Immunology 1978 35 167

In vitro macrophage chemotactic generation from serum immunoglobulin G by neutrophil neutral seryl protease

M. ISHIDA, M. HONDA & H. HAYASHI Department ofPathology, Kumamoto University Medical School, Kumamoto, Japan

Received 13 October 1977; acceptedfor publication 1 December 1977

Summary. A neutral protease with a molecular weight of about 14,000 was separated at acid pH from rabbit neutrophils and then partially purified by elution on DEAE-Sephadex, CM-Sephadex and Sephadex G-75 in that order. This enzyme was inactivated by diisopropyl fluorophosphate (DFP), phenylmethyl sulphonylfluoride (PMSF), soybean trypsin inhibitor (SBTI), or elastatinal, suggesting a seryl protease resembling elastase, but it failed to digest elastin-orcein. The enzyme seemed different from histonase of rabbit neutrophils because of its haemoglobin (3HHb)-degrading ability and of inactivation by heparin. The protease generated in vitro macrophage chemotactic activity from guineapig serum IgG. This chemotactic factor had a molecular weight similar to that of IgG and its chemotactic generation was accompanied by release of dialysable peptide(s). No generation of macrophage chemotactic activity from IgG was induced in vitro by elastase from pig pancreas or by neutral thiol protease from rabbit neutrophils.

neutrophil chemotactic factor was generated in vitro from serum IgG by neutral thiol protease from inflammatory tissue or neutrophils; and its generation was accompanied by release of dialysable peptide(s) from the IgG molecule. However, this protease failed to generate in vitro macrophage chemotactic activity from IgG (Hayashi et al., 1974; Hayashi, 1977). This type of neutrophil chemotactic factor (leucoegresin) was isolated from inflammatory tissue (Yoshinaga, Yoshida, Tashiro & Hayashi, 1971; Maeda, Yoshinaga & Hayashi, 1975); it was active for neutrophils but not for macrophages. The purpose of the present communication is to describe the characterization of a neutral seryl protease associated with in vitro production of a macrophage chemotactic factor from the IgG molecule.

MATERIALS AND METHODS Collection of neutrophils Male albino rabbits, weighing 3 -03 -5 kg, were used. Neutrophils were collected from peritoneal exudates withdrawn 4 h after instillation of 0-1 % glycogen. The final leucocyte pellets after low speed centrifugation contained over 96 % neutrophils. If necessary, contaminating red cells were removed from the neutrophil pellets by hypotonic lysis.

INTRODUCTION

As previously described (review by Hayashi, Yoshinaga & Yamamoto, 1974; Hayashi, 1975), a

Correspondence: Professor H. Hayashi, Department of Pathology, Kumamoto University Medical School, Kumamoto 860, Japan. 0019-2805/78/0700-0167 $02 00 © 1978 Blackwell Scientific Publications

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168

M. Ishida, M. Honda & H. Hayashi

Extraction ofneutrophils In a preliminary experiment, proteases were extracted from neutrophil granules or from whole cells at neutral pH, but it was found that the amount of proteases extracted was clearly smaller than that of proteases extracted at acid pH as described below. Accordingly, in the present experiment, neutrophils (5 x 109) were suspended in 25 ml of 0 34 M sucrose and then disrupted by a Teflon homogenizer at 1200 rev/min for 15 min in an ice bath. The disrupted cells were extracted with 25 ml of 0 02 M citric acid (pH 2 8) at 20 for 4 h and centrifuged at 15,000 g for 20 min. The supernatant fluid was used as the neutrophil extract.

