Life Sciences Vol . 19, pp . 1199-1210, 1976 . Printed in the U .S .A .
Pergamon Press
A ROLE OF CATHEPSIN BI * IN POLYMORPHONUCLEAR LEUKOCYTES CHEMOTA%IS Kazu Kobayashi, Yoko Endo, Keiichi Matsuda, Kiichiro Tanaka and Eiichi Misaka Central Research Laboratories, Sankyo Co ., Ltd ., Shinagawa-ku, Tokyo, 140 (Received in final form August 30, 1976)
Summary Cathepsin BÎ was purified from rat liver lysosomal fraction by ammonium sulfate fractionation, followed by chromatography on Sephadex G-200 and DEAF-Sephadex . Formation of chemotactic factor for guinea pig polymorphonuclear (PMN) leukocytes was demonstrated in vitro when guinea pig serum was incubated with cathepsin HI . This factor formation was dependent on SH-reagent dithiothreitol (DTT) and was maximal at pH 6 .0 . ZnS04, an inhibitor of cathepsin BI, inhibited the chemotactic factor formation likewise . Lysosomal enzymes, cathepsin D in particular, may play an important role in the development of inflammatory reactions . Ali and Evens (1) and Sapolsky et al (2, 3) have demonstrated that cathepsin D activity increased in cartilage of osteoarthritic joints . Poole et al (4) have shown that the release of cathepsin D was seen in synovia adjacent cells and pannua tissue cells from rheumatoid patients . Thus, these authors suggest that cathepsin D may be an important autolytic enzyme responsible for the breakdown of joint cartilage . On the other hand, Muirden and Phillips (5) have reported the increased cathepsin D activity in the articular cartilage in the experimental arthritis induced by intra-articular injection of filipin in rabbit, and Lazarus (6) has also shown that cathepsin D activity increased in turpentin-induced inflammatory skin . As another role of tissue proteases in inflammatory processes, there are many reports concerning their participation in producing PMN leukocytes chemotactic factors . Chemotactic factor formation from complements by some tissue proteases was reported (7-10) . However the enzymes used in these investigations are not purified ones . There are also cases using purified enzymes for demonstrating production of chemotactic factors . Hayashi et al (11-13) purified inflammatory SH-dependent protease from Arthus-type akin lesion, and demonstrated to form leukoegresin, a chemotactic factor, from IgG at neutral pH . Snyderman et al (14) reported the fact that the purified acid protease from macrophages was also capable to give some chemotactic factors from complement . In these cases, however, enzymes used were unclear of their * Benzoyl-DL-arginine-p-nitroanilide hydrolyzing enzyme (cathepsin Bl) . 1199
1200
PHId-Chemotaaia and Cathapain B1
Vol . 19, No . 8
subcellular localizations . Apart from chemotactic factor ßormation, Boyle et al (15) reported that purified cathepsin D ßrom human rheumatoid synovium had chemotactic activity by itselß . The present report deals with the possible chemotactic ßactor formation by highly purified rat liver lysosomal cathepsin BI using guinea pig serum as substrate . Materials and Methods N-Carbobenzoxy-a-L-glutamyl-a-L-phenylalanine (substrate for cathepsin A) was obtained Brom the Protein Research Foundation, Osaka . Benzoyl-DL-arginine-4-nitroanilide (substrate ßor cathepsin BI) waa purchased Brom Merck, Darmstadt, and benzoyl-Largininamide (substrate ßor cathepsin B) from Fluke Chemische Fabrik, Bucks, Switzerland . Glycyl-tyrosinamide acetate (substrate ßor cathepsin C) was obtained Brom Sigma Chemical Co ., St . Louis, Mo ., and hemoglobin (substrate for cathepsin D) Brom Nutritional Biochemicals Co ., Cleveland, Ohio . DTT was purchased from Seikagaku Kogyo Co ., Tokyo, and sodium caseinate Brom Dißco Lab ., Detroit, Mich . Bovine serum albumin was obtained Brom Armour Pharmaceutical Co . Leupeptin was a generous gift from Dr . Umezawa of the Institute oß Microbial Chemistry, Tokyo . Peritoneal PMN leukocytes preparation . The peritoneal PMN leukocytes were prepared as described by Kakinuma (16) with a slight modification . Hartley male guinea pigs (300-350 g) were injected with 30 ml oß 1~ sterile casein in pyrogen-ßree saline eritoneally . Eighteen hours later, 30 ml oß heparinized saline ~6 units/ml) were introduced into the peritoneal cavity and the exudate withdrawn . The cells, 95~ of which were PMN leukocytes, were collected by centrißugation and suspendeg in 2~ bovine serum albumin-medium at a concentration oß 1 .5 x 10 cells/ml . Medium . Gey's medium was used with a slight modification . In order to stabilize pH in the chemotactic assay, phosphate bußßer (0 .02 M) was used substituting ßor NaHC0 3 . Enzyme ~renaration . Lysosomal cathepsins oß rat liver were prepared by the method of Matsuda and Misaka (17) and subnumbers of cathepsins (such as AI, AII, BI, BIIr etc .) correspond with their report . Lysosomal supernatant was fractionated by ammonium sulfate ßollowed by Sephadex G-200 chromatography . The mixéd ßraction oß cathepsin BI and DI on the gel-chromatography was further purified with DEAF-Sephadex column . One unit oß enzyme activity is deßined as that amount which produces 1 ~ mole oß reaction product per minute . Enzyme ease . Assays oß cathepsin activities were carried out according to the methods reported by Matsuda and Misaka (17) . Chemotactic ßactor ßormation . Unless otherwise stated, guinea pig serum used was heated at 56o ßor 30 min and was dialysed against 0 .001 M acetate bußßer pH 4 .5 ßor 18 hr . The re tentate was centrifuged at 20,000 x g ßor 20 min to remove precipitate and was diluted with 0 .01 M phosphate bußßer or with 0 .005 M acetate bußßer at desired pH . Chemotactic factor ßormation was carried out in an incubation mixture containing enzyme, heated serum and DTT (125 }iM) in a ßinal volume oß 1 .5 ~ml at acidic pH . Aßter 30 or 60 min incubation at 37 ° , the mixture was
Vol . 1201 19, No . 8 P2~T-Chemotaaie and Cathepein B1 adjusted to pH 7 .2 with cold medium (ßinal volume, 5 .0 ml) and then subjected to the chemotactic assay . As a blank ßor chemotactic assay, reaction mixture was adjusted to pH 7 .2 without the incubation step ßor chemotactic factor ßormation at acidic pH .
