Eur. J. Biochcm. 204. 501 - 508 (1992) FEBS 1992

Immunological characterization of rat kininogens with monoclonal antibodies to T-kininogen Distinction between the different domains of T-kininogen and the multiple rat kininogens

',

Suzanne LESAGE ', Jacob BOUHNIK Jean-Pierre RICHOUX2, Thierry BAUSSANT ', Francis GAUTHIER3, Kendra EAGER4, Pierre CORVOL and Franpois ALHENC-GELAS I

*

'

INSERM U36 and U367, Paris, France Laboratoire d'Histologie, Facultt de Medecine, Vandoeuvre-les-Nancy, France Laboratoire de Biochimie, Facultt de Medecine, Tours, France The Bristol-Myers-Squibb Pharmaceutical Research Institute, Princeton, NJ, USA

(Received June 26/October 10, 1991) - EJB 91 0830

A panel of 16 monoclonal antibodies (mAb) were produced against rat T-kininogen to characterize this family of proteins. These mAbs bound '251-T-kininogen by radioimmunoassay as well as reacting strongly with immobilized T-kininogen in an enzyme-linked immunosorbent assay (ELISA). The reactivity of these antibodies with proteolytic fragments of T-kininogen demonstrated the recognition of several different epitopes. One antibody was specific for the domain 1 of the heavy chain and/or the light chain, twelve antibodies were specific for domain 2 and three antibodies were specific for domain 3. All monoclonal antibodies recognized the two forms of T-kininogen encoded by the two different T-kininogen genes, T, and TIIkininogen, except antibody TK 16-3.1 which uniquely reacted with TI, kininogen. Two antibodies recognizing domain 2 cross-reacted with the high-molecular-mass kininogen (H-kininogen), whereas all the other monoclonal antibodies were specific to T-kininogen and did not recognize the heavy chain of H-kininogen. None of the antibodies tested altered the thiol protease inhibitory activity of T-kininogen, its partial proteolysis by rat mast cell chymase or the hydrolysis of H-kininogen by rat urinary kallikrein. The use of these antibodies in the development of sensitive ELISA to measure T-kininogen levels in plasma, urine, liver microsomes and hepatocytes is described. Two different forms of T-kininogen were distinguished by these monoclonal antibodies in Western blotting using rat plasma. The localization of T-kininogen was defined using these monoclonal antibodies by immunohistochemistry in rat liver hepatocytes and rat kidney.

Kininogens belong to the cystatin superfamily. They are single-chain plasma glycoproteins synthesized in the liver and contain the sequence of bradykinin in their structure. Kininogen molecules contain three cystatin-like repeat domains (DI, D2, D3) constituting the so-called heavy chain, followed by the kinin sequence and an additional carboxyterminal peptide sequence, the so-called light chain, which is unrelated to the cystatins. Two of the three heavy-chain repeat sequences (D2 and D3) carry the reactive site believed to be involved in cysteine proteinase inhibition [l, 21. All the mammalian species studied so far have two types of kininogens, a high-molecular-mass kininogen (H-kininogen) and a low-molecular-mass kininogen (L-kininogen). These proteins have similar heavy chains but different light chains, different sensitivities to plasma and glandular kallikreins and different physiological functions [3]. H-kininogen possesses Correspondence to F. Alhenc-Gelas, INSERM U367, 17, rue du Fer-A-Moulin. F-75005Paris, France Ahbrrviufions. H-kininogen, high-molecular-mass kininogen; Lkininogen, low-molecular-mass kininogen; CPI, cysteine protease inhibitor; MAP, major acute-phase protein. Enzymes. Chymase (EC 3.4.21.39); kallikrein K10, endopcptidase K (EC 3.4.21.-); urinary kallikrein (EC 3.4.21.35); papain (EC 3.4.22.2);trypsin (EC 3.4.21.4).

an additional histidine-rich C-terminal sequence involved in the contact phase of blood coagulation [4, 51. Okamoto and Greenbaum have identified a third kininogen, T-kininogen, which has been found so far only in the rat [6]. T-kininogen has an apparent molecular mass similar to that of L-kininogen, but unlike the other kininogen, it is not sensitive to kallikrein hydrolysis. T-kininogen releases Ile-Ser-bradykinin (T-kinin) in vitro when treated with high concentrations of trypsin, cathepsin D [7] or endopeptidase K [8]. It is the most abundant kininogen in the rat. Whereas H-kininogen and L-kininogen are chiefly the precursors of kinins, T-kininogen seems to behave only as a cysteine protease inhibitor (CPI) and is typically a major acute phase protein (MAP). Esnard and Gauthier reported that rat al-MAP and a,-CPI are homologous, based on physicochemical and immunochemical properties [9]. The similarity between T-kininogen, a,-MAP and al-CPI was subsequently established by cloning of cDNAs for T-kininogen and a,-MAP [lo, 111. T-kininogen levels increase markedly in plasma during all types of experimental inflammation [12]and after tissue effraction, while the two other kininogens are not altered under these conditions [13]. H-kininogen and Lkininogen are encoded by a single gene, whereas two different but structurally closely related genes encode for two forms of

