells.

After secretion, DPPI may cleave proteins in the extracellular space. Protein targets include matrix proteins as well as small bioactive peptides. In this report we show that DPPI cleaves the extracellular matrix proteins fibronectin and types I, III, and IV collagen. Fibronectin and types I and III collagen are found at higher levels in asthmatic airways, where they are hypothesized to contribute to the irreversible airways obstruction found in some asthmatics (23). Extracellular cleavage of these proteins by DPPI alone, or in concert with other mast-cell proteases tryptase, chymase, or the gelatinases (25), may facilitate the passage of mast cells into the asthmatic airway. Alternatively, by cleaving extracellular matrix proteins, DPPI and the other mast-cell proteases may remodel matrix in the asthmatic airway (25, 30). As noted earlier, gelatinase B has 500-fold greater activity for hydrolysis of gelatin than do DPPI and chymase. Therefore, although DPPI can endoproteolytically hydrolyze matrix proteins, this activity is weak relative to other matrix degrading proteases. Despite this fact, mast cell-mediated cleavage of matrix proteins by DPPI is likely to be significant because mast cells appear to store larger amounts of DPPI than gelatinase A and B (our unpublished observations). Also, because DPPI's peak activity is at an acidic pH and that of gelatinases A and B is at neutral pH, DPPI may be more active than the gelatinases when the environment outside of a degranulating mast cell is acidic. Finally, DPPI may participate in matrix remodeling by secondarily removing NH₂-terminal dipeptides from matrix proteins that have been cleaved endoproteolytically by other proteases, thereby solubilizing the degraded matrix components and facilitating their tissue clearance.

Endoproteolytic cleavage of fibronectin and types I, III, and IV collagen by DPPI is demonstrated by the large cleavage products generated by DPPI hydrolysis of fibronectin. It is unlikely that these fragments were generated by processive removal of dipeptide fragments because of their discrete appearance and because both fibronectin and collagen have a large number of prolines near their NH₂-termini that will block DPPI's dipeptidyl peptidase activity. Further supporting DPPI's endoproteolytic activity is an earlier report that DPPI endoproteolytically cleaves lysozyme and NH₂-terminally blocked synthetic dipeptide subtrates (6). Dual-functioning cysteine proteases are not without precedent inasmuch as two other cysteine proteases, cathepsins B and H, also exhibit both endoproteolytic and exoproteolytic activities. Cathepsin B has both carboxydipeptidase and endoprotease activity (31). Its carboxydipeptidase specificity is generated by an occluding loop, which blocks the prime portion of the active site and provides a positively charged anchor for the CO₂H-terminus portion of protein substrates (32). Cathepsin H has both aminopeptidase and endoprotease activity (33). The aminopeptidase specificity is determined by a covalently attached octapeptide (34), which blocks the substrate-binding site, allowing only the NH₂-terminus of the substrate access to the active site. Cathepsin B's occluding loop and cathepsin H's octapeptide appear to have some degree of wobble inasmuch as they allow some substrates full access to the active site with subsequent endoproteolytic cleavage (31, 33). We predict that DPPI is similar to cathepsins B and H in that a region of the protein (possibly the residual propeptide) blocks part of the active site, restricting substrate access and allowing only the NH₂-terminal dipeptide to be cleaved in some substrates. This blocking peptide may then be displaced by selective substrates (such as fibronectin), which are then endoproteolytically cleaved. Alternatively, a portion of the blocking peptide may be cleaved and removed, unblocking the active site. Also, a subpopulation of DPPI may lack the blocking peptide entirely and function exclusively as an endoproteolytic enzyme.

The DPPI-immunoreactive cells in dog lung have characteristic features of alveolar macrophages, including their number, mononuclear morphology, and presence in the alveolar space, suggesting that they are in fact alveolar macrophages. This finding is supported by previous studies showing that alveolar macrophages express high levels of DPPI mRNA (15). Interestingly, levels of DPPI mRNA are higher in alveolar macrophages than in their precursor monocytes (15), suggesting that alveolar macrophages require greater quantities of DPPI as they mature. The high levels of DPPI expression in alveolar macrophages also suggest that the enzyme may play a role in macrophage functions. One possibility is participation in antigen presentation, a speculated macrophage function. Cysteine proteases such as cathepsins L and S degrade the invariant chain, an essential process for antigen presentation by major histocompatibility complex (MHC) class II proteins (35). Also, the cysteine proteases legumaine (38) and cathepsin B (39) cleave antigens into fragments for presentation. The optimal length of an antigen for MHC class II presentation is 15 to 22 amino acids (40). One possible role for DPPI in antigen presentation is to process antigens into fragments suitable for presentation by MHC.

In summary, this report shows that mast cells and macrophages are the major sources of DPPI in dog airways and alveoli, respectively. DPPI from these cells can cleave extracellular molecules such as fibronectin and collagen types I, III, and IV. These findings suggest that DPPI may be involved in remodeling of the extracellular matrix in airway diseases such as asthma.