the cytokine-protease connection: identification of a 96-kd thp-1 gelatinase and regulation by...

THE CYTOKINE-PROTEASE CONNECTION: IDENTIFICATION OF A 96-kD THP-1 GELATINASE

AND REGULATION BY INTERLEUKIN-1 AND CYTOKINE INDUCERS

Marc Van Ranst, Koen Norga, Stefan Masure, Paul Proost, Filip Vandekerckhove, Johan Auwerx, Jo Van Damme,

Ghislain Opdenakker*

The induction of proteolytic enzymes is an important mechanism in the migration of monocytes into tissues and body fluids. The monocytic cell line THP-1 was used as a model system to study the production of a particular gelatinase. Upon stimulation with phorbol myristate acetate (PMA) the cells differentiated to the adherent phenotype and produced significant amounts of a 96-kD gelatinase in a dose-dependent way. The secretion rate was maximal between 12 and 24 h after induction. Study of gelatinase mRNA steady state levels showed that the synthesis of THP-1 gelatinase is regulated by PMA at transcriptional or posttranscriptional levels. Stimulation of signal transduction pathways with other substances, including calcium ionophore A 23187, dibutyryl cyclic AMP, and dexamethasone, were ineffective in inducing gelatinase mRNA or enzyme activity. However, THP-1 cells were responsive to the cytokine interleukin (IL)-li!!, to bacterial lipopolysaccharide (LPS), and the lectin concanavalin A (Con A), the kinetics of gelatinase induction being similar to those of induction by PMA. The THP-1 cells did not synthesize and/or secrete detectable levels of IL-6 after stimulation with PMA, Con A, LPS, or IL-lp. The 96-kD monocytic THP-1 gelatinase was shown to be a neutral metalloproteinase that cross-reacted with hepatoma-derived and neutrophil gelatinases in immunoprecipitation experiments. The active enzyme produced by THP-1 cells consistently showed, however, a molecular mass different from that of normal granulocyte-, monocyte-, and tumor cell-derived gelatinases. The THP-1 enzyme was therefore purified to homogeneity by substrate affinity chromatography, and its amino- terminal amino acid sequence was determined and compared to those of known gelatinases. The terminus was identical to that of the 92-kD gelatinase from fibrosarcoma cells but was eight residues longer than that of the gelatinase from normal human blood granulocytes and monocytes. The activity of the gelatinase was also found to be regulated at the extracellular level by simultaneous production and secretion of the tissue inhibitor of metalloproteases (TIMP-1) by the THP-1 cells. This inhibitor was copurified and unequivocally identified by amino-terminal sequence analysis. Copyright o 1991 by W.B. Saunders Company

Monocytes/macrophages are key regulators of the humoral and cellular immune responses. The cells are responsive to various stimuli including bacterial products and viruses, double-stranded RNA, and particu-

Rega Institute for Medical Research and Laboratory for Experimen- tal Medicine, Department of Developmental Biology, University of Leuven, Belgium.

*Address correspondence and reprint requests to G. Opdenakker, Rega Institute, Minderbroedersstraat 10, B-3000, Leuven, BeI- gium.

Copyright o 1991 by W.B. Saunders Company 1043-4666/91/0303-0003$05.00/0

KEY WORDS: gelatinase/interleukin-l/metalloproteinase/monocyte

CYTOKINE, Vol. 3, No. 3 (May), 1991: pp 231-239

late material. In response to such stimuli they process and present non-self antigens to the lymphocytes and secrete primary cytokines, e.g., interleukin (IL)-1 and tumor necrosis factor (Y (TNF-ar), as well as secondary cytokines, e.g., IL-6 and IL-%’ Another important class of macrophage secretion products includes the proteolytic enzymes.* Amongst these are plasminogen activa- torq3 both urokinase and tissue-type, and also metalloproteinases.4 The former are believed to play a regulatory role in the cleavage to (in)activation of cytokines (e.g., chemotactic factors)’ and of other proteolytic enzymes (e.g. metalloproteinases).” They also mediate plasmin-dependent lysis of fibrin in clot- ted blood and fibrin deposits at, e.g., inflammatory sites

