Detection of granuloma-associated plasminogen activator in experimental murine schistosomiasis

Detection of granuloma-associated plasminogen activator in experimental murine schistosomiasis

EXPERIMENTAL AND MOLECULAR PATHOLOGY 43, 82-89 (1985) Detection of Granuloma-Associated Plasminogen Experimental Murine Schistosomiasis S. IzAKI,...

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EXPERIMENTAL

AND

MOLECULAR

PATHOLOGY

43, 82-89 (1985)

Detection of Granuloma-Associated Plasminogen Experimental Murine Schistosomiasis S. IzAKI,‘~*

L. HORNBECK,] M. HIGUCHI,~ K. FUKUYAMA,~ S. TOKAIRIN,* AND 0. MATSUO~

‘Department of Dermatology, University of California School California 94143; 2Department of Dermatology, Iwate Medical Morioka, Iwate, Japan; and 3Department of Physiology, Kinki Sayama, Osaka, Japan Received

November

Activator

in

W. L. EPSTEIN,’

of Medicine, San Fruncisco, University School of Medicine, University School of Medicine,

5, 1984

Hypersensitivity granulomas induced by infection with Schistosoma mansoni were isolated from the livers of BALBic mice after 7. 8, 10, and 12 weeks. The parasite egg-granulomas were sequentially extracted with a Tris-buffered saline (soluble fraction) and 2 M KSCN (bound fraction). Fibrinolytic enzyme activity measured with both synthetic substrates and fibrin plates demonstrated an elevated level of plasminogen activator activity in the bound fraction 7-8 weeks after infection when mature granulomas first began to appear, followed by a gradual decrease lo-12 weeks after infection. An electrophoretic enzymography technique revealed multiple molecular species of plasminogen activator at M, = 95K. 74K, 6OK, 45K, and 24K. The bands with M, = 45K and 24K were found compatible with the electrophoretic pattern of macrophage-plasminogen activator. When the granulomas reached maximum size after 10 to 12 weeks, the plasminogen activator with 45K and 24K diminished, while plasminogen activator activity at M, = 95K, 74K, and 60K remained unchanged suggesting the presence of both vascular and tissue types of plasminogen activators. There was no urokinase-type plasminogen activator detectable in granulomas at any time. In the soluble fraction no enzymatic activity was found, whereas regulating inhibitor activity for plasminogen activator was consistently detectable. 0 1985 Academic Prey. Inc.

INTRODUCTION Activation of clotting factors has been demonstrated in delayed hypersensitivity (Cohen er al., 1967; Colvin et al., 1973). We have previously shown that fibrin deposition and subsequent fibrin clearance occur during the development of hepatic granulomas in thymus intact mice with schistosomiasis, but not in congenital athymic mice (Izaki et al., 1979b). In addition, the presence of plasminogen activator (PA) was detected in a recent study of hypersensitivity-type murine lepromas (Izaki et al., 1983). However, early attempts at quantifying the fibrinolytic enzyme activity in schistosome egg-granulomas was unsuccessful, although elevation of inhibitor activity for urinary PA (urokinase) was associated with granulomatous inflammation (Izaki et al., 1979a, b). In this communication we report the extraction and partial characterization of PA of the hepatic granulomas of mice infected with Schistosomu munsoni. For this purpose we separated egggranulomas from the rest of the hepatic tissue (Moore et al., 1977) and sequentially extracted first the inhibitors of fibrinolysis and then PA. Changes in the PA activity, as well as inhibitor activity for urinary PA was measured. The PA extracted from granulomas (gPA) was further analyzed by an electrophoretic enzymography technique (Heussen and Dowdle, 1980) in order to compare gPA with urokinase-type (White et al., 1966), tissue-type (Rijken and Collen, 1981), vascular endothelial-type (Izaki and Kitaguchi, 1977), and macrophage-type (Unkeless ef al., 1974) PAS by molecular weight. 82 0014-4800/85 $3.00 Copyright All rights

0 198s by Academic Press. Inc. of reproduction in any form reserved.

