1991 77: 841-848  

PH Hart, GF Vitti, DR Burgess, GA Whitty, K Royston and JA Hamilton activitystimulating factor: increased urokinase-type plasminogen activator Activation of human monocytes by granulocyte-macrophage colony- 

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Activation of Human Monocytes by Granulocyte-Macrophage Colony-Stimulating Factor: Increased Urokinase-Type Plasminogen Activator Activity

By Prue H. Hart, Gerard F. Vitti, Diana R. Burgess, Genevieve A. Whitty, Keith Royston, and John A. Hamilton

Granulocyte-macrophage colony-stimulating factor (GM- CSF) raised the plasminogen activator (PA) activity of cul- tured human monocytes. This activity was characterized to be urokinase-PA (u-PA) by incubation with specific IgG and by sodium dodecyl sulfate-polyacrylamide gel electrophore- sis zymography. Increased U-PA activity reflected GM-CSF- induction of U-PA mRNA levels. The stimulatory properties of GM-CSF for monocyte PA activity differed from those of interleukin-4, which induced monocyte tissue-type PA (+PA) activity, and of interferon-y (IFN-y), which alone was not

RANULOCYTE-MACROPHAGE colony-stimulat- G ing factor (GM-CSF) was initially recognized as the glycoprotein that stimulated the proliferation and differen- tiation of GM precursor cells from bone marrow (BM).' However, additional biologic roles for this and other CSFs are becoming apparent and include the activation of ma- ture cells of the hematopoietic lineages."

For peripheral blood (PB) human monocytes, high af- finity receptors for GM-CSF have been well character- i~ed .~ , ' GM-CSF activates human monocytes in vitro by increased monocyte tumoricidal activity: HLA-DR expres- sion,' and killing of Leishmania donovani' and Trypanosoma cruzi.' We have previously found that GM-CSF alone does not stimulate interleukin-1 (IL-1) or tumor necrosis factor a (TNFa) activities, or prostaglandin E, (PGE,) levels of human monocytes," disagreeing with a study in which unfractionated mononuclear cells were used." However, our findings are consistent with those of others who reported GM-CSF-enhanced monocyte tumoricidal activ- ity only when a second stimulatory agent, viz endotoxin, was present." Large numbers of activated macrophages have been reported in the peritoneal and pleural cavities of GM-CSF transgenic mice and it has been suggested that products from these macrophages may be responsible for many of the biologic abnormalities observed in these ani mal^.'^"^

Plasminogen activators (PAS) are serine proteases that convert plasminogen to another serine protease, plasmin." There are two types of PA, urokinase-type PA (u-PA) and tissue-type PA (t-PA); the u-PNplasmin system is impli- cated in inflammation, tissue remodeling, and cell migra- tionI6 while the t-PNplasmin is considered to be responsi- ble for the removal of fibrin clots because the t-PA activity is potentiated by fibrin." WeLs and have found that unstimulated human monocytes produce u-PA; how- ever, its activity is not detected in culture supernatants without prior removal biochemically of a monocyte-derived u-PA inhibitor, presumably PA-inhibitor type 2 (PAI-2).2L322 The lymphokine, interferon-y (IFN-y), has been reported t o increase the PA activity of human monocytes cultured on a fibrin ~ubstrate.'~ We have reported that monocytes can also produce t-PA when stimulated with lipopolysaccharide (LPS)," with LPS and IFN--y together," or with another lymphokine, IL-4.24

Because PA expression by the macrophages of GM-CSF transgenic mice might explain much of their pathology, eg,

stimulatory but augmented lipopolysaccharide-induced t-PA activity. GM-CSF alone did not stimulate detectable mono- cyte t-PA activity but combined with IFN-7 to promote this activity. Plasmin formation arising from GM-CSF-induced u-PA in monocytes may contribute to the matrix turnover involved in, eg, cell migration and inflammation, and may explain some of the pathology seen in GM-CSF transgenic mice. o 1991 by The American Society of Hematology.

muscle wasting and peritoneal bleeding,".I4 and because we wanted to explore further the actions of GM-CSF on human monocytes, the PA activities of GM-CSF-treated human monocytes, with and without electrophoresis to remove u-PA inhibitors, were measured and characterized. We report here that for monocytes cultured on regular tissue culture surfaces, GM-CSF alone ( 2 10 U/mL; 2 10 pmol/L) can stimulate u-PA production, thus distinguishing this lymphokine from IFN-y" and IL-4F and distinguishing u-PA from the other monocyte mediators, viz IL-1, TNFa, and PGE,." However, incubation of monocytes with GM- CSF together with IFN-y resulted in the expression of t-PA activity. Unlike IFN-y" and IL-424 again, GM-CSF did not provide a strong, reproducible signal with LPS for en- hanced t-PA activity.

MATERIALS AND METHODS

Monocyte Isolation leukocyte-rich fractions collected after leuko-

phoresis of 500-mL donations of venous PB were obtained from the Melbourne Red Cross Blood Bank. Mononuclear cells were selected by centrifugation (17Og, 40 minutes) of the leukocyte fractions on pyrogen-tested Lymphoprep (Nycomed, Oslo, Nor- way). After multiple washings, the cells (3 x 108 to 8 x lo8) were suspended in Hanks Balanced Salt Solution (Commonwealth Serum Laboratories, Melbourne, Australia) containing 0.21% sodium citrate, 1 pg/mL polymyxin B sulfate (Sigma Chemical Co, St Louis, MO), and 70 pg/mL neomycin sulfate (Upjohn, Rydalm- ere, Australia) and separated by countercurrent centrifugal elutri- ation (Beckman J E d B elutriation system, Palo Alto, CA) with constant rotor speed (2,000 rpm) but increasing pump rates from 8 (loading flow rate) to 22 muminute. Monocytes were collected at

As

From the University of Melboume, Department of Medicine, Royal

Submitted November 20, 1989; accepted October 19, 1990. Supported by grants from the National Health and Medical Re-

search Council of Australia and the Arthritis Foundation of Australia. Address reprint requests to P.H. Hart, PhD, Clinical Microbiology

Unit, School of Medicine, Flinders University of South Australia, South Australia 5042, Australia.

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. section 1734 solely to indicate this fact.

Melboume Hospital, Parkville, Victoria, Australia.

0 I991 by The American Society of Hematology. 0006-4971 I91 17704-001 2$3.00/0

84 1 Blood, Vol77, No 4 (February 15). 1991: pp 841-848

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842 HART ET AL

flow rates between 14.5 and 22 mL/minute; monocyte enrichment was confirmed by May-Griinwald-Giemsa staining of cytocentri- fuged smears and by nonspecific esterase staining.

Monocyte Culture

Cell fractions containing 2 90% monocytes were cultured as previously in a-Modified Eagle's Medium (a-MEM; Flow Laboratories, McLean, VA) supplemented with 20 mmol/L 3-(N-morpholino) propane sulfonic acid (Sigma), 13.3 mmol/L NaHCO,, 2 mmol/L glutamine, 5 x lo-' mol/L 2-mercaptoethanol, and 70 & n L neomycin sulfate (complete a-MEM), osmolarity 290 mmolkg. Cells (0.8 x lo6 to 1.0 x lo6 in 1 mL of medium supplemented with 1% fetal calf serum [FCS; Flow]) were cultured in 2-cm2 wells (Linbro 76-063-05) for up to 18 hours in 5% CO, at 37°C; triplicate cultures for each variable were examined. When indicated, the following reagents were added; recombinant human GM-CSF (rhuGM-CSF) from 1.0 to lo4 U/mL, rhu IFN-7 from 10 to 100 U/mL, and LPS from Escherichiu coli Olll:B4, purified by the Westphal method (Difco, Detroit, MI), at 100 ng/mL. To cultures to which LPS was not added, polymyxin B sulfate was added to a final concentration of 1 pg/mL. Cultures were termi- nated by removal of the supernatant, which was then centrifuged and stored at -20°C until assay. Cell monolayers were treated in one of two ways. For quantitation of the number of cells after 18 hours in culture, cell monolayers together with pelleted cells from the culture supernatants were lysed with Zaponin (Coulter Electron- ics, Bedford, UK) and nuclei measured in a Coulter Sampling Stand; for measurement of cell-associated PA, the same cells were washed twice with phosphate-buffered saline (PBS) before lysis with 0.5 mL 0.2% Triton X-100 (Ajax Chemicals, Sydney, Austra- lia) in Ca2+- and Mg2'-free PBS.