D C B
FIG . 1 Chemotactic chamber consists of two titanium compartments (A and D) separated by a micropore filter membrane (B) held in position by silicone rubber ring (C) and ring (E) . Chemôtactic activity assa . Chemotactic activity was determined in vitro by a modißied method of the micropore filter technique by Boyden (18) . The chemotactic chamber devised by Ward (19) was employed with a slight reßormation . A schematic drawing of the chamber is illustrated in Fig . 1 . A micropore ßilter (3 .0 Fore size, SSWP01300 ; Millipore Co ., New Bedßord, Mass .) was used to separate these two compartments . The lower compartment oß the chamber was filled with the test solution ßor chemotactic activity and the upper compartment was with 7 .5 x 10 5 PMN leukocytes . After an incubation ßor 3 hr at 37° in a moist atmosphere of 5~ C02 in air, the ßilter was removed, Yixed in methanol, stained with Harris' hematoxylin, dehydrated, and cleared in xylene . The number oß cells migrating through to the lower surßace of the ß}lter were counted microscopically ßor an unit field (10 x 0 .13 mm ) and the mean value of four ßilters was calculated .
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PI~Pi-Chemotaxis and Cathepein B 1
Vol . 19, No . 8
Chemotactic activity was expressed as this mean value of migrated cell numbers per unit after subtracting the value in blank . Results Requirement for cathepsin BI in formation of chemotactic factor . As cathepsin D has reported to participate in inflammatory reactions, a possible role of cathepsin D in chemotaxis was studied in the first place . In preliminary experiments, cathepsin DI fraction of Sephadex G-200 chromatography was found to form PMN leukocytes chemotactic factor with guinea pig serum as substrate . This fraction alone showed no chemotactic activity . However, cathepsin DI fraction was overlapped with cathepsin BI In order to know which fraction as was reported previously (17) . cathepsin namely BI or DI is responsible for formation of chemotactic factor with guinea pig serum as substrate, a further trial for separation of them was done . Namely, mixed fraction of BI and DI obtained in the Se hadex G-200 chromatography was concentrated with Diaflo membrane p (UM-2 ; Amicon Co ., Lexington, Mass .) and applied onto DEAF-Sephadex column . As shown in Fig . 2, these two enzymes were separated each other considerably . Then, cathepsin BI rich fraction (fraction No . 4) and cathepsin DI rich fraction (fraction No . 37) were tested for the formation of chemotactic factor with serum in various conditions . It is well known that cathepsin B is activated in the presence of SHreagents and cathepsin D is not . And also pH optimum of cathepsin B is around 6, while that of cathepsin D is more acidic range . Then, a series of experiments was carried out in the presence or absence of DTT at pH 6 .0 or 4,5 . OD~ s .o
NaCI (MI LO ~ O .I o .os
FIG . 2 DEAE-Sephadex chromatogram of lysosomal cathepsin BI and DI . Cathepsin DI fraction of Sephadex G-200 containing cathepsin BI was concentrated and applied to DEAF-Sephadex column (2 x 20 cm) in 0 .02 M sodium acetate, pH 5 .0, with a linear gradient of NaCl (0 .02 to 0 .1 M, total volume ; 800 ml, 10 ml~tube) .
PMN-Chemotaais aad Cathepain Bl
Vol . 19, No . 8
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1204
PMN-Chamotaxia and Cathapsin B1
Vol . 19, No . 8
As shown in Table I, an incubation of cathepsin BI with heated serum at pH 6 .0 caused a ~rominent chemotactic activity ßormation in the presence oß DTT , while cathepsin BI itselß showed practically no activity without substrate . Enzymation of cathepsin DI, on the other hand, demonstrated only weak activity ßormation in various conditions with the combination of pH and DTT addition . Chemotactic factor formation as _a function of dose _of serum and cathe sin _BI . Guinea pig 20),rabbit 21,2 and human serum 10,23 are reported to possess chemotactic âctivity . Migrated cell numbers in the case oß the ßresh guinea pi serum in our experiment was linear with the amount oß serum Fig . 3), but migration by the heated guinea pig serum was not Bound . In order to exclude chemotaxis by the ßresh serum itself, the fresh serum could be used only at low concentration (over 27-fold dilution) . Using a definite amount oß cathepsin BI (0 .96 milliunit/ml), chemotactic activity with heated serum was higher than that with ßresh serum at any amount used (Fig . 4) . Maximum activity was achieved at 27-fold dilution oß heated serum . Larger amounts oß heated serum tended to yield decreased activity . Figure 5 indicates that, with a definite amount oß heated serum, chemotactic activity was linear with the amount oß cathepsin BI up to the concentration oß 1 milliunit/ml in the incubation mixture .
