Lipopolysaccharide induces release of a metallo-protease from hemocytes of the ascidian, Halocynthia roretzi

Developmental and Comparative Immunology,Vol 15, pp 1-7. 1991 Printed in the USA All rights reserved

0145-305X/91 $3 00 + 00 Copyright © 1991 PergamonPress plc

LIPOPOLYSACCHARIDE INDUCES RELEASE OF A METALLO-PROTEASE FROM HEMOCYTES OF THE ASClDIAN, HALOCYNTHIA RORETZI K a o r u A z u m i , H i d e y o s h i Y o k o s a w a , * a n d S h i n - i c h i Ishii Department of Biochemistry, Faculty of Pharmaceutical Sciences, Hokkaido University, Sapporo 060. Japan

(Received November 1989; Accepted Februa~, 1990) 7qAbstract--Hemocytes of the solitary ascidian, Halocynth& roretzi, released a succinylLeu-Leu-VaI-Tyr-4-methylcoumaryl-7-amide hydrolyzing enzyme in response to lipopolysaccharide treatment. The response was dependent on the temperature for incubating hemocytes. The protease release reaction was not triggered by IM-3 glucan. The protease released showed strict substrate specificity and its activity was inhibited by EDTA and ophenanthroline, but not by phosphoramidon, diisopropylfluorophosphate, N-ethylmaleimide, or p-chloromercuribenzoic acid. Thus, the enzyme was characterized as a phosphoramidon-insensitive metallo-protease. Calcium ionophore, phorbol myristate acetate, concanavalin A, and thrombin also induced the release of the same protease from H. roretzi hemocytes. ~:Keywords--Ascidian; Hemocyte; Metalioprotease; Lipopolysaccharide.

Introduction In response to various stimuli that may be encountered in h e m o l y m p h (blood) of animals, h e m o c y t e s (blood cells) release p r o t e a s e s as a cellular response. Protease release has important roles in defense since thc reaction occurs usually in localized sites of an a n i m a l ' s body, and the proteascs which are released inThis work was supported in part by a Grantin-Aid for Scientific Research from the Ministry of Education, Science, and Culture of Japan. * Author to whom correspondence should be addressed.

duce a series of cascade reactions of limited p r o t e o l y s e s a n d p r o d u c t i o n o f chemical mediators. For example, a m o e b o c y t e s of horseshoe crabs release serine proteases, which can trigger the coagulation system in the h e m o l y m p h , when they are exposed to bacterial endotoxin (lipopolysaccharide, LPS) (1). In l o c a l i z e d i n f l a m m a t o r y s i t e s o f m a m m a l s , a chymotrypsin-like e n z y m e and a trypsin-like one are released from mast cells: the former e n z y m e acts on immunoglobulin G to p r o d u c e chemotactic factors for neutrophils, while the latter activates prothrombin which triggers fibrin formation (2). During o u r studies on the d e f e n s e m e c h a n i s m s o f the a s c i d i a n , Halocynthia roretzi, we have already isolated two lectins of different sugar specificities ( 3 - 5 ) , two t r y p s i n inhibitors (6) from plasma, and two antibacterial substances (7), an LPS-binding hemagglutinin (8) from hemocytes. Recently, we found a new cellular response of H. roretzi h e m o c y t e s : they released a protease in response to bacterial LPS treatment. In this paper, we describe the cellular reaction of p r o t e a s e release and characterize the protease released from hemocytes.

Materials and Methods

Preparation o f Hemocytes Solitary ascidians, H. roretzi, Type C, were h a r v e s t e d in Mutsu Bay, Japan.

