Mitogenic proteinases from human leukocytes

0161-5890/84$3.00+O.OO IC’1984Pergamon Press Ltd

Molecular Immunolog?.Vol. 21, No. 4. pp. 31I-320. 1984 Printed in Great Britain

MITOGENIC

PROTEINASES

FROM HUMAN

LEUKOCYTES

JOHN D. FRASER* and G. KENNETH SCOTT? Department of Biochemistry, The University of Auckland. Private Bag. Auckland, New Zealand (First received

I July 1983; accepted in rerised,form 1 Nooember 1983)

Abstract-Serine proteinase activity has been identified in purified human lymphocyte membranes, and the corresponding enzymes have been isolated from human leukocyte extracts by affinity chromatography. The localization of these enzymes on lymphocyte and granulocyte membranes has been immunochemically demonstrated. Mitogenic activity towards human lymphocytes has been demonstrated for these enzymes, and anti proteinase antibodies inhibit the growth of transformed lymphocytes. The proteinases are similar in many properties to enzymes previously isolated from human leukocytes, and reported to be involved

in a wide variety of leukocyte functions.

INTRODUCTION There are now several independent lines of evidence to support the view that proteinases operating at or near the cell surface have a vital role in the growth and division of normal and tumour cells in culture (reviewed by Vasiliev and Gelfand. 1981). Some of the earliest evidence for this view was based on the mitogenic ability of exogenous proteinases, and in fact this phenomenon was first observed with trypsin or chymotrypsin acting on human lymphocytes (Mazzei et al.. 1966). Trypsin has a similar effect on mouse B lymphocytes (Vischer, 1974) and Vischer also showed that, although a potent mitogen for B cells, trypsin had no effect on mouse T lymphocytes. Subsequently it was shown that pronase was as effective a mitogen as trypsin at much lower concentrations (Kaplan and Bona, 1974), and that both enzymes stimulated DNA synthesis in human lymphocytes. Thrombin was also demonstrated to be mitogenic for B cells but not for T cells (Chen et al., 1976). An intrinsic surface proteinase activity on rodent lymphocytes has been reported by TiikCs and Kiefer (1976), but not further characterized. Further evidence for such an enzyme on human cells comes from studies with a-1-antitrypsin, which inhibits a neutrophil surface proteinase (Wintroub et al., 1974, 1977) and also binds to the surface of lymphocytes (Bata et u/., 1981). Chemical inhibitors of serine proteinases inhibit the effect of B cell-specific mitogens but not of concanavalin A, a T cell-specific mitogen (Ku et al., 1981). The inhibitors used in this study could affect cytoplasmic enzymes following diffusion through the plasma membrane, but a similar effect has been observed with insolubilized soy bean trypsin inhibitor (SBTI), which also blocks B cell mitogenesis (Moreau et ul.. 1975).

Proteinases assayed by their ability to activate plasminogen to plasmin have been reported on the membranes of rodent thymocytes (Fulton and Hart, 1981) and of B cells (Maillard and Favreau. 1981). There is also some evidence for secreted enzymes which could be involved in mitogenesis. Recently it has been shown that the mitogenic effect of concanavalin A in human leukocytes involves the stimulation of secretion of proteinase (Hatcher and Norin, 1982). Many other activities of blood cells depend upon proteolysis at the cell surface. These include the activation of kininogens by neutrophils (Wintroub et al., 1974), the inhibition of leukocyte migration (Bentzen, 1979), the generation of other lymphokines (Kishimoto et al.. 1979), chemotaxis (Thomas et al., 1977). and the cytocidal action of killer lymphocytes (Allison and Ferluga. 1976). The last-mentioned phenomenon may be related to the cell-surface chymotrypsin-like proteinase in human polymorphonuclear leukocytes which is involved in superoxide production by these cells (Kitagawa et al., 1979). A recent report shows that proteinases stimulate the differentiation of mouse erythroleukaemia cells (Scher et al., 1982). In many of these studies, the evidence for proteinase involvement is indirect, and the enzyme(s) involved have been characterized in few of the remainder. We have attempted to isolate and characterized intrinsic leukocyte enzymes involved in the stimulation of cell growth, but it is clear that a fuller understanding of these enzymes may illuminate many other aspects of cellular immunology.

MATERIALS AND

METHODS

Proteinase assays A highly sensitive assay was employed to detect small amounts of neutral proteolytic activity. Highly purified a-casein was iodinated by the chloramine-T method (Hunter and Greenwood, 1962). Typically,

*Present address: Dana-Farber Cancer Institute. Harvard Medical School, Boston. Mass., U.S.A. tTo whom correspondence should be addressed. 311

311

Joti> D. FKAS~Kand G.