Maintenance of LPS-Free Conditions During monocyte isolation and culture, intense care was taken to

limit LPS c ~ n t a m i n a t i o n . ' ~ ~ ' ~ ~ ~ ~ All equipment was of a plastic disposable nature whenever possible. Glassware was soaked in 1% E-Toxiclean (Sigma) and, after multiple washings, heated to 240°C. All buffers and media were filtered through Zetapor membranes (AMF Cuno, Meriden, CT). LPS levels of less than 50 pg/mL in all reagents were confirmed in the Limulus lysate assay (Common- wealth Serum Laboratories) and by assay of induced procoagulant activity (ie, decreased plasma clotting time in the presence of 0.03 mol/L CaCI,) by mononuclear cells incubated with the reagents in question.lu

Assay of PA Activity By methods previously r e p ~ r t e d , ' ~ . ~ ~ 0.05 mL of culture superna-

tant or cell lysate were added to 0.28-cm2 wells, previously coated with I2'I-fibrin. Plasminogen (0.8 kg dissolved in 0.1 mL Tris-HC1, 0.1 mol/L, pH 8.1) was also added. The plasminogen was prepared from human plasma by Sepharose-lysine affinity chromatography.*' Monocyte-derived plasminogen-independent fibrinolytic activity was assayed in all experiments and was always 1 5 % of the plasminogen-dependent activity. Aliquots were withdrawn after 2 to 3 hours and soluble '"I-fibrin degradation products measured in a gamma counter; one or two further aliquots were taken at later times until significant levels of fibrinolysis were seen. The activities of both one- and two-chain forms of PA were measured because of low levels of contaminating plasmin in plasminogen preparations." PA activity was expressed according to the activity of u-PA standards (0.001 to 2.0 IU/mL; Leo Pharmaceutical Products, Ballerup, Denmark), dissolved in either culture medium or 0.2% Triton X-100 (in Ca*+- and Mg2'-free PBS). In the present experiments, allowances were not made for the lag period of

activation of single-chain to two-chain u-PA16 and activities have been expressed as International Units Equivalent (IUE). PA activities were also expressed according to the number of cells initially plated. As previously reported, in response to GM-CSF there was no significant change in the number of monocytes in any treatment group after 18 hours in culture.Iu

Immunologic Characterization of Monocyte PA Activity IgGs were isolated from rabbit antisera to human u-PA and to

human t-PA using Protein A Sepharose CL-4B (Pharmacia) as previously des~ribed'~.'~; the mouse myeloma IgG, HOPCY, was used as an irrelevant antibody.'* PA standards (0.1 IU/mL; u-PA from Leo Pharmaceutical Products, t-PA as a culture supernatant from the MM138 melanoma cell line26) and pooled supernatants from duplicate 18-hour monocyte cultures were incubated with the IgGs (1 pg/mL final concentration) for 1 hour at 37°C before assay of residual PA activity by the techniques outlined above. PA standards from the National Institute for Biological Standards and Control, London, UK, were used to confirm the definition of PA units.

Characterization of PA by Sodium Dodecyl Sulfate-Polyacylamide Gel Electrophoresis (SDS-PAGE) Zymography

SDS-PAGE was performed essentially as p~b l i shed . '~~~ ' The resolving gel (10%) contained casein (2 mg/mL, Sigma) and human plasminogen (6 kg/mL) and was pre-electrophoresed at 15 mA for 1 hour at room temperature. After loading onto a 4% stacking gel, the samples (0.02 mL), equilibrated in 0.0625 mol/L Tris-HC1, pH 6.8,1.25% SDS, 10% glycerol, were electrophoresed at 12 mA for 2 hours using the buffer system of Laemmli.'x After electrophoresis, the gels were washed for 1 hour in 2.5% Triton X-100, then rinsed and incubated in 0.1 mol/L Tris, pH 8.0, for 18 hours at 37°C. Gels were stained by immersion in 0.25% Coomassie Blue R-250 in 50% methanol/ 7% acetic acid for 60 minutes, then destained for 2 hours in 30% methanol/ 10% acetic acid. Clear bands of lysis appeared where PA had converted plasminogen to plasmin, which then degraded the casein. The hands provided a semi-quantitative measure of PA levels; this was particularly pertinent for superna- tant u-PA, whose activity upon conversion to the two-chain form in the presence of low levels of plasmin" was inhibited in nonelectro- phoresed samples by PAI-2." Low molecular weight markers (Pharmacia) were used to identify the PA standards (0.1 IU/mL); the main lytic bands for u-PA and for t-PA were detected at 52 Kd and 70 Kd, respectively. As published previously,'* immunoprecipi- tation experiments using the specific IgGs described above also confirmed the identity of the standard PA bands.