231

232 I Van Ranst et al. CYTOKINE, Vol. 3, No. 3 (May 1991: 231-239)

and atherosclerotic lesions. The metalloproteinases are essential enzymes to solubilize the extracellular molecular network. This network consists of a complex mixture of matrix molecules such as collagens, lami- nins, proteoglycans, and entactin.’ The solubilization of this molecular meshwork facilitates not only the migration of leukocytes to the inflammatory focus but also the migration of fibroblasts and the development of capillary sprouts in scar formation and angiogenesis. The matrix metalloproteinases (MMP) have therefore been widely studied in invasion and metastasis of tumor cells.’ The migration of monocytes/macrophages through endothelial and basement membrane barriers and ground substance into tissues and body fluids necessitates the activity of a similar molecular machin- ery in chemotaxis. The family of metalloproteinases can be divided into three groups of enzymes: the collagenases, the gelatinases (e.g. type IV collagenase), and the proteoglycanases (also called transin or stromel- ysin).’ The activity of these enzymes is controlled by specific inhibitors.” Until recently little was known about the structure and natural activation process of these latent enzymes. Apparently, the on/off mechanism referred to as the “cysteine switch” plays a crucial role in the natural activation of metalloproteinases and serves to explain the diversity of mechanisms active in different cell types and with different activating substances.” The human monocytic leukemia cell line THP-1 resembles mature monocytes in many aspects.‘* Furthermore, these cells differentiate into a macrophage-like cell after treatment with phorbol esters. This differentiation process is associated with several changes in gene expression which also occur during the differentiation of monocytes. These changes include the induction of oncogenes and genes involved in lipid metabolism.13

A gelatinase with an apparent molecular mass of 96 kD was found to be regulated in THP-1 cells by tumor promotors, IL-1B, lipopolysaccharide (LPS), and mitogen. This study demonstrates that this neutral proteinase resembles, serologically, structurally, and functionally, natural monocytic gelatinase. In analogy with other cell types, a possible model for a protease cascade in this cell line is discussed.

RESULTS

Induction of Gelatinase by Pill4 in THP-I Cells

Cultures of the monocytic cell line THP-1 were treated with phorbol myristate actetate (PMA) or left untreated. After 48 h the supernatant culture fluids were collected and analyzed by substrate zymography for the presence of gelatinases. Different dilutions of each sample were subjected to SDS/PAGE zymography and quantitated by scanning densitometry. Figure

1A shows that within a loo-fold range a linear relation existed between the amount of gelatinase (96 kD) and the analyzed activity expressed in scanning units. When samples from phorbol ester-treated and control cell supernatants were analyzed in such a way, a reproduc- ible dose-response relationship was obtained (Fig. 1B) with effective doses ranging from 10-l’ to lo-’ M of PMA. Next, cells were incubated for different time intervals and with an optimal stimulatory concentration of 100 rig/ml (10m8 M) of PMA. This kinetic analysis (Fig. 1C) resulted in cumulative production of gelatinase for at least 72 h with a maximal production rate between 12 and 24 h. Gelatinases present in the serum and constitutively produced by the THP-1 cells were useful as a control for sample preparation and processing. These migrated at an apparent molecular weight of 70 kD (see inset to Fig. 1C). Counting of cells (untreated suspension cultures as well as trypsinized PMA-treated cells) indicated that at optimal stimulatory doses of PMA cell viability and absolute cell numbers remained unaltered. This indicates that PMA increased the gelatinase yields per cell. Together with the gelatinase induction, the cells changed morpholog- ically from round suspension cells when untreated to fusiform adherent cells after PMA treatment (data not shown).

Since addition of PMA to cells results in the activation of the protein kinase C signal transduction pathway, it was next investigated whether activation of cells via other pathways also resulted in gelatinase induction. Activation of protein kinase A by the addition of dibutyryl cyclic adenosine 3’-5’ monophosphate (db-CAMP) to a final concentration of 1 uM did not result in augmented gelatinase activity in 48-h culture fluids. Similarly, an increase in intracellular calcium provoked with 10 &ml of the Ca2+ ionophore A-23187, as well as 10 ~.LM of dexamethasone, did not induce gelatinase in the THP-1 cells.