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The tunic m a t r i x was e x t e n s i v e l y washed with seawater, cut without injuring the internal organs, and the hemolymph collected from individual animals into 50 mL of 0.1% EDTA-5/9 M NaCI (EDTA solution) to prevent aggregation of hemocytes. EDTA solution was then added to yield a final volume equal to that of the hemolymph. After centrifugation (800 ×g, 10 min) of this 2-fold diluted hemolymph, the resulting pellet (hemocytes) obtained from individual animals was gently washed with 5 mL of Ca 2+, Mg2+-free Herbst's artificial sea water (F-HASW; 450 mM NaCI-9.4 mM KCI-32 mM Na2SO4-3.2 mM NaHCO 3, pH 7.6) and was suspended in 50 mL of F-HASW.

lnhibitors Phosphoramidon, chymostatin, clastatinal, and E-64 were obtained from the Peptide Institute (Japan). Diisopropylfluorophosphate (DFP) was purchased from Sigma Chemical Co. (U.S.A.). oPhenanthroline, p-chloromercuribenzoic acid (PCMB), and N-ethylmaleimide were obtained from Nakarai Tesque (Japan). Bestatin and leupeptin were generous gifts of Dr. W. Tanaka of Nippon Kayaku Co. (Japan).

Measurement of'Protease Release from Hemocytes E n z y m e release was initiated by adding 25 ILL of a Boivin preparation of LPS from Escherichia coli 026:B6 (Difco Lab., U.S.A.) at an indicated concentration to 1 mL of cell suspension (about 107 cells) containing 10 mM CaCI 2. The suspension was gently mixed and then allowed to stand for 2.5 h at 20°C. After hemocytes were removed by centrifugation (8000 × g, 5 min), the resulting supernatant was used for enzyme assays.

K. Azumi et al.

Effect of 131-3 glucan (Wako Pure Chemical Ind., Japan) was measured under the same conditions. Inducing effects of other stimuli were measured by dissolving various reagents as follows: 25 ttL each of 5 mg/mL LPS in F-HASW: 2 mg/mL concanavalin A [Con A, prepared in this laboratory (9)] in F-HASW; 2.5 m g / m L t h r o m b i n (Green Cross Corp., Japan) in F-HASW or 5 ~L each of 5 mM ionomycin (Calbiochem Corp., U.S.A.) in methanol: 2.5 mg/mL phorbol 12-myristate 13-acetate (PMA, Sigma C h e m . Co.) in d i m e t h y l s u l f o x i d e (DMSO). They were then added to 500 I~L of the cell suspension containing I0 mM CaCi2. The mixture was incubated for 2 h at 20°C, and the supernatant collected as described above. Protease activity of each supernatant was measured as described below.

Assay of Protease Activity Protease activity was measured at 25°C in 50 mM Tris-HCi, pH 8.0, containing 10 mM CaCI2 and 0.2 mM bestatin using succinyl-Leu-Leu-VaI-Tyr-4methylcoumaryl-7-amide (MCA) (20 ttM, the Peptide Institute) as a substratc. Activities towards various peptidylMCAs (the Peptide Institute) were measured under the same conditions. The appearance of fluorescence due to 7amino-4-methylcoumarin was monitored with excitation at 380 nm and emission at 460 nm.

Preparation of Enzyme Solution Crude enzyme solution was obtained as the supernatant after incubation for 2 h at 20°C in the presence of 0.2 mg/mL LPS and 5 mM CaCI z, and used for characterization and gel filtration of the enzyme.

Ascidian hemocyte response to LPS

3

Gel Filtration Crude enzyme solution in a volume of 2 mL was applied to a column (I.5 × 72 cm) of Sephadex G-50 (Pharmacia Fine Chemicals, Sweden) previously equilibrated with 50 mM Tris-HCI, pH 8.0, containing 10 mM CaCI 2, 0.5 M NaCI, and 0.005% Brij 35 (Pierce, U.S.A.). Brij 35 was added as an effective stabilizer. The column was developed with the same buffer at a flow rate of 20 mL/h. Effluents were collected in a volume of 1.4 mL, and protease activity was assayed as described above. The molecular weight was estimated using blue dextran (void volume), chymotrypsinogen A (26 K, Worthington Biochemical Corp., U.S.A.), cytochrome c (12 K, Sigma Chem. Co.), and aprotinin (6.5 K, Seikagaku Kogyo Co., Japan) as standards.

