 |
INTRODUCTION |
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
and
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
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
and
chains by specific serine proteases at an
Arg-X bond (17), in which X is most frequently
Val. The 
chain junction of MSP is
Arg483-Val484 (18). The
chain
of MSP is homologous to the
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
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 
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
1-antitrypsin, C1-inhibitor, anti-thrombin III, and
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 |
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.
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
-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
-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 
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 ± 30
(S.E.) for
normal mice and 900 ± 30
30 min after intraperitoneal
serotonin. The 900
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 |
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."

View larger version (16K):
[in this window]
[in a new window]
|
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).

View larger version (43K):
[in this window]
[in a new window]
|
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
1-antichymotrypsin. Travis et al. (24)
reported that the apparent molecular mass of purified human
1-antichymotrypsin on SDS-PAGE gels was 68 kDa.

View larger version (70K):
[in this window]
[in a new window]
|
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.
|
|
1-Antichymotrypsin Preferentially Inhibits the
Macrophage Pro-MSP Degrading Enzyme--
The inhibition of macrophage
pro-MSP cleavage by
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
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
-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
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
1-antichymotrypsin (lane
4). At a concentration of 5 µM,
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).

View larger version (85K):
[in this window]
[in a new window]
|
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
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
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.

View larger version (121K):
[in this window]
[in a new window]
|
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
1-antichymotrypsin (Fig. 6D). These findings
correlate with the data in Fig. 4, showing that inhibition of the
degrading enzyme by STI or
1-antichymotrypsin resulted
in conversion of pro-MSP to MSP.

View larger version (135K):
[in this window]
[in a new window]
|
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
1-antichymotrypsin.
|
|
 |
DISCUSSION |
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
1-antitrypsin, C1-inhibitor, anti-thrombin III, and
2-macroglobulin (22). We have now shown that
the inhibitor in human plasma is
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
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 
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
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
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
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.