In order to determine the mechanism of the induction of the 96-kD gelatinase by PMA, gelatinase mRNA was measured by dot and Northern blot analysis. Cultures of THP-1 cells were treated for different time intervals with 1.6 x lo-’ M of PMA and used as a source of mRNA. Figure 2A shows the scanning analysis of the induction of gelatinase mRNA as determined by dot blot analysis. The mRNA was induced within 6 h, the synthesis rate was maximal between 6 and 12 h (sixfold increase) and doubled once between12and24handonceagainbetween24and48 h. The steady-state levels increased up to 48 h. The kinetics of appearance of gelatinase mRNA are thus in accordance with the data for enzyme titrations. From a Northern blot analysis of RNAs from PMA-treated and unstimulated THP-1 cells (Fig. 2B) it was clear that only one mRNA band appeared after PMA treatment. The molecular size corresponded to the

THP-1 gelatinase regulation by cytokine / 233

3200

2 i = 2400

is i 5 1600

ul 600

, , ” I ” “ ,

1 10 100

DILUTION FACTOR

2400

F 2 2 1600

t2 i s 1200

z 600

i/’ PMA CONCENTRATION (M)

0 I I I / I 0 15 30 45 60 75

INCUBATION TIME (hours)

Figure 1. Induction of 96-kD THP-1 gelatinase by PMA.

(A) THP-1 cells were treated for 48 h with 100 rig/ml of PMA. Supernatant culture fluids were analyzed for gelatinase activity by densitometric scanning analysis of a dilution series. The graph shows the processed scanning analysis and the zymogram is shown in the inset. (B) THP-1 cells were treated for 48 h with different doses of PMA and culture fluids from separate experiments analyzed for the presence of gelatinase activity. Means and standard errors are indicated for three different experiments. (C) THP-1 cells were treated for different time intervals with an optimal stimulatory dose of PMA and culture supernatants analyzed for cumulative gelatinase content. The 96-kD gelatinase is expressed in absolute scanning units.

92-kD protein gelatinase mRNA.14 These data together with the results of Fig. 1 suggest that the induction of gelatinase is regulated at the transcriptional or the posttranscriptional level.

Regulation of THP-1 Gehtinase by IL-l, LPS, and Mitogen

THP-1 cells were treated with IL-l and culture fluids collected and titrated after several time intervals. IL-l induced the 96-kD gelatinase in a dose-dependent way, 1 to 10 U/ml being a minimal effective dose (Fig. 3A). IL-l stimulation also resulted in prolonged cumulative gelatinase secretion. In another type of experiment two other monocyte activating substances, endo- toxin and concanavalin A (Con A), were used to stimulate the production of THP-1 gelatinases. Similar

dose-response curves were reproducibly obtained with both lipopolysaccharide (Fig. 3B) and lectin (Fig. 3C). The minimal concentrations needed for optimal gelatinase production in THP-1 cells were 2 pg/rnl and 30 kg/ml for LPS and Con A, respectively. In all instances the gelatinase that was induced showed the same apparent molecular mass as the phorbol ester- inducible 96-kD gelatinase (insets). Cell morphology was not changed towards the adherent type by either LPS or Con A, although the latter substance caused agglutination of the cells.

It was observed that in normal peripheral blood monocytes gelatinase was coproduced with IL-6 after stimulation with the aforementioned inducers. A statis- tically significant correlation was found between IL-6 and gelatinase titers in normal monocytes. When IL-6

234 I Van Ranst et al.

A

CYTOKINE, Vol. 3, No. 3 (May 1991: 231-239)

5000

4000

z z 2 3000

P z 5 2000

cn 1000

0 0 4 0 12 16

DILUTION FACTOR

Figwe 2. Induction of gelatinase/type IV collagenase mRNA by PMA.

