activation and proliferation signals in murine macrophages

Activation and Proliferation Signals in Murine Macrophages. Biochemical Signals Controlling the Regulation of Macrophage Urokinase-Type Plasminogen

Activator Activity by Colony-Stimulating Factors and Other Agents

By John A. Hamilton, Gin0 Vairo, Kenneth R. Knight, and Benjamin G. Cocks

Purified hematopoietic growth factors such as colony- stimulating factor-1 (CSF-1) or macrophage CSF, granulocyte- macrophage CSF, and interleukin-3 or multi-CSF, stimulate the urokinase-type plasminogen activator (u-PA) activity of murine bone marrow-derived macrophages (BMM) and resident peritoneal macrophages. Granulocyte-CSF was inactive. The increases in BMM u-PA activity were inhibited by the glucocorticoid dexamethasone, and by agents that raise intracellular cyclic adenosine monophosphate levels, including prostaglandin E, and cholera toxin. These changes in U-PA

S FOR OTHER mature hematopoietic cells, macro- A phages derive from progenitor cells in the bone marrow through a coordinated process of proliferation and differentiation under the control of a group of glycoprotein hormones known as colony-stimulating factors or CSFs.’ The shortage of adequate numbers and/or sufficient purity of hematopoietic progenitor cells currently hinders attempts to determine the intracellular signals mediating CSF action. Cell lines are being used to study the mechanism of action of CSFs, including CSF-1 (or macrophage- CSF [M-CSF],’ which acts on committed hematopoietic cells of the mononuclear phagocyte lineage? However, murine bone marrow-derived macrophages (BMM) are an easily obtainable homogeneous normal cell population useful for the study of the biochemistry of the action of CSFs.’” CSF-1 is responsible for the survival, proliferation, differentiation, and activation of macrophages and macrophage precursors at various stages of their development.’ As well as CSF-1, granulocyte-macrophage CSF (GM-CSF) and interleukin-3 (IL-3) (or multi-CSF) are also mitogenic for BMM, although not to the same degree as CSF-1.5 All of the CSFs synergize with each other in stimulating BMM DNA synthesis.’

The actions of CSF-1 are mediated via binding to a specific surface receptor that has been shown to be the tyrosine kinase product of the c-fms proto-oncogene,8 and it is likely that the kinase activity is required for the cellular responses to CSF-1.2 The murine IL-3 receptor does not

From the Department of Medicine, University of Melbourne, Royal Melbourne Hospital, Parkville; and Microsurgery Research Centre, St Encent’s Hospital, Melbourne, Australia.

Submitted May 22, 1990; accepted October 5, 1990. Supported by grants from the National Health and Medical Re-

search Council ofAustralia (Program Grant), the Anti-Cancer Coun- cil of Victoria, and St Vincent’s Hospital, Melbourne.

Address reprint requests to John A. Hamilton, PhD, University of Melbourne, Department of Medicine, Royal Melbourne Hospital, Parkville, 3050, 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.

0 1991 by The American Society of Hematology. 0006-4971 /91/7703-0029$3.00/0

activity were paralleled by corresponding changes in u-PA mRNA levels. Evidence was obtained for protein kinase C and phospholipase C-mediated stimulation of BMM u-PA activity and mRNA levels; however, no evidence was found for an involvement of Na+/H+ exchange or Na+,K+-ATPase activity, Caz+ fluxes, or pertussis toxin-sensitive G proteins. Several findings point to a dissociation between macrophage u-PA expression and DNA synthesis. o 1991 by The American Society of Hematology.

contain a consensus sequence for a tyrosine kinase in the cytoplasmic domain, although there is evidence for involvement of such a kinase in the signal transduction pathway.’ In murine hematopoietic cells the binding of GM-CSF to its receptor is not directly competed for by IL-3, although it can be indirectly down modulated at 37°C by IL-3.” Despite the biochemical differences in the structures of these CSFs and their receptors, they all stimulate in BMM a variety of early responses that precede the onset of DNA synthesis, including stimulation of Na+/H+ exchange and/or Na+,K+- ATPase activity,6 an elevation of glucose uptake: c-jios, and c-myc mRNA levels,”.’2 however, none of them activate the phosphatidylinositol-Ca2+-diacylglycerol ca~cade . ’~*’~ The biochemical basis for these common actions is unknown.