Detection of mRhLA in Human Monocytes As described,18 total RNA was prepared using the method of

Chirgwin et al.2R RNA (5 pgllane) was fractionated on a formalde- hyde-containing 1% agarose gel before transfer to Genescreen Plus nylon membrane (DuPont, Boston, MA). The filter was hybridized overnight at 60°C in a standard hybridization buffer containing greater than 2 x lo6 cpm/mL of "P-labeled u-PA cDNA, or t-PA cRNA. For u-PA cDNA, hybridization was essentially as previously described? except that it was performed at 45°C. The t-PA cRNA was prepared as before," and after hybridization, the filter was washed and treated with 1 pg/mL RNaseA.'* Loading of mRNA for all lanes was generally shown to be approximately equal by probing with a "P-labeled cDNA for glyceraldehyde-3-phosphate dehydrogenase.

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GM-CSF INCREASES MONOCYTE U-PA ACTIVITY 843

Reagents

Bacterially-synthesized huGM-CSF was a kind gift of Dr J.F. DeLamarter, Biogen SA, Geneva, Switzerland; it had been pre- pared and purified as described by Burgess et al.” Batches CAE 392-32 and AS 5/5 were used; the stock solutions of 100 pg/mL and 20 pg/mL had activities of 8 x lo6 U/mL and lo6 U/mL, respec- tively, as judged by day 14 colony growth of normal human BM samples.”’ With a molecular weight of 14.5 Kd, 1,000 U GM- CSF/mL approximated 1.1 nmol/L or 16 ng/mL. The following reagents were kindly supplied by those listed: rhu IFN-.I (1.4 X lo7 U/mg protein), Dr E. Hochuli, Hofkann-La Roche, Basel, Switzer- land; rabbit antisera to human PAS, Dr W-D. Schleuning, Schering AG, Berlin, Germany; MM138 melanoma cell line, Dr R. White- head, Ludwig Institute for Cancer Research, Melbourne, Austra- lia; mouse myeloma IgG, Dr A. Burgess, Ludwig Institute for Cancer Research, Melbourne, Australia; cDNA probe for u-PA and for t-PA, Dr F. Blasi, University of Copenhagen, Denmark, and Dr W-D. Schleuning, respectively.

Expression of Results Unless otherwise indicated, mean values f SEM are shown. The

significance of differences was assessed using a two-tailed Student’s t-test; results were considered significantly different ifP < .05.

RESULTS

PA Activity of GM-CSF-Treated Human Monocytes

To measure (this section) and to characterize (see below) cell-associated PA activity, either a net intracellular activity that allows for inactivation by PA inhibitors, or one associ- ated with specific cell membrane receptor^,'^." lysates of cells treated for 18 hours with GM-CSF were prepared using 0.2% Triton X-100 as the lysing agent. Table 1 shows the mean cell-associated PA activities for triplicate cultures from each of 15 monocyte donors. Although there was variability between donors for the low levels of detectable cell-associated PA activity in both control and stimulated

Table 1. PA Activities of GM-CSF-Treated Monocytes From Several Donors

Addition to Monocyte Cultures.

IFN-y GM-CSF GM-CSF + IFN-y LPS LPS + GM-CSF LPS + IFN-y

PA Activity (IUE/106 cells)t Mean ? SEM

Cell Lysates (n = 15)

0.004 f 0.001 0.005 2 0.002 (NS) 0.036 f 0.017 (P < .01) 0.021 * 0.010 (P < .01) 0.004 f 0.002 (NS) 0.005 t 0.002 (NS) 0.004 -c 0.002 (NS)

Culture Supernatants (n = 19)

ND ND ND

0.14 f 0.04 0.16 f 0.04 0.20 t 0.05 (P* < .05) 0.46 -c 0.07 (P* < .01)

Abbreviations: ND, not detected; NS, not significant (P < .05). *Monocytes were cultured for 18 hours with no added stimulus, or

with the following alone or in combination: IFNy, 100 U/mL; GM-CSF. 1,000 U/mL; LPS, 100 ng/mL.

tPA activities were assayed as described in Materials and Methods and expressed according to the activity of U-PA standards. P values were determined by comparison with control cultures. PS values were determined by comparison with LPS-treated cultures. For the cell lysates, the differences between the mean PA activities forthe GM-CSF- treated and the GM-CSF + IFN-y-treated groups were not statistically significant.

cells, GM-CSF (1,000 U/mL; - 1 nmol/L) consistently enhanced such PA activity. The effects of other monocyte- stimulating agents were also examined; IFN-y (100 U/mL) was inactive by itself and did not alter the stimulatory effect of GM-CSF (Table 1). As we have reported previously,” LPS (100 ng/mL) did not increase the net PA activity. There was no significant difference between LPS-treated cultures and those incubated with LPS and GM-CSF, ie, LPS completely inhibited the stimulatory effect of GM-CSF for cell-associated PA activity. The combination of LPS + IFN-y, which is potent at increasing the synthesis of other monocyte products,’o,L8 was not stimulatory for cell- associated PA activity (Table 1).