tivity was detected after incubation at the higher temperature (20°C), indicating a temperature dependence of the protease release. Upon incubation at 20°C, protease was released even in the absence of LPS. But the release without LPS treatment was not observed in the absence of CaCI z (data not shown). We then investigated the dependence of protease release on LPS concentration. As shown in Fig. 2, LPS induced release, dependent on concentrations of LPS. In this experiment, trypsin-like enzyme activity was also measured using Boc-ValPro-Arg-MCA as a substrate, but the activity could not be detected. In contrast to the action of LPS, 13I-3 glucan did not induce the release of the protease which was assayed using the two substrates. Thus, H. roretzi hemocytes respond to LPS, measured by the release of a protease from hemocytes.

Results

Substrate Specificity

LPS-lnduced Protease Release LPS induced the release of succinyiLeu-Leu-VaI-Tyr-MCA hydrolyzing activity from H. roretzi hemocytes (Fig. !). Although a substantial amount of the enzyme was released at 4°C, more ac-

To ascertain the nature of the released protease, we first examined substrate specificity of the protease released from hemocytes by the addition of LPS. Table 1 shows the activities of the protease toward various substrates. SuccinyI-Leu-

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TIME ( h ) Figure 1. Time course of release of succinylLeu-Leu-VaI-Tyr-MCA hydrolyzing activity from H. roretzi hemocytes with LPS (0.2 mg/mL) at 20°C (O) or 4°C (11), and without LPS at 20°C (©) or 4°C ([]).

i

0

100

200

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CONCENTRATION(pg/ml) Figure 2. Release of succinyI-Leu-Leu-VaI-TyrMCA hydrolyzing activity from H. roretzi hemocytes induced by various concentrations of LPS (O) and 131-3glucan (©). The vertical coordinate indicates the enzyme activity minus the value in the absence of LPS or fll-3glucan.

K Azumi et al.

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Table 1. Substrate Sl)eclflclty of metallo-protease released from H. roretzl hemocytes Substrate

Activity (%)

S uc-Leu-Leu-VaI-Tyr-MCA Ac-Ala-Ala-Tyr-MCA Su c-Ala-Ala-Pro-Phe-MCA GIt-Gly-Gly-Phe-MCA Suc-Ala-Pro-Ala-MCA

100, 3 2 0 1

Boc-VaI-Pro-Arg-MCA Leu-MCA

7 1~

• The activity toward succinyI-Leu-Leu-VaI-Tyr-MCA was defined as 100%. b The activity toward Leu-MCA was measured in the absence of bestatin. Suc, succinyl; Ac, acetyl; GIt, glutaryl; Boc, t-butyloxycarbonyl.

Leu-VaI-Tyr-MCA was most efficiently hydrolyzed. Other substrates for chymotrypsin-like enzyme, together with t h o s e for t r y p s i n - l i k e e n z y m e and aminopeptidase, were hardly hydroiyzed, Thus, the protease which was released from hemocytes in response to LPS treatment showed highly strict substrate specificity.

Protease Inhibitor Susceptibility The effects of various protease inhibitors on the succinyl-Leu-Leu-Var-TyrMCA hydrolyzing activity of the enzyme were examined (Table 2). Metal chelators, EDTA and o-phenanthroline, were strong inhibitors for the protease. Phosphoramidon, another inhibitor for Table 2. Effects of various reagents on the activity of metallo-protease released from H. roretz/hemocytes Inhibitors EDTA o-Phenanthroline Phosphoramidon DFP Leupeptin Chymostatin Elastatinal E-64 N-Ethylmaleimide PCMB

Concentration (raM)

Inhibition (%)

1.0 0.5 0.1 0.1 0.2 0.1 0.1 0.1 O.1

94 98 97 4 0 14 0 5 0

0.1

0

0.1

12

metallo-protease, as well as inhibitors for serine protease and cysteine protease, showed little inhibitory effect. The fact that the protease completely inhibited by EDTA or o-phenanthrolin was restored to full activity by the addition of I mM Zn z+ ion indicated that the succinyI-Leu-Leu-VaI-Tyr-MCA hydrolyzing protease was classified as a metallo-protease, which was insensitive to phosphoramidon.