(A) THP-1 cells were treated for different time intervals with 100 r&ml of PMA and cellular RNAs were analyzed for the presence of gelatinase RNA by dot blot hybridization to a cDNA probe.” The graph represents the cumulative increase of gelatinase mRNA levels expressed in scanning units. A representative dot blot is shown in the inset. (B) THP-1 cells were treated for 24 h with 100 @ml of PMA or were left untreated. RNA was then separated by electrophoresis in a denaturing agarose gel, blotted to a nylon membrane, and probed with radioactive gelatinase cDNA. The arrowheads indicate the 28s and 18s ribosomal RNA markers, respectively. The arrow indicates the gelatinase mRNA.

was measured in the THP-1 cell line, no activity could be detected in conditioned media from control cells and cells treated with IL-lp, Con A, LPS, and PMA.

Characterization of the 96-kD Gelatinme of the THP-I Cells

In order to characterize the 96-kD THP-1 gelatinase, several types of experiments were performed.

The enzymatic activity was measured at different pH values, resulting in a curve with optimal activities around neutral pH (Fig. 4A). Several types of inhibitors for specific classes of proteinases were tested (Table 1). Since PMSF did not, but EDTA, phenanthroline, and reducing agents did, block the activity, the gelatinase can be classified as a neutral metalloproteinase.

Previously, a polyclonal antibody directed against purified human tumor cell-derived gelatinase was generated.14a With this antibody, cross-reactivity between THP-1 cell derived and monocyte and neutro- phi1 gelatinases was observed (data not shown). The 96-kD gelatinase was precipitated with the immune but not with the preimmune serum. Similarly to the human 65 to 70-kD endogenous Malavu gelatinase, the 70-kD gelatinase from THP-1 cells was also precipitated. For further chartacterization, sufficient amounts of THP-1 gelatinase were produced to allow structural analysis.

” 1 10 100

IL-l dose bJ/mlI

LPS CONCENTRATION (pg/ml]

C 4000

1

is 2000

z

z w 1000

O/ 1 10 100 1000

Con A CONCENTRATION (pg/mlI

Figure 3. Regulation of 96.kD gelatinase by IGll3, LPS, and Con A.

(A) Confluent cultures of THP-1 cells were treated for different time intervals with various concentrations of IL-Q and the 96-kD gelatinase measured as described. Data points represent means and standard errors of three separate cell cultures. (B) THP-1 cells were treated for 48 h with different doses of LPS and gelatinase activity was quantitated by zymographic analysis. (C) Effect of different doses of Con A on 48-h production of THP-1 96-kD gelatinase.


3 4 5 6 7 8 9 10 11 12

PH

Figure 4. The THP-196&D gelatinase is a neutral proteinase.

Multiplicates of a standard gelatinase sample were separated in a zymography gel. Then the gel was sliced and individual gel lanes incubated during the development of the enzymatic activity in buffers at various pH values. Residual enzyme activity was plotted as absolute scanning units. Insets show the corresponding zymograms.

Pmfication of THP-1 Gehtinase and Sequence Analysis

THP-1 gelatinase was purified from approximately 5 x lo9 cells, cultured and stimulated for 48 h with 100 rig/ml of PMA. The culture fluid (1,000 ml) was then filtered and processed by affinity chromatography on a gelatin-sepharose column. The recovery of this one- step purification procedure was 96.5%. The resulting material (200 Fg) was extensively dialyzed and concen-

Table 1. Inhibition of THP-1 gelatinase. THP-1 gelatinase was separated in multiple lanes on a zymography gel. Individual gel lanes were then processed for zymography in incubation buffer supplemented with the indicated proteinase inhibitors at the indicated concentrations. Residual activity was determined by scanning analysis and expressed as the percentage of activity in control samples.