502 main of the heavy chain of T-kininogen plus a part of the third domain 1171. A 43-kDa peptide was obtained by proteolysis with rat submaxillary gland kallikrein K10 and included D1 TK domains 1 and 2 [20]. Two 20-kDa peptides (A and B) were obtained by digestion with rat mast cell chymase and separated by C4 reverse-phase HPLC. They correspond to the third D 2 TK 15-19.3 domain of T-kininogen bearing an inhibitory site (Fig. 1). The .I33 TK 15-1.1 TK 15-3.1 TK 16-28.5b.2 TK 15-11.2 20-kDa A and 20-kDa B peptides were derived from kininogen TK 16-3.1 TK 17-1.2 TIIand TI, respectively [20]. TK 17-8.1 Two other T-kininogen synthetic fragments of the light TK 17-17.1 TK 17-19.1 chain of TI-kininogen were synthesized by Dr Fehrentz TK 21-1.1 (CNRS-INSERM, Montpellier). These are peptide 420 - 430 TK 22-1.3a (numbered according to the T-kininogen sequence [ 111) correTK 22- 6.10 sponding the last 11 C-terminal amino acids of TI-kininogen, ;;;,':410a and peptide 387 - 397, which corresponds to the amino acid Fig. 1. Schematic representation of T-kininogen and its proteolytic frag- sequence just after the C-terminus ofthe bradykinin sequence. ments and classification of the 16 mAbs according to their specificity. The heavy chain of H-kininogen was obtained after hydrolysis (-----) 43-kDa fragment; (--- -) 20-kDa fragment; (-----) 24-kDa by rat urinary kallikrein by a procedure adapted from that fragment; (A) postulated inhibitory site; ( 00 0 ) bradykinin se- used for the preparation of the heavy chain of human quence; (++)arrows delineating the three domains (D1,Dz and D3) kininogen 1211. Bradykinin and T-kinin (Tle-Ser-bradykinin) of thc heavy chain. were purchased from Peninsula.

1;

T-kininogen, TI and TII[ll]. These two kininogens also differ in their sialic acid content 1141 and their electrophoretic mobility on polyacrylamide gels. Monoclonal antibodies (mAbs) against T-kininogen were produced to characterize rat kininogens better and to investigate their regulation and biological functions. They were tested against T-kininogen by radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA). Cross-reactivities with other related kininogens or kininogen fragments were examined in order to define the epitopes recognized. The influence of these mAbs on some biological activities of kininogens was studied, as was their influence on limited proteolysis of T-kininogen. The antibodies were used to characterize rat kininogens in plasma by Western blotting and in liver and kidney by immunohistochemistry. An ELISA for quantitating T-kininogen in plasma, tissues and biological fluids has been developed.

MATERIALS AND METHODS Enzymes

Rat mast cell chymase was purified from peritoneal cells [I 51, kallikrein K10, formerly reported as endopeptidase K from rat submaxillary gland homogenate [8] and rat urinary kallikrein was purified from male Wistar rat urine [16]. Papain and bovine trypsin were purchased from Boehringer Mannheim. Preparation of rat and bovine kininogens

T-kininogen was purified from inflamed Wistar rat serum and H-kininogen from Wistar rat plasma as described 117, 181. Bovine kininogen was partially purified from plasma 1191. Preparation of kininogen fragments

Four different T-kininogen fragments were isolated by limited enzymatic proteolysis and purified by FPLC on a Superose 12 column and reverse-phase HPLC [20] (Fig. 1). A 24-kDa peptide was obtained by limited bovine trypsin hydrolysis and corresponded to the second cystatin-like do-

Polyacrylamide gel electrophoresis

Sodium dodecylsulfate gcl electrophoresis (SDS/PAGE) was carried out on 15% mini-slab gels at 20 mA for 1 h [22]. Proteins were stained with Coomassie brilliant blue G-250 (Sigma). Commercial protein size markers were used for calibration (Pharmacia). Determination of protein concentrations

Proteins were assayed by the procedure of Lowry et al. [23] using bovine serum albumin as standard. Production and characterization of monoclonal antibodies Production of monoclonal antibodies

The procedure for the production of hybridoma cells was that of Kohler and Milstein [24] as modified by Kennett et al. [25]. Spleen cells were fused with the myeloma cell line sp2/0Ag14 [26]. Supernatants of hybridoma cells were screened by ELTSA. Immunoglobulins purified from ascites were characterized using class and sub-class specific anti-mouse immunoglobulins [27]. Binding of'monoclonals to T-kininogen

Monoclonal antibodies were characterized first by their capacity to bind '251-kininogen in the T-kininogen RIA [28] and to immobilized T-kininogen in an ELISA 1291. The crossreactivity of each mAb was examined by ELISA with partially denatured T-kininogen, rat H-kininogen, heavy chain of Hkininogen (all at I pg/ml). The mAbs were also tested against human transferrin (Sigma) and ovalbumin (Sigma) since these proteins may contain glycan structures in common with Tkininogen [14]. In this ELISA, the plates were coated with the antigen to be tested. Effect of mABs on the thiolprotease inhibitory activity of T-kininogen and the 20-kDa peptide

Papain and T-kininogen were titrated as previously described [20]. The cysteine proteinase inhibitory activities of T-