Molecular Weight The result of gel filtration on Sephadex G-50 is shown in Fig. 3. The protease activity emerged as two peaks: one was eluted at a void fraction, while another was eluted as a rather broad peak with a molecular weight of approximately !1,000-13,000. Substrate specificities and protease inhibitor susceptibilities of both enzymes eluted early and later were almost identical and also similar to those of the original enzyme before gel filtration (data not shown). In this experiment, we added a non-ionic detergent, Brij 35, as a good stabilizing agent. The protease activity during storage without Brij 35 decreased to the level less than 50% of the original activity only overnight at 4°C.

Response to Other Stimuli We investigated whether H. roretzi h e m o c y t e s would respond to other stimuli in addition to LPS by measuring the cellular response on the basis of the release of metallo-protease. As shown in Fig. 4, Con A (100 p.g/mL), thrombin (125 ~,g/mL), ionomycin (0.5 ~.M, a calcium ionophorc), and PMA (0.25 p.g/mL, a protein kinase C activator) induced protease release from hemocytes. Methanol (!%) and DMSO (!%) were added as control media for ionomycin and PMA, respectively. F-HASW was used as a medium for LPS, Con A, and

Ascidian hemocyte response to LPS

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FRACTION NUMBER Figure 3. Gel filtration on Sephadex G-50 of protease released from H. roretzi hemocytes. Absor-

bance at 280 nm(

), succinyI-Leu-Leu-VaI-Tyr-MCA hydrolyzing activity (-(3-).

thrombin. In either case, the level of protease activity detected in the presence of the stimulant was higher than that in medium alone. The protease released by either of the stimuli including LPS seems to be characterized as the same metalio-protease on the basis of its substrate specificity and protease inhibitor susceptibility (data not shown). Thus, H. roretzi hemocytes can respond to various stimuli such as LPS, Con A, and thrombin, releasing the metailo-protease possibly through the increase of intracellular calcium concentration and the activation of protein kinase C.

F-HASW

con A~ ~ J Thrombin ~:~~ Methanol Ionomycln ~

DMSO ~ PMA ~ 0

~

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~ ~ ~ 100 200 300 400 RELATIVE ACTIVITY(%)

Figure 4. Effects of various stimuli on protease release from H. roretzi hemocytes.

Discussion

We showed that H. roretzi hemocytes respond to LPS, releasing a metallo-protease into the extracellular environment. LPS is the major component of cell walls of Gram-negative bacteria. LPS triggers activation of the clotting system in Limulus, its cellular defense mechanism, through a sequential activation of serine protease zymogens (1); it induces activation and secretory responses of mammalian macrophages (10) and neutrophils (! I), both of which are involved in inflammation. H. roretzi hemocytes recognize LPS when they were in contact with foreign bacteria containing LPS as a component of their cell walls and attack them. Thus, they function in the defense mechanisms of H. roretzi. This is the first report of a cellular response of ascidian hemocytes to LPS. In general, the mechanism for LPSinduced enzyme release has not been well elucidated. Two possibilities can be assumed. One of them is that lipid A. a component of LPS, acts on the surface membrane of h e m o c y t e s to change membrane fluidity. Such a change in