5 mg of Y-casein W;I~ heated to 80 C’ for 30 min to destroy any contaminating protrinase activity. then iodinated with I mC’i of carrier-free “‘-iodine and 5 ITI~ oE chloramine-T in 0.3 ,U sodium phosphate bulter pH 7.4. After 10 sec. 15 mg of sodium hisuphite \+;I!, added and the reaction products were separated on a I Y 20 cm column of Sephadex G-35. The labelled casein M;I\ then diluted kvith the addition of I5 mg cold. heat-tl-eatcd casein and made up to 70 ml in 0. I .U sodium phosphate butler pH 7.4. The specitic actlvit! 01‘ the substrate varied bet\veen 6 .I 10J and 1 x IO’cprn pg when freshly prepared. Vials containing I5 x 10h dpm were stored at - 20 C until use or until the specific activity fell belo& 1 2’ lO”dpm:~~p. The assay itself in\ olved the addition of 50 /~g of “‘I-casein to the ample to he assayed in ;i total volume of 0.1 ml oi‘(1.7)5 \I sodium phosphate buffer pH 7.0. and incubating at 37 C for between 15 min and 5 hr. The reaction was then terminated with the addition of 0.5 mp of‘ ice-cold. heat-treated, unlabelled casein. folIoNed immrdiatel) by the addition of 0.25 ml of ice-cold IO”,, (w’v) trichloroacetic acid (TCA). The vials were left for I hr at 4 C before being centrifuged and 0.15 ml of the supernatant was then counted in a ;‘-ray spectrometer (Beckman). Blank tubes (without added protcinase) showed no more than I”,, of the total radioacti\it\ hccnming acidsoluble. If any vials showed greater than ZO”,, zolubilization of the total radioactivity, then the assa> was repeated with either ;I reduction In sample size or incubation time. The hydrolysis of I pg 01‘ cassin substrate in 1 hr was defined as one unit of proteinase activity. Peptide substrates linked to ;I I’-nitroaniline chromophore (Kabi Diagnostics) lvere used as precIousi) described (Allen c/ t/l.. 19X1: A11en and Scott. 1983). Nitrophenyl esters of carbobenzoxy-substituted lysine. phenylalanine and glycylglycine (Sigma) were made up as 1mg;‘ml stock solutions in acetonitrile, diluted 40-fold into 0.1 )2f sodium phosphate hulTer pH 6.0 for assay. Hydrolysis wa:, followed by an soluincrease in il,,,,j,ln and standard p-nitrophenol tions were used for calibration. Plasminogen :!cti\,ation \\;I:; assayed using the standard cascinolytic assa). with the addition of 5 /lg human plasminogen purified by the method of Deutsch and Mertz (1970).

Human I>mphocytcs were isolated from fresh peripheral venous blood as previously described (Allen and Scott. 19X2). Membrane purification followed the method of Schmidt-Cillrich (‘I I(/. (1976). with moditication>. Lymphocytes v,ere suspended in modified balanced salt solution (BSS: no calcium) at a concentration of X x IO’ cells,‘ml. This suspension was then placed in a precooled nitrogen cavitation bomb and with gentle stirring was incubated for 30 min at 4 C under ;I pressure of 450 psi nitrogen. To release the

KEWETH

SCOTT

pressure, the bomb was inverted und the valle openc~! slowly so that the vessel was slow1> voided of I(\ contents. The homogenate wab imnredi;r!ei\. n~;td< 0.0025 M in disodium ethylenediaminet~t~~l~~cet,~. acid (EDTA) to prevent clumping of orfanellcs ‘\ phase contrast microscope \\\a~ used to dctcrrnlm~ 111~ ratio of intact nuclei over intact cclI\ T’hl\ ‘,,II. usually about 70:30 on a per cent hasi\. \ialui.. higher than this indicated that the )loni,‘gerli/:ttli,r: procedure had been too sevcrc and L;LIUC’\ /o\\er ttl;lli this indicated that the etlicienq 01 the ic>chniqtlc could be increased. It was important that the dc\trriition of internal organellex such a\ 11uc.l~ and I\ sosomal granules was kept to a minimulli. The homogenate was centrifuged fur !o*R.II:~ ii! a bench centrifuge and the pellet washed and recentrifuged to sediment intact cells ;IIIJ IIU+~. .rhc pooled supernatant was centrifuged .II : \ irl g.min at 4 C. The pellet l‘rom ttu< sp171 (‘\!I \\,1. washed. recentrifuged and stored at 4 ( 10 he WC% later. The supernatant was centrifuged for ;I Sinai tlmc at lO’g.min at 4 C to sediment remainlug prirt~cul:~tt: material (P). The M and P pellets were each suspendcci Ifi 15ml 25”,, (w/v) Ficoll (dlj= 1.08) butfcred \+ith it.001 .\I N-2-hydroxyethylpiperazine-,~-7-ethanesulphonatr (HEPES). 0.001 M MgC1, pH X.0 using a IO(W fitting Dounce homogenizer. Each SLIS~CIISIOI? \\;I\ ~LIC‘C‘C~sivelj overlayed with 1 ml I i”,, I\\ 1 I Fic~~ll (dy = 1.05) then X ml modified BSS. Thc~ ~I\C,IIItinuous gradients were then subjected to tquillhrlun1 centrifugation for lO”a.min. Resulting Interfac,c\ were removed along with the overiying Iayer. diluted to three times their volume with distilled \+:tter then centrifuged at IO’g.min. Pellets were resuspended 1~ a small amount of BSS ;md assayed for ihc I’,tllrl\\inF cytochemical marker snzyme\: Alkaline phosphatase Mg’ ’ (Na K ’ ) .4TPase Glucose-h-phosphatase

(Emmelot and t&l\. I %>(JI (Jorgensen. 19741 (Eylar and H+X~~I;IIL

N-acetyl glucosaminidase Succinate-INT-reductase

(Touster ot LII.. iF0) (Eylar and Hapqmn.

Protein

( Lowr\

l’J71)

107i)

c/ II/.. ! L)>I ).

5’-Nucleotidase activity was found TV be ICI ;, IOU !:I lymphocyte plasma membranes and W;I< not I’OLItinely assayed.