503 kininogen (16 pg/ml final) and the 20-kDa peptide (10 pg/ml final) to papain were examined after incubating them for 2 h at 30°C with serial dilutions of mAb in 0.15 M NaCl, 0.003 M KC1, 0.008 M Na2HP04, 0.002 M K H 2 P 0 4 (NaCI/Pi) at antibodylantigen molar ratios ranging over 0.1 - 10. Papain inhibition was measured with N-benzyloxycarbonyl-phenylalanyl-arginine 4-methylcoumarin (Bachem) as substrate. Effect of'mAB on the 1imitedproteoIysi.s of T-kininogen bv rat niast cell chymase

The effect of the three mAbs which recognized the third domain of the T-kininogen heavy chain (see Results) was examined on the limited proteolysis of T-kininogen by chymase, which liberates biologically active fragments of Tkininogen including the 20-kDa peptide containing the inhibitory domain 3. The antibodies were preincubated for 1 h at 37"C with T-kininogen (0.6 mg/ml final) to obtain antibody/ T-kininogen molar ratios of 1.5 and 2.5. The mixtures were then incubated for 30min at 37°C with purified chymase (chymase/T-kininogen molar ratio = 0.1) in 80 mM potassium phosphate pH 8.0 containing 5% ammonium sulfate. The reaction was stopped by adding reducing buffer and boiling the mixtures. The proteolytic activity of chymase on Tkininogen, in absence or in presence of mAbs, was followed by SDSjPAGE under reducing conditions. Ej]ec[ of mAb on the hydrolysis of rat H-kininogen hq' rat urinary knllikrein

The influence of the two mAbs recognizing H-kininogen (see Results), on hydrolysis of rat H-kininogen by rat urinary kallikrein and the release of kinins was examined as follows. Increasing amounts of antibody were preincubated overnight at 4°C with a constant amount of pure H-kininogen (1 pg) in 0.2 M Tris/HCl pH 8.2 containing 3 mM EDTA, 3 mM (rphenanthroline and lysozyme (1 mg/ml) to obtain antibody/ H-kininogen molar ratios ranging over 0.1 - 10. The enzymatic reaction was started by adding 1 ng purified kallikrein and continued for 30 min at 31°C; the reaction was stopped by boiling for 3 min. Kinin liberation was measured by radioimmunoassay [19]. Sandwich enzyme immunoassay for T-kininogen Microtiter plates were coated with mAb solution (10 pg/ ml); the samples to be tested (or pure T-hninogen) were then added to the wells. The ELISA was carried out essentially as described previously I291 using polyclonal rabbit anti-Tkininogen antiserum [28] (final dilution 1 :5000) as second antibody. The intra-assay and inter-assay precision was evaluated according to Rodbard [30] by assaying pooled samples of normal or inflamed rat plasma. Recovery was checked by adding known plasma standards. The ELISA was used to determine T-kininogen in plasma and 17-h collection urines from Wistar rats before and after turpentine-induced inflammatory reaction, tissue extracts and cultured hepatocytes. Wistar rat liver microsomes were prepared as previously reported [31] and solubilized by treatment with 8 mM Chaps (Serva). T-kininogen was also measured in homogenates of Wistar rat kidneys. Kidneys were perfused in situ with saline, homogenized in 0.1 M phosphate pH 7.0, centrifuged at 30000 g for 1 h and the supernatant tested for T-kininogen. The Fao rat hepatoma cell line which secretes large amounts ofT-kininogen was cultured and tested as previously described

[32]. Cultures treated with dexamethasone (0.1 - 10 nM) were also tested. Immunological characterization of T-kininogens in rat plasma by Western blotting Polyacrylamide gel electrophoresis (in the absence of SDS) was done according to Laemmli [22] using 8% gel. Gels were loaded with 0.5 pg T-kininogen or 2 p1 rat plasma. Proteins were then transferred electrophoretically [33] to nitrocellulose (Amersham) and revealed after incubation with mAb by using the Protoblot system (Promega-Biotec, Madison, USA). Immunohistochemical localization of kininogen in rat liver and kidney Normal rats, rats injected intraperitoneally with colchicine (2.5 mg/kg body mass, 8 h before killing) and turpentinetreated rats (0.5 m1/100 g48 h before killing) were anesthetized with ether and perfused intracardiacally or via the portal vein with 0.15 M NaCl, followed by 4% paraformaldehyde in 0.1 M phosphate pH 7.4. Livers were rinsed under running water, dehydrated, embedded in paraffin and sectioned at 5 pm. Kidneys were cut into slices approximately 5 mm thick, rinsed with phosphate buffer containing saccharose (5 - 2O0/0) for 48 h, embedded in OCT (Miles), frozen in isopentane at - 110°C and sectioned at 5 Fm. Immunostaining was performed as described [34] with mAb TK 21-1 .I diluted I / 200-400 and incubated with the tissue sections for 2 h at room temperature. The avidin-biotin peroxidase technique was used for amplication, carried out as previously described [34]. The specificity of immunostaining was established by inhibition tests using pure T-kininogen immobilized on glutaraldehyde-preactivated gel (approximately 25 pg pure Tkininogen/ml gel). RESULTS Generation and screening of monoclonal antibodies to T-kininogen The hybridomas were produced from a fusion with a hyperimmunized BALB/c mouse. Supernatants from 156 wells were tested for the presence of T-kininogen antibodies by an ELISA procedure in which purified T-kininogen was immobilized on polystyrene plates. Forty clones with the highest reactivity to T-kininogen were identified and chosen for further expansion, subcloning and characterization. Sixteen hybridomas lines are described in this report. The immunoglobulin class and subclass were determined by ELISA with IgGl being the class identified for all hybridomas. With the exception of hybridoma TK17-17.1 which secretes lambda light chain, all of the remaining hybridomas are kappa light-chain producers. Immunoglobulins were purified from ascites fluids obtained from the intraperitoneal injection of the hybridoma cell lines into histocompatible mice. Characterization of mAbs Reactivity with kininogens