GRANULOMA-PLASMINOGEN

MATERIALS

ACTIVATOR

83

AND METHODS

Infection and sequential extraction of egg-granulomas. Groups of 30 BALB/c mice weighing 20-25 g were infected with 50 cercariae of Schistosoma mansoni, NIH strain, freshly hatched from snails. Mice were sacrificed at 6, 7, 8, 10, and 12 weeks after infection. The livers obtained were perfused with two changes of 10 ml of 0.15 M NaCl from the portal vein at 4°C and homogenized with 10 ml/ g tissue of 0.05 M Tris-HCl + 0.1 M NaCl, pH 8.0 (Tris-saline), in a Waring blender for 60 set at low speed. The homogenate was settled in an ice bath. The floating liver tissue debris and hepatic cell extract were collected as the “granuloma-poor fraction” and separated from the sedimented egg-granulomas. The isolated egg-granulomas were washed three times with cold Tris-saline and used as the “granuloma-rich fraction.” Reliability of the fractionation technique was examined histologically (Nishimura et al., 1982). The granuloma-rich fraction was homogenized in 10 ml/g pellet of Tris-saline with a glass homogenizer. After centrifugation at 20,OOOg for 20 min at 4”C, the supernatant (soluble fraction) was removed and the pellet reextracted with 10 ml/g pellet of 2 M KSCN with a Polytron cell disruptor, for 30 set, three times, followed by freezing-thawing, three times. The supernatant (bound fraction) collected after ultracentrifugation at 105,OOOg for 30 min at 4°C was dialyzed against 0.05 M Tris-HCl + 1M NaCl, pH 8.0, at 4°C. The granuloma-poor fraction was centrifuged, and the pellet treated with 2 M KSCN under the same procedures was used as well, for comparison. Peritoneal macrophage culture. Mouse (BALB/c) peritoneal cells elicited by injection of Thioglycollate medium, (Brewers, Oxoid, Hants, England) were cultured on a petri dish (10’ cells/dish 5 cm in diameter) in 3 ml of Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% heat-inactivated fetal calf serum (FCS) (Unkeless et al., 1974). After 4 hr the adherent cells were incubated with DMEM + FCS for 24 hr and further with DMEM supplemented with 10% lactoalbumin hydrolysate (LH) solution (DMEM + LH). The culture medium was collected every 24 hr and stored frozen. Cells were collected by Days 3 or 4, washed, and sonicated in 1 ml of 0.02 M phosphate buffer + 0.14 M NaCl, pH 7.4 (PBS). Melanoma cell culture. A Bowes human melanoma cell line that secretes high level of PA (tissue-type PA) (Rijken and Collen, 1981; Matsuo et al., 1981) was cultured to confluency in Earle’s minimum essential medium (EMEM) supplemented with 0.12% NaHCO,, 2 mM glutamine, and 10% heat-inactivated FCS. The culture medium was changed to EMEM without FCS and collected after 24 hr. Vascular endothelial PA. A hind leg of dog was isolated under anesthesia and perfused from the femoral artery with Hanks’ solution, oxygenated with 5% oxygen + 95% air, pH 7.4, at 37°C by a pulsatile pump to replace the blood component. After 30 min acetylcholine or histamine (5 kg/ml at final concentration in Hanks’ solution) was infused for 1 hr. The perfusate in which PA activity was released (Izaki and Kitaguchi, 1977) was collected from the femoral vein, and condensed by lyophilization. Fibrinolytic enzyme and inhibitor assay. For calorimetric assay chromogenic peptide substrates (Friberger, 1982), L-pyroglutamyl-glycyl-L-arginine-p-nitroanilide (s-2444) (Kabi, Stockholm, Sweden) and N-o-valyl-L-leucyl-L-lysine-p-nitroanilide (s-2251) (Kabi) were used, since gPA has been found to show high

84

IZAKI

ET AL.