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w
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FIG . 3 Relationship between concentration of ßresh guinea pig serum and PMN leukocytes chemotaxis . Diluted ßresh serum was placed in lower chamber as chemotactic ßactor . Other methods were the same as noted in "METHODS" .
In the experiments, the ßinal concentration oß DTT in chemotaxis chamber was 37 .5 yM at which concentration DTT showed no eßßect on PMN leukocytes chemotaxis .
Vol . 19, No . 8
PHI~1-Chemotaais aad Cathepain B1
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FIG . 4 Eßßect oß serum concentration on chemotactic factor ßormation . Cathepsin BI (0 .96 milliunit/ml) was incubated for 60 min at 37 ° , pH 6 .0, with heated serum (--o-), and with ßresh serum (-~--) . Other procedures were noted in "METHODS" .
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Eßfect oß cathepsin BI concentration on chemotactic ßactor ßormation . The designated amount of cathepsin BI was treated with heated serum (1 :27 dilution) ßor 60 min at 37 ° , pH 6 .0 . Other procedures were the same with "METHODS" .
1205
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PMN-Chemota~da and Cathepsin Bi
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Effect of incubation time on chemotactic factor formation . Cathepsin BI (0 .64 milliunit/ml) was treated with heated serum (1 :27 dilution) at pH 6 .0, 37 0 . Other methods were shown in "METHODS" . Chemotactic factor formation as a function of incubation time . Chemotactie activity was linear up to 60 min when heated serum was incubated with Cathepsin HI at 37 ° , pH 6 .0 (Fig . b) .
FIG . 7 Effect of pH on chemotactic factor formation . Cathepsin BI (0 .64 milliunit/ml) was treated with heated serum (1 :27 dilution) at indicated pH, 37o for 60 min . Other procedures were shown in "METHODS" .
Vol . 19, No . 8
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PIß1-Chemotaxia and Cathepsin B1
Optimal BFI ßor chemotactic ßactor formation . Optimal pH for chemotactic factor ßormation was determined by incubating the mixture containing cathepsin BI, heated serum and DTT for 60 min at 37 o at varie,ble pH . Chemotactic factor ßormation was dependent upon pH, with maximal activity at pH 6 .0 (Fig . 7) . Eßßects oß inhibitors . From the above studies, it appeared likely that the enzyme to form the chemotactic factor from guinea pig serum was cathepsin BI because chemotactic factor formation by cathepsin BI rich ßraction, not by DI rich ßraction, was enhanced in the presence of DTT . In order to identify the chemotactic ßactor ßorming enzyme more clearly, efßects oß inhibitors oß cathepsin activity on the factor formation were investigated . As shown in Table II, ZnS04 inhibited cathepsin BI and BII activities and leupeptin inhibited cathepsin BII and DI activities when synthetic substrates (for BI and BII) and denatured hemoglobin (ßor DI~ were used . Efßects oß these two cathepsin inhibitors on chemotac is ßactor ßormation were then investigated (Table III) . When cathepsin BI was preincubated with ZnS04 ßor 10 min at 37 ° pH 6 .0 before addition oß heated serum, ZnS04 inhibited the chemotactic factor formation at the concentration oß 167 ~. Once chemotactic ßactor was formed, the addition oß ZnS04 had little or no eßßect . Treatment oß cathepsin BI with leupeptin was without significant eßßect . These data also suggest that cathepsin BI was the enzyme to ßorm chemotactic factor from heated serum at pH 6 .0 . Discussion
It has recently been proposed that cathe sins participate in the initiation oß inßlammatory reactions (1-6~ . Some proteases were reported to ßorm chemotactic ßactors from complement at neutral pH (7-10), or acidic pH (14) . In acidic protease, Snyderman et al (14) have reported that partially purified protease oß macrophage cleaved CS into a number oß ßragments possessing chemotactic activity at pH 3 .8 . This protease may be similar to cathepsin D because its activity was assayed at pH 3 .8 using TABLE II Eßßects oß Zn ++ and Leupeptin on Lysosomal Cathepains Residual activity of cathepsins
Inhibitor None ZnS0 4 Leupeptin
100 50
5 .0 0 .5
(~M) (~g~ml)
AI
AII
AIII BI
BII
C
100
100
100
100
100
100
99 100
99
100
99 100
97 100
99 100
98 100
100
9 2
100 100
99
100
100 29
34
70
99
11
17 0 1
100
Purißied enzymes were preincubated at 37 ° for 10 min with inhibitors ßollowed by addition oß substrates .