We have previously shown that there is no detectable PA activity in the supernatants of unstimulated or IFN-y- treated monocytes cultured for 18 hours on plastic tissue culture dishes.” We now report that PA activity was also not detected in the culture medium of monocytes from 19 donors treated with 1,000 U GM-CSF/mL (Table 1). Even though IFN-y (100 U/mL) was also not stimulatory, simulta- neous incubation of monocytes with GM-CSF and IFN-y gave detectable PA activity in the supernatants of cells from 16 of the 19 donors investigated with a mean activity (+SEM) of 0.14 (k0.04) IUE/106 cells. LPS alone was active for all donors. GM-CSF augmented, although weakly, LPS-induced activities (P < .05, Table 1); however, this increase was significant for only 7 of 19 donors examined. In contrast, IFN-y (100 U/mL) consistently interacted strongly with LPS for enhanced PA activity (Table 1).

Thus, the detectable responses to GM-CSF varied de- pending whether cell-associated or supernatant PA activity was monitored.

Kinetics and Dose Response to GM-CSF

A significant enhancement by GM-CSF of cell-associated PA activity was found in response to 10 U GM-CSFlmL (10 pmol/L) (P < .02), with maximal stimulation detected for this, and other donors, in response to 100 U GM-CSF/mL (Fig 1). For the two donors investigated, increases in cell-associated PA activity were first detected after expo- sure to GM-CSF (1,000 U/mL) for 4 and 6 hours, respec- tively.

PA activity was not detected in the culture medium over a concentration range of 1 to lo4 U GM-CSF/mL (data not shown). When the effects of varying both GM-CSF and IFN-y concentrations on the supernatant PA activity were monitored for two donors, IFN-y at 10 U/mL was less potent than at 100 U/mL for combining with GM-CSF (100 and 1,000 U/mL) (data not shown).

Characterization of the PA Activities of GM-CSF-Treated Human Monocytes

We next determined the type(s) of PA activity that were being measured in the cell lysates and supernatants of monocytes subjected to the various treatment regimes.

We have previously reported that cell- associated PA activities are predominantly u-PA for un- treated monocytes and for those incubated with IFN-y

Antigenicity.

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844

h

m aJ d

U

0 0 - \

m I 0

X W 3

7

H Y

)r + > + U

.-

.-

Q

2

Fig 1.

5

4

3

2

1 T n

0 1 10

I.

-

1

- i o 2 lo3 IO*

GM-CSF W m l ) Dose resDonse to GM-CSF. Monocytes from a representa-

tive donor were incubated for 18 hours with.0 to 10' U/mL GM-CSF before harvest of the supernatants and, after two washes, lysis of the monocytes with 0.2% Triton X-100. Mean PA activity t SEM in the cell lysates is shown.

and/or LPS.'* Using IgGs specific for u-PA and t-PA,'* and the irrelevant IgG, HOPCY," the increased PA activity in the lysates of GM-CSF-treated monocytes is predomi- nantly u-PA because its activity is blocked by anti-u-PA IgG (Fig 2A). The same result was found for monocytes incubated with GM-CSF together with IFN-y, as well as with LPS + GM-CSF (Fig 2A). However, the PA activity in the supernatants of monocytes incubated with GM-CSF and IFN-y together, or GM-CSF with LPS, was t-PA-like (Fig 2B), agreeing with our previous that the PA activity detected in the supernatants of plastic-adhered monocytes is always t-PA. Similar results to those provided in Fig 2 for a single donor were obtained for five additional donors.

Cell lysates were also exam- ined by SDS-PAGE zymography. As previously rep~rted, '~ ' '~ a band of 52 Kd, corresponding to the molecular weight of u-PA, was observed in the sample from control monocytes and from the lysate of cells incubated with IFN-y (lanes 4 and 5, respectively, Fig 3A). For the lysates from GM-CSF- treated monocytes, and from monocytes treated with GM- CSF together with IFN-y, similar but quantitatively greater bands of 52 Kd were detected (lanes 6 and 7, respectively,

SDS-PAGE zymogruphy.