Inhibitor Concentration Percent

(mW inhibition

EDTA 10 1 0.1

l,lO-Phenanthroline 10 1 0.1

PMSF 2 0.2 0.02

Dithiothreitol 10 1 0.1

P-Mercaptoethanol 10 1 0.1

100 89 79

100 100 76 0 0 0

100 68 15 41

6 3

trated as described. Samples of gelatinase (1 kg), both with or without the addition of ovalbumin, were processed for staining and zymographic analysis (Fig. 5A). One major protein band was visible at 96 kD and thus comigrated with the gelatinase activity. A second band with an apparent molecular weight of approximately 27 kD was also stained. The bulk of the dialyzed gelatinase was separated by SDS-polyacrylamide gel electrophoresis and electroblotted onto a solid support, and the stained protein bands were directly processed for microsequence analysis (Fig. 5B). The resulting sequences are indicated at the left hand side in Fig. 5C. Again, in addition to the ovalbumin, two major protein bands were observed. The one corresponding to the 96-kD gelatinase showed a sequence at the 12 picomole level (top) and was found to be identical to fibrosarcoma-derived gelatinase14 (lower sequence in the alignment). The other protein band, migrating at 27 kD, consisted of pure TIMP-1 protein as deduced from the obtained sequence (200 picomole level, top) and its alignments with the cDNA-derived protein sequences of TIMP” or EP016 (bottom).

DISCUSSION

The present study describes the production, regulation, and purification of a 96-kD gelatinase originating from the monocytic cell line THP-1. The enzyme is characterized as a neutral metalloproteinase and cross- reacts in immunoprecipitation experiments with monocyte and neutrophil gelatinases. Furthermore, the enzyme was purified and its amino-terminal amino acid sequence determined.

The sequence analysis showed that the core protein of the THP-1 gelatinase has the same structure and NH, terminus as the type IV collagenase from fibrosarcoma and U937 cells.14 Peripheral blood monocyte gelatinase is at least eight amino acid residues shorter. Perhaps in a natural environment proteolytic processing enzymes cleave the molecule to shorter forms.

Recently, the THP-1 cell line was described to produce urokinase as well as a deficient plasminogen activator inhibitor-2.” Since activation of metalloproteinases can result from proteolytic cleavage by, e.g., trypsin but also-and perhaps more physiologically-by plasmin, it is possible to extrapolate the enzymatic cascade previously described in bone tissue’* to the THP-1 cell line. It should be emphasized that enzymes other than those mentioned and other activation mechanisms might be involved in the regulation of metallo- protease activity.” Of special interest is the fact that monocytes/macrophages are rich in different classes of proteases and also can produce, e.g., superoxides that might contribute to the activation of metalloproteinases in the natural cellular microenvironment.


A

200000

150000

100000

50000

WASH ELUTION

2M NaCl zM NaC’ 5% DMSO

j. j. * *

100 1050 1150 1200 1230 1240 1250 1260 1270 1280

COLUMN EFFLUENT VOLUME (ml)

/r

.l4

Figure 5. Purification of 96-kD THP-1 gelatinase.

(A) Alhnity chromatography. THP-1 cells were stimulated with 100 @ml of PMA for 48 h. Then the culture fluid was collected, filtered, and applied to a gelatin substrate affinity chromatography column. The elution method as well as the enzymatic activities of eluate fractions are indicated. The input material of the column contained activity out of the lower range of the graph. The inset shows the zymographic analysis of the enzymatically active fractions. (B) Staining analysis. Samples of 10 ug of purified gelatinase were separated by SDS/PAGE and stained with Coomassie blue. Molecular weight markers are indicated in kD. Arrows indicate the gelatinase (gel), ovalbumin (ov), and the tissue-inhibitor of metalloproteinases (TIMP), respectively. (C) Electrotransfer and sequence analysis. A sample of 200 ug of purified THP-1 gelatinase was supplemented with ovalbumin (ov.), extensively dialyzed, separated by SDS/PAGE, transferred to Pro-Blott membrane, and stained (right panel). Then the purified proteins were sequenced on a gas-phase sequencing apparatus (left panel) and the sequences were aligned with those of fibrosarcoma gelatinase14 and TIMP. 15,16 The drawn lines connect the stained protein bands with their respective experimentally determined amino acid sequences. Below these, the corresponding cDNA derived protein sequences are aligned. Residues between brackets were present in lower amount than the next following residue. Open positions indicate undetermined residues.

c- E

G d

100 5 F U- E 0

75 E i 00 =-

50 8 z

25 ii 5

Invasion is known to be facilitated by the secretion of proteinases such as plasminogen activator and metalloproteinases. Moreover, from recent studies on the urokinase receptor,” it is becoming evident that cell migration by solubilization of the extracellular matrix depends on multiple interactions of several proteases” with their inhibitors, receptors, and substrates. Thus the production of metalloproteinases

might also contribute to invasiveness, especially in view of the induction of these enzymes by tumor-promoting agents such as PMA and their activation by plasmin.