For the routine preparation of the plasn~a ~ncnlbrane proteinase. leukocytes were used ;I\ ‘tilrting material instead of lymphoq tes. Approximately IO cells were obtained from six ‘buR;i coat? ;1\ prim viously described (Lim 6’1 (ri.. 19X2) and \\ere w\pended in 500 ml of distilled water and \tlrrcd Lit -i ( for 6 hr. Disruption was aided by 3 .: .30 set hur\th c:I 8 pi4 ultrasonic signal from an MSE sonic;Itor \\~til large probe attached. The homogenate u ;jm\i’cniri-

Mitogenic leukocyte proteinases fuged at 2.4 x lO”g.min at 4 C. The resulting pellet was again suspended in 200 ml of distilled water, sonicated and stirred at 4 C for 1 hr before being centrifuged as before. The resulting plasma membrane enriched pellet was suspended in lOOmI of 0.05 ,!V sodium phosphate buffer pH 7.4 containing I ,\I sodium chloride and 0.003 hi EDTA and stirred overnight at 4 C. After centrifuging for lO’a.min the pellet wits re-extracted with the same buffer. The two pooled supernatants were then dialysed extensively against distilled water and the precipitate that formed was remov*ed by centrifugation. The extract was then made SO”,, saturated with the slow addition of finely powder-cd ammonium sulphate at 0 C and left for I hr at 0 C before being centrifuged for 2 x 10’ g.min. The supernatant was then raised to 70”” saturation with the slow addition of finely powdered ammonium sulphate at 0 C and again left for 1 hr before centrifugation as before. The pellet was dissolved in, and dialysed against, distilled water. The extract was then freeze-dried and redissolved in 20 ml 0.01 ,%I sodium phosphate buffer pH 7.4 containing 0.003 !ld EDTA and passed through a 10 ml column of IysincSepharose which had been equilibrated at room temperature with the same buffer. Flow rate was maintained at about I mlimin and the column washed with phosphate buffer until the AISOnmof the eluate was less than 0.01. Bound protein was eluted with I :Zl sodium chloride in the starting buffer. This peak was dialysed against distilled water and freezedried. After this, the purified enzyme could be stored dry or dissolved in a minimum of phosphate-buffered saline (PBS) and stored at -20 C, at which temperature the enzyme appeared to be quite stable. The remaining salt extracted membrane enriched particulate pellet was suspended in 50 ml 0. I M sodium phosphate pH 7.4 containing 0.4 M sodium chloride and 0.5”,, Triton X-100 and stirred overnight at 4 C. Detergent insoluble material was removed with centrifugation at IO’ R. min then re-extracted with another 50 ml of Triton buffer. The two supernatants were pooled and extensively dialysed. This extract was also passed through 1Oml of lysineSepharose and non-specific and specifically bound proteins removed under the same conditions as those described for the salt extract. Lysinr- Sepharose was prepared by the method of Cuatrecasas ( 1970). Sepharose 4B (Pharmacia; lOOmI packed volume) was activated with 30g of cyanogen bromide (Sigma) and reacted overnight with 100 g of r.-lysineeHC1 in 150 ml 0.1 M sodium bicarbonate buffer pH 8.9 at 4 C. ArginineSepharose was prepared by an identical procedure.

Diisopropyhluorophosphate (DFP: BDH) was used as previously described (Scott and Tan, 1979) and was 10 mill in tinal proteinase assay mixtures, as were iodoacetamide and EDTA. Soy bean trypsin inhibitor (SBTI: Sigma) was used at IOpg/ml in the

313

final assay mixtures. Assays were also done in the presence of heparin (100 U; Weddel Pharmaceuticals, London), of human plasma (SO”,;) and both of these reagents together. SDS polym$arnide

gel electrophoresis

Using the procedure of Laemmli (1970). samples were first solubilized in 0.5 M TrissHCl pH 6.8 containing 3”” sodium dodecyl sulphate (SDS). then heated to 1OO~Cfor 3 min. Treated samples were then run on lo”, acrylamide gels of 0.5 mm thickness in the presence of O.l”, SDS. For the detection of [IH]DFP labelled prbteinases. samples were incubated for 10 min at 37 C with 2 PCi [“H]DFP (6Ci/mol; Amersham) prior to SDS solubilization. then run as normal on lo”, gels. After completion of the run, either before or after staining with Coomassie blue. the gel was soaked in I M sodium salicylate pH 7.0 in 509; (v/v) ethanol: water for 10 min according to the method of Chamberlain (1979), then dried and overlayed with a sheet of X-ray film (Kodak X-Omat S). Fluorograms were developed I day and at -80 C over a period of between 1 month depending on the amount of radioactivity incorporated into protein. In~tnunochemicuI

techniques

The 75K leukocyte proteinase, the immune rabbit ?;-globulin prepared against it, and the corresponding Fab fragment were prepared as previously described (Allen and Scott, 1983). Identical immunization and purification procedures were used to prepare a rabbit y-globulin specific for the “triplet proteinases” purified in the present study. None-immune rabbit ;‘-globulin was also prepared for control experiments. For proteinase localization experiments. human lymphocytes and granulocytes were fractionated as before (Allen and Scott, 1983) and suspended at 2 x 10’ cells/ml in phosphate-buffered isotonic saline (PBS). The cells were sedimented and to the packed pellet was added 0. I ml of ice-cold PBS and 0. I ml of either antiproteinase y-globulin (3 mg/ml w/v) or normal rabbit g-globulin. Cells were resuspended by gentle vortexing and incubated on ice for 30 min before being washed three times with 1 ml ice-cold PBS. To the packed cell pellet was then added 0.1 ml of fluorescein-labelled goat anti-rabbit IgGH antiserum (Wellcome) diluted one-hundred-fold in PBS. After incubating the resuspended cells on ice for a further 30min. they were again washed three times with 1 ml cold PBS and finally suspended in 0.02 ml PBS containing loo/;, (v/v) glycerol. Fluorescence was observed with a Zeiss fluorescent microscope at 1000 x magniffcation. ;I-globulin (50 mg) was attached to Anti-proteinase 5 ml packed washed Sepharose 4B by the carbonyl diimidazole method of Bethel1 et (11. (1979). To this 5 ml column was added a proteinase extract buffered with 0.01 M TrissHCl pH 8.0 and the column then washed until the AzBOnmwas below 0.01. The column