DI
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PIß~I-Chemota~d.s and Cathapsia B1
Vol . 19, No . 8
TABLE III Inhibition oß Chemotactic Factor Formation Inhibitor ZnS04 Leupeptin
Concen . 50 167
(~M)
0 .5 (y.g~ml)
f Inhibition Inhibitor added to incubation mixture Before* Aßter* 26 .5 70 .0
-1 .7 8 .8
22 .6
30 .3
Cathepsin BI (0 .71 milliunit~ml) was preincubated with inhibitor and DTT ßor 10 min at 37 ° , pH 6 .0, ßollowed by addition of heated serum (1 :9 dilution) . Reaction mixtures were incubated ßor 60 min at 37 ° , pH 6 .0, and then adjusted at pH 7 .2 ßor chemotactic assay . * "Before" or "Aßter" reßers to the presence or absence oß inhibitor, respectively, during the 60 min incubation period at 37 0 , PH 6 .0 . urea-denatured hemoglobin as substrate . As the alternative possibility, however, the protease might be also cathepsin BI because Otto (24), Barrett (25) and Swanson _et al (26) have reported that cathepsin BI along with cathepsin can hydrolyze denatured hemoglobin . The present report indicates that purified rat liver lysosomal cathepsin BI ßraction, which lacks chemotactic activity by itselß, forms PMN leukocytes chemotactic factor ßrom heated guinea pig serum in vitro . This factor ßormation was activated with SHreagent DTT, maximal at pH 6 .0, and was inhibited by Zn50q. (inhibitor oß cathepsin BI and BII) but not by leupeptin (inhibitor oß cathepsin BII and DI) . These results support the fact that the enzyme to ßorm chemotactic ßactor from heated serum is cathepsin BI . The chemotactic activity was found to be much higher in thin present system when the heated serum was used as substrate in place oß ßresh serum. As one of the reason ßor this ßact, it could be speculated that cathepsin BI can act on heated serum more easily because oß some conßormational changes of substrate, or that some cathepsin inhibitors present in serum are destroyed by heating . Concerning with cathepsin inhibitors in serum, Starkey and Barrett (27) reported that human cathepsin BI was inhibited by human a -macroglobulin and IgG . Hill and Ward (~) and Weksler and Coupal (10) reported the chemotactic factor ßormation from fresh serum by neutral protease, in which the chemotactic ßactor was not ßormed at all ßrom the heated serum . This apparent discrepancy might be caused, partly at least, by the dißference in sorts oß enzymes and substrates because they used neutral protease as the enzyme and C3 or C5 as the substrate . The present studies suggested that cathepsin BI may act on a certain denatured substrate in serum at inßlammatory sites to produce some chemotactic ßactor for PMN leukocytes . Although ßurther investigations are clearly necessary, it would appear that cathepsin BI participates through this ßunction in some
D
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PAß~T-Chemotaxie and Cathepain B1
1209
stages oß inßlammation in vivo . Acknowledgements The authors wish to thank Dr . K . Arima, Director oß Central Research Laboratories, Sankyo Co ., Ltd ., ßor his couragement of this work . Thanks are also due to Mr . T . oß the Laboratories for his kind support in designing oß tactic chamber .
the enTakahashi chemo-
Reßerences 1. 2. 3. 4. 5. 6. 7. 8. 9. 10 . 11 . 12 . 13 . 14 . 15 . 16 . 17 . 18 . 19 . 20 . 21 . 22 . 23 . 24 . 25 . 26 . 27 .
S .Y . Agi and L, Evans, Fed . Proc . 32 1494-1498 (1973) A .I . Sapolsky, R .D . Altman, and D .S, Howell, Fed . Proc . 32 1489-1493 (1973) A .I . Sapolsky, R .D . Altman, J .F, Woessner, and D .S . Howell, J . Clin . Invest . ~ 624-633 (1973) A,R . Poole, R .M . Hembry, J .T . Dingle, I . Pinder, E,F,J . Ring, and J . Cosh, Ann . Rheum . Disease _33 405-406 (1974) K .D, Muirden and M . Phillips, Ann . Rheum . Disease ~ 251-261 (1973) G .S . Lazarus, J . Invest . Dermatol . 62 367-371 (1974) J .H . Hill and P .A . Ward, J . Exp . Med . 130 505-518 (1969) P .A . Ward and J,H, Hill, J, Immunol . 104 535-543 (1970) S .B . Taubman P,R, Goldschmidt, and I,H. Lepow, Fed . Proc . 2~ 434 (1970 B,B . Weksler and C .E, Coupal, J . Exp . Med . ~ 1419-1430 (1973) M . Yoshinaga, K . Yoshida, A . Tashiro, and H . Hayashi, Immunol . _21 281-298 (1971) S . Yamamoto, M . Yoshinaga, H . Hayashi, and S, Maeda, Immunol . 20 803-808 (1971) M . Yoshinaga, S . Yamamoto, S . Maeda, and H . Hayashi, Immunol . 20 809-815 (1971) R . Snyderman, H .S . Shin, and A .M, Dannenberg, J . Immunol . 10~ 896-898 (1972) J,T . Boyle, F .E . Tabachnick, and J .L . Granda, Arthritis Rheum . 1~ 431 (1972) K . Kakinuma, Japan J . Exp . Med . ~8 165-169 (1968) K . Matsuda and E . Misaka, J . Biochem . ~ 639-649 (1974) S . Boyden, J . Exp . Med . 11~, 453-466 (1962) P,A . Ward, Biochem . Pharmacol . Supplement 99-105 (1968) R . Snyderman, H .S . Shin, J .K . Phillips, H . Gewurz, and S .E . Mergenhagen, J . Immunol . 1~ 413-422 (1969) P,C . Wilkinson, J .F . Borel, V .J . Stecher-Levin, and E . Sorkin, Nature 222 244-247 (1969) J .F . Borel and E . Sorkin, Experientia ~ 1333-1335 (1969) A .P . Kaplan A,B . Kay, and K,F . Austen, J . Exp . Med . 81-97 (1972 ; K . Otto, Tissue Proteinases, (p .1-28) North-Holland Publishing Co ., Amsterdam (1971) A .J . Barrett, Biochem . J . 1~ 809-822 (1973) A .A . Swanson, B .J . Martin, and S,S, Spicer, Biochem . J, 223-228 (1974) P .M . Starkey and A .J . Barrett, Biochem . J . 1~ 823-831 (1973)
Pergamon Press
A ROLE OF CATHEPSIN BI * IN POLYMORPHONUCLEAR LEUKOCYTES CHEMOTA%IS Kazu Kobayashi, Yoko Endo, Keiichi Matsuda, Kiichiro Tanaka and Eiichi Misaka Central Research Laboratories, Sankyo Co ., Ltd ., Shinagawa-ku, Tokyo, 140 (Received in final form August 30, 1976)