Plasminogen activators (PAS) are serine proteases that cleave plasminogen to generate another serine protease, plasmin; there are two types of PA, urokinase-type PA (u-PA) and tissue-type PA (t-PA).I4 A relationship between cellular proliferation and elevated u-PA mRNA levels has been proposed, being independent of the cell type and origin.’’ BMM16,” and murine peritoneal macrophages” increase their PA activity in response to CSF-1, but the relationship to DNA synthesis is not completely clear. How CSF-1 regulates PA expression at the biochemical level is unknown.

PA activity has also been correlated with cell migration and tissue remodeling in a number of cellular system~,’~ although there is little information on the signaling pathways involved in the control of PA synthesis by growth factors and hormones; in the particular case of the macrophage, PA activity has been correlated with inflammati~n’~~’~ and cell-mediated immunity.” Uncharacterized lymphokine samples have been shown to elevate macrophage PA activity.Z2-’’ The availability of purified recombinant cytokines has now made it possible to define which lymphokines or cytokines can activate macrophages by this criterion.

We report here on the effects of a number of purified recombinant murine CSFs on BMM u-PA mRNA expression and activity; this system is also used to analyze mechanisms and signaling pathways controlling both the increase and decrease in the expression of cellular u-PA activity. The relationship of u-PA activity to BMM DNA synthesis is also explored.

616 Blood, Vol77, No 3 (February 1). 1991: pp 616-627

For personal use only.on January 1, 2019. by guest www.bloodjournal.orgFrom

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MACROPHAGE PLASMINOGEN ACTIVATOR REGULATION 617

MATERIALS AND METHODS

Mice Cells were obtained from male or female CBA or endotoxin-

hyporesponsive C3H/HeJ mice (8 to 12 weeks old; Walter and Eliza Hall Institute, Parkville, Australia).

BMM BMM were obtained from precursor cells in bone marrow as

described beforez6 and grown to confluence for 5 to 6 days in RPMI-1640,15% (volhrol) fetal bovine serum (FBS), 20% (vol/vol) L-cell conditioned medium (CM) (see below). BMM were generally “starved” of growth factor for 18 to 20 hours before use to render cells in a quiescent G$G, phase of the cell cycle?’ BMM for PA assays were generally cultured in 24-well tissue culture dishes (Linbro; Flow Laboratories, McLean, VA) yielding - 1.5-2 X 10’ adherent cells/well. Where indicated, some experiments were performed with BMM cultured in 12-well dishes ( -3 to 4 X 10s adherent cellshell). For measurement of PA activity, the cells were washed twice with phosphate-buffered saline, pH 7.4, depleted of calcium and magnesium (PD), and resuspended in RPMI-1640/5% acid-treated, heat-inactivated (1 hour, 56°C) FBS from which plasminogen had been depleted (ATFBS-P).% After the various additions, supernatants were collected at the indicated times and stored frozen until required for measurement of PA activity.

Resident Peritoneal Macrophages Resident peritoneal cells were obtained from the peritoneal

cavity as described” and plated at -1.5 x lo5 cells per well in ‘“I-fibrin-coated 96-well tissue culture plates (Linbro; Flow Labo- ratories) in RPMI-1640/15% FBS.” The cultures were incubated overnight to allow the macrophages to adhere. Before use, the cultures were washed twice with PD to remove nonadherent cells and incubated in RPMI/5% ATFBS with additions to a final volume of 200 ILL.

PA Activity PA activity released into the BMM culture medium was assayed

essentially as described earlier.= Briefly, plasminogen-dependent fibrinolysis was measured as the release of soluble ’%fibrin degradation products from 1Z51-fibrin-coated wells of 96-well plates. PA activity in the supematants was expressed relative to the fibrinolytic activity of urokinase standards (0.06 to 2.0 Ploug U/well). Plasminogen-independent fibrinolysis was always I 10% of plasminogen-dependent fibrinolytic activity.