Most of the mAbs reacted strongly in ELISA with immobilized T-kininogen. The antibody titer for each clone was determined by serial dilutions using the antibody at an

504 Normal rat plasma (nl)

-

Liver microsomesolg protein) n - - n

-

100

40 58 88130200 290 440 660

Inflamed rat plasma (nl) 4

200

400

&-------,------A

Supernatant of cultured hepatocytes(p1)0

--

-0

5.88.8 13 20 29 44 66 A

2.0 E K

1.0

2 * 4.-

m a V

K K

m

g

0.1

v)

a

0.01 12.5 25

50

100 200

-

12.5

T-Kininogen(ng/ml)

25

50

100 200

Fig. 2. Standard curve for T-kininogen in ELISA using mAb 16-28.5b2. Serial dilutionsof normal or inflamed rat plasma (A), or liver microsome cxtract and supernatant of cultured hepatocytes (B) are compared to the standard T-kininogen curve.

initial concentration of 1 mg/ml. It was defined as the last dilution of antibody giving a signal in ELISA at least double that of the blank. The highest titer ( 2 1/100000) were found for antibodies TK 15-3.1, TK 15-11.2, TK 15-19.3, TK 1727.410 A, TK 20-4.1, TK 21-1.1, TK 22-1.3A and TK 22-6.10, followed by antibodies TK 16-21.1, TK 17-1.2 and TK 17-8.1 ( 21/10000). The ability of the purified mAbs to bind pure T-kininogen was also tested in the soluble phase by RIA, The binding of all antibodies to 1251-T-kininogenin RIA was low compared to polyclonal antibodies [28] and did not exceed 36% at a concentration of 2 pg/ml. The cross-reactivities with other kininogens or kininogen fragments are shown in Table 1. Most of the antibodies recognized T-kininogen partially denaturated by dithiothreitol reduction and alkylation [21]. All 16 mAbs recognized at least one of the four T-kininogen fragments obtained by limited proteolytic hydrolysis with trypsin, kallikrein K10 or rat mast cell chymase (Fig. 1). The cross-reactivities of antibodies with these T-kininogen fragments were used to classify the 16 mAbs into three groups corresponding to the three domains of the T-kininogen heavy chain (Table 1 and Fig. 1). Group 1 comprised one mAb (TK 16-21.1) which was specific to domain 1, or the light chain, as it recognized only the 43-kDa N-terminal fragment. Group I1 was composed of twelve mAbs which recognized T-kininogen heavy-chain domain 2, as they reacted with both the 24-kDa and the 43-kDa fragments. Group I11 included three mAbs which recognized the 20-kDa peptide corresponding to domain 3 of T-kininogen. Of these, TK 16-3.1 was specific for a determinant in the 20-kDa peptide unique to TII-kininogen (Table 1). Two group I1 mAbs (TK 17-27.410A and TK 20-4.1) crossreacted with the heavy chain of rat H-kininogen (Table I). They also reacted with partially purified bovine kininogen, as did another mAb, TK 17-17.1. None of the mAbs detecting domain 2 reacted with bradykinin, T-kinin or the synthetic peptides corresponding to the light chain of T-kininogen. N o

cross-reactivity was observed with human transferrin or ovalbumin.

Effect qf untibodies on thiol proteuse inhibitory activity The 16 mAbs were tested for their ability to modify the inhibitory activity of T-kininogen to papain. The effect of the threemAbs (TK 15-1.1, TK 16-3.1 andTK 16-28.5b.2),which recognize T-kininogen domain 3 was examined for inhibitory activity towards the 20-kDa peptide. Residual cysteine proteinase inhibitory activity was measured by incubating various amounts of mAb with T-kininogen or the 20-kDa fragment, which were used at concentrations giving approximately 50% inhibition of papain. None of the mAbs, tested at an antibody T-kininogen molar ratio ranging over 1-20, altered the cysteine-proteinase inhibitory activity of the native kininogen or its inhibiting fragment. By comparison, polyclonal anti-Tkininogen suppressed about 40% of the inhibitory activity of T-kininogen to papain at an antibody/T-kininogen molar ratio of 10 (data not shown).

Ejfects of TK 1.5-1.1, TK 16-28.56.2 and TK 16-3.1 on limited T-kininogen proteolysis by rut must cell chymuse The proteolytic fragmentation of T-kininogen produced by chymase was analysed by SDSIPAGE. An enzymelsubstrate molar ratio of roughly 0.10 was found to generate three T-kininogen fragments, of 60, 40 and 20 kDa. None of the three mAbs recognizing the T-kininogen heavy chain domain 3 interfered with the hydrolysis of the T-kininogen molecule by chymase at antibody/T-kininogen molar ratios of 1.5 and 2.5 (not shown).