Summary Cathepsin BÎ was purified from rat liver lysosomal fraction by ammonium sulfate fractionation, followed by chromatography on Sephadex G-200 and DEAF-Sephadex . Formation of chemotactic factor for guinea pig polymorphonuclear (PMN) leukocytes was demonstrated in vitro when guinea pig serum was incubated with cathepsin HI . This factor formation was dependent on SH-reagent dithiothreitol (DTT) and was maximal at pH 6 .0 . ZnS04, an inhibitor of cathepsin BI, inhibited the chemotactic factor formation likewise . Lysosomal enzymes, cathepsin D in particular, may play an important role in the development of inflammatory reactions . Ali and Evens (1) and Sapolsky et al (2, 3) have demonstrated that cathepsin D activity increased in cartilage of osteoarthritic joints . Poole et al (4) have shown that the release of cathepsin D was seen in synovia adjacent cells and pannua tissue cells from rheumatoid patients . Thus, these authors suggest that cathepsin D may be an important autolytic enzyme responsible for the breakdown of joint cartilage . On the other hand, Muirden and Phillips (5) have reported the increased cathepsin D activity in the articular cartilage in the experimental arthritis induced by intra-articular injection of filipin in rabbit, and Lazarus (6) has also shown that cathepsin D activity increased in turpentin-induced inflammatory skin . As another role of tissue proteases in inflammatory processes, there are many reports concerning their participation in producing PMN leukocytes chemotactic factors . Chemotactic factor formation from complements by some tissue proteases was reported (7-10) . However the enzymes used in these investigations are not purified ones . There are also cases using purified enzymes for demonstrating production of chemotactic factors . Hayashi et al (11-13) purified inflammatory SH-dependent protease from Arthus-type akin lesion, and demonstrated to form leukoegresin, a chemotactic factor, from IgG at neutral pH . Snyderman et al (14) reported the fact that the purified acid protease from macrophages was also capable to give some chemotactic factors from complement . In these cases, however, enzymes used were unclear of their * Benzoyl-DL-arginine-p-nitroanilide hydrolyzing enzyme (cathepsin Bl) . 1199
1200
PHId-Chemotaaia and Cathapain B1
Vol . 19, No . 8
subcellular localizations . Apart from chemotactic factor ßormation, Boyle et al (15) reported that purified cathepsin D ßrom human rheumatoid synovium had chemotactic activity by itselß . The present report deals with the possible chemotactic ßactor formation by highly purified rat liver lysosomal cathepsin BI using guinea pig serum as substrate . Materials and Methods N-Carbobenzoxy-a-L-glutamyl-a-L-phenylalanine (substrate for cathepsin A) was obtained Brom the Protein Research Foundation, Osaka . Benzoyl-DL-arginine-4-nitroanilide (substrate ßor cathepsin BI) waa purchased Brom Merck, Darmstadt, and benzoyl-Largininamide (substrate ßor cathepsin B) from Fluke Chemische Fabrik, Bucks, Switzerland . Glycyl-tyrosinamide acetate (substrate ßor cathepsin C) was obtained Brom Sigma Chemical Co ., St . Louis, Mo ., and hemoglobin (substrate for cathepsin D) Brom Nutritional Biochemicals Co ., Cleveland, Ohio . DTT was purchased from Seikagaku Kogyo Co ., Tokyo, and sodium caseinate Brom Dißco Lab ., Detroit, Mich . Bovine serum albumin was obtained Brom Armour Pharmaceutical Co . Leupeptin was a generous gift from Dr . Umezawa of the Institute oß Microbial Chemistry, Tokyo . Peritoneal PMN leukocytes preparation . The peritoneal PMN leukocytes were prepared as described by Kakinuma (16) with a slight modification . Hartley male guinea pigs (300-350 g) were injected with 30 ml oß 1~ sterile casein in pyrogen-ßree saline eritoneally . Eighteen hours later, 30 ml oß heparinized saline ~6 units/ml) were introduced into the peritoneal cavity and the exudate withdrawn . The cells, 95~ of which were PMN leukocytes, were collected by centrißugation and suspendeg in 2~ bovine serum albumin-medium at a concentration oß 1 .5 x 10 cells/ml . Medium . Gey's medium was used with a slight modification . In order to stabilize pH in the chemotactic assay, phosphate bußßer (0 .02 M) was used substituting ßor NaHC0 3 . Enzyme ~renaration . Lysosomal cathepsins oß rat liver were prepared by the method of Matsuda and Misaka (17) and subnumbers of cathepsins (such as AI, AII, BI, BIIr etc .) correspond with their report . Lysosomal supernatant was fractionated by ammonium sulfate ßollowed by Sephadex G-200 chromatography . The mixéd ßraction oß cathepsin BI and DI on the gel-chromatography was further purified with DEAF-Sephadex column . One unit oß enzyme activity is deßined as that amount which produces 1 ~ mole oß reaction product per minute . Enzyme ease . Assays oß cathepsin activities were carried out according to the methods reported by Matsuda and Misaka (17) . Chemotactic ßactor ßormation . Unless otherwise stated, guinea pig serum used was heated at 56o ßor 30 min and was dialysed against 0 .001 M acetate bußßer pH 4 .5 ßor 18 hr . The re tentate was centrifuged at 20,000 x g ßor 20 min to remove precipitate and was diluted with 0 .01 M phosphate bußßer or with 0 .005 M acetate bußßer at desired pH . Chemotactic factor ßormation was carried out in an incubation mixture containing enzyme, heated serum and DTT (125 }iM) in a ßinal volume oß 1 .5 ~ml at acidic pH . Aßter 30 or 60 min incubation at 37 ° , the mixture was
Vol . 1201 19, No . 8 P2~T-Chemotaaie and Cathepein B1 adjusted to pH 7 .2 with cold medium (ßinal volume, 5 .0 ml) and then subjected to the chemotactic assay . As a blank ßor chemotactic assay, reaction mixture was adjusted to pH 7 .2 without the incubation step ßor chemotactic factor ßormation at acidic pH .