Chromatography This was done on columns of the following materials: DEAE-Sephadex A-50 (3 5 mEq/g, Pharmacia, Uppsala, Sweden), CM-Sephadex C-50 (4 5 mequiv/g,. Pharmacia, Uppsala, Sweden) and Sephadex G-75 (Pharmacia, Uppsala, Sweden). Protein concentration during chromatography was estimated by measuring the absorbancies at 260 and 280 nm. Measurement ofproteolytic activity This was performed essentially according to the method of Hille, Barrett, Dingle & Fell (1970) and Bernacki & Bosmann (1972) with [3H-]acetyl haemoglobin ([3H]Hb) as a substrate. The substrate showed a specific activity of 350 Ci per mol on the basis of a molecular weight of 68,000. The reaction mixtures, consisting of 100 ,ul of enzyme solution, 200,cp1 of 0 1 M phosphate buffer or Britton Robinson's buffer, and 100,ul (300,ug; 4A4nmol; 342x 104d.p.m.; 1-54 uCi) of [3H]Hb, were kept at 370 for 60 min. The reaction was terminated in the cold by adding 2004ul of 2-5 % cold haemoglobin and 100 ul of 50 % trichloroacetic acid (TCA). After centrifugation and filtration, 200 p1 of the filtrate were used for estimation of TCA soluble radioactivity. Protease samples heated at 1000 for 15 min or buffer were used at the enzyme blank or reagent blank, respectively. Proteolytic activity was expressed as nmol of [3H]Hb degraded per hour. For calculation of specific protease activity, protein concentration was estimated by the method of Groves, Davis & Sells (1968) after measuring the absorbancies at 224 and 233 nm; crystalline bovine serum albumin (Sigma, St Louis, Missouri) was used as standard. In some experiments, proteolytic activity was measured by a modification (Hayashi, Miyoshi, Nitta & Udaka,

1962) of the casein digestion method of Kunitz (1947); one proteolytic unit represents 0-06 absorbance units at 276 nm (Koono & Hayashi, 1969). Protease inhibitors The neutral protease separated was tested with the following protease inhibitors: N-a-p-tosyl-L-lysine chloromethyl ketone HC1 (TLCK; Sigma, St Louis, Missouri), L-1-tosylamide-2-phenylethyl chloromethyl ketone (TPCK; Sigma, St. Louis, Missouri), diisopropyl fluorophosphate (DFP; Sigma, St Louis, Missouri), phenylmethyl sulphonylfluoride (PMSF; Sigma, St Louis, Missouri), elastatinal (Institute of Microbial Chemistry, Tokyo) (Umezawa, Aoyagi, Okura, Morishima, Takeuchi & Okami, 1973), leupeptin (Institute of Microbial Chemistry, Tokyo) (Aoyagi, Takeuchi, Matsuzaki, Kawamura, Kondo, Hamada, Maeda & Umezawa, 1969), chymostatin (Institute of Microbial Chemistry, Tokyo) (Umezawa, Aoyagi, Morishima, Kunimoto, Matsuzaki, Hamada & Takeuchi, 1970), trasylol® (Bayer, Leverkusen, Germany), soybean trypsin inhibitor (SBTI; Sigma, St Louis, Missouri), heparin (Sigma, St Louis, Missouri), pchloromercuribenzoate (Wako Chemical, Tokyo), ethylenediaminetetraacetic acid disodium salt (Wako Chemical, Tokyo) and N-ethylmaleimide (Wako Chemical, Tokyo). The protease (in 0-1 M phosphate buffer, pH 7-0) and inhibitor (in the same buffer) were mixed in a ratio of 2: 1 (V/V) and allowed to stand at 370 for 20 min before adding [3H]Hb. Estimation of macrophage chemotactic activity Chemotactic assays for macrophages were performed in vitro by a previously described method (Hirashima, Honda & Hayashi, 1976) using the chambers containing two 1-ml compartments with Nuclepore filters (GE-500, pore size 5 pm) (General Electric Company, New York). After incubation at 370 for 90 min, the cells that had migrated through to the lower surface of the filter were stained and counted in five higher power fields (10 x 40) randomly selected. Guinea-pig macrophages were collected from the peritoneal cavity 4 days after instillation of mineral oil. The cells (75-85 % macrophages) were suspended in RPMI 1640 solution, pH 7-2 (Grand Island Biological Company, Grand Island, New York) containing 10% foetal calf serum

(Microbiological Associates Incorporated, Bethesda, Maryland) and 5 U heparin (Sigma, St Louis, Missouri) at a concentration of 5 x 105 cells per ml.

Macrophage chemotactic generation from serum IgG

169

there were two proteolytic peaks, one being active at pH 3-5 and the other at pH 7 0. Total proteolytic activity at pH 7 0 was calculated to be approximately 350 nmol/h, while that at pH 3 5 was calculated to be 1650 nmol/h. The cell extract was fractionated at 70% saturation with ammonium sulphate at 2° for 4 h, the precipitate produced being dialysed against 0-02 M phosphate buffer (pH 7 4) and centrifuged at 15,000 g for 20 min; most (about 87%) of the proteolytic activity (at pH 7 0) of the cell extract was concentrated in the precipitate fraction.