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membrane structure would promote fusion of its membrane with the membrane of granules which contain enzymes, resulting in enzyme release. According to another explanation there are receptors for LPS on the surface membranes of hemocytes. Signal transduction from the receptor into intracellular molecules would trigger enzyme release. In our preliminary experiments, we could not detect protease release from H. roretzi hemocytes by the addition of lipid A. Thus the former possibility seems irrelevant. It is necessary, therefore, to make clear whether a receptor for LPS is present on the cell membrane of H. roretzi hemocytes. It should be noted that It. roretzi hemocytes did not respond to [31-3 glucan, at least measured on the basis of the release of metalio-protease. It has been reported that [31-3 glucan can trigger activation of the clotting system of horseshoe crabs (I) and of the melanogenesis of insects (12). Although evidence for the receptor molecule for 131-3 glucan in H. roretzi hemocytes has not been presented, there is a possibility that the response to 131-3 glucan could be demonstrated by experimental criteria other than the release of metallo-protease. In addition to LPS, we demonstrated that H. roretzi hemocytes can respond to Con A and thrombin, both of which have been reported as stimulants for mammalian blood cells (13,14). Furthermore, H. roretzi hemocytes release the metallo-protease by the addition of ionomycin, a calcium ionophore, and PMA, a protein kinase C activator. In our preliminary experiment, we found that the presence of extracellular calcium was required for the action of the former reagent, and that the effect of the latter was inhibited by inhibitors of protein kinase C. Thus, the increase of intracellular calcium concentration and the activation of protein kinase C may be involved in the mechanisms of protease release. In connection with the above

K Azumi et al.

possible mechanisms, it should be noted that the temperature-dependent protease release takes place in the presence of extracellular calcium, although to the less extent in comparison with the result with LPS treatment. The mechanism, however, remains unknown. Although direct evidence for "exocytosis" has not been presented, we assume that the metallo-protease would be released from tt. roretzi hemocytes through exocytosis. During the protease release reaction, hemocytes do not undergo lysis as observed microscopically. It will be necessary to demonstrate the presence of the metallo-protease in intracellular compartments of hemocytes and its disappearance after exposure to stimuli. In contrast, it has been reported that H. roretzi hemocytes undergo lysis. which can be clearly observed microscopically, when they contact hemocytes originating from another ascidian (15). This is called a "contact reaction." We have preliminary evidence that protease activity could be detected in the medium of cell suspensions after a "contact reaction" and that the protease detected in the "'contact reaction" was quite different from the protease released upon I,PS stimulation on the basis of substrate specificity and protease inhibitor susceptibility. Thus, we assume that the metallo-protease release reaction of H. roretzi hemocytes against LPS or other stimuli is a different cellular reaction from the "'contact reaction" of hemocytes. From the viewpoint of defense mechanisms of H. roretzi, it is important to make clear whether intact bacteria can induce the release of the metallo-protease from hemocytes. We examined in preliminary experiments the inducing effect of heat (95°C, 15 min) treated E. coli BE, in which I.PS was estimated to be contained at the concentration of approximately 2 p.g/mL, but could not detect protease release from hemocytes. Further investigations using active bac-

Ascidian hemocyte response to LPS

7

teria in higher amounts or other kinds of bacteria will be necessary to answer the above question. Partial characterization of the protease released from H. roretzi hemocytes gave evidence for a novel metalloprotease with highly strict substrate specificity. The activity emerged on gel filtration as two peaks with almost identical substrate specificities and protease inhibitor susceptibilities. Therefore, we can assume the presence of two kinds of proteases, a high molecular weight one and a low molecular weight one, both of which show similar enzymatic properties. Alternatively, the possibility that the high molecular weight protease is an

aggregate formed by the lower molecular weight one could not be ruled out. Purification of the protease would clarify this point. The physiological roles of this metallo-protease remain to be elucidated. Perhaps it may act on hemolymph proteins to activate them or to produce chemical mediators. Alternatively, it may activate the hemocytes participating in defense mechanisms. In any case, it may play a role in the defense mechanisms of ascidians against invading bacteria. Further purification and characterization of the metallo-protease are now in progress to determine its natural substrate(s) and to define its physiological roles.