D C B
FIG . 1 Chemotactic chamber consists of two titanium compartments (A and D) separated by a micropore filter membrane (B) held in position by silicone rubber ring (C) and ring (E) . Chemôtactic activity assa . Chemotactic activity was determined in vitro by a modißied method of the micropore filter technique by Boyden (18) . The chemotactic chamber devised by Ward (19) was employed with a slight reßormation . A schematic drawing of the chamber is illustrated in Fig . 1 . A micropore ßilter (3 .0 Fore size, SSWP01300 ; Millipore Co ., New Bedßord, Mass .) was used to separate these two compartments . The lower compartment oß the chamber was filled with the test solution ßor chemotactic activity and the upper compartment was with 7 .5 x 10 5 PMN leukocytes . After an incubation ßor 3 hr at 37° in a moist atmosphere of 5~ C02 in air, the ßilter was removed, Yixed in methanol, stained with Harris' hematoxylin, dehydrated, and cleared in xylene . The number oß cells migrating through to the lower surßace of the ß}lter were counted microscopically ßor an unit field (10 x 0 .13 mm ) and the mean value of four ßilters was calculated .
1202
PI~Pi-Chemotaxis and Cathepein B 1
Vol . 19, No . 8
Chemotactic activity was expressed as this mean value of migrated cell numbers per unit after subtracting the value in blank . Results Requirement for cathepsin BI in formation of chemotactic factor . As cathepsin D has reported to participate in inflammatory reactions, a possible role of cathepsin D in chemotaxis was studied in the first place . In preliminary experiments, cathepsin DI fraction of Sephadex G-200 chromatography was found to form PMN leukocytes chemotactic factor with guinea pig serum as substrate . This fraction alone showed no chemotactic activity . However, cathepsin DI fraction was overlapped with cathepsin BI In order to know which fraction as was reported previously (17) . cathepsin namely BI or DI is responsible for formation of chemotactic factor with guinea pig serum as substrate, a further trial for separation of them was done . Namely, mixed fraction of BI and DI obtained in the Se hadex G-200 chromatography was concentrated with Diaflo membrane p (UM-2 ; Amicon Co ., Lexington, Mass .) and applied onto DEAF-Sephadex column . As shown in Fig . 2, these two enzymes were separated each other considerably . Then, cathepsin BI rich fraction (fraction No . 4) and cathepsin DI rich fraction (fraction No . 37) were tested for the formation of chemotactic factor with serum in various conditions . It is well known that cathepsin B is activated in the presence of SHreagents and cathepsin D is not . And also pH optimum of cathepsin B is around 6, while that of cathepsin D is more acidic range . Then, a series of experiments was carried out in the presence or absence of DTT at pH 6 .0 or 4,5 . OD~ s .o
NaCI (MI LO ~ O .I o .os
FIG . 2 DEAE-Sephadex chromatogram of lysosomal cathepsin BI and DI . Cathepsin DI fraction of Sephadex G-200 containing cathepsin BI was concentrated and applied to DEAF-Sephadex column (2 x 20 cm) in 0 .02 M sodium acetate, pH 5 .0, with a linear gradient of NaCl (0 .02 to 0 .1 M, total volume ; 800 ml, 10 ml~tube) .
PMN-Chemotaais aad Cathepain Bl
Vol . 19, No . 8
1203
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1204
PMN-Chamotaxia and Cathapsin B1
Vol . 19, No . 8
As shown in Table I, an incubation of cathepsin BI with heated serum at pH 6 .0 caused a ~rominent chemotactic activity ßormation in the presence oß DTT , while cathepsin BI itselß showed practically no activity without substrate . Enzymation of cathepsin DI, on the other hand, demonstrated only weak activity ßormation in various conditions with the combination of pH and DTT addition . Chemotactic factor formation as _a function of dose _of serum and cathe sin _BI . Guinea pig 20),rabbit 21,2 and human serum 10,23 are reported to possess chemotactic âctivity . Migrated cell numbers in the case oß the ßresh guinea pi serum in our experiment was linear with the amount oß serum Fig . 3), but migration by the heated guinea pig serum was not Bound . In order to exclude chemotaxis by the ßresh serum itself, the fresh serum could be used only at low concentration (over 27-fold dilution) . Using a definite amount oß cathepsin BI (0 .96 milliunit/ml), chemotactic activity with heated serum was higher than that with ßresh serum at any amount used (Fig . 4) . Maximum activity was achieved at 27-fold dilution oß heated serum . Larger amounts oß heated serum tended to yield decreased activity . Figure 5 indicates that, with a definite amount oß heated serum, chemotactic activity was linear with the amount oß cathepsin BI up to the concentration oß 1 milliunit/ml in the incubation mixture .