substrate specificity for both s-2444 and s-2251 (Izaki et al., 1984). All assays were done in duplicate. The tissue extract (0.1 ml) and 0.8 ml of 0.1 M Tris-HCl, pH 8.0 (Tris-buffer) were mixed with 0.05 ml of 4 mM s-2444 or 6 mM s-2251 and incubated at 37°C for 60 min. Reaction was stopped by the addition of 0.05 ml of acetic acid and precipitated materials were removed by centrifugation at 3OOOg for 15 min. Absorbance was read at 405 nm for freed p-nitroaniline (E = 10,500). For a fibrinolysis assay, a fibrin plate method (Astrup and Mtillertz, 1952) was used. Bovine fibrinogen (Miles Laboratories, Elkhard, Ind.) (48 mg clottable protein/8 ml 0.01 M borate buffer + 0.14 M NaCl, pH 7.5) with or without rat or dog plasminogen purified by a lysine-Sepharose column technique (Deutsch and Mertz, 1970) was clotted in petri dishes, 8.5 cm in diameter, with 0.05 ml of 20 NIH U/ml Pentex bovine thrombin (Miles). A drop of 20 r~.l tissue extract was placed on the plate and incubated at 37°C for 18 hr. The lysed area was measured and converted to urokinase activity (Green Cross, Osaka). PA inhibitor activity of the tissue extract was determined by measuring colorimetrically the inhibition of urokinase activity using s-2444, since urokinase inhibitor in the granulomatous tissue extract showed regulating function for gPA in murine leprosy (Izaki et al., 1984). Tissue extract (0.1 ml) was mixed with 0.75 ml of Tris-buffer and 0.05 ml of 120 IU/ml urokinase. After 60 min at room temperature 0.05 ml of 4 mM s-2444 was mixed and incubated at 37°C in a water bath for 30 min. Reaction was stopped by 0.05 ml acetic acid. The decrease in level of liberation of p-nitroaniline (nmole/min) was calculated (Izaki et al., 1983). Protein concentration of each fraction was assayed with Lowry’s method (Lowry et al., 1951) and enzymatic and inhibitor activity was expressed as the activity per milligram protein. Electrophoretic enzymography. The method developed by Heussen and Dowdle (1980) was applied. Polyacrylamide gel (10 or 14%) was copolymerized with 0.12 mg of rat or dog plasminogen and 0.1% (w/v) a-casein (Sigma) in TrisHCl buffer, pH 8.8, containing 0.1% sodium dodecyl sulfate (SDS). A gel without plasminogen was also prepared. The gel plates sized 50 x 85 x 1 mm were overlayed by 3% polyacrylamide stacking gel containing 0.1% SDS, pH 6.8. Protein samples, dialyzed and lyophilized, were resolved in 5% SDS, 2% sucrose, and 8 kg/ml phenol red. Approximately 0.04 IU of PA activity was applied on the stacking gel. Electrophoresis was carried out in a Micro Slab Gel Electrophoresis Apparatus (Marysol Ind., Co., Tokyo) with Tris-glycine buffer, pH 8.3, containing 0.1% SDS, in a cold room, as described by Laemmli (1970). The current was set at 6 mA in the stacking gel and 12 mA in the separation gel. After electrophoresis, the gel was immersed in two changes of 2.5% Triton X-100 with gentle shaking at room temperature for 2 hours to remove SDS. The gel was then incubated in a bath containing 0.1 M Glycine-NaOH buffer, pH 8.3, at 37°C for 24 hr. The gel was fixed in 50% trichloroacetic acid for 1 hr and 7% acetic acid for 1 hr, and stained with Coomassie brilliant blue. RESULTS gPA Activity

The granuloma-rich fraction was collected from homogenates of livers older than 7 weeks after infection, when granuloma development was identifiable histologically. Percentage granuloma weight [(weight of granuloma-rich fraction/ weight of whole liver) x 1001 increased rapidly between 7 weeks [9.6 ? 1.5%