Preparation of immunoglobulin G The IgG2 fraction was separated in the cold from fresh sera of healthy guinea-pigs according to the method of Kapusta & Halberstam (1964), using sodium sulphate precipitation, DEAE-cellulose chromatography (eluted in 0 005 M phosphate buffer, pH 8 0) and Sephadex G-200 gel filtration; the isolated IgG sample was homogeneous as judged by immunoelectrophoresis and immunodiffusion with anti-guinea-pig serum.

Preparationi of '251-labelled immunoglobulin G This was performed by the chloramine-T method (McConahey & Dixon, 1966). After elution on Sephadex G-200 column (1-5 x 40 cm), the IgG fraction was dialysed against 0-067 M phosphate buffer (pH 7 2) at 2° for 8 h; it showed a specific activity of about 6-0 x 106 c.p.m. (5 x 10-3 mCi) per mg of IgG.

Chromatography on DEAE-Sephadex The above precipitate fraction (40 ml containing 145 mg protein in 0-02 M phosphate buffer, pH 7 4) was applied to a DEAE-Sephadex column equilibrated with the same buffer at 20. Elution at pH 7-4 was done as follows: (1) 0-02 M phosphate buffer; (2) 0 067 M phosphate buffer; (3) 0-067 M plus 0-2 M NaCl; and (4) 0-02 M plus 1-0 M NaCl. Flow rate was 20 mI/h and 6 g effluent fractions were

RESULTS

collected. As seen in Fig. 2, proteolytic activity was concentrated in the first component corresponding to 50 % of applied samples, measured as the absorbancy at 280 nm. Total proteolytic activity (at pH 7-0) was approximately 285 nmol/h, correspond-

Experiment I. Separation and purification of neutral seryl protease Extraction andfractionation Proteolytic activity of the neutrophil extract (50 ml containing 300 mg protein) was assayed against [3H]Hb at different pH values. As shown in Fig. 1,

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100 150 200 250 300 Elution volume (ml)

Figure 2. Chromatography of proteolytic fraction from neutrophil extract on DEAE-Sephadex. Changes in the ionic strength of eluants at pH 7-4 are indicated by arrows: (1) 0-02 M phosphate buffer; (2) 0-067 M phosphate buffer; (3) 0-067 M phosphate buffer plus 0-2 M NaCl; and (4) 0-02 M phosphate buffer plus 1 0 M NaCl. The first component (stippled area) exhibiting most of the protease activity was used for further chromatography. Solid column, neutral protease activity (nmol/h) x 10-2; hatched column, acid protease activity (nmol/h) x 10-2.

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M. Ishida, M. Honda & H. Hayashi

170

ing to about 93 % of that of the applied samples or to 81 % of that of cell extract, while proteolytic activity (at pH 3 5) was approximately 820 nmol/h, corresponding to about 82% of that of the applied samples or to 50 % of that of cell extract.

Chromatography on CM-Sephadex After dialysing against 0-02 M phosphate buffer (pH 6-8) at 2° for 8 h, the proteolytic fraction (stippled area in Fig. 2; 120 ml containing 70 mg protein) was eluted on a CM-Sephadex column equilibrated with the same buffer. Elution at pH 6-8 was performed as follows: (1) 0-02 M phosphate buffer; (2) linear gradient with NaCl (0 5 M NaCl in the same buffer in terminal); (3) 0-5 M NaCl in the same buffer; and finally (4) 1 0 M NaCl in the same buffer. Flow rate was 20 ml/h and 6 g effluent fractions were collected. As illustrated in Fig. 3, five chromatographic components were obtained. The total yield was about 95% of the applied samples, measured as the absorbancy at 280 nm; the first comprised 26%, the second 10 %, the third 16 %, the fourth 26 % and the fifth 17 %. Most of the proteolytic activity at pH 7 0 was in the component (stippled area) eluted in 0-5 M NaCl; its total proteolytic activity was calculated to be about 225 nmol/h, corresponding to about 79 % of that of the applied samples or to about 64% of that of cell extract. No proteolytic activity (at pH 3 -5) was detected in this component. Acid protease activity was revealed in the second, third