2000
v
w
1000
1 :40 1 :20 Strum Dilution
I~10
FIG . 3 Relationship between concentration of ßresh guinea pig serum and PMN leukocytes chemotaxis . Diluted ßresh serum was placed in lower chamber as chemotactic ßactor . Other methods were the same as noted in "METHODS" .
In the experiments, the ßinal concentration oß DTT in chemotaxis chamber was 37 .5 yM at which concentration DTT showed no eßßect on PMN leukocytes chemotaxis .
Vol . 19, No . 8
PHI~1-Chemotaais aad Cathepain B1
ws 300 u
a u
200
û v
0
~, 100 c~
FIG . 4 Eßßect oß serum concentration on chemotactic factor ßormation . Cathepsin BI (0 .96 milliunit/ml) was incubated for 60 min at 37 ° , pH 6 .0, with heated serum (--o-), and with ßresh serum (-~--) . Other procedures were noted in "METHODS" .
o .a
I .o
Cath. 8~ Im Unite/ ml ) FIG . 5
Eßfect oß cathepsin BI concentration on chemotactic ßactor ßormation . The designated amount of cathepsin BI was treated with heated serum (1 :27 dilution) ßor 60 min at 37 ° , pH 6 .0 . Other procedures were the same with "METHODS" .
1205
1206
PMN-Chemota~da and Cathepsin Bi
Yo1 . iß, No . B
s,
20
~0
80
lecubatian Tim* {min) FIG . 6
Effect of incubation time on chemotactic factor formation . Cathepsin BI (0 .64 milliunit/ml) was treated with heated serum (1 :27 dilution) at pH 6 .0, 37 0 . Other methods were shown in "METHODS" . Chemotactic factor formation as a function of incubation time . Chemotactie activity was linear up to 60 min when heated serum was incubated with Cathepsin HI at 37 ° , pH 6 .0 (Fig . b) .
FIG . 7 Effect of pH on chemotactic factor formation . Cathepsin BI (0 .64 milliunit/ml) was treated with heated serum (1 :27 dilution) at indicated pH, 37o for 60 min . Other procedures were shown in "METHODS" .
Vol . 19, No . 8
1207
PIß1-Chemotaxia and Cathepsin B1
Optimal BFI ßor chemotactic ßactor formation . Optimal pH for chemotactic factor ßormation was determined by incubating the mixture containing cathepsin BI, heated serum and DTT for 60 min at 37 o at varie,ble pH . Chemotactic factor ßormation was dependent upon pH, with maximal activity at pH 6 .0 (Fig . 7) . Eßßects oß inhibitors . From the above studies, it appeared likely that the enzyme to form the chemotactic factor from guinea pig serum was cathepsin BI because chemotactic factor formation by cathepsin BI rich ßraction, not by DI rich ßraction, was enhanced in the presence of DTT . In order to identify the chemotactic ßactor ßorming enzyme more clearly, efßects oß inhibitors oß cathepsin activity on the factor formation were investigated . As shown in Table II, ZnS04 inhibited cathepsin BI and BII activities and leupeptin inhibited cathepsin BII and DI activities when synthetic substrates (for BI and BII) and denatured hemoglobin (ßor DI~ were used . Efßects oß these two cathepsin inhibitors on chemotac is ßactor ßormation were then investigated (Table III) . When cathepsin BI was preincubated with ZnS04 ßor 10 min at 37 ° pH 6 .0 before addition oß heated serum, ZnS04 inhibited the chemotactic factor formation at the concentration oß 167 ~. Once chemotactic ßactor was formed, the addition oß ZnS04 had little or no eßßect . Treatment oß cathepsin BI with leupeptin was without significant eßßect . These data also suggest that cathepsin BI was the enzyme to ßorm chemotactic factor from heated serum at pH 6 .0 . Discussion
It has recently been proposed that cathe sins participate in the initiation oß inßlammatory reactions (1-6~ . Some proteases were reported to ßorm chemotactic ßactors from complement at neutral pH (7-10), or acidic pH (14) . In acidic protease, Snyderman et al (14) have reported that partially purified protease oß macrophage cleaved CS into a number oß ßragments possessing chemotactic activity at pH 3 .8 . This protease may be similar to cathepsin D because its activity was assayed at pH 3 .8 using TABLE II Eßßects oß Zn ++ and Leupeptin on Lysosomal Cathepains Residual activity of cathepsins
Inhibitor None ZnS0 4 Leupeptin
100 50
5 .0 0 .5
(~M) (~g~ml)
AI
AII
AIII BI
BII
C
100
100
100
100
100
100
99 100
99
100
99 100
97 100
99 100
98 100
100
9 2
100 100
99
100
100 29
34
70
99
11
17 0 1
100
Purißied enzymes were preincubated at 37 ° for 10 min with inhibitors ßollowed by addition oß substrates .