GRANULOMA-PLASMINOGEN

ACTIVATOR

85

(mean + SE) of four to six animals] and 8 weeks (34.7 + 1.6%) after infection and plateaued at 10 weeks (40.7 + 2.8%) and 12 weeks (38.7 ? 3.1%) after infection. Hydrolytic activities for s-2251 and s-2444 were detectable in ‘I-weekold granulomas (Fig. 1). In the soluble fraction the activities were noticeably lower than in the bound fraction throughout the period of observation and the activities decreased promptly as granulomas enlarged. In the bound fraction the fibrin plate assay with plasminogen confirmed the PA activity, revealing a parallel relationship with the activity change that was measured using synthetic substrates. The gPA activity peaked during the early stage of granuloma development in size between 7 and 8 weeks after infection, and diminished when granulomas reached a plateau level in size. Fibrin plates without plasminogen did not demonstrate lytic activity (data not shown). In contrast, both the soluble and bound fractions in the granuloma poor fraction showed low-grade hydrolytic activities for the two synthetic substrates (Table I) but no fibrinolytic activity was detectable regardless of plasminogen content in the fibrin plates. Inhibitor for PA Activity Inhibitor activity for urokinase was shown mainly to localize in the soluble fraction of isolated granulomas (Fig. 2). A high level of the inhibitor activity was detected after 7 weeks, and it decreased after 12 weeks of infection. A low level of the inhibitor activity was observed between 8 and 12 weeks after infection in the bound fraction. Additionally, the soluble fraction of granuloma-poor liver extract demonstrated a weak but constant level of PA inhibitor activity (Table I), except 6 weeks after infection. In the bound granuloma-poor fraction no inhibitor activity for PA was seen before infection, whereas a weak level of PA inhibitor activity was observed when the inflammation was elicited. Electrophoretic

Enzymography

of gPA and Other PAS

In 14% polyacrylamide gel without plasminogen, weak proteolytic activity was demonstrated between 7 and 10 weeks after infection at M, = 76K, and 61K (Fig.

6

7

a Weeks after

10

12

Infection

FIG. 1. Activity of gPA measured in the soluble and bound fractions of isolated egg-granulomas using s-2251 (a) and s-2444 (H), and fibrin plates with plasminogen (pig) (column). The results are means f SE of samples of four to six mice.

86

IZAKI ET AL. TABLE I Enzyme and Inhibitor Activities in Granuloma-Poor

Fraction

Weeks after infection 0"

6

8

12

Enzyme activityb Soluble fraction s-2444

0.19

1 Bound fraction s-225

s-2444 s-225

1

0.15 2 0.02 0.28 2 0.01

rtr 0.02'

0.31 ? 0.02

0.14 5 0.04

0.15 * 0.02 * 0.01

0.33

2

0.05

0.33

0.04 0.08

+ 0.02 !z 0.05

0.10 f 0.02 k 0.29

0.01 0.06

f f

0.03 0.02

0.03

0.23

2 0.09

0

0.25

+

0.14

0.51 z!z 0.12

0.40

0.10 * 0.11 2 0.02

Inhibitor activity for PAd Soluble fraction Bound fraction 0

0.12 2 0.09

0.19 2 0.16

0.16 “_ 0.15

a Noninfected mouse livers. b nmole/min/mg protein. c Means 2 SE of four to six animals. d nmole/min/mg protein.

3), however, the detection of activity was inconsistent. In the gel with plasminogen, bands representing PA activity of M, = 95K, 74K, 60K, 45K, and 24K were demonstrated after 7 weeks of infection. It was found that the PA with M, = 24K diminished after 8 weeks, and the PA with M, = 45K decreased after 10 weeks. In 10% polyacrylamide gel with plasminogen, the gPA obtained at 7 weeks after infection showed PA activity at positions of M, = 95K, 74K, 60K, 45K, and 24K (Fig. 4), as well. Culture media and cell lysates of mouse peritoneal macrophages showed PA activity at 45K and 24K, identical positions with the two species of

A,

Soluble

Weeks

after

Fraction

Infection

FIG. 2. Inhibitor activity for urinary PA in the soluble (0) and bound (m) fractions of isolated egggranulomas. The results are means 2 SE of four to six animals.

GRANULOMA-PLASMINOGEN

WA 7~

aw

uPA

YPA 1OW

87

ACTIVATOR

7~

aw

HMW LMW

low

95k 74k 60k 45k 24k

without

with

pig

with

plg

plg

FIG. 3. Enzymographic pattern of the bound fraction of isolated egg-granulomas and urinary PA (UPA) in 14% polyacrylamide-SDS gels containing a-casein with and without plasminogen (pig).

gPA that modulated during the development of granulomas. Vascular endothelial PA demonstrated PA activity at 92K and 74K, while tissue-type PA secreted by cultured melanoma cells was detected at 65K. The urinary PA activity was found at 55K and 33K, representing high- and low-molecular-weight urokinase. No proteolytic bands were identified in the gel without plasminogen for the PA samples prepared from macrophages, vessel wall, melanoma cells, or urokinase (data not shown). DISCUSSION Detection of PA activity was biochemically achieved by the step-wise extraction that separated the enzyme activity in the bound granuloma-rich fraction, from PA inhibitor activity contained in the soluble granuloma-rich fraction, as well as @A

niPAl

niPA

7W

cul cell imed lys

cul med

with

vPA

tPA

UPA

cul lmcd

HMW LMW

plg

FIG. 4. Comparison of enzymographic pattern of gPA obtained 7 weeks after infection with different lots of macrophage PA (mPA, and mPA2) from both culture medium (cul med) and cell lysate (cell lys), vascular endothelial PA (vPA), tissue-type PA @PA) from melanoma cell culture medium and high- and low-molecular-weight (HMW and LMW) urinary PA (UPA) in a 10% polyacrylamide-SDS gel with cu-casein and plasminogen (pig).

88

IZAKI ET AL.

the soluble and bound granuloma-poor fractions. The gPA activity was shown to be elevated during the early stages of granuloma development, between 7 and 8 weeks after infection. The modulation in the gPA activity is compatible with our previous immunohistochemical findings which showed fibrin clearance between 8 and 10 weeks after infection (Izaki et al., 1979b). The results of electrophoretic enzymography revealed that the gPA consists of several molecular species with M, = 95K, 74K, 60K, 45K, and 24K. The enzyme species with M, = 95K, 74K, and 60K similar to vascular and tissue-type PAS, respectively, remained unchanged as granulomas developed. However, PAS with M, = 45K and 24K which are molecular weight of macrophage PA diminished from older granulomas. The molecular weight of macrophage PA in the present study is consistent with previous results by Unkeless et al. (1974); [3H]diisopropyl fluorophosphate-labeled PA demonstrated M, = 48K and 28K in polyacrylamide gel electrophoresis. Data from culture studies of mouse peritoneal macrophages by Nogueira et al. (1977) and Gordon and Cohn (1978) have revealed that exposure to sensitized T lymphocytes stimulates secretion of PA in BCG and protozoa1 infections. We assume that activation of macrophages in situ in granulomatous inflammation induces secretion of PA that modulates further development of the inflammatory processes. The other species of gPA probably due to vascular endothelial and tissue-type PAS may be responsible for the basal level of PA activity in granulomatous tissue. However, the present study showed that involvement of urokinase-type PA is less likely. Further studies to characterize the gPA are in progress. Previous studies with murine schistosomiasis (Izaki et al., 1979b; and Goldstein et al., 1979) have shown that inhibitor activity for urokinase and plasmin also are associated with granulomatous inflammation. The urokinase inhibitor was found more specific for granulomatous inflammation, whereas plasmin inhibitor was detected in both infected and noninfected murine tissues. Since the present study disclosed that there is no urokinase-type PA in granulomas, the specificity of the inhibitor is required to be further investigated. However we consider that the inhibitor activity for urokinase may represent gPA inhibitor activity, since a recent study of murine leprosy (Izaki et al., 1984) demonstrated that urokinase inhibitor of granulomas inactivated gPA extracted from murine lepromas, as well as urokinase, in a time-dependent fashion. ACKNOWLEDGMENTS This study was supported by grants from the National Institute of Arthritis, Diabetes, Digestive and Kidney Diseases (AM31853), Minister of Education of Japan (58770751), and Iwate Medical University, Keiryokai Research Foundation.

REFERENCES ASTRUP, T., and M~LLERTZ, S. (1952). The fibrin plate method for estimating fibrinolytic activity. Arch. Biochem. Biophys. 40, 346-351. COHEN, S., BENACERRAF, B., MCCLUSKEY, R. T., and OVARY, 2. (1967). Effect of anticoagulants on delayed hypersensitivity reactions. .Z. Immunol. 98, 351-358. COLVIN, R. B., JOHNSON, R. A., MIHM, M. C., JR., and DVORAK, H. E (1973). Role of the clotting system in cell-mediated hypersensitivity. I. Fibrin deposition in delayed skin reactions in man. J. Exp. Med. 138, 686-698. FRIBERGER, P. (1982). Chromogenic peptide substrates: their use for the assay of factors in the fibrinolytic and the plasma kallikrein-kinin systems. Stand. J. C/in. Lab. Invest. 42, (Suppl. 162), 9-98.