References I. Armstrong, E B. Adhesion and motility of the blood cells of Limulus. In: Cohen, W. D., cd. Blood cells of marine invertebrates: experimental systems in cell biology and comparative physiology. New York: Alan R. I,iss. Inc.: 1985; 77-124. 2. Katunuma, N.; Fukusen, N.: Kido, H. Biological functions of serine proteases in the granules of rat mast cells. Adv. E n z y m e Regul. 25:241-255; 1986. 3. Yokosawa, H.; Sawada, H.; Abe, Y.: Numakunai, T.; lshii, S. Galactose-specific lectin in the hemolymph of solitary ascidian, Halocynthia roretzi: isolation and characterization. Biochem. Biophys. Res. Commun. 107:451457; 1982. 4. Yokosawa, H.; Harada, K.; lgarashi, K.; Abe, Y.; Takahashi, K.; Ishii, S. Galactose-specific lectin in the hemolymph of solitary ascidian. Halocynthia roretzi. Molecular, binding and functional properties. Biochim. Biophys. Acta 870:242-247; 1986. 5. Harada-Azumi, K.; Yokosawa, H.; Ishii, S. N-Acetyl-galactosamine-specific lectin, a novel lectin in the haemolymph of the ascidian Halocynthia roretzi: isolation, characterization and comparison with galactose-specific lectin. Comp. Biochem. Physiol. 88B:375381; 1987. 6, Yokosawa, H.: Odajima, R.; lshii, S. Trypsin inhibitor in the hemolymph of a solitaty ascidian, Halocynthia roretzi. Purification and characterization. J. Biochem. 97:1621-1630; 1985. 7. Azumi. K.; Yokosawa, H.; Ishii, S. Halocyamines: novel antimicrobial tetrapeptide-like substances isolated from the hemocytes of the solitary ascidian Halocynthia roretzi. Biochemistry. 29:159-165; 1990.

8. Azumi, K.; Ozeki, S.; Yokosawa, H.; lshii, S. A novel lipopolysaccharide-binding hemagglutinin isolated from hemocytes of the solitary ascidian, Halocynthia roretzi: it can agglutinate bacteria. Dev. Comp. Immunol. 15:9- 16: 1991. 9. Oda, Y.; Kasai, K.; lshii, S. Studies on the specific interaction of concanavalin A and saccharides by affinity chromatography. Application of quantitative affinity chromatography to a multivalent system. J. Biochem. 89:285-296; 1981. 10. Morrison, D. C.: Ulevitch, R. J. The effects of bacterial cndotoxins on host mediation systems. Am. L Pathol. 93:527-617; 1978. I I. Fittschen, C.; Sandhaus, R. A.; Worthen, G. S.; Hensom R M. Bacterial lipopolysaccharide enhances chemoattractant-induced elastase secretion by human neutrophils. 3. Leukocyte Biol. 43:547-556; 1988. 12. Soderh/-ill, K. Prophenoloxidase activating system and m e l a n i z a t i o n - - a recognition mechanism of arthropods? A review. Dev. Comp. lmmunol. 6:601-61 I; 1982. 13. Detwiler, T, C.; Wasiewski, W. W. The equilibrium reaction of thrombin with platelets. In: l,undblad, R. L.: Fenton, J. W., ll; Mann, K. G., eds. C h e m i s t r y and b i o l o g y of thrombin. Ann Arbor, Ml: Ann Arbor Science Publishers, Inc.; 1977; 465-478. 14. Baggiolini, M.: Dewald, B. Exocytosis by neutrophils. In: Snyderman, R., ed. Regulation of leukocyte function (Contemporary topics in immunobiology, Vol. 14). New York: Plenum Press; 1984; 221-246. 15. Fuke, M. T. "'Contact reactions" between xenogeneic or allogeneic coelomic cells of solitary ascidians. Biol. Bull. 158:304-315; 1980.