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and fourth components, but neutral protease activity was negligible in these components. The first component, as previously described by Kouno (1971), exhibited neutral caseinolytic activity which was inactivated by 1 mm p-chloromercuribenzoate but reactivated by 1 mm cysteine; this component was used as the thiol protease fraction. Gel filtration on Sephadex G-75 After concentration by Diaflo membrane, UM-2, the proteolytic fraction (stippled area in Fig. 3; 3 ml containing 10 mg protein) was applied to a Sephadex G-75 column equilibrated with 1-0 M NaCl in 0-02 M phosphate buffer, pH 7 0. Flow rate was 15 ml/h and 2-5 g effluent fractions were collected. As seen in Fig. 4, one proteolytic peak was revealed. Total proteolytic activity was calculated as about 46 nmol/h; the recovery of proteins eluted was about 90 % and that of proteolytic activity was about 80 %. Each step of purification of the neutral protease is summarized in Table 1. Properties

(a) Effect of pH. As seen in Fig. 5, the protease (Fig. 3) was found to be optimally active at pH 7 0 when assayed against [3H]Hb. No proteolytic activity was detected at acid pH.

(b) Effect of protease inhibitors. As summarized in Table 2, the protease (Fig. 4) was strongly inacti-

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Figure 3. Chromatography of proteolytic fraction on CM-Sephadex. Changes in the ionic strength of eluants at pH 6-8 are indicated by arrows: (1) 0-02 M phosphate buffer; (2) linear gradient with NaCl (0-5 M NaC1 in 0-02 M phosphate buffer in terminal); (3) 0-5 M NaCI in 0-02 M phosphate buffer; and finally (4) 10 M NaCI in 0-02 M phosphate buffer. Most of neutral protease activity (stippled area) was used for further chromatography. Solid column, neutral protease activity (nmol/h) x 10-1; hatched column, acid protease activity (nmol/h) x 10-2.

171

Macrophage chemolactic generation from serum IgG 20

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vated by DFP, PMSF, SBTI, elastatinal, or heparin, but little or no inhibition was observed with EDTA, TLCK, TPCK, leupeptin, chymostatin, or trasylol. No inhibition was revealed with 1 mm p-chloromercuribenzoate or 5 mm N-ethylmaleimide. These results suggest that the protease may be a neutral seryl protease resembling elastase. (c) Effect of heat. The protease (stippled area in Fig. 3) was thermolabile, because its activity was decreased by 60 % on heating at 60° for 15 min, and completely abolished at 800 for 15 min. (d) Estimation of molecular weight. The protease (Fig. 3) was eluted on a Sephadex G-75 column (1 5 x 95 cm) equilibrated with 1 0 M NaCl in 0-02 M

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phosphate buffer, pH 7-0 (Andrews, 1965). Flow g effluent fractions were collected. As standard substances, blue dextran and ovalbumin from Pharmacia, Uppsala, Sweden, or a-chymotrypsinogen, cytochrome c, and bacitracin from Sigma, St Louis, Missouri were used. The elution volume of the protease was 122 ml; its molecular weight was assumed to be 14,000± 3000. rate was 15 ml/h and 2-5

Experiment II. In vitro generation of macrophage chemotactic activity from serum IgG by neutral seryl protease The seryl protease (Fig. 4) was dialysed against

Protein (mg)

Total activity (nmol/h)

Mean specific activity (nmol/h/mg)

Yield (%)

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with ammonium

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Figure 5. Effect of pH on activity of the neutral protease after elution on CM-Sephadex. Activity of the proteolytic fraction (stippled area in Fig. 3) was assayed against [3H]Hb at various pH conditions. Protein concentration of the sample was 135 pg/mI.

Table 1. Purification of the neutral protease

Steps of

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Table 2. Effect of inhibitors on the neutral protease activity

Agents EDTA

TLCK TPCK

DFP PMSF

SBTI

Trasylol Elastatinal Leupeptin

Chymostatin N-ethylmaleimide Heparin

Concentrtions 10mM

Changes in proteolytic activities (%)

50 0-3 mM 3-0 0 03 mm 03 1-0 mM 01 mM 1*0 50 pg/ml 100 100 U/ml 1000 18 pg/mI 180 10 pg/ml 100 15 pg/ml

+2 -19 +8 +2 0 0 -98 -61 - 95 -70 - 81 +5 +7 -35 - 89 -7 - 11 - 12

5 mM 5 U/ml 50

-3 -71 - 84

The neutral protease (Fig. 4) was assayed against [3H]Hb. In the absence of inhibitors, the activity of the neutral protease was between 0 5 and 1 0 nmol/h/ml. Changes in the proteolytic activities were expressed as mean values of three assays.