Effect of TK 17-27.410~and T K 20-4.1 on H-kininogen hydrolysis by rut urinury kallikrein

The two mAbs, TK 17-27.410a and TK 20-4.1, which recognized the rat H-kininogen were tested for their ability to

505 'Table 1. Cross-reactivity of mAb against T-kininogen with different kininogens and with T-kininogen fragments. Each mAb was tested by ELISA at 1 pg/ml concentration. The two 20-kDa fragments (A and B) correspond to the two forms of T-kininogen (see text). ( 0.6. Kg = kininogen, T-Kg = T-kininogen, H-Kg = high-molecular-mass kininogen.

+

Anti body

43-kDa fragment

24-kDa fragment

20-kDa fragment A

TK 15-1.1 TK 15-3.1 TK 15-11.2 TK 15-19.3 TK 16-3.1 TK 16-21.1 TK 16.28.5B-2 TK 17-1.2 TK 17-8.1 T K 17-17.1 TK 17-19.1 TK 17-27.410a TK20-4.1 TK21-1.1 TK22-1.3A TK22.6.10

-

+++ +++ +- + +

+- + + +++ +++ +++ +++ +++ +++ +++ +++ +++

-

+++ +++ ++ -

++ +++ +++ +t+ +++ +++ +++ ++t ++

+++

+++

-

-

-

-

-

-

~

+++

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

interfere with the kinin-releasing activity of urinary kallikrein on H-kininogen. The mAb TK 15-19.3, which did not crossreact with rat H-kininogen, was used as a control. Hkininogen (1 pg corresponding to a bradykinin equivalent to 12 ng) was incubated for 30min with 1 ng rat urinary kallikrein. The amounts of liberated kinin represented approximately 20% of the amounts of kinins contained in Hkininogen. None of the three mAbs tested inhibited the proteolytic activity of urinary kallikrein on H-kininogen at antibody/H-kininogen molar ratios ranging over 0.1 - 10 (not shown). Sandwich enzyme immunoassay

The 16 mAbs were tested after adsorption to microtiter plates, as indicated in Materials and Methods, in order to develop a sensitive sandwich enzyme immunoassay for quantifying T-kininogen. The group 111 mAb TK 16-28.5b.2 was the most sensitive for quantifying T-kininogen. The sensitivity of the assay, defined as the lowest T-kininogen concentration giving an absorbance at least twice the background, was 6.2 ng/ml. The mAb TK 16-28.5b.2, tested by ELISA, did not crossreact with rat H-kininogen or bovine kininogen (up to 100 pg/ i d ) or with mouse or human plasma. This antibody recognized the 20-kDa peptide; when increasing amounts of the 20-kDa peptide (0.03- 3 pg/ml) were incubated with TK 16-28.5b.2, a curve parallel to that of native T-kininogen was obtained ( n o t shown). When molar concentrations were compared, cross-reactivity was roughly 8%. The ELISA detected Tkininogen in rat plasma, urine, tissue extracts and the culture media of Fao cells. In all cases, serial dilutions of sample to be tested gave a dose/absorbance line that was parallel to standard T-kininogen (Fig. 2). The intra-assay coefficients of variation was 7.9% for the pool of normal rat plasmas and 7.2% for the pool of turpentine-treated rat plasmas ( n = 10). The corresponding inter-assay coefficients of variation were 10.0% and 10.4% (n = 8). Recoveries of 89 8 % (n = 7) and 90 6 % (n = 7) respectively, were obtained when varying amounts of pure T-

Rat H-Kg

Heavy chain of rat H-Kg

Bovine Kg

-

-

-

B

-

+++ +++

Denatured T-Kg -

++ ++

-

-

-

-

-

+ +++ -t+ + +++ +++ ++ +++ +++

-

-

-

-

+++ ++ -

~

~

-

-

+ -

+++

t+

-

-

-

-

+-

+ ~

Table 2. Measurement of T-kininogen by ELISA in different Wistar rat media or tissue extracts. Results are mean f SEM except for the control Fao cell medium where two results from individual cxpcrirnents are presented. Numbers in brackets refer to numbersof animals or samples tested. Total urine refers to a 17-h collection.

Sample

Kininogen content

Plasma: male (7) female (7) male turpentine-treated (9)

w/ml 214 k 22 1282 74 7898 f 618

Urine: malc ( 5 ) male turpentine-treated ( 5 )

pg/total urine 25.4 8.5 217 116

Tissues: male, liver microsomes (6) male, kidney homogenate (3)

ng/ml protein 1350 270 89.4 i 15.8

Fao cell medium : control (2) 0.1 nM dexamethasonc (3) 1.0 nM dexamethasone (3) 10.0 n M dexamethasone (3)

ng/1O6 cells 839, 1166 856 k 25 1658 5 199 4168 858

+

+

kininogen (1.5 - 100 ng/ml) were added to the pool of normal plasmas or the pool of turpentine-treated plasmas. T-kininogen was measured by ELISA in plasma, urine, kidney and liver microsomes as well as in Fao cell culture medium (Table 2). The results obtained by ELISA were compared with those given by T-kininogen RIA using a polyclonal antibody [28]. Fig. 3 shows a n excellent correlation between the results of these two assays. Western blotting of rat plasma with mAbs

In rat plasma TK 16-3.1 recognized only one of the two forms of T-kininogen corresponding to the T,,-kininogen,

506 DISCUSSION

10000

1000

1

1

10

100

1000

10000

ELISA i pg/ml)

Fig. 3. Correlation between results of measurements of T-kininogen by ELlSA and by RIA. ( 0 )Plasma; (+) kidney extract; ( A ) microsomes;).( urine; (0)supernatants of cultured hepatocytes.