The THP-1 cell line is not only an interesting cell line to study the malignant phenotype, but by its resemblance to mature monocytes also represents a model system for the study of macrophages. Neverthe- less, it was found that, in comparison with native


monocytes, the expression of the IL-6 gene was defec- tive. From our study we can conclude that the regulated induction of gelatinase is a differentiation marker as are other gene products.13 Migration of monocytes is an essential part of their life cycle. As is evident from this and other studies,” these cells use proteolytic enzymes to solubilize the extracellular matrix on their way to the inflammation site. Since substances such as IL-l and LPS induce gelatinase and monocyte chemotactic factor’l the migratory activity of the macrophage is enhanced during inflammation. Modulation of the migratory capacity of macrophages with cytokines or other immunostimulants might thus be beneficial for the host. In the malignant phenotype, however, the facilitation of the solubilization of the extracellular matrix by “cytokine” therapy (e.g., IL-1)22 might also result in invasion-promoting effects.

MATERIALS AND METHODS

Reagents Phorbol 12-myristate 13-acetate and gelatin were from

Sigma Chemical Company, St. Louis, MO, USA. Tris, Triton X-100, and EDTA were from Merck (Darmstadt, Germany). Human serum albumin was from Boehringer Mannheim (Mannheim, Germany). Sodium dodecyl sulphate and pure ovalbumin were from Serva (Heidelberg, Germany). IL-1B was purified from human huffy coat supernatants as described.23

Isolation, Culture, and Induction of Cells THP-1 cells, a human monocytic cell line,” and a human

hepatoma-derived tumor cell line (Malavu)‘4a were grown in stationary culture flasks (Falcon Plastics, Oxnard, CA, USA) in Eagle’s minimum essential medium with Earle’s salts (EMEM), supplemented with 10% (v/v) fetal calf serum. Occasionally, the THP-1 cells were cultured in serum containing RPMI-1640 medium or under serum-free conditions. Cell numbers and viability were determined by microscopic counting in Biirker chambers after vital staining with trypan blue. Human neutrophils from heparinized human peripheral blood from single or multiple donors (Belgian Red Cross Blood Transfusion Centers of Leuven and Antwerp) were isolated as previously described.*l For the production of gelatinases, 24 multiwell plate cultures (Nunc, Roskilde, Denmark) were treated with different inducers and cell culture supernatants were harvested after indicated time intervals.

Detection of Gelutinase Activity by Zymography Gelatinase activity in cell culture supernatants was

determined by sodium dodecyl sulphate/polyacrylamide gel electrophoresis (SDS/PAGE) zymography according to a modificatior? of the method of Heussen and Dawdle.*“ Briefly, samples were run without prior denaturing on 7.5% (w/v) polyacrylamide/l% (w/v) SDS gels to which 0.1% (w/v) of gelatin was added and copolymerised. Stacking gels were

5% (w/v) polyacrylamide/OS% (w/v) SDS and did not contain gelatin substrate. Gels were run at 4°C for 16 h at 85 V. Molecular weight markers (Bio-Rad, Richmond, CA, USA) used were phosphorylase b (M, 97,400) bovine serum albumin (BSA) (M, 66,200), ovalbumin (M, 42,700) carbonic acid anhydrase (M, 31,000), and soybean trypsin inhibitor (M, 21,500).

After electrophoresis, gels were washed to remove SDS, then incubated for development of enzyme activity, stained with Coomassie brilliant blue R250, and destained in 30% (v/v) methanol/lo% (v/v) acetic acid.