0-067 M phosphate buffer (pH 7.2) at 2° for 8 h. Equal volumes (0 5 ml) of the protease (1-3 nmol/ h/ml) and IgG (400 ,g/ml in 0-067 M phosphate buffer, pH 7 2) were mixed and incubated at 370 for 1, 3, and 6 h, respectively. As summarized in Table 3, IgG itself had no chemotactic activity for macrophages, but it became clearly chemotactic for macrophages after treatment with the protease; the intensity of ictivity generated from IgG was found to be maximal after 6 h digestion with the protease. No generation of chemotactic activity for macrophages from IgG was revealed with heat-inactivated protease. The protease itself, before or after heating at 800 for 15 min, had no chemotactic activity on macrophages. The above proteolytic activity, measured against [3H]Hb, corresponded to 0 33 units/h/ ml in caseinolytic activity. The thiol protease (first component in Fig. 3) was dialysed against 0-067 M phosphate buffer (pH 7 2) at 20 for 8 h and its activity was measured against casein in the presence of cysteine (10-3 M). Equal volumes (0 5 ml) of the protease (0 33 units/h/

ml) and IgG (400 ,ug/ml) were mixed and incubated at 37° for 3 and 6 h, respectively. No generation of chemotactic activity for macrophages from IgG was revealed with the thiol protease, as seen in Table 3. The protease itself had no chemotactic activity on macrophages. Based on the above observations, IgG (4 mg) was incubated with the seryl protease (13 0 nmol/h in 2-5 ml of 0-067 M phosphate buffer, pH 7 2) at 370 for 6 h under sterile condition and then the reaction mixture (4 ml) was eluted on a Sephadex G-75 column (1-5 x 95 cm) equilibrated with 0-067 M phosphate buffer, pH 7-2. As shown in Fig. 6a, three components at 280 nm were observed after 6 h digestion. The first component was indistinguishable from the elution volume of untreated IgG. The second component seemed to correspond to the protease, because its elution volume was similar to that of the protease eluted in 0-067 M phosphate buffer. The third component seemed to be composed of peptide(s), because the optical density at 280 nm of the component was apparently decreased after dialysis against 0-067 M phosphate buffer. Chemotactic activity for macrophages was revealed only in the first component. No chemotactic activity was found in other components. The observations described above were confirmed using 1251-labelled IgG. Labelled IgG (400,ug/ml) was incubated with the above protease (1-3 nmol/h in 1 ml of 0-067 M phosphate buffer, pH 7 2) at 370 for 6 h and then eluted on Sephadex G-75, as mentioned above. As seen in Fig. 6b, two radioactive components were detected; the first corresponded to the IgG fraction and the second to that of dialysable peptide(s). These findings suggest that the protease may degrade IgG as a substrate and release dialysable peptide(s) without any production of 3 5 S fragments such as Fab and Fc.

Experiment III. Failure of generation of macrophage chemotactic activity from serum IgG by pancreatic elastase On the basis of observations that the above protease may resemble elastase, elastase from pig pancreas (Worthington Biochemical, Freehold, New Jersey) was tested for generation of macrophage chemotactic activity from IgG. Equal volumes (0 5 ml) of pancreatic elastase (0-32, 0-65, 1 3, and 2-6 nmol/h/ ml in 0-067 M phosphate buffer, pH 7 2) and IgG (400,ug/ml in 0-067 M phosphate buffer, pH 7 2)

Macrophage chemotactic generation from serum IgG

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Table 3. Macrophage chemotactic generation by the neutral seryl protease of serum IgG

IgG

(pg) 200 200 200 200 Buffer* 200 Buffer* 200 Buffer* 200 200 Buffer*

Protease activities

Duration of digestion (h)

0-65 nmol/h 0-65 nmol/h 0 65 nmol/h Heated at 800 for 15 min Heated at 800 for 15 min Buffer* 0-65 nmol/h 0-65 nmol/ht 0-65 nmol/ht 0 16 units/h§ 0-16 units/h§