Fig. 4. Western blot analysis of rat plasma. The transferred protein bands of T-kininogen were revealed by incubation with mAb TK 171.2 (A) or mAb TK 16-3.1 (€3).

whereas all the other mAbs tested, including T K 17-1.2, recognized the two forms (Fig. 4).

lmmunohistochemistry of T-kininogen in rat liver and kidney Most of the mAbs could be used at dilutions of 1 :200 400 to detect T-kininogen in rat liver and kidney. Only a few hepatocytes containing T-kininogen were detected in normal rat liver (Fig. 5, 1). Colchicine greatly enhanced T-kininogen concentration in all hepatocytes through its inhibitory role on protein release (Fig. 5, 2). Immunoreactive material appears as fine granular formations scattered throughout the cytoplasm. By contrast to normal liver, most of the cells from turpcntine-treated rats contained granular immunostaining around the nuclei (rough endoplasmic reticulum) and along the plasma membrane in the Golgi apparatus (Fig. 5, 3). The immunostaining was inhibited by preadsorption of the mAb with the T-kininogen gel (Fig. 5, 4). In rat kidney, the immunostaining appeared as granular formations in the cells of the proximal convoluted tubules (Fig. 5, 5); no staining appeared in the distal tubule; preadsorption with T-kininogen gel specifically abolished the immunostaining (Fig. 5, 6).

A large number of monoclonal antibodies to rat Tkininogen were obtained by a single fusion, probably because of the highly antigenic nature of this protein in the mouse, which lacks T-kininogen. These antibodies were highly reactive with immobilized T-kininogen when tested by ELlSA but their capacity to bind '251-T-kininogen in an RIA was low. This discrepancy may be due to one or more of the following factors: (a) incorporation of 1251 into T-kininogen can induce conformational changes; (b) the ELISA allows more sensitive detection of the antigen-antibody complex; (c) the culture supernatants after fusion were screened by ELISA using immobilized T-kininogen. Similar discrepancies between the recognition of the soluble and the immobilized antigen have been reported for other T-kininogen antibodies [35, 361 and for renin antibodies [37]. Most of the mAbs reacted with the two forms of Tkininogen and with partially denaturated T-kininogen, but only two of them recognized the rat H-kininogen, although the sequence of the heavy chain of H-kininogen is more than 80% identical to that of T-kininogen with large stretches of identical amino acids [ll]. This suggests that specific highly antigenic epitopes are present on the T-kininogen molecule, probably because of structural differences between the heavy chains of kininogens related to some specific amino acids. Another factor involved in antibody recognition is the overall conformation of the molecule which differs between Tkininogen and H-kininogen as they possess light chains of different sizes and structures [I 11. However the observation that the isolated heavy chain of H-kininogen is, like the native H-kininogen molecule, not recognized by these antibodies indicates that structural differences in the heavy chain of kininogens are indeed the major factor explaining antibody specificity. This also suggests that these antibodies do not cross-react with L-kininogen, which has the same heavy chain as H-kininogen [38]. For the same reason, the two antibodies TK 17-27.a and TK 20-4.1 that recognize the H-kininogen heavy chain probably cross-react with the L-kininogen. Antibodies specific to each of the three T-kininogen domains were identified by comparing their specificity toward different proteolytic fragments. Competitive binding experiments (data not shown) suggest that the antibodies TK 15-3.1, TK 15-11.2, TK 15-19.3, TK 17-1.2, TK 17-8.1, TK 21-1.1, TK 22-1.3a and TK 22-6.10 recognize either the same epitope on domain 2 or share parts of an antigenic region. One of the antibodies, TK 16-3.1, specifically recognized TI,-kininogen. The corresponding epitope was shown to be localized within cystatinlike domain 3. This is in agreement with the observation that all amino acid substitutions between TI- and TI,-kininogens are located in this particular domain [lI]. Recently, Lalmanach et al. have described an antibody which specifically recognized domain 3 of T,-kininogen, but not the native molecule [39]. T K 16-3.1 is a valuable tool for studying possible differences in the distribution or regulation of these two kininogens (see below). Contrary to the polyclonal antibody, none of the 16 mAbs tested altered the cysteine protease inhibitory activity of either the intact T-kininogen or its biologically active fragment. It is possible that the inhibitory site, although probably located at the surface ofthe protein since accessible to its target enzymes, was poorly antigenic, and/or that, if T-kininogen inhibits by steric hindrance, this was not altered by antibody binding. The same observation was made by Lalmanach et al. using their specific TI-kininogen domain-3 mAb [39] and by

507

Fig.S. Detection of T-kininogen using monoclonal antibodies in the rat liver (1-4) and kidney (5, 6). (1) Liver of control rat; (2) liver of 21 colchicinc-injected rat; (3) T-kininogen in the liver of a turpentine-treatcd rat (pbs = portobiliary space); (4)preabsorption of monoclonal antibody with an excess of T-kininogen gel; ( 5 ) T-kininogen in the kidney (the presence of specific staining in granular formations locatcd under the brush border in proximal tubules (pt) is indicated by arrows; dt = distal tubule); (6) specificity of T-kininogen immunostaining in the kidney demonstrated after preincubation of diluted monoclonal antibody with T-kininogen gel. Magnification: x 460.