JOHN D. FRASER and G. KENNETH SCOTT

314

Table

Marker

enzyme

Mg’+ (Na + K - ) ATPase Alkaline phosphatase Glucose-6-phosphatase N-acetyl glucosaminidase Succinate-INT-reductase Protein

I. Lymphocyte

plasma

membrane

M,

Ml

MI

P,

P,

PI

17 I5

?I) 14

OU6 I5

II 6.5

I .4

(1.8

75

0.9

I

0.5

2

3

3

2.3

0.6 0

I4 I.1

25 15

U.3 0

0 3 0

0.4 17

Total recover> (“/,I 82.5 7x xi x5 88 99

purdic&ion Specific actwitiei

Lymphocyte5 (4.3 r 10’) were disrupted by nitrogen cavitation and the particulate material fritctionuted on discontinuous Ficoll density gradients ( 10X,q.min). The interfaces from the developed gradients plus the overlying layer were removed. numbered from top of the gradient. diluted at least three-fold wth aster and concentrated by centrifugation. The resulting pellets were resuspended in a small quantity of BSS and assayed for the five cytochemical markers. Fractions having the htghrst specific actiwt!. in units mg-’ protein. (M,. M7 and P,) of the two plasma membrane markers (Mg’+ (Na *K’) ATPase and alkaline phosphatase) were pooled. washed with BSS and stored in BSS at 70 C. Protem content was monitored throughout the purificatton and the final pooled membrane preparation was found to have 6.6mg total protein (approximately 4”,, recovery of total protein).

was then successively washed with 1 M sodium chloride and 1 M potassium thiocyanate. Eluted fractions were immediately dialysed against water and freezedried. Cell

culture

antrprotemase y-globulin (Allen et ~11..1981).

on monolayer

cell cultures

RESULTS

techniques

Fresh human lymphocytes were suspended at 1 x 10hcells/ml in RPM1 1640 medium (Gibco) supplemented with 1”” (w/v) glutamine. Cells were cultured under a 7”;, CO, atmosphere, without the addition of antibiotics. From the investigation of the mitogenic properties of the proteinase. the enzyme was dissolved at 100 units/ml in culture medium and added to lymphocyte cultures in varying amounts. Cells were then cultured at 37’ C for 50 hr, after which time 2 p Ci of [3H]thymidine (Amersham: 26 Ci/mol) was added and the cells left for a further 18 hr at 37’C. Cells were harvested on Millipore pre-filters (AW 03) under suction, washed three times with 2 ml cold PBS followed by 2 x 2 ml lo”,, (w/v) TCA. Filters were then counted for ‘H-incorporation into TCA-insoluble materials as previously described (Scott and Tan. 1979). An adherent transformed human lymphocyte strain (RAJI; Pulvertaft. 1964) was a gift from Mr E. J. Carolan, Department of Biochemistry, University of Glasgow. It was cultured in RPM1 1641 medium (Gibco) lo”, (v/v) in foetal calf serum and buffered with 0.025 M HEPES at 37’ C. Initially, the cells were suspended at 10’ cells/ml and 0.1 ml aliquots dispensed into 96-well plate. After 24 hr. 0.05 ml of medium was removed from test wells and replaced with 0.075 ml of medium containing antiproteinase ;t-globulin (150 ng/ml). Other wells were similarly treated with antiproteinace Fab (100 pg/ml) or normal rabbit y-globulin (150 ng/ml) instead of the intact immune ~-globulin. This procedure was repeated twice at 48 and 72 hr. Cells were then counted 24 hr later on the inverted microscope. Reported cell counts are the mean (*SD) of eight counts of random high-power fields. A basically similar protocol has been used to test the effects of

Plasma membranes were puritied from a lymphocyte lysate (4.3 x 10” cells). Cytochemical marker enzymes demonstrated that fractions M,. M, and P, represented a relatively pure plasma membrane preparation (Table I). They were pooled. washed with PBS, concentrated by centrifugation, resuspended in PBS at 0.7 mg protein ml ’ and stored in 1 ml had a proaliquots at -80 C. This preparation teolytic activity of 12.9 units mg ’ protein, measured with the caseinolytic assay. This activity was enhanced by a factor of 3.2 in the presence of I”, Triton X-100. DFP at a concentration of 0.2 mM caused a 50”” inhibition of this activity but it was necessary to use 30mM DFP to obtain complete proteinase inhibition. The inhibitory effect of DFP was not affected by l”,, Triton X-100. Autoradiography of [jH]DFP-treated lymphocyte membranes indicated a closely-spaced triplet group of isotopically-labelled proteins, with apparent molecular weights of approximately 25, 27 and 29K. The extent of labelling was roughly equivalent in the 25K and 29K components, with the 27K protein being more lightly labelled (Fig. 1). Incubation of lymphocyte membranes with various peptide-chromogen substrates indicated a trypsin- or thrombin-like proteinase specificity (Table 2). It was thus decided to use arginine- or lysine-agarose affinity chromatography to effect further purification of the enzyme(s) from the membrane preparation. It was decided to use a total leukocyte preparation for larger-scale experiments. Lrukoq~tr proteinuse

c~.~tructior7,

purification

untl

characterizutior7

Lysine-agarose chromatography of salt and Triton X-100 extract of the human leukocyte particulate