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S. M., IZAKI, S, and EPSTEIN, W. L. (1979). Inhibition of plasminogen activator associated with chronic granulomatous inflammation. Thromb. Res. 16, 727-735. GORDON, S., and COHN, Z. (1978). Bacille Calrnette-Gukrin infection in the mouse. Regulation of macrophage plasminogen activator by T-lymphocytes and specific antigen. J. Exp. Med. 147, I1751187. HEUSSEN, C., and DOWDLE, E. B. (1980). Electrophoretic analysis of plasminogen activators in polyacrylamide gels containing sodium dodecyl sulphate and copolymerized substrates. Anal. Biochem. 102, 196-202. IZAKI, S., FUKUYAMA, K., and EPSTEIN, W. L. (1979a). Modulation of anti-thrombin and anti-fibrinolytic activities in tissue during the development of granulomas induced by Schistosoma mansoni. J. Reticuloendothel. Sot. 26, 507-514. IZAKI, S., GOLDSTEIN, S. M., FUKUYAMA. K., and EPSTEIN, W. L. (1979b). Fibrin deposition and clearance in chronic granulomatous inflammation: Correlation with T-cell function and proteinase inhibitor activity in tissue. J. Invest. Dermutol. 73, 561-565. IZAKI, S., HIBINO, T., ISOZAKI. Y., Hsu, P. S.. IZAKI, M., and MATSUO, 0. (1984). Plasminogen activator and plasminogen activator inhibitor associated with granulomatous inflammation: A study with murine leprosy. Thromb. Haemostas. 52, 243-249. IZAKI, S., ISOZAKI, Y.. SATOH, M., HIBINO. T., KON, S., and IZAKI, M. (1983). Comparative study with two polar types of murine leprosy: An involvement of plasminogen activator and its possible regulating factor in the granulomatous tissue reaction. J. Invest. Dermatol. 80, 81-95. IZAKI, S., and KITAGUCHI. H. (1977). Calcium dependent and independent release of plasminogen activator from the vascular wall. Thromb. Res. 10, 765-770. LAEMMLI, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227, 680-685. LOWRY, 0. H., ROSEBROUGH,N. J., FARR, A. L., and RANDALL, R. J. (1951). Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265-275. MATSUO, 0.. RIJKEN, D. C., and COLLEN, D. (1981). Comparison of the relative fibrinogenolytic. fibrinolytic and thrombolytic properties of tissue plasminogen activator and urokinase in vitro. Thromb. Haemostas. 45, 225-229. MOORE, D. L., GROVE, D. I., and WARREN, K. S. (1977). The Schistosoma mansoni egg granuloma: Quantitation of cell populations. J. Pathol. 121, 41-50. NISHIMURA, M., EPSTEIN, W. L., and FUKUYAMA. K. (1982). Autotransplantation of hepatic granulomas into the skin of mice with Shistosoma mansoni infection. J. Invest. Dermatol. 79, 153-157. NOGUEIRA, N., GORDON, S., and COHN, Z. (1977). Trypanosoma cruzi: The immunologic induction of macrophage plasminogen activator requires thymus-derived lymphocytes. J. Exp. Med. 146, 172-183. RIJKEN, D. C., and COLLEN, D. (1981). Purification and characterization of the plasminogen activator secreted by human melanoma cells in culture. J. Biol. Chem. 256, 7035-7041. UNKELESS, J. C., GORDON, S., and REICH, E. (1974). Secretion of plasminogen activator by stimulated macrophages. J. Exp. Med. 139, 834-850. WHITE, W. F., BARLOW, G. H., and MOZEN, M. M. (1966). The isolation and characterization of plasminogen activator (urokinase) from human urine. Biochemistry 5, 2160-2169. GOLDSTEIN,