For resident peritoneal macrophages cultured directly on ’=I- fibrin-coated wells, PA activity was expressed as release of soluble ’“I-fibrin degradation products directly into culture medium relative to the total radioactivity released by 0.25% (wthrol) trypsin (ie, % fibrinolysis),29 the fibrinolytic activity from these cells has been shown to be plasminogen-dependent by depletion of the plasminogen from the ATFBS used in the cultures during the assay period.”

DNA Synthesis DNA synthesis was measured as the incorporation of ’H-

thymidine as described before? Quiescent BMM, incubated in 12-well plates (Linbro) with the various treatments for 20 hours, were then pulsed with ’H-thymidine (2.5 pCi/mL) for a further 2 hours. Uptake was stopped, the cells solubilized in 0.2 mol/L NaOH, and the incorporation of label into trichloroacetic acid- precipitable material measured?

u-PA mRNA Expression Isolation of total RNA. Quiescent BMM in 6-well plates (Cos-

tar, Cambridge, MA) were treated with the respective agents as indicated and the total RNA was isolated as described previously.” Each sample represented the pool of the total RNA from three wells treated identically, 2 to 3 X lo6 cells total.

Northem analysis was performed as described previously.” Samples of RNA were analyzed by formalde- hyde agarose gel electrophoresis. RNA was transfered to Gene- screen Plus (DuPont, Boston, MA) according to the manufacturer’s instructions and hybridized to DNA probes radiolabeled by ran- dom primingM The murine urokinase probe was a 0.65-kb PstI- Hind111 fragment of the cDNA plasmid pDB4501 kindly provided by D. Belin and J.D. Vassalli.)’ The filters were washed and then exposed to Kodak XARS film (Eastman Kodak, Rochester, NY). The amount of RNA applied to each lane was monitored with a ”P-labeled cDNA to murine &-microglobulin (the 0.3-kb PstI fragment from the murine P,-microglobulin gene).

Northem analysis.

Statistical Analysis of Data Where indicated, statistical significance of measurements was

determined by a two-tailed, unpaired Student’s t-test. In all cases the results represent the average of triplicate determinations (2SEM).

Reagents For the establishment of BMM cultures, serum-

containing CM from mouse L60T L cells (L-cell CM) was used as a source of CSF-1. This medium was essentially as described.z6 Undiluted L-cell CM typically contained 8,000 to 12,000 U/mL CSF-1 (see below).

Purified recombinant human CSF-1 (rhCSF-1), which is biologically cross-reactive in the murine system, was used.’ This material was a gift of the Department of Process and Product Development, Cetus Corporation (Emeryville, CA), and the stock solution at 5 x lo6 U/mL (see below) contained less than 0.015 ng/mL of endotoxin. In some experiments another source of CSF-1 was used that was derived and purified from mouse placental CM by affinity chromatography using antibody as described.)2 This was kindly provided by T.R. Bradley and E.R. Stanley (Department of Developmental Biology and Cancer, Albert Einstein College of Medicine, Bronx, NY). Also, in some experiments, concentrated serum-free L-cell CM, subjected to calcium phosphate gel absorp- tion (stage 2 L-cell CSF-l), was used as a source of CSF-1.’’

Bacterially synthesized recombinant murine GM- CSF (rGM-CSF), purified to homogeneity: was provided by A. Burgess (Ludwig Institute for Cancer Research, Parkville, Austra- lia).

Two sources of murine IL-3 were used, being either recombinant or native material. The recombinant material (rIL-3) used was partially purified material produced by J. Delamarter (Biogen SA, Geneva, Switzerland) and further purified to homogeneity by N. Nicola (Walter and Eliza Hall Institute) as described before? The native material (nIL-3) derived from the clonal T-cell line LB-3 was purified to homogeneity as presented previ~usly.~

Murine G-CSF was purified to homogeneity from lung conditioned medium by N. Nicola.”

CSF bioactivity was measured using C57B1/6 bone marrow cells in semisolid agar medium assigning 50 U/mL to the concentration giving half-maximal colