alpha 1-Antichymotrypsin Is the Human Plasma Inhibitor of Macrophage Ectoenzymes That Cleave Pro-macrophage Stimulating Protein*

Alison Skeel and Edward J. LeonardDagger

From the Immunopathology Section, Laboratory of Immunobiology, NCI-Frederick Cancer Research and Development Center, Frederick, Maryland 21702

Received for publication, January 24, 2001, and in revised form, March 22, 2001


    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Macrophage stimulating protein (MSP) is secreted as 78-kDa single chain pro-MSP, which is converted to biologically active, disulfide-linked alpha beta chain MSP by cleavage at Arg483-Val484. Murine resident peritoneal macrophages have two cell surface proteolytic activities that cleave pro-MSP. One is a pro-MSP convertase, which cleaves pro-MSP to active MSP; the other degrades pro-MSP. The degrading protease is inhibited by soybean trypsin inhibitor or by low concentrations of blood plasma, which allows the convertase to cleave pro-MSP to MSP. Using pro-MSP cleavage as the assay, we purified the inhibitor from human plasma. The bulk of the plasma protein was removed by salting out and by isoelectric precipitation of albumin. Highly purified inhibitor was then obtained in three steps: dye-ligand binding and elution, ion exchange chromatography, and high performance liquid chromatography gel filtration. After SDS-polyacrylamide gel electrophoresis and transfer to a polyvinylidene membrane, N-terminal sequencing of the product identified it as alpha 1-antichymotrypsin. The mean concentration of alpha 1-antichymotrypsin in human plasma is 7 µM. At this concentration, alpha 1-antichymotrypsin inhibits both macrophage enzymes. A concentration of 0.4 µM, which is in the expected concentration range in extracellular fluid, preferentially inhibits the degrading enzyme, which allows for cleavage to active MSP by the pro-MSP convertase.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Macrophage stimulating protein (MSP)1 is a pleiotropic 78-kDa growth and motility factor that is structurally related to several proteins of the coagulation system (1, 2). MSP acts on a number of cell types including tissue macrophages, epithelia, and hematopoietic cells (3). Actions on macrophages include stimulation of motility (4), induction of phagocytosis of serum complement-coated erythrocytes (1), and inhibition of the up-regulation of NO synthase by inflammatory stimuli (5, 6). MSP induces adhesion, motility, and replication of epithelial cells, and it can prevent the apoptosis that occurs when epithelial cells are prevented from attachment to a substrate (7). MSP mediates its effects by binding to and activating a cell receptor tyrosine kinase known as RON in humans (8, 9) and STK in mice (10, 11).

Human MSP is a disulfide-linked heterodimer comprising an alpha  and beta  chain with molecular masses (not including N-linked carbohydrate) of 53 and 25 kDa, respectively. It is a member of a plasminogen-related family of proteins characterized by multiple copies in the alpha  chain of a highly conserved triple disulfide loop structure (kringle). The kringle protein family includes plasminogen (12), prothrombin (13), urokinase (14), and hepatocyte growth factor/scatter factor (HGF/SF) (15, 16). These proteins are secreted as single-chain precursors, which have no biological activity until the protein is cleaved into alpha  and beta  chains by specific serine proteases at an Arg-X bond (17), in which X is most frequently Val. The alpha beta chain junction of MSP is Arg483-Val484 (18). The beta  chain of MSP is homologous to the beta  chain catalytic domain of the serine protease members of the kringle family. However, MSP and HGF/SF are devoid of proteolytic activity, because of amino acid substitutions of the three beta  chain protease active site residues His, Asp, and Ser. They are thought to have evolved from an ancient coagulation protein (19) to become growth and motility factors, while retaining the protease-dependent activation mechanism of the zymogens of the family.

Like kringle proteins of the coagulation system, MSP is constitutively synthesized by hepatic parenchymal cells and is secreted into the circulating blood as biologically inactive pro-MSP. The mean concentration of pro-MSP in the plasma of a series of normal human subjects is 5 nM (20). The level is not changed in the course of an acute phase reaction (21). To act on target cells in extravascular sites, pro-MSP must diffuse into tissues and be proteolytically cleaved to the biologically active disulfide-linked alpha beta chain heterodimer (22). The EC50 for the action of the MSP heterodimer on macrophages is 0.25 nM (1).

The Arg483-Val484 scissile bond of pro-MSP is a typical cleavage site for trypsin-like serine proteases. Several such proteases of the coagulation system, including factors XIa and XIIa and serum kallikrein cleave pro-MSP to active MSP in vitro (23). However, cleavage is minimal when blood clots, indicating that pro-MSP is not a preferred substrate for these enzymes (22). We have described two pro-MSP convertase activities in extravascular sites, one in wound fluid exudates (20) and the other associated with murine resident peritoneal macrophages (22).

Cleavage of pro-MSP by resident peritoneal macrophages involves a minimum of two surface proteolytic activities: one, a pro-MSP convertase, cleaves pro-MSP to active MSP; the other degrades pro-MSP (22). When pro-MSP is added to macrophages in culture wells in serum-free medium, the activity of the degrading enzyme predominates. However, if soybean trypsin inhibitor (STI) is present, the degrading enzyme is preferentially inhibited, and the pro-MSP convertase cleaves the protein to active MSP, as shown by SDS-PAGE of the products and by induction of characteristic shape changes in the macrophages. The effect of STI could be reproduced by adding human or mouse serum to the cultures. A number of human serum protease inhibitors, including alpha 1-antitrypsin, C1-inhibitor, anti-thrombin III, and alpha 2-macroglobulin, had no effect on macrophage cleavage of pro-MSP (22). Identification of the serum inhibitor and approaches to determining its physiological role are the subjects of this article.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Reagents and Chromatography Columns-- Recombinant human pro-MSP and MSP were supplied by Drs. W. Yoshikawa and T. Takehara of Toyobo, Osaka, Japan. STI was from Roche Molecular Biochemicals, Indianapolis, IN. AEBSF and bovine albumin, Fraction V, fatty acid-poor, nuclease- and protease-free were from Calbiochem, Palo Alto, CA. alpha 1-Antichymotrypsin was from Athens Research and Technology, Athens, GA. 125I-Labeled Bolton-Hunter Reagent (NEX120) and Enlightning were from PerkinElmer Life Sciences. Serotonin was from Sigma-Aldrich. [3H]Dextran, 70,000 kDa, was from Amersham Pharmacia Biotech. Affi-Gel blue gel was from Bio-Rad, Hercules, CA. Immobilon-P transfer membranes (polyvinylidene fluoride) were from Millipore/Waters, Bedford, MA. LeukoStat stain was from Fisher Scientific. HPLC-DEAE, Protein Pak DEAE5PW was from Millipore/Waters. HPLC TSK-3000, TSK gel, Type G3000SW was from TosoHaas, Montgomeryville, PA.

Labeling of Human Pro-MSP with 125I-- Ten micrograms of pure recombinant human pro-MSP in 30 µl of 0.1 M borate buffer, pH 8.5, were added to 250 µCi of 125I-labeled Bolton-Hunter reagent and equilibrated on ice for 60 min. The reaction was terminated by addition of 300 µl of borate buffer containing 0.5 M glycine. After 10 min on ice the reaction mixture was applied to an Excellulose GF-5 desalting column (Pierce, Rockford, IL) that had been equilibrated with phosphate-buffered saline containing 0.25% gelatin. The iodinated protein was eluted with 1 ml of phosphate-buffered saline-gelatin buffer, and counted in gamma -counter (Gamma 5500, Beckman). The specific activity of the labeled protein was ~400 Ci/mmol.

Partial degradation of iodinated pro-MSP occurred during storage at -20 °C, as shown by the appearance on SDS-PAGE of a band that migrated to about the same position as the MSP alpha -chain and a band corresponding to small fragments (Fig. 2, lane C). To detect the capacity of the macrophage pro-MSP convertase to cleave pro-MSP to the alpha beta chain heterodimer, we used 125I-pro-MSP stored for a limited time (Fig. 4) or freshly prepared (Fig. 5).

Assay for Pro-MSP Cleavage by Macrophages-- Resident peritoneal macrophages were obtained from C3H/HeN male mice by lavage of the peritoneal cavity with DMEM containing 2% fetal bovine serum. Cells were washed twice and resuspended in DMEM without serum at a concentration of 8 × 105 total cells/ml. Wells of a 96-well microtiter plate (Costar 3596, Corning, Inc., Corning, NY) were loaded with 200-µl volumes of cell suspension. After 30 min for cell settling and adherence, well fluid was replaced with 50 µl of DMEM containing 1 mg/ml nuclease-protease-free bovine serum albumin, about 150 fmol of 125I-pro-MSP, and fractions being tested for inhibition of pro-MSP cleavage by macrophages. The plate was incubated at 37 °C for 3-4 h. Proteins of the well fluids were separated by SDS-PAGE under reducing conditions on a polyacrylamide gel. MSP, pro-MSP, and Bio-Rad pre-stained protein standards were also applied to the gel as reference markers. Radioautographs of the dried gel were made to determine the presence of 125I-pro-MSP cleavage products.

For determination of morphological effects of pro-MSP cleavage products, murine resident peritoneal cells in DMEM were distributed into wells of a 48-well tissue culture plate (400,000 cells/well). After incubation at 37 °C for 1 h, well fluids were replaced by 400 µl of DMEM containing 3 nM MSP as a positive control or 5 nM pro-MSP with protease inhibitors being tested. The plate was incubated for 3-4 h, and then the adherent cells were stained with LeukoStat for microscopic evaluation and photography.

Protease Inhibitor Activity in Mouse Peritoneal Fluid-- Into the peritoneal cavity of a series of mice, we injected 4 ml of 0.15 M NaCl with or without 4 × 10-5 M serotonin (5HT). After an interval of 1 min for the NaCl mice and 30 min for the 5HT mice, the animals were killed by cervical dislocation and fluid was aspirated from the peritoneal cavity. The recovered peritoneal cavity washout fluids were concentrated by ultrafiltration. A280 was measured before and after ultrafiltration, and fluids were tested for capacity to affect the pattern of 125I-pro-MSP cleavage by mouse peritoneal macrophages. After incubation, reaction products were resolved by SDS-PAGE and visualized by radioautography.

To estimate the volume of fluid in the peritoneal cavity of normal mice or 30 min after intraperitoneal serotonin, we injected 0.5 ml of 70,000-kDa [3H]dextran into a series of 3 mice each. After massage of the abdomen, the mice were killed and peritoneal fluid was aspirated and counted in a scintillation counter. From a comparison of the aspirate counts/min and the injected counts/min, we calculated that the mean peritoneal fluid volume was 140 ± 30lambda (S.E.) for normal mice and 900 ± 30lambda 30 min after intraperitoneal serotonin. The 900lambda figure for the serotonin-treated mice was obtained after subtraction of the injected serotonin volume. From the recovered A280 in the peritoneal cavity washouts and the pre-washout peritoneal fluid volumes, we calculated values for pre-washout A280.

Initial Steps to Isolate Inhibitor from Human Plasma-- To 200 ml of pooled outdated human blood plasma were added 53 g of (NH4)2SO4 to make a molarity of 1.5. The slurry was stirred at 20 °C for 30 min, and centrifuged at 25,000 × g for 30 min. The precipitate was discarded. To precipitate albumin from the supernatant, 0.3 N HCl was added until the pH was 4.1. The precipitate was removed by centrifugation, and the supernatant was ultrafiltered on an Amicon YM-10 filter from a volume of about 250 to 150 ml. The solution was dialyzed at 4 °C against water to remove the (NH4)2SO4 and then against 0.02 M, pH 7.1, sodium phosphate buffer.

Column Chromatography of Albumin-free Inhibitor-- The 150 ml of albumin-free inhibitor solution was applied to a 3 × 23-cm column containing 75 ml of Affi-Gel blue gel. After unbound protein was washed through the column, bound protein was eluted with 1.4 M NaCl. Total A280 of the pooled eluate was 26% of the applied A280. The 100-ml eluate was dialyzed against 0.02 M, pH 8.0, Tris buffer, and concentrated on an Amicon YM 10 and Centricon 10 filter to a volume of about 3 ml, and an A280 of 42. A 2.5-ml volume of this solution was applied to an HPLC DEAE column, and bound proteins were eluted with a NaCl gradient from 0 to 0.3 M in starting buffer over a period of 60 min. Fractions with inhibitor activity were pooled, ultrafiltered to a volume of 2.5 ml, dialyzed against 0.02 M, pH 8.0, Tris buffer, and rechromatographed on the HPLC DEAE column under the same conditions. In the final chromatography step, the peak DEAE inhibitor fraction was dialyzed against DMEM and applied to an HPLC TSK 3000 gel filtration column.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Initial Purification Steps-- A flow chart that outlines the initial steps in purification of the inhibitor is shown in Fig. 1. The rationale was to separate the bulk of the plasma proteins from the inhibitor prior to final purification. In the first step, about 70% of the total A280 was precipitated at a concentration of 1.5 M (NH4)2SO4, leaving the inhibitor and plasma albumin in the supernatant. Exploratory experiments showed that in two different high resolution fractionation trials, Affi-Gel blue dye-ligand chromatography and ion exchange chromatography, albumin, and the inhibitor behaved similarly. Therefore, albumin was removed by precipitation near its isoelectric point at pH 4.1, leaving the inhibitor in the supernatant. The inhibitor was then purified by sequential chromatography on Affi-Gel blue and HPLC DEAE columns as described under "Experimental Procedures."


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Fig. 1.   Flow chart for initial purification of inhibitor. Fractions with inhibitor activity are in the right leg of the flow chart.

Final Purification and Analysis-- A relatively large amount of protein was eluted from the HPLC DEAE column just prior to the inhibitor. To minimize admixture of this protein, pooled inhibitor fractions were equilibrated with starting buffer and re-run on the HPLC-DEAE column (DEAE-2). Proteins were eluted with a linear NaCl gradient. A range of fractions from 38 to 54, corresponding to a portion of the NaCl gradient from 0.15 to 0.23 M was assayed for capacity to inhibit proteolysis of 125I-pro-MSP by macrophages. SDS-PAGE and autoradiography after incubation with macrophages showed that proteolysis of pro-MSP was inhibited by fractions in the 44-48 range (Fig. 2).


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Fig. 2.   Purification of inhibitor on anion exchange column. Re-run of DEAE-1 inhibitor pool on HPLC DEAE column (DEAE-2). Assay of fractions for capacity to inhibit cleavage of pro-MSP by macrophages. After 125I-pro-MSP was incubated with murine macrophages, reaction products were resolved by SDS-PAGE in an 8% gel under reducing conditions. Arrows on the right point to pro-MSP, a 53-kDa marker, and small mass proteins not resolved on the 8% gel (F). Lane C, control incubation without macrophages. Macrophages caused disappearance of the pro-MSP band unless inhibitor was present in fractions. Fraction 44 was selected for final purification, illustrated in Fig. 3.

SDS-PAGE under reducing conditions of fractions 44-48 showed a major band at about 66 kDa and a number of less intense bands over the 14-97-kDa marker range (data not shown). To determine if the major band was the inhibitor, we applied DEAE-2 fraction 44 to an HPLC TSK-3000 gel filtration column. Fig. 3 shows maximal inhibitor activity in fractions 35 and 36, which corresponded to a protein peak that eluted at the position of serum albumin (68 kDa). After SDS-PAGE of the peak fractions, the gel was lightly stained with Coomassie Blue to locate the 68-kDa band (data not shown). This was repeated with a pool of fractions 35-37, the protein band was transferred to Immobilon P, and a piece of membrane corresponding to the 68-kDa band was cut out for N-terminal sequencing. The sequence of the first 15 N-terminal residues (NSPLDEENLTXENXD) had 87% identity to a 15-residue sequence of human alpha 1-antichymotrypsin. Travis et al. (24) reported that the apparent molecular mass of purified human alpha 1-antichymotrypsin on SDS-PAGE gels was 68 kDa.


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Fig. 3.   Final purification of inhibitor. Gel filtration of DEAE-2 inhibitor fraction 44 on an HPLC TSK-3000 column. Assay of fractions for capacity to inhibit cleavage of 125I-pro-MSP by macrophages. SDS-PAGE in an 8% gel under reducing conditions. Maximal inhibitor activity was detected in the A280 elution peak, which was at the serum albumin marker location (fraction 35). Densitometry values in relative units for the pro-MSP band in fractions 33-39 were 3, 16, 21, 21,18, 16, and 13, respectively.

alpha 1-Antichymotrypsin Preferentially Inhibits the Macrophage Pro-MSP Degrading Enzyme-- The inhibition of macrophage pro-MSP cleavage by alpha 1-antichymotrypsin is shown in Fig. 4. After a control incubation of 125I-pro-MSP without cells for 180 min, SDS-PAGE under reducing conditions shows by autoradiography a predominant 78-kDa band that represents uncleaved pro-MSP (Fig. 4, lane 6). The small band below it reflects a pro-MSP degradation product that occurs on storage of the iodinated protein. In all other lanes, which are autoradiographs of pro-MSP after incubation with macrophages, the two bands of interest are at 53 and 46 kDa. The 53-kDa band corresponds to the alpha  chain of MSP, generated by the macrophage pro-MSP convertase; the 46-kDa band is a fragment generated by the macrophage pro-MSP degrading enzyme (22). Without inhibitors of the macrophage enzymes, the 46-kDa degradation product band is more intense than the 53-kDa MSP alpha -chain band (Fig. 4, lane 1). STI, a selective inhibitor of the degrading enzyme, allows exclusive cleavage by the macrophage pro-MSP convertase to MSP (Fig. 4, lane 2, 53 kDa band). The action of 3 different concentrations of alpha 1-antichymotrypsin is shown in Fig. 4, lanes 3-5. Lane 3 shows detectable inhibitor action at a concentration of 0.07 µM as shown by an increase, compared with lane 1, in the 78- and 53-kDa bands and a decrease in the 46-kDa band and small fragments (F). These changes are more pronounced for 0.4 µM alpha 1-antichymotrypsin (lane 4). At a concentration of 5 µM, alpha 1-antichymotrypsin almost completely inhibited both enzymes, as shown by the large amount of uncleaved pro-MSP (lane 5) and a band pattern comparable to the incubation control (lane 6).


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Fig. 4.   Effect of STI and ACT on cleavage of pro-MSP by macrophages. After 125I-pro-MSP was incubated with murine macrophages, reaction products were resolved by SDS-PAGE in a 10% gel under reducing conditions. Lane 6, 180-min incubation control without cells; all other lanes were from 180-min incubations with macrophages. Lane 1, no inhibitor. Lanes 3-5, 0.07, 0.4, and 5 µM alpha 1-antichymotrypsin, respectively. Lane 2 (from a different gel of the same experiment), 5 µM STI. Molecular mass markers are on the left. F, small fragments.

Macrophage Protease Inhibitor Activity in Mouse Peritoneal Fluid-- The above data show that over the concentration range from 0.07 to 5 µM alpha 1-antichymotrypsin preferentially inhibited the macrophage pro-MSP degrading enzyme at the low end of the range and completely inhibited both the degrading enzyme and the convertase at 5 µM, a value that approaches the mean human serum concentration of 7 µM (25). Thus, the effect of this inhibitor on macrophage proteolytic activity in extravascular sites will be critically dependent on its concentration. Using the peritoneal cavity as an accessible extravascular site, we assayed inhibitor activity of peritoneal fluid from normal mice and from mice treated with intraperitoneal serotonin to induce a moderate increase in vascular permeability. The results are shown in Fig. 5. Lane 1 shows almost complete degradation of 125I-pro-MSP by macrophages in medium alone. Three percent mouse serum (A280 = 1.2) is in the partial inhibition range, which reveals cleavage by the convertase to MSP (53 kDa line) and by the degrading enzyme to the 46-kDa product (lane 2). Fluid washed out of the peritoneal cavity and ultrafiltered to an A280 of 2.2 (lane 3) also allowed partial cleavage to MSP (53 kDa line). Lane 4 shows the effect of concentrated (A280 6.1) peritoneal fluid from serotonin-treated mice. It minimized activity of the pro-MSP degrading enzyme (note low amount of the fragment band) and favored the action of the convertase as shown by the relative sizes of the 53- and 46-kDa bands.


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Fig. 5.   Effect of peritoneal fluid from normal or serotonin-treated mice on cleavage of pro-MSP by macrophages. Into the peritoneal cavity of a series of mice, we injected 4 ml of 0.15 M NaCl with or without serotonin (5HT) as described under "Experimental Procedures." The recovered fluids were concentrated by ultrafiltration and tested for capacity to affect the pattern of 125I-pro-MSP cleavage by mouse peritoneal macrophages. After incubation, reaction products were resolved by SDS-PAGE and visualized by autoradiography. Macrophage incubation medium composition was as follows. Lane 1, diluent. Lane 2, 3% mouse serum. Lane 3 (from a different gel), normal peritoneal fluid, A280 2.2. Lane 4, 5HT peritoneal fluid, A280 6.1. Lane 5, 125I-pro-MSP incubated without cells. Molecular mass markers are on the left. F, small fragments.

Inhibition of the Macrophage Pro-MSP Degrading Enzyme Allows Sufficient Cleavage of Pro-MSP to MSP to Stimulate Macrophage Shape Change-- Pro-MSP, with or without protease inhibitors, or MSP as a positive control, was added to murine peritoneal macrophages in tissue culture wells, and the cells were periodically observed by phase microscopy during a 4-h incubation at 37 °C. In the presence of pro-MSP without protease inhibitors (Fig. 6A), macrophages did not change their rounded morphology, characteristic for cells in DMEM alone or DMEM and protease inhibitors without pro-MSP (not shown). MSP induced macrophages to become elongated in shape (Fig. 6B). Similar macrophage morphology was observed in wells with pro-MSP in the presence of STI (Fig. 6C) or alpha 1-antichymotrypsin (Fig. 6D). These findings correlate with the data in Fig. 4, showing that inhibition of the degrading enzyme by STI or alpha 1-antichymotrypsin resulted in conversion of pro-MSP to MSP.


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Fig. 6.   Morphology of macrophages in the presence of MSP, pro-MSP, or pro-MSP plus protease inhibitors. After 4 h in culture, cells were stained and observed by phase microscopy with a ×40 objective. A, 5 nM pro-MSP; B, 3 nM MSP; C, 5 nM pro-MSP and 5 µM STI; D, pro-MSP and 0.4 µM alpha 1-antichymotrypsin.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

We reported that murine resident peritoneal macrophages have two cell surface proteolytic activities that can cleave pro-MSP. One cleaves pro-MSP to active MSP, the other degrades pro-MSP (22). The degrading protease was inhibited by STI and by normal human or mouse serum. It was not inhibited by a number of plasma protease inhibitors, including alpha 1-antitrypsin, C1-inhibitor, anti-thrombin III, and alpha 2-macroglobulin (22). We have now shown that the inhibitor in human plasma is alpha 1-antichymotrypsin. It is therefore probable that the macrophage pro-MSP degrading enzyme is a serine protease with chymotrypsin or cathepsin-G-like substrate preferences, which cleaves at sites with aromatic or large aliphatic residues. Inasmuch as the initial major macrophage inactivating cleavage product of pro-MSP is a 46-kDa fragment (Ref. 22 and Fig. 4, lane 1), our results suggest that this fragment is formed by cleavage at Phe418, which would yield a polypeptide with a calculated mass of 45.7 kDa. Phe418 is located within the triple disulfide loop structure of kringle 4, and therefore release of the fragment would require reduction. Consistent with this prediction, the amount of 46-kDa fragment was minimal after SDS-PAGE under nonreducing conditions (data not shown).

Inhibition of the macrophage pro-MSP degrading enzyme by STI (Fig. 4, lane 2) led to the expectation that the degrading enzyme would be a trypsin-like serine protease. It was thus a surprise to find that the serum inhibitor of the enzyme is alpha 1-antichymotrypsin. A review of the literature showed that STI can bind and inhibit chymotrypsin as well as trypsin. In fact, the inhibitor has two binding sites, one for trypsin or chymotrypsin and another for chymotrypsin (26).

Since the macrophage pro-MSP convertase generates active MSP, the cleavage site is expected to be Arg483-Val484, the alpha beta chain junction (18). This is a preferred cleavage site for trypsin-like serine proteases. However, the inhibitor profile for the macrophage pro-MSP convertase is unusual, in that it is not inhibited by various inhibitors of trypsin-like serine proteases, including leupeptin, aprotinin, FPR-chloromethylketone, FFR-chloromethylketone (data not shown), and STI. And it is completely inhibited, along with the pro-MSP degrading enzyme, by 5 µM alpha 1-antichymotrypsin, a preferential inhibitor of chymotrypsin-like proteases. Purification and characterization of the macrophage pro-MSP convertase would therefore be of interest. It is possible that the convertase and the degrading activities reflect dual specificities of a single protein. An example is human cathepsin G, which has both trypsin- and chymotrypsin-like specificities (27), and binds both trypsin and chymotrypsin inhibitors (28). In addition to its well known location in leukocyte azurophilic granules, cathepsin G with dual specificity has been detected on cell membranes isolated from the U-937 human promonocytic cell line (29). These cells, with or without the maturational stimulus of phorbol esters, had no protease activity for pro-MSP (data not shown).

Like the coagulation factors from which MSP is thought to have evolved (19), regulation of its activity depends on proteolytic cleavage of the single chain precursor pro-MSP, which is present in human circulating blood at a concentration of 5 nM (20), about 20 times the EC50 of 0.25 nM (1). If pro-MSP and alpha 1-antichymotrypsin diffuse from the circulation into the extravascular space, inhibition of pro-MSP degradation by tissue macrophages might allow the macrophage pro-MSP convertase to generate active MSP, which could act on cells expressing the RON MSP receptor, including macrophages themselves as well as epithelial cells (30).

Using the murine peritoneal cavity as an example of extravascular space, we showed that peritoneal fluid from normal or serotonin-treated mice could preferentially inhibit the macrophage pro-MSP degrading enzyme and allow cleavage by the convertase to MSP (Fig. 5, lanes 3 and 4). From the A280 of peritoneal washouts and determination of pre-washout peritoneal fluid volume by radioactive dextran dilution (as described under "Experimental Procedures"), we estimated that the mean A280 in peritoneal fluid of normal and serotonin-treated mice was 6.8 ± 0.6 and 4.4 ± 0.3, respectively, for a series of three mice each. The A280 of the fluids tested in the experiment illustrated in Fig. 5 was 2.2 for lane 3 and 6.1 for lane 4. These data suggest that in both normal mice and mice with a moderate increase in vascular permeability, the concentration of inhibitor in this extravascular space is in the range that can inhibit proteolytic degradation of pro-MSP by macrophages, and allow generation of MSP by the pro-MSP convertase.

In contrast, since the concentration of plasma proteins in acute, severe inflammatory exudates approaches that of proteins in the circulation, the mean alpha 1-antichymotrypsin concentration of 7 µM (25) is in the range that completely inhibits macrophage pro-MSP convertase activity (Fig. 4, lane 5). Mature MSP is present in these exudates (20), generated by a unique fluid phase pro-MSP convertase, purification of which is in progress. In contrast to the exudate fluid phase convertase, the potential significance of the macrophage pro-MSP convertase may be to generate MSP locally to act on cells expressing the RON receptor under physiological conditions, in the absence of inflammation.

    ACKNOWLEDGEMENTS

We are grateful to Dr. David Leonard for useful discussions about estimations of peritoneal fluid volume and protein concentration, Dr. Philip Askenase for suggesting the use of serotonin for increasing vascular permeability in the mouse, and Dr. Alla Danilkovitch for her computer software wizardry.

    FOOTNOTES

* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed. Tel.: 301-846-1560; Fax: 301-846-6145; E-mail: leonarde@mail.ncifcrf.gov.

Published, JBC Papers in Press, March 23, 2001, DOI 10.1074/jbc.M100652200

    ABBREVIATIONS

The abbreviations used are: MSP, macrophage stimulating protein; STI, soybean trypsin inhibitor; PAGE, polyacrylamide gel electrophoresis; AEBSF, 4-(2-aminoethyl)benzenesulfonyl fluoride; HPLC, high performance liquid chromatography; DMEM, Dulbecco's modified Eagle's medium.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1. Skeel, A., Yoshimura, T., Showalter, S., Tanaka, S., Appella, E., and Leonard, E. (1991) J. Exp. Med. 173, 1227-1234[Abstract]
2. Yoshimura, T., Yuhki, N., Wang, M.-H., Skeel, A., and Leonard, E. J. (1993) J. Biol. Chem. 268, 15461-15468[Abstract/Free Full Text]
3. Leonard, E. J. (1997) in Plasminogen-related Growth Factors (Bock, G. R. , and Goode, J. A., eds) , pp. 183-191, John Wiley & Sons, New York
4. Leonard, E. J., and Skeel, A. (1976) Exp. Cell Res. 102, 434-438[Medline] [Order article via Infotrieve]
5. Wang, M.-H., Cox, G. W., Yoshimura, T., Sheffler, L. A., Skeel, A., and Leonard, E. J. (1994) J. Biol. Chem. 269, 14027-14031[Abstract/Free Full Text]
6. Correll, P. H., Iwama, A., Tondat, S., Mayrhofer, G., Suda, T., and Bernstein, A. (1997) Genes and Funct. 1, 1-15
7. Danilkovitch, A., and Leonard, E. J. (1999) J. Leukocyte Biol. 65, 345-348[Abstract]
8. Ronsin, C., Muscatelli, F., Mattei, M. G., and Breathnach, R. (1993) Oncogene 8, 1195-1202[Medline] [Order article via Infotrieve]
9. Wang, M.-H., Ronsin, C., Gesnel, M.-C., Coupey, L., Skeel, A., Leonard, E. J., and Breathnach, R. (1994) Science 266, 117-119[Medline] [Order article via Infotrieve]
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