Olafsson et al. with their monoclonal antibodies directed against the heavy-chain domain 3 of human H-kininogen [40]. Ishiguro et al. however have produced two mAbs against human H-kininogen which can suppress the inhibitory activity of this kininogen, even though they did not recognize the predictive sequence of the reactive site [41]. Preferential cleavage of kininogens by proteinases occurs at their interdomain regions [20, 42, 431. However, none of the three antibodies recognizing the domain 3 interfered with the cleavage of T-kininogen by chymase, suggesting that they recognized epitopes located some distance from the cleavage site. Rat urinary kallikrein hydrolyses H-kininogen to liberate bradykinin [16, 191. The two mAbs recognizing the Hkininogen did not, however, inhibit the kininogenase activity of kallikrein. This was not unexpected, as they indeed recognize epitopes located in domain 2, far from the bradykinin sequence. Using a specific sandwich enzyme immunoassay, Tkininogen was quantified in rat plasma and urine, where the well-documented effect of turpentine-induced T-kininogen stimulation was observed. Male/female differences in Tkininogen concentration were confirmed [44]. The ELISA was also suitable for quantifying T-kininogen content in liver microsomes and in F a o cells in culture before and after stimulation by dexamethasone [32]. These results correlated well with our previously described RIA for T-kininogen [29]. However, the ELISA is faster and need no radiolabeled kininogen. All the antibodies detected two forms of T-kininogen in rat plasma by immunoblotting, except T K 16-3.1 which recognized only the T,,-kininogen. Similar results were obtained for normal and inflamed rat plasma (unpublished results). Enjyoji

et al. reported that the plasma of non-inflamed rats contained a mixture of two kinds of T,-kininogen, whereas TII-kininogen is not present in significant amounts, but is generated in adjuvant-treated rats [45]. Our results suggest, however, that the plasma of both normal and inflamed rats contains TI- and TI[kininogens. A monoclonal antibody specific to T-kininogen is especially useful for immunohistochemistry as it does not crossreact with other kininogens present in the same cell, as polyclonal antibodies may do. Only a few hepatocytes stain in the normal rat possibly because T-kininogen, like angiotensinogen [46], is normally not stored in liver cells. The inflamed rat liver contained more stained hepatocytes reflecting increased T-kininogen synthesis. T-kininogen was found in the kidney proximal convoluted tubules as granular formations, most likely as a result of tubular reabsorption by micropinocytosis of filtered kininogen. These antibodies may also be used to detect kininogen fragments in tissues and biological fluids and to distinguish between kininogens in tissues where they are coexpressed, such as in liver and brain

WI. We would like to thank FranGoise Savoie for expert technical assistance, Florence Lopez for typing the manuscript and Annie Boisquillon for artwork. This work was supported by the /nstifu/ National de la Suntt. c I a'u Iu Recherche Mi.dicale and by a grant from the Bristol-Myers-Squibb Laboratories Inc.

REFERENCES 1. Muller-Estcrl, W. (1987) Semin. Throtnb. HemoJfusis 13, 115-

126.

508 2. Salvescn, G., Parkes, C., Abrahamson, M., Grubb, A. & Barrett, A. (1986) Biochem. J. 234,429-434. 3. Movat, H. Z. (1979) in Handbook of experimental pharmacology (Erdos, E. G., ed.) vol. XXV (Suppl.) pp. 1-71, SpringerVerlag, Berlin. 4. Saito, H., Ratnoff, 0. D., Waldmann, R. &Abraham, J. P. (1975) J . Clin. Invest. 55. 1082- 1089. 5. Wucppcr, K. D., M i l k , P. R. & Lacombe, M. J. (1972) J. Clin. Invest. 56, 1663- 1672. 6. Okamoto, H. & Greenbaum, L. M. (1983) Life Sci 32, 2007201 3. 7. Okamoto, H. & Greenbaum, L. M. (1983) Biochem. Biophys. Res. Commun. 112,701 - 708. 8. Gutman, N., Moreau, T., Alhenc-Gelas, F., Baussant, E., El Mouiahed, A,, Akpona, S. & Gauthier, F. (1988) Eur. J . Bioclzem-. 171, 577-582. 9. Esnard, F. & Gauthier, F. (1983) J . Biol. Chem. 258, 1244312447. 10. Anderson, K. P. & Heath, E. C. (1985) J . Biol. Chem. 260, 12065- 12071. 11. Furuto-Katol S . , Malsumolo, A., Kitamura, N. & Nakanishi, S . (1985) J . Biol. Chem. 260,12054-12059. 12. Barlas, A,, Okamoto, H. & Greenbaum, L. M. (1985) Biochem. Biojdi.pss. Res. Commun. 129, 280 - 286. 13. Suzuki, H., Alhcnc-Gclas, F., Bouhnik, J., Menard, J. & Corvol, P. (1988) Endorrinologji 122,2809 - 281 5. 14. Baussant, T., Alonso, C., Wieruszcski, J.-M., Strecker, G., Montrcuil, J., Alhenc-Gelas, F. & Michalski, J.-C. (1992) Biochern. J., in the press. 15. Kido, J., Fukusen, N. & Katunuma, N. (1985) Biochem. fnt. 10, 863 - 871. 16. Girolaini, J. P., Alhenc-Gelas, F., Dos-Reis, M. L., Bascands, J. L., SUC,J . M., Corvol, P. & Menard, J. (1986) Adv. Exp. Med. Biol. 17, 137- 145. 17. Moreau, Th., Cutman, N., El Moujahed, A,, Esnard, F. & Gauthier, F. (1986) Eur. J. Biochem. 159, 341 -346. 18. Reis, M. L., Alhenc-Gelas, F., Alhenc-Gelas, M., Allcgrini, J., Kerbiriou-Nabias, D., Corvol, P. & Menard, J. (1985) Biochim. Biophys. Acta 831, 106 - 113. 19. Alhenc-Cielas, F., Marchetti, J., Allegrini, J., Corvol, P. & Menard, J. (1981) Biochim. Biophys. Acta 677,477-488. 20. Moreau, T., Gutman, N., Faucher, D. & Gauthier, F. (1989) J . Biol. Chom. 264,4298-4303. 21. Kerbiriou, D. M. & Griffin, T. H. (1979) J . Biol. Chem. 254, 12020-12027. 22. Lacmmli, U. K. (1970) Nature 227, 680-685. 23. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. (1951) J . Biol. Chem. 193,265-275.

24. Kohler, G . & Milstein, C. (1975) Nature 256.495 -497. 25. Kennett, R. H. (1979) Methods Enzymal. 58, 345-359. 26. Shulman, M., Wilde, C. D. & Kohler, G. (1978) Nutzire276,269270. 27. Eager, K. B. (1991) Oncogene 6,819-824. 28. Bouhnik, J., Baussant, T., Savoie, F., Alhenc-Gelas, F., Gauthier, F. & Esnard, F. (1987) Biochem. Biophys. Res. Cummun. 144, 1090- 1097. 29. Mknard, J., Guyenne, T. T., Corvol, O., Pau, B., Simon, D. & Roncucci, R. (1985) J . Hypertens. 23 (suppl3), 275-278. 30. Rodbard, D. (1974) Clin. Chem. 20,1255- 1270. 31. Baudry, M., Bouhnik, J., Michel, 0. & Michel, R. (1972) Biochimie (Paris) 54,219 -227. 32. Baussant, T., Michaud, A,, Bouhnik, J., Savoie, F., Alhenc-Gelas, F. & Corvol, P. (1988) Biochem. Biophys. Res. Cornmun. 154, 1160- 1166. 33. Towbin, H., Staehelin, T. & Gordon, J. (1979) Proc. Natl Acad. Sci. USA 76, 4350-4354. 34. Richoux, J. P., Gelly, J. L., Bouhnik, J., Baussant, Th., Grignon. G., Alhenc-Gelas, F. & Corvol, P. (1991) Histochemistry 96. 229 - 243. 35. Bedi, G. S. & Back, N . (1987) Hybridornu 6, 521 -526. 36. Utsunomiya, I., Oh-Ishi, S., Hayashi, I., Maruhashi, J., Tsuji, N., Yamamoto, N. & Yamashina, S . (1988) J. Biochem. (Tokyo) 103,225-230. 37. Bouhnik, J., Galen, F. X., Menard, J., Corvol, P.,Seyer, R., Fehrentz, J. A., Le Nguyen, D., Fulcrand, P. & Castro. B. (1987) J . Biol. Chem. 262, 2913-2918. 38. Kitagawa, H., Kitamura, N., Hayashida, H., Miyata, T. & Nakanishi, S . (1986) J . Biol. Chem. 262, 2190-2198. 39. Lalmanach, G., Adam, A., Moreau, T., Gutman, N. & Gauthier, F. (1991) Eur. J . Rioehem. 196, 73-78. 40. Olafsson, I., Lotberg, H., Abrahamson, M. & Grubb, A. (1988) Scand. J. Clin.Lab. Invest. 48, 573-582. 41. Ishiguro, H., Higashiyama, S . , Ohkubo, 1. & Sadaki. M. (1987) Biochemistry 26, 7021 - 7029. 42. Salvescn, G., Parkes, C., Abrahamson, M., Grubb, A . & Barret, A. J. (1986) Biochem. J . 234,429-434. 43. Vogel, R., Assfdg-Machleidl, I., Ester], A., Machleidt, W. & Muller-Ester1 W. (1988) J . Biol. Chem. 263, 12661 - 12668. 44. Bouhnik, J., Savoie, F., Michaud, A., Baussant, Th., AlhencGelas, F. & Corvol, P. (1 989) Life Sci. 44, 1859- 1866. 45. Enjyoji, K . , Kato, H., Hayashi, I., Oh-Ishi, S. & Iwanaga, S. (1 988) J . Biol. Chem. 263,973 - 979. 46. Richoux, J . P., Cordonnier, J. L., Bouhnik, J., Clauser, E., Corvol, P., Menard, J. & Grignon, G. (1983) Cell. Tissue Res. 233, 439 -451.