HART ET AL

Fig 3A). LPS induced a smaller increase in the u-PA band (lane 8, Fig 3A). No 70-Kd band corresponding to t-PA was noticed in any of the cell lysates including those obtained from cells stimulated with LPS alone, or with LPS + IFN-y or with LPS + GM-CSF (lanes 8 through 10, Fig 3A). The absence of a 70-Kd band in the lysates, together with the inactivation of cell-associated activity by anti-u-PA IgG, suggests that t-PA, if produced, is secreted and not stored within the monocyte.

For the monocyte supernatants, a 52-Kd band was again seen for control monocytes and those treated with IFN-y, as previously reported18 (lanes 4 and 5, respectively, Fig 3B). In the supernatants of GM-CSF-treated monocytes, a similar but quantitatively greater band was once again found (lane 6, Fig 3B). However, when monocytes were co-treated with GM-CSF and IFN-y, a 70-Kd band migrat- ing in a manner characteristic of the t-PA standard also appeared (lane 7, Fig 3B). This 70-Kd band was also apparent in the culture medium of monocytes incubated

A U-PA t-PA C GMCSF GMCSF LPS LPS std std + I F N - X +GM-CSF

3 I

? 0,

e - 2

x E

x c .- c Y

a 1 B

B

ant i - t -PA I&

m

u- PA t - P A GM-CSF LPS L P S std std + IFN-X +GM-CSF

Fig 2. Immunologic characterization of monocyte PA activities. (A) Cell lysates. (B) Culture supernatants. Monocytes from a representa- tive donor were incubated for 18 hours with no added stimulus (C), or with the following, alone or in combination: GM-CSF, 1,000 U/mL; IFN-y.100 U/mL; LPS, 100 ng/mL. Cell supernatants and lysates were harvested as in Fig 1 and treated with HOPCY IgG, anti-u-PA IgG, or anti-t-PA !.go as described in Materials and Methods. PA standards (0.1 IU/mL) sewed as specificity controls for the IgGs. PA levels are expressed as '%fibrinolytic activity (cpm); mean activities t SEM are shown for triplicate measurements of pooled aliquots from duplicate cultures. ND, not detected.

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GM-CSF INCREASES MONOCYTE U-PA ACTIVITY 845

A kD

1 2 3 4 5 6 7 8 9 1 0

B

Fig 3. Characterization of monocyte PA activity by SDS-PAGE rymography. (A) Cell lysates. (B) Culture supernatants. Lane 1, cell-free cultures (in A showing no lytic activity in Triton X-100. in B showing the caseinolytic bands due to 1 % fetal calf serum); lane 2, U-PA standard (0.1 IU/mL); lane 3, t-PA standard (0.1 IU/mL). Pooled lysates/supernatants from duplicate cultures were obtained from monocytes incubated with: lane 4, no added stimulus; lane 5,100 U/mL IFN-y; lane 6,1,000 U/mL GM-CSF; lane 7, GM-CSF (1,000 U/mL) with IFN-y (100 U/mL); lane 8, LPS, 100 ng/mL; lane 9, LPS (100 ng/mL) with IFN-y (100 U/mL); lane 10, LPS (100 ng/mL) with GM-CSF (1.000 U/mL). SDS-PAGE zymography was performed as described in Materials and Methods; clear bands of lysis appeared where PA had converted plasminogen to plasmin, which then degraded the casein. The main caseinolytic bands for U-PA and t-PA standards were detected at 52 Kd and 70 Kd, respectively, as defined by the migration pattern of low molecular weight markers.

with LPS, LPS + IFN-y, and LPS + GM-CSF (lanes 8 through 10, Fig 3B). We have previously confirmed by precipitation with anti-t-PA IgG that the 70-Kd band in culture supernatants from LPS-treated monocytes is indeed t-PA.I8 In all supernatants from control and stimulated cells, caseinolytic bands of greater than 90 Kd were de- tected and reflected the lytic activity of serum components in the culture medium as shown in lane 1 of Fig 3B.

PA mRNA Levels in GM-CSF-Treated Human Monocytes Both u-PA and t-PA mRNA levels were investigated by

Northern analysis in GM-CSF-treated monocytes. An in- crease in u-PA mRNA with GM-CSF was first detected after 2 hours of exposure; a greater increase was detected after 18 hours (Fig 4A). For another donor, a stimulatory effect of LPS on u-PA mRNA, but not that of IFN-y, was also observed (Fig 4B). The increase by LPS for u-PA activity is not seen in the results of Table 1 possibly because of induction of PAI-2 by LPS?1.22 Furthermore, to support LPS-induction of u-PA, but not expression of its activity because of an inhibitor, treatment with LPS can give rise to a larger lytic band of u-PA activity under the conditions of SDS-PAGE zymography (Fig 3A). For the donor shown, low, but detectable levels of t-PA mRNA were observed for monocytes incubated for 4 hours with GM-CSF or IFN-y alone (Fig 4C). However, the potentiation of t-PA tran- scripts by GM-CSF together with IFN-y was observed (Fig 4C). For other donors, t-PA transcripts were not always detected in response to the lymphokines alone."