1 3 6 6 6 6 6 6 6 3 6

Number of macrophages

migrated: 45 80 135 15 5 20 21 13 11 25 32 5

After dialysis against 0-067 M phosphate buffer (pH 7 2), the neutral seryl protease (Fig. 4) and the neutral thiol protease (first component in Fig. 3) were assayed. The activity of the protease added was 1-3 nmol/h/ml when tested against [3H]Hb and 0 33 units/h/ml when tested against casein. * 0-067 M phosphate buffer, pH 7-2. t Pancreatic elastase. $ Mean values of four assays in duplicate. § Neutral thiol protease in the presence of cysteine (10-3 M).

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Figure 6. Gel filtration of the neutral protease-treated IgG on Sephadex G-75. (a): IgG (4 mg) was treated with the neutral protease (13 nmol/h) at 370 for 6 h and eluted on this column (1-5 x 95 cm). (b): 125I-labelled IgG (400 pg/ml) was treated with an equal volume (0 5 ml) of the neutral protease and eluted. ( ), Treated with 1-3 nmol/h/ml of the protease. (- - -), Not treated with the protease. Hatched column, number of migrated macrophages.

M. ishida, M. Honda & H. Hayashi

174

were mixed and incubated at 370 for 1, 3, and 6 h. As seen in Table 3, the enzyme induced no generation of macrophage chemotactic activity from IgG, in spite of IgG digestion at various activities of the enzyme. The results suggest a different hydrolytic action of the above protease and pancreatic elastase on IgG as a substrate. In order to analyse the different hydrolytic action of these proteases, elastin-orcein was used as a substrate according to the method of Sacher, Winter, E C

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Figure 7. Effect of the neutral protease on elastin-orcein. The neutral protease (Fig. 4) and pancreatic elastase were assayed against [3H]Hb at pH 7 0 for 60 min and elastinorcein at pH 8-8 or pH 7 0 for 3 h. (0) Neutral protease; (0) pancreatic elastase.

Sicher & Frankel (1955). As shown in Fig. 7, the above protease clearly digested [3H]Hb in parallel with its concentration, but not elastin-orcein. In contrast, pancreatic elastase clearly digested both [3H]Hb and elastin-orcein in parallel with its concentration. DISCUSSION The present results demonstrate that a neutral protease, extracted at acid pH from rabbit neutrophils, was partially purified by chromatography with DEAE-Sephadex, CM-Sephadex and Sephadex G-75 in that order; it had a molecular weight of about 14,000 daltons and was optimally active at pH 7 0 when tested against [3H]Hb. No acid protease was contained in the enzyme preparation. This en-

zyme was considered to be a seryl protease resembling elastase because of strong inactivation by DFP, PMSF, or elastatinal, but it failed to digest elastinorcein as an elastase substrate. This strongly suggests that the enzyme closely resembles that from bone marrow cells of human which was described as a new limited proteolysis-inducing enzyme by Aoki, Urata, Takaku & Katunuma (1975); their seryl protease was also inactivated by elastatinal but did not digest elastase substrate. The present enzyme seemed to differ from histonedegrading protease (histonase) separated from rabbit neutrophils by Davies, Rita, Krakauer & Weissmann (1971), because their protease failed to digest haemoglobin but was activated by polyamines such as heparin or dextran sulphate, while the present enzyme digested haemoglobin ([3H]Hb) but was inactivated by heparin. Recently, it has been found that a neutral seryl protease, released from neutrophils in culture, strongly accelerates thymocyte activation by PHA, as shown by increased DNA synthesis (Yoshinaga, Nakamura & Hayashi, 1975; Nakamura, Yoshinaga & Hayashi, 1976). This enzyme seemed to be different from the present enzyme, because it was inactivated by trasylol but not by elastatinal; its molecular weight was about 19,000 daltons. The present enzyme was inactivated by elastatinal but not by trasylol; and it failed to accelerate thymocyte activation by PHA (unpublished data, Kuratsu, Ishida & Hayashi). Much interest has been concentrated on neutral proteases from human neutrophil granules, especially elastase-like enzymes (Janoff, 1973; Ohlsson & Olsson, 1974; Feinstein & Janoff, 1975; Baugh & Travis, 1976) or chymotrypsin-like enzymes (Gerber, Carson & Hadorn, 1974; Schmidt & Havemann, 1974; Rinder-Ludwig & Braunsteiner, 1975); these enzymes were separated by differential salt concentration, affinity chromatography or cationic ion exchange chromatography. Schmidt & Havemann (1974) have described a separation of each enzyme on CM-Sephadex, the elastase-like enzyme being eluted in 0-46 M NaCl, while the chymotrypsin-like enzyme was eluted in 0-78 M NaCl. It is of special interest that the present enzyme, although separated from rabbit neutrophils, is eluted in 0 5 M NaCl on CM-Sephadex and that the enzyme is insensitive to chymostatin. More distinct characterization of the present enzyme awaits further purification of the enzyme.

Macrophage chemotactic generation from serum IgG The present observations demonstrate that the present protease produces a macrophage chemotactic factor from the IgG molecule in vitro. Its molecular weight was similar to that of IgG, suggesting a minor structural change in the IgG molecule; and its chemotactic generation was shown to be accompanied by release of dialysable peptide(s) from the IgG molecule without concomitant release of Fab or Fc fragments. In contrast, no generation of macrophage chemotactic activity from IgG was induced in vitro by elastase from pig pancreas or by neutral thiol protease from rabbit neutrophils, though these enzymes were tested at a proteolytic activity similar to that of the present enzyme. It is thus assumed that generation of macrophage chemotactic activity from IgG is mediated via the characteristic hydrolytic activity for IgG of the present enzyme. In relation to the generation of chemotactic activity from IgG possibly due to structural change, it is of interest that human serum albumin (HSA), when denatured with alkali, is more chemotactic for macrophages than native HSA; its chemotactic effect may be associated with structural changes in the monomeric molecule, as shown by physicochemical evidence of unfolding which was measured by the increase in the specific viscosity (Wilkinson & McKay, 1971) and by increased surface activity (Wilkinson, 1974). Similarly haemoglobin and myoglobin molecules can be unfolded by removing the haem group from the globin molecule by acidacetone treatment and then become chemotactic (Wilkinson, 1973). On the other hand, it has been shown that the neutral thiol protease from inflammatory tissue or from neutrophils induces generation of neutrophil chemotactic activity from IgG in vitro, accompanied by release of dialysable peptide(s) from the IgG molecule occurring exclusively at the Fc portion (Hayashi et al., 1974; Hayashi, 1977). However, the site of the structural change of IgG in the generation of macrophage chemotactic activity by the present enzyme has not yet been clarified. Recently, it has been demonstrated that three different macrophage chemotactic factors (a, b, and c) can be separated from DNP-ascaris extractinduced skin lesions in guinea-pig (Hirashima, Honda & Hayashi, 1976). Chemotactic factor a has a molecular weight similar to that of IgG, shares common antigenicity with guinea-pig serum IgG, and its activity is completely absorbed by anti-IgG and anti-light chain antibodies (Honda, Hirashima, M

175

Nishiura & Hayashi, 1977), suggesting that this chemotactic factor may resemble the above in vitro chemotactic factor from the IgG molecule.

ACKNOWLEDGMENTS This study was supported in part by grants from the Japanese Ministry of Education, Culture and Science (No. 148131 and No. 244031). REFERENCES ANDREWS P. (1965) The gel-filtration behaviour of proteins related to their molecular weight over a wide range. Biochem. J. 96, 595. AOKI Y., URATA G., TAKAKU F. & KATUNUMA N. (1975) A new protease inactivating 3-aminolevulinic acid synthetase in mitochondria of human bone marrow cells. Biochem. biophys. Res. Commun. 65, 567. AOYAGI T., TAKEUCHI T., MATSUZAKI A., KAWAMURA K., KONDO S., HAMADA M., MAEDA K. & UMEZAWA H. (1969) Leupeptins, new protease inhibitors from actinomycetes. J. Antibiotics, 22, 283.

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In vitro macrophage chemotactic generation from serum immunoglobulin G by neutrophil neutral seryl protease.

Immunology 1978 35 167 In vitro macrophage chemotactic generation from serum immunoglobulin G by neutrophil neutral seryl protease M. ISHIDA, M. HON...
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