Gelatinase activity was detected as unstained bands on a blue background. Quantitative determination of gelatinase activity was achieved by computerized image analysis through two-dimensional scanning densitometry. Gelatinase activity was expressed in scanning units representing the scanning area under the curves, which is an integration ratio that takes into account both brightness and width of the substrate lysis zone. Titration of standard gelatinase samples and plotting of the scanning units versus gelatinase dilution showed a sigmoi- da1 relationship. Ratios of gelatinase induction were calculated from the shift in the parallel linear parts of the sigmoidal plots. Titrations of parallel sample sets from repeated experiments showed standard errors of means always less than 10% of the mean value.14a

Purijication of Standard Hepatoma Gelutinase and Production of Polyclonal Antiserum

Gelatinolytic activity from crude supernatants of Malavu cells treated with 100 rig/ml of phorbol 12-myristate 13- acetate’4a was concentrated and purified by substrate affinity chromatography on gelatin-sepharose (Pharmacia, Uppsala, Sweden). For this purpose, batches of approximately 500 ml of serum-free cell culture fluids were filtered, supplemented with 0.02% sodium azide, 10 mM EDTA, and 0.4 M NaCl and loaded on a gelatin-sepharose column equilibrated with 50 mM Tris HCl, pH 7.6, 10 mM EDTA, 0.02% NaN,, 0.01% Tween 20 (equilibration buffer) supplemented with 0.5 M NaCl. The bound enzyme was eluted with 1 M NaCl in equilibration buffer. Test samples of input, wash, and elution fractions were analyzed for protein content and gelatinolytic activity. Protein content was determined by the method of Bradford” with albumin as a standard protein and gelatinolytic activity was determined by zymography. The peak elution fractions of each chromatography run contained two major gelatinolytic enzymes with apparent molecular weights of 67 and 85 kD, respectively.14”

This gelatinolytic activity was used to raise a polyclonal antiserum in a rabbit. After collection of preimmune serum the rabbit was first immunized with approximately 10 pg of antigen in complete Freund adjuvant. The rabbit received, at two week intervals, several boosters of approximately 10 pg of antigen in incomplete Freund adjuvant. One week after the fourth booster, the immune serum was collected and tested.

Immunoprecipitation and Inhibition Experiments For the immunoprecipitation of gelatinases, culture

fluids from antigen-producing cell types were reacted with


the preimmune or immune serum originating from the same rabbit. In a typical experiment, 10 ~1 of culture fluid from monocytes or from a laboratory standard of hepatoma gelatinase were supplemented with 20 ~1 of a solution of 30 mg/ml BSA and 2.5% (v/v) Triton X-100 and with 20 ~1 of a 1:lOO dilution of the rabbit preimmune serum or of the polyclonal immune serum. Samples were adjusted to a final volume of 90 ~1 with bidistilled water. After a reaction time of 15 min at O”C, 10 ~1 of pretreated Pansorbin cells (Calbiochem, La Jolla, CA, USA) were added and reacted with the formed immune complexes for approximately 2 h at 4°C. After centrifugation of the bacterial cells coated with the immune complexes (30 set, 12,OOOg), the pellets were washed three times with 100 ~1 of buffer containing 2.5% (v/v) Triton X-100, 0.15 M NaCl, 5 mM EDTA, 50 mM Tris-HCl, pH 7.4, 0.02% (w/v) NaN,, and 1 mg/ml BSA. Samples were finally resuspended in loading buffer for SDS/PAGE and immunoprecipitated gelatinase activity was determined by zymography as described.

For the determination of the pH optimum of the gelatinase, a standard sample of THP-1 gelatinase was separated in multiple lanes on a zymography gel. The gel was then sliced and individual lanes incubated for 16 h in buffers of various pH before staining and scanning analysis. The pH values were controlled before, several times during, and after the incubation period.

Sensitivity to inhibitors was tested in a similar way at neutral pH, but in the presence of proteinase inhibitors during the incubation period.

Determination of Human Interleukin-6

IL-6 levels were determined using the hybridoma growth factor (HGF) assay on mouse hybridoma 7TDl cells, as previously described.26 HGF titers were expressed as log,, U/ml, defined as the dilution giving half-maximal prolifera- tion of the hybridoma cells.