DISCUSSION

We have reported here that GM-CSF increased the levels of u-PA activity and of u-PA mRNA in purified human monocytes. In these studies in which strict precau- tions were followed to limit LPS contamination, the GM- CSF-induced u-PA activity was detected under conditions for which other monocyte mediators, viz TNFa, IL-1, and PGE2," were not found. Therefore, expression of u-PA activity can be dissociated from that of these other mono- cyte products, which have also been implicated in inflamma- tory processes, and points to a relatively specific effect of GM-CSF on monocyte product synthesis. Two other lym- phokines, I F N y (Table 1 and ref 18) and IL-4Y did not have the same effect under the culture conditions used. However, co-culture of monocytes with GM-CSF and IFN-y resulted in expression of t-PA activity despite the fact that neither cytokine could induce detectable t-PA activity by itself (Table 1). We have shown previously that IL-4 alone can dramatically induce human monocyte t-PA a~tivity.2~ Thus, different lymphokines have varied actions on monocyte u-PA and t-PA expression. Whether the same cell can produce both types of PA or whether monocyte subsets are involved is as yet undetermined. Furthermore, it is unknown whether there is a quantitative increase in the response by all monocytes, or whether GM-CSF induction of u-PA activity, for example, reflects the recruitment of a subpopulation not inherently producing this activity."

For the experiments above, only u-PA activity was detected in the cell lysates. Part of this low activity is likely to be bound to a characterized 55-Kd u-PA receptor on

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HART ET AL 846

2h A

B

4h responsible, at least in part, for the disparate results for U-PA activity detected in cell lysates, before and after SDS-PAGE zymography (Table 1, Fig 3) and on Northern blotting (Fig 4). Cell-associated u-PA activity can be de- tected under our isolation conditions possibly because of insufficient inhibition by the less-glycosylated cell-associ- ated PAI-2." Unlike the u-PA activity, t-PA activity is readily detectable in supernatants after appropriate mono- cyte activation (Table 1, Figs 2 and 3), possibly on account of the lower affinity of PAI-2 for t-PA.34

18h

U-PA

CJ& 6 &

CJ

C

The actions of the lymphokines were also different when the monocytes were co-cultured in the presence of LPS. GM-CSF significantly increased, although weakly and irreg- ularly, the t-PA activity of LPS-treated monocytes (Table 1). In contrast, IFN-y augmented considerably LPS- induced t-PA activity (Table l) , while the t-PA activities induced by LPS and IL-4 together were approximately additi~e.2~ Such influence of LPS highlights once again the U-PA importance of minimizing LPS levels in monocyte cultures if correct information on the actions of lymphokines is to be obtained. The strong interaction between LPS and IFN-y for t-PA production may explain the reported effects of IFN-y on PA production for cells that were cultured on fibrin." In this and our previous study,'" we found no increase by IFN-y on PA production by monocytes cultured on regular plastic tissue culture surfaces. However, with the addition of 2 1 ng/mL LPS, IFN-y was stimulatory." Either the fibrin itself could be altering the properties of the cells or LPS might have contaminated the fibrin in the previous report?

This study has shown that lymphokines differentially regulate both the amount and the type of monocyte PA. We have also found a very similar differential effect of GM- CSF, IFN-y, and IL-4 on the activities of other monocyte mediators, including TNFa, IL-1 and PGE,. Although none of the lymphokines alone was able to induce the synthesis of any of these monocyte products, GM-CSF with IFN-y consistently activated the monocytes for TNFa activity and

t-PA

Fig 4. mRNA levels in human monocytes. (A) U-PA mRNA. Mono- cytes from a representative donor were incubated for 2, 4, and 18 hours with no added stimulus (C) or with GM-CSF (1.000 U/mL). (E) U-PA mRNA. Monocytes from another donor were incubated for 14 hours with no added stimulus (C); with IFN-y (100 U/mL); with LPS (100 ng/mL); or with GM-CSF (1.000 U/mL). (C) t-PA mRNA. Mono- cytes from a representative donor were incubated for 4 hours with no added stimulus (C); with IFN-y (100 U/mL); with GM-CSF (1.000 U/mL), or with IFN-y (100 U/mL) together with GM-CSF (1.000 U/mL).