Mitogenic

leukocyte

proteinases

Fig. 1. SDS polyacrylamide gel electrophoresis cf proteinase preparations. Electrophoresis was carried out as described in Materials and Methods. The origin of the gel is at the top of the photograph in each case. Tracks B--H are all approximately to the same scale. Track A is to a larger scale. A, Purified lymphocyte membranes treated with 13H]DFP and subjected to autoradiography after electrophoresis. B, Purified, salt-extracted triplet proteinase preparation, stained with Coomassie blue. C, As B, but treated with [%]DFP, and autoradiographed after electrophoresis. D, Purified, detergent-extracted triplet proteinase preparation, stained with Coomassie blue. E, Purified, salt-extracted proteinase stained with Coomassie blue and showing the presence of a 75 K band. This band was seen in both salt- and detergent-extracted proteinase preparations, but not consistently. F, Material eluted from antibodyaffinity chromatography by 1 A4 saline. G, Material eluted from antibody-affinity chromatography by 1A4thiocyanate. H, Standards. From top of the gel; transferrin, serum albumin, chymot~psin, lysozyme.

Table 2. Lymphocyte plasma membrane activity against chromogenic substrates Substrate S-2444 s-2251 s-2222 S-2 160

Peptide sequences &lU-giy-arg v&leu-lys ile-gly-gly-arg phe-val-arg

Specific activity (nmol~min/Ing) 0.620 0.294 0.265 undetected

Lymphocyte plasma membrane protein (34pg) was assayed agamst four Kabi chromogenic substrates at pH 7.4. Each substrate has a C-terminal nitroanilide chromophore, which was monitored at 405 nm.

fraction is summarized in Table 3. In both cases, most of the protein passed directly through the column and the proteinase fraction was eluted as a sharp peak of 1 M sodium chloride. Despite the additional ammonium sulphate fractionation, and the much higher purification factor achieved, the specific activity of the purified, salt-extracted proteinase was almost four times less than that of the purified enzyme which had been extracted in the presence of Triton X-100. If the salt-extracted proteinase was diluted and assayed in

Table 3. Leukocvte txoteinase wrification

Fraction Salt extract Ammonium sulphate fraction Lysine-Sepharose pooled fractions Detergent extract Lysine-Sepharose oooled fractions

Protein (mg) 349

Specific activity (units/mg) 33.4

Total activity (units) 11.360

Yield (:a) 100

Purification factor

15.7

14

525

5356

47

0.44 164

4932 820

1120 134.726

IO 199

19.122

66.927

3.5

The purification of proteinases from both the salt and detergent Proteinase activity was assayed by the “‘1-casein method.

I

148 I

50

23.3

exfrdCtS

of leukocyte pellet.

316 67K

0 aI

N

25K

14K

0.2

a 0.1

4

8

12

16

fraction

20

24

28

no.

Fig. 2. Gel tiltration 01‘protrinase. Purilied proteinase was chromatographrd on ;i I .O x 60 cm column of Sephadcx G75 eauilibrated at 4 C with 0.01 AI Tria-Cl pH 7.4. The two major peaks monitored by AzBllnmwere freeze-dried then assayed for proteolytic activity and analysed by SDS gel electrophoresis. Peak I consisted only of the triplet bands corresponding to protcolqtic activity.

the Triton X- 100 extraction but‘fer following purification. the actlvlty was identical to that of a sample diluted and assayed in a Tris -HCI bult‘er without Triton X-100. Arginine-agarose was also used (data not shown) but resulted in lower purifications. The low overall recovery of cnzymic activity indicates that other proteinases were present in the crude leukocyte extracts. The SDS gel electrophoresis profiles for a number of preparations are shown in Fig. I. It is clear that a triplet group of proteins. with identical molecular weights to the lymphocyte membrane proteinase\. was always present in purified samples from both salt and detergent extracts. Sometimes. but not consibtently, a 75K band was also seen in these preparations. Preparations in which the 75K component could not be detected were used for further characterization and. in particular, to raise an *‘anti-tripletproteinase” antiserum. Gel filtration of the purified triplet proteinases is summarized in Fig. 2 and it is clear that the enzymes form an excluded, high-molecular-weight aggregate at low ionic strength. Attempts to fractionate the three components of the triplet by ion-exchange chromatography and by isoelectric focusing were not successful (data not shown). The salt-extracted purified triplet proteinase preparation hydrolysed all the amino-acid-nitrophenyl esters. A specific activity of 37 pm01 min ’ mg ’ Was obtained Lvith the lysine ester. Corresponding, values for the phenylalanine and glycylglycine esters mere I5 and 0.7 respectively. Thus the enzyme displayed both trypsin-like and chymotrypsin-like esterase activity. Addition of human plasminogen to caseinolytic assal samples showed no enhancement of activity. The caseinolytic activit> of samples of the triplet prepara-

tion (5 log; 4.8 units activity) was totally inhibited by IOmM DFP. There was u ZO”,, activation in the presence of IO mM EDTA. Iodoacetamide did not affect the activity. SBTI (IO ~~g,ml) reduced t.he activity to IO”(i of the control. Heparin (I 00 U) reduced the activity to 73”” and plasma ( W,) reduced it to 34”,,: these two inhibitory factors together caused ;I reduction to I7”,, of the initial activity. The antiserum prepared against the purified triplet proteinascs formed two precipitin lines with the immunogen in a double diirusion experiment (not shown). CLunma globulin prepared from this antiserum was used to make an antibody affinity column. The triplet protrinase preparation was applied to this column and it was found that the 29K component had a higher affinity for the column. being eluted with I M potassium thiocyanate, following the elution 01 the 25 and 27K components with I M sodium chloride (Fig. I) Similar results were found with proteinases from both salt and detergent extracts. In each case the 79K component had a significant11 higher specitic activity. The 29K component purltied in this way gave :I single precipitin line against the original antiserum. and also against an antiserum specific for the 75K proteinase pre\,iouxlq puritied from human leukocytez. The two prcclpitin lines fused completely (Fig. 3). The fraction cluted from the antibody aI?init> column with I M sodium chloride, and containing the 25 and 17K proteinasez. also gave 3 single precipitin line with the original antiserum (data not shown) and with the anti-75!< antiserum (Fig. 3). Ciamma globulins from both antisera were used in experiments to loc;iiiLe the proteinases on intact human Iymphoqteh and pranulocytes. Following treatment with the specific ;,-globulins and then fluorescein-labelled goat anti-rabbit IgG. the photographs shown in Figs 4a and b were taken with the fluorescence mitt-oscopr. These diagrams show that ;‘-globulin bound to most granuthe ant]-triplet locytes but to relati\.ely few lymphocyte\. Exactly parallel rchult\ \\crc obtained \\ith the anti-75K ~~-gl<~bulln. C’ontrol cellx treated with normal :,-globulin hcrc not fuorrsccntly labelled (data not shown ).

The mitogenic elfect of the triplet proteinase on lymphocytes freshly isolated from peripheral blood was measured in terms of the incorporation of [‘Hlthytnidine into acid-insoluble DNA and the results are summarired in Fig. j. The stimulation of DNA synthesis was maximal at about 3 pg,:ml of the proteinase and declined at higher concentrations. The harvested cell pellets were. in fact. considerably smaller where high protelnase concentrations had been used. and it IS possible that the enzyme i\ cytotoxic at these high concentrations. Similar results were obtained with salt and detergent-extracted en7ymes.

Mitogenic

leukocyte

proteinases

(a) Fig. 3. Precipitin formation hetween puritied proteinases and antibodies to triplet species and 75K species. (a) Purified proteinase from the antibody column (1 1Mthiocyanate eluted) showing only the 29K species was tested for its precipitin formation against antibodies raised to the purified triplet enzyme and also against antibodies to the 75K dalton proteinase. Approximately IO b(g of 29K proteinase was placed in SO/‘g of each specitic rabbit IgG placed in opposing wells. The a I”, agar well and approximately Ouchterlony diffusion pattern was then developed for three days at room temperature. (h) A similar 25 and 27K components. and the experiment using the 29K preparation. the preparation containing anti-7SK proteinase :s-globulin.

Fig. 4. Localization of proteinases to the surface of granulocytes and lymphocytes by indirect antibody labelling. Peripheral leukocytes were isolated and separated into constituent granulocytcs and lymphocytes, then treated as described in Materials and Methods with anti-triplet or anti-75K IgG followed by fluorescein labelled goat anti-rabbit IgG. Cells were photographed under U.V. irradiation using a Zeiss fluorescent microscope. Magnification was 1000 x and exposure time was 120 sec. a. anti-triplet IgG binding to granulocytes. h. anti-triplet IgG binding to lymphocytes. c. anti-75 K- proteinase IgG binding to granulocytes. d, anti-75 K proteinase IgG binding to lymphocytes.

317

318

JOHN D. FRAS~K and G. KENNETHSCOTT

20

40

a0

60

100

proteinase(tJ/ml) Fig. 5. Effect of purified proteinase on the growth of cultured lymphocytes. Peripheral lymphocytes were isolated from human venous blood and cultured at IO6 cell/ml in RPM1 medium supplemented with 1”; glutamine. Lymphocytes were cultured in medium containing 0, 25. 50. 75 or 100 units of proteinase activity in triplicate for 50 hr after which time the uptake of [‘Hlthymidine was determined.

It

was

considered

likely

that

antiproteinasr

be used as an inhibitor of proteinase on intact, growing cells. In view of its higher specificity, the anti-75K-proteinase preparation (Allen and Scott, 1983) was used, in multivalent form and as Fab fragments. Transformed human lymphocytrs were used as the target. Under the conditions used, control cultures in normal medium gave a count of 42.3 (k4.3). In the presence of 150 pg antiproteinase 7 -globulin. the corresponding count was 23.6 (k 2.3). In the presence of 100 pg ml ’ antiproteinase Fab, the value was 16.5 (k3.0). It is clear that both normal antiproteinase antibodies and the corresponding monoinhibited lymphocyte muitivalent fragments plication, relative to a normal control. ;I-globulin

could

DISCUSSION

Purified human lymphocyte membranes contain a protein triplet which can be isotopically labelled with DFP. An identical triplet pattern. also sensitive to DFP and possessing strong proteolytic activity. can be purified by salt extraction of a human leukocyte particulate fraction followed by affinity chromatography on lysine-agarose. Using immune )I-globulin. prepared using the purified triplet proteinase fraction as the antigen, and linked to agarose to form an affinity column, it was possible to separate the 29K proteinase, which had a higher affinity for the column, from the 27 and 25K components of the triplet. The purified 29K proteinase and the fraction containing the smaller enzymes each gave a single precipitin line with the original y-globulin preparation, and also with a y-globulin specific for 75K proteinase previously isolated from human leukocytes (Allen and Scott, 1983). In each case. the two precipitin lines fused completely. In the light of these results, it is not clear why two precipitin lines were observed with the triplet proteinases and the original antiserum.

Both y-globulin preparations, used in conjunction with a fluorescent second antibody. showed the respective proteinases to be widely distributed on the surface of most human granulocytes, and present at lower levels on a small proportion of human lymphocytes. This result confirms the presence of the triplet enzymes in lymphocyte membranes, and the previous report of lymphocytes and granulocytes by the anti75K-proteinase preparation (Allen and Scott, 1983). It is clear from these immunochemical crossreactions that there must be close structural homology between the three small proteinases and the 75K leukocyte proteinase previously described. This is consistent with the observations that these enzymes are all serine proteinases found on the outer surfaces of both lymphocytes and granulocytes. It is possible that the 75K proteinase is a precursor of the smaller enzymes. which are generated by partial proteolysis. This situation has been observed with a proteolytically-active. high-molecular-weight precursor of porcine tissue plasminogen activator (Wallen c( ul. 1982). There is no evidence for an autolytic conversion of the larger into the smaller species when it is incubated alone, or in the presence of the smallci enzymes (H. F. Seow. personal communication). The triplet proteinases show a marked loss of activity when incubated at 37 C but there is no apparent autodigestion detected by gel electrophoresis (data not shown). Salt and detergent extracts ultimately yielded proteinases which were indistinguishable immunologically and by SDS gel electrophoresis: the nature of the higher specific activity of the detergent-extracted enzyme is known. The much higher yield with detergent extraction may imply an intrinsic membrane localization for most of the enzymes (Singer and Nicholson. 1972). The results with inhibitors contirm that ail the triplet proteinase species are probably serine proteinases. It is significant that plasma can inhibit these enzymes; it suggests that the proteinase inhibitors in plasma could act as physiological modulators of the enzymes in situ on leukocyte membranes. Heparin slightly enhances this effect. which may be due to co-operation with antithrombin III. We have previously shown that heparin and antithrombin I11 are both required to inhibit the 75K leukocyte membrane proteinase (Long et al., 1982). In the present case. heparin itself is also inhibitory, and synergism is absent. The triplet proteinases are not piasminogen activators. We have established that the majority of human granulocytes, and some human lymphocytes. possess on their cell surface an enzyme or group of enzymes which can catalyse mitogenesis. The mitogenic property of the proteinase is not of particular significance in itself, since proteinases of widely different origins and specificity have this effect. Trypsin. chymotrypsin. pronase and thrombin are all active in this role (Mazzei et ul., 1966: Kaplan and Bona. 1974:

Mitogenic leukocyte proteinases Chen et al., 1976). The optimal mitogenic concentrations of these enzymes are typically of the order of 2 pgml- ’ but pronase is effective as a mitogen on human lymphocytes at 0.03 pg ml--’ (KapIan and Bona, 1974). Thus the mitogenic activity of the leukocyte proteinases is not particularly high, but they are appropriately located for an “intrinsic” mitogenic role. We have not tested the ability of antiproteinase y-globulin to effect DNA synthesis in human Iymphocytes, but in human fibroblasts the y-globulin inhibits this process (G. K. Scott and H. F. Seow, unpublished results). Confirmatory evidence for these enzymes having a role in leukocyte growth comes from the experiments with the anti-75K-proteinase antibodies and a transformed human lymphocyte strain. There is clearly some inhibition of cell multiplication under these circumstances. The inhibition is not merely a consequence of the cross-linking of cell-surface proteinase molecules. because Fab fragments have a similar effect. Using a variety of human normal and tumour cells on monolayer culture, we have previously demonstrated similar findings, and have shown that they can be correlated with the inhibition of cell surface proteinase activity and are not due to complement activation in the foetal calf serum which forms part of the growth medium (Allen et al., 1981: Pitts and Scott, 1983). Thus. the antibodies prepared against a leukocyte enzyme clearly recognize and inhibit a similar enzyme on the surface of hbroblasts and normal and tumour glial cells, astrocytes and colon cells. The immunochemical proteinase inhibition is coincident with the inhibition of cell multiplication. It seems likely that we are dealing with a fundamental biochemical aspect of cell growth and division, since closely-related enzymes are found in a wide variety of cell types. The apparent absence of these enzymes from the majority of human lymphocytes is thus a surprising discovery. It may correlate with the observation that proteinases are not T cell mitogens (Vischer, 1974). However, we have observed that the growth of some tumour and transformed ceils is not affected by the anti-75K proteinase antibodies, perhaps due to the presence of other, unrelated cell-surface proteinases (Pitts and Scott, 1983). The smaller leukocyte enzymes are similar in size to the SBTI-sensitive serine proteinase isolated from mito~enicaIly-stimulated lymphocytes, and other cells and tissues, and shown to be involved in chemotaxis (Thomas et al., 1977). These workers have also demonstrated a cytotoxic role for their enzyme (Hatcher et al., 1978), which may correlate with our observations of the effects of higher proteinase concentrations on DNA synthesis by lymphocytes. However, cytotoxicity was demonstrated with picogram quantities of proteinase by Hatcher er ul. (1978). which is in marked contrast to our results. There is a high degree of correlation between the properties of the enzyme reported here. and those

319

reported for a neutrophil plasma membrane proteinase (Wintroub et al., 1977). In particular, the neutrophil enzyme co-purified with 5’-nucleotidase could readily be extracted with strong salt solutions, had a molecular weight of approximately 20,000 but aggregated readily at low ionic strength, and was readily inhibited by DFP and SBTI. In intact cells. the neutrophil proteinase was not totally accessible to macromolecular proteinase inhibitors, which may correlate with the peculiar distribution of the enzyme on lymphocyte surfaces as seen in the present study, although granulocytes did not exhibit this effect. There is one discrepancy between the two reports: much shorter exposures to saline solutions liberated most of the proteinase in the earlier study (Wintroub et al.. 1977). However, the experi~nental conditions were not strictly comparable between the two reports, and we can conclude that this is a minor discrepancy.

thank Mrs J. Herbert and Mrs R. J. Allen for technical assistance, Mesdames E. Simanke, M. G. Hudspith and M. R. Mackie for secretarial assistance and the Auckland regional Blood Transfusion Centre for the supply of buffy coats. The prqject was partly supported by the Auckland Medical Research Foundation, and bv scholarshias (to J.D.F.) from the Medical Research Council and the ‘University Grants Committee. We also thank Dr J. D. Pitts of the University of Glasgow and the Cancer Research Campaign (U.K.) for the facilities to carry out the transformed Iymphocyte experiments.

Acknowledgements---Wc

REFERENCES

Allen R. J. and Scott G. K. (1983) A neutral proteinase from human leukocyte membranes. In?. .I. Bi~e~ze~~. 15, 151-154. Allen R. J.. Rsttray S. and Scott G. K. (1981) Preliminary evidence that thrombin may mimic a naturally occurring proteinase in cultured cells. Biosci. Rep. 1, 881-884. Allison A. C. and Ferluga J. (1976) How lymphocytes kill tumour cells. Ne,t Engl. J. Med. 295, 165-167. Bata J.. Deviller P. and Revillard J. P. (1981) Binding of alpha 1 antitrypsin (al proteinase inhibitor) to human lymphocytes. B&hem. biophys. Res. Commun. 98, 709-7 16. Bendtzen K. (1979) Leukocyte migration inhibiting factor. A serine esterase released by stimulated human lymphocytes. Kinetic analysis and inhibition by cyclic- GMP. Biochinr. bio&s. Acta 566, 183-191. Bethell G. S.,. Ayers J. S.. Hancock W. S. and Hearn M. 7. W. (1979) A novel method of activation of crosslinked aparoses with 1.1’~carbonvl diimidazole which gives a matrix for affinity chromatography devoid of additional charged groups. J. hiol. Chem. 254,2572Z1574. Chamberlain J. P. (1979) Fluorographic detection of radioactivity in polyacrylamide gels with the water-soluble Ruor. sodium salicylate. &U/IV. Biochern. 98, 132-135. Chen L. B.. Teng M. N. H. and Buchanan J. M. (1976) Mitogenicity of thrombin and surface alterations on mouse splenocytes. Expi Cell Rex 101, 41-46. Cuatrecasas P. (1970) Protein purification by affinity chromatography. Derivitisations of agarose and nolvacrylamide beads. J. biol. Chem. 245; 3059-3065. L . Deutsch D. G. and Mertz E. T. (1970) Plasminogen: . .ourification from human plasma by affinity chromatography. Science 170, 1095--l096.

320

JOHK D. FRASEKand G. KENNETHSCOTI

Emmelot P. and Bos C. J. (1966) Studies on plasma membranes. III Mg’ ’ -ATPase. (Na + K + Mg’+ )-ATPasc and 5’-nucleotidase activity of plasma membranes isolated from rat liver. Biochim. hioph?,.r. .-ictti 121, 369-382. Eylar E. H. and Hagiopan A. (1971) Isolation of plasma membranes from mammalian cells. hfc,llt. ,%:~n~o/. 22, 123~ 130. Fulton R. J. and Hart D. A. (1981) Characterisation of a plasma membrane-associated plasminogen activator on thymocytes. Biuclrin~. hioph~~s. Acrrr 642. 345-364. Hatcher V. B. and Norin A. J. (1982) Expression of protease and protease-inhibitory activity in human mononuclear leukocyte cultures. Effect of concanavalian A. Espl ~‘r/l Re.v. 142, 471-476. Hatcher V. B.. Oherman M. S.. Lararuc G. S. and GraqLel A. I. (1978) A cqtotouic proteinase isolated from human lymphocytes. J. Imnzurl. 120, 665-670. Hunter W. M. and Greenwood F. C. ( 1962) Preparation of iodine-l 31 labelled human growth hormone of high specific activity. Nature. Land. 194, 495496. Jorgensen P. L. (1974) Isolation of (Na’ + K’ )-ATPase. Meth. En:ymol. 32, ?77?290. Kaplan J. G. and Bona C. (1974) Proteases as mitogenb. The effect of trypsin and pronase on mouse and human lymphocytes. E.~pl Cell Res. 88, 38% 394. Kishimoto T.. Kikutani H.. Nishizawa Y.. Sakaguchi N. and Yamamura Y. (1979) Involvement of anti-I activated serine protease in the generation of cyloplasmic factor(s) that are responsible for the transmission of Ig-receptormediated signals. J. It~wzun. 123, 1504-l 510. Kitagawa S.. Takaku F. and Sakamoto S. (1979) Possible involvement of proteases m quperoxide production by human polymorphonuclear leukocytes. FEHS L~II. 99, 1755778. Ku G. S. B.. Qulglev J. P. and Sultzer B. M. ( 1981) Time-dependent mhihition of tuberculin induced Iqmphocyte DNA \yntheais by a srrinr protease inhlbitor. J. fn2/?7u!1. 126, X09%1:! 11. Laemmli Ll. K. (1970) Cleavage of structural protcmn during the assembly of the head of bacteriophage T4. Nuturr. Lonrl. 221, 6X%685. Lim A. K.. Scott G. K. and Scott R. (1981) Neutral proteinases from human blood cells. ht. J. Biochm. 14. 367-370. Long W. F.. Williamson F. B., Fraser J. D. and Scott G. K. (1982) Human leukocyte surface proteinasecatalysed amidolysis is inhibited by anionic polysaccharide-modulated antithrombin III. IRCS Med. Sci. Biochenz. 10, 208. Lowry 0. H.. Rosebrough N. J.. Farr A. L. and Randall R. J. (1951) Protein measurement with the Folin phenol reagent. J. hid C‘htvn 193. 265-175. Maillard J. I.. and Favreau C. (1981) Plasminogen actikation by normal B lymphocytes; a function associated with the cell membrane. J. Immtrn. 1lX+l 130.