Purification of THP-1 Gelutinase and Amino Acid Sequence Analysis

For the production of THP-1 gelatinase, about 5 x lo9 cells were treated for 48 h with 100 r&ml of PMA at 37°C. Then the supernatant fluids (1,000 ml) were supplemented with 400 mM NaCl, 10 mM EDTA, and 0.02% NaN, and filtered (Whatman paper, 3MM, Whatman, Maidstone, En- gland), and then refiltered through a 1.2~pm filter (Gelman Acrodisc, Gelman Sciences, Ann Arbor, MI, USA). Crude material was purified by substrate affinity chromatography on gelatin sepharose (Pharmacia, Uppsala, Sweden). The column was equilibrated with 50 mM Tris . HCI, pH 7.6,lO mM EDTA, 0.02% NaN,, 0.5 M NaCl, and 0.01% Tween 20. After extensive washing, stepwise elution of bound activity was done with equilibration buffer supplemented with 2 M NaCl and with 2 M NaCl/S% DMSO. Samples of input, wash, and elution fractions were assayed for gelatinase activity. Two elution fractions contained more than 85% of the total gelatinolytic activity. These were pooled and supplemented with 200 kg of ovalbumin (five times crystalized) in 5 ml of bidistilled water and extensively dialyzed against bidistilled water. A control sample was dialyzed in the absence of

ovalbumin. The addition of ovalbumin enhanced protein recoveries after dialysis and did not interfere with subse- quent amino-terminal sequence analysis since ovalbumin is N-terminally blocked. After dialysis, the volume was reduced to 50 ~1 by freeze-drying in siliconized glass tubes. Then the sample was separated by SDS/PAGE in a 10% gel with a 4% stacking gel. The proteins in the gel were electrotransferred in a semi-dry blotting apparatus” for 4 h at 200 mA onto a Pro-blott membrane (Applied Biosystems, Inc, Foster City, CA) following the recommendations of the manufacturers. The membrane was then stained with Coomassie blue R-250. Molecular weights were calculated from included standards (BioRad, SDS-Page Molecular Weight Standards-Low, Bio- Rad Laboratories, Richmond, CA and Rainbow Markers, Amersham RPN756, Aylesbury, UK). Fragments of the filter showing the stained protein bands were excised, and the amino-terminal sequences of the corresponding proteins were determined in a modified version of the sequence cycle (BLOlT-1) on an automated gas-phase sequencer (Applied Biosystems, model 477A). The adapted cycle for use in pulsed liquid chemistry yields higher sequencing efficiency with Pro-blott membranes in the absence of polybrene.

Northern Blot and Dot Blot Analysis

For RNA blot analysis, cultures of untreated and PMA- treated THP-1 cells were collected at indicated time intervals. Total RNAwas isolated by the guanidine isothiocyanate- CsCl method and analyzed either by electrophoresis through 1% agarose-formaldehyde gels followed by capillary transfer to nylon membranes, or by dot blot hybridization exactly as described previously.” Gelatinase mRNA was detected by hybridization experiments with the complete cDNA insert from the fibrosarcoma gelatinase.14 Before labeling by ran- dom priming,29 the insert was excised from the Bluescript vector PBS-92 with XbaI. The relative concentrations of specific mRNA were determined by densitometric scanning of dot blot autoradiograms and expressed in relative absor- bance units. For this purpose serial dilutions of 12.5 pg of total cellular RNA, obtained from cells that were treated for various time intervals with 100 rig/ml of PMA, were applied to solid supports and processed as described.

Accession Number

The accession number in the SWISS-PROT protein sequence database is P14780 for gelatinase.

Acknowledgments

The present work is supported by the cancer fund of the ASLWCGER and FGWO 3.0027.90 and ILSI grants to J.A., G.O., J.A., and J.V.D. are research associates of the national fund for scientific research. The technical assistance of Pierre Fiten, Willy Put, and Jean-Pierre Lenaerts is acknowledged. The authors thank Dr. G.I. Goldberg for the PBS-92 plasmid and Dr. A. Billiau for support.


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the cytokine-protease connection: identification of a 96-kd thp-1 gelatinase and regulation by...

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