human monocytes""'; elevated u-PA receptor levels have been found in human monocytes treated with IFN-y and TNFa, but not with GM-CSF..'j It has been suggested that u-PA is secreted from cells only when this receptor is saturated." Our results suggest that t-PA, once produced, does not remain cell associated. u-PA activity was detected in the monocyte supernatants only on SDS-PAGE zymogra- phy, ie, using a technique that allows PA detection in the presence of inhibitor^"^^'; monocytes can produce large amounts of PAI-2,2',22 which preferentially inhibits u-PA? ' .~ LPS is a potent inducer of monocyte PAI-2*' and must be

PGE, production," but not for IL-1 activity.'" IL-4 did not cooperate with other lymphokines for production of these mediators." When cooperative interactions of the lympho- kines with LPS were investigated, GM-CSF interacted weakly and irregularly with LPS for enhanced IL-1 activity, but not for PGE, levels or TNFa activity." In contrast, IFN-y strongly augmented LPS-induced IL-1 and TNF-a activities, but not PGE,." We have also shown that IL-4 inhibits the induction of TNFa, IL-1, and PGE, in LPS- treated monocytes.'s Thus, the regulation of the synthesis of monocyte products, including U-PA and t-PA, by different lymphokines is complex and follows no set pattern.

Plasmin formation catalyzed by u-PA is believed to contribute to the matrix turnover occurring, for example, as part of the tissue destruction and cell migration present at sites of inflammation and tumor invasion.j6 In particular, injection of inflammatory stimuli into the peritoneal cavity of mice has been shown to induce macrophages with elevated PA acti~ity,~'.'' and it was proposed that this heightened proteolytic activity might allow the cells to

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GM-CSF INCREASES MONOCYTE U-PA ACTIVITY 847

transgress from the bloodstream into neighboring tissue by degrading interstitial matrix. It has also been shown that GM-CSF activates murine peritoneal macrophages in vitro to produce more PA activity." Elevated levels of GM-CSF have been found in the synovial fluids of patients with rheumatoid arthritis7 Because raised u-PA levels have also been detected in the synovial fluids of such patients:' GM-CSF may contribute to the pathogenesis of this disease by activating monocyte u-PA production, leading to en- hanced monocyte/macrophage migration, cartilage destruc- tion, and inflammation. The same possibility would exist at other inflammatory sites.

It has been suggested that the various complications present in GM-CSF transgenic mice were induced by GM-CSF-stimulated products of macrophages, such as TNFa, IL-1, or PA.'3,14 Our results above showing that GM-CSF by itself stimulates u-PA production, but not that of TNFa or IL-1," indicates that u-PA activity might be of more significance in this regard. GM-CSF alone also does not stimulate IL-1 or TNFa activity or mRNA levels in cultured murine macrophage^.^' PA production by macroph- ages in the GM-CSF transgenic mice may contribute to the necrotic lesions of the eye and striated muscle; a role for PNplasmin in the degradation of the adhesive basement membrane macromolecules of muscle has been ~uggested.~' In certain GM-CSF transgenic mice, there was the develop-

ment of ascites with evidence of chronic bleeding associated with the presence of unusually large numbers of peritoneal macrophage^.'^ Cells produce u-PA, in fact, as a latent or single-chain form (scu-PA) that is activated by trace amounts of plasmin to the two-chain form.43 Because scu-PA re- quires fibrin for its activity, it is being used clinically, in addition to t-PA, as a thrombolytic agent to lyse clots formed, for example, during myocardial in far~ t ion .~~ The bleeding noted in the GM-CSF transgenic miceI4 could be because of plasmin arising from the scu-PA, and therefore u-PA, produced by the activated macrophages. This obser- vation with the transgenic mice suggests that controlled short-term in vivo infusion of GM-CSF might lead to elevated scu-PA levels around monocyte/macrophages which, because of its fibrin-dependent a~tivity:~ could lead to lysis of local fibrin clots. In this connection, we have proposed before that IL-4, on account of its ability to stimulate monocyte production of the thrombolytic t-PA? might have a role in thrombolysis; GM-CSF may function likewise.

This study highlights the complexities of the activation processes in monocytes; further experimentation into the control of expression of PA in monocytes may contribute to a better understanding of GM-CSF-mediated control of inflammatory mediator production by monocytes.

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