DI
1208
PIß~I-Chemota~d.s and Cathapsia B1
Vol . 19, No . 8
TABLE III Inhibition oß Chemotactic Factor Formation Inhibitor ZnS04 Leupeptin
Concen . 50 167
(~M)
0 .5 (y.g~ml)
f Inhibition Inhibitor added to incubation mixture Before* Aßter* 26 .5 70 .0
-1 .7 8 .8
22 .6
30 .3
Cathepsin BI (0 .71 milliunit~ml) was preincubated with inhibitor and DTT ßor 10 min at 37 ° , pH 6 .0, ßollowed by addition of heated serum (1 :9 dilution) . Reaction mixtures were incubated ßor 60 min at 37 ° , pH 6 .0, and then adjusted at pH 7 .2 ßor chemotactic assay . * "Before" or "Aßter" reßers to the presence or absence oß inhibitor, respectively, during the 60 min incubation period at 37 0 , PH 6 .0 . urea-denatured hemoglobin as substrate . As the alternative possibility, however, the protease might be also cathepsin BI because Otto (24), Barrett (25) and Swanson _et al (26) have reported that cathepsin BI along with cathepsin can hydrolyze denatured hemoglobin . The present report indicates that purified rat liver lysosomal cathepsin BI ßraction, which lacks chemotactic activity by itselß, forms PMN leukocytes chemotactic factor ßrom heated guinea pig serum in vitro . This factor ßormation was activated with SHreagent DTT, maximal at pH 6 .0, and was inhibited by Zn50q. (inhibitor oß cathepsin BI and BII) but not by leupeptin (inhibitor oß cathepsin BII and DI) . These results support the fact that the enzyme to ßorm chemotactic ßactor from heated serum is cathepsin BI . The chemotactic activity was found to be much higher in thin present system when the heated serum was used as substrate in place oß ßresh serum. As one of the reason ßor this ßact, it could be speculated that cathepsin BI can act on heated serum more easily because oß some conßormational changes of substrate, or that some cathepsin inhibitors present in serum are destroyed by heating . Concerning with cathepsin inhibitors in serum, Starkey and Barrett (27) reported that human cathepsin BI was inhibited by human a -macroglobulin and IgG . Hill and Ward (~) and Weksler and Coupal (10) reported the chemotactic factor ßormation from fresh serum by neutral protease, in which the chemotactic ßactor was not ßormed at all ßrom the heated serum . This apparent discrepancy might be caused, partly at least, by the dißference in sorts oß enzymes and substrates because they used neutral protease as the enzyme and C3 or C5 as the substrate . The present studies suggested that cathepsin BI may act on a certain denatured substrate in serum at inßlammatory sites to produce some chemotactic ßactor for PMN leukocytes . Although ßurther investigations are clearly necessary, it would appear that cathepsin BI participates through this ßunction in some
D
Vol . 19, No . 8
PAß~T-Chemotaxie and Cathepain B1
1209
stages oß inßlammation in vivo . Acknowledgements The authors wish to thank Dr . K . Arima, Director oß Central Research Laboratories, Sankyo Co ., Ltd ., ßor his couragement of this work . Thanks are also due to Mr . T . oß the Laboratories for his kind support in designing oß tactic chamber .
the enTakahashi chemo-
Reßerences 1. 2. 3. 4. 5. 6. 7. 8. 9. 10 . 11 . 12 . 13 . 14 . 15 . 16 . 17 . 18 . 19 . 20 . 21 . 22 . 23 . 24 . 25 . 26 . 27 .
S .Y . Agi and L, Evans, Fed . Proc . 32 1494-1498 (1973) A .I . Sapolsky, R .D . Altman, and D .S, Howell, Fed . Proc . 32 1489-1493 (1973) A .I . Sapolsky, R .D . Altman, J .F, Woessner, and D .S . Howell, J . Clin . Invest . ~ 624-633 (1973) A,R . Poole, R .M . Hembry, J .T . Dingle, I . Pinder, E,F,J . Ring, and J . Cosh, Ann . Rheum . Disease _33 405-406 (1974) K .D, Muirden and M . Phillips, Ann . Rheum . Disease ~ 251-261 (1973) G .S . Lazarus, J . Invest . Dermatol . 62 367-371 (1974) J .H . Hill and P .A . Ward, J . Exp . Med . 130 505-518 (1969) P .A . Ward and J,H, Hill, J, Immunol . 104 535-543 (1970) S .B . Taubman P,R, Goldschmidt, and I,H. Lepow, Fed . Proc . 2~ 434 (1970 B,B . Weksler and C .E, Coupal, J . Exp . Med . ~ 1419-1430 (1973) M . Yoshinaga, K . Yoshida, A . Tashiro, and H . Hayashi, Immunol . _21 281-298 (1971) S . Yamamoto, M . Yoshinaga, H . Hayashi, and S, Maeda, Immunol . 20 803-808 (1971) M . Yoshinaga, S . Yamamoto, S . Maeda, and H . Hayashi, Immunol . 20 809-815 (1971) R . Snyderman, H .S . Shin, and A .M, Dannenberg, J . Immunol . 10~ 896-898 (1972) J,T . Boyle, F .E . Tabachnick, and J .L . Granda, Arthritis Rheum . 1~ 431 (1972) K . Kakinuma, Japan J . Exp . Med . ~8 165-169 (1968) K . Matsuda and E . Misaka, J . Biochem . ~ 639-649 (1974) S . Boyden, J . Exp . Med . 11~, 453-466 (1962) P,A . Ward, Biochem . Pharmacol . Supplement 99-105 (1968) R . Snyderman, H .S . Shin, J .K . Phillips, H . Gewurz, and S .E . Mergenhagen, J . Immunol . 1~ 413-422 (1969) P,C . Wilkinson, J .F . Borel, V .J . Stecher-Levin, and E . Sorkin, Nature 222 244-247 (1969) J .F . Borel and E . Sorkin, Experientia ~ 1333-1335 (1969) A .P . Kaplan A,B . Kay, and K,F . Austen, J . Exp . Med . 81-97 (1972 ; K . Otto, Tissue Proteinases, (p .1-28) North-Holland Publishing Co ., Amsterdam (1971) A .J . Barrett, Biochem . J . 1~ 809-822 (1973) A .A . Swanson, B .J . Martin, and S,S, Spicer, Biochem . J, 223-228 (1974) P .M . Starkey and A .J . Barrett, Biochem . J . 1~ 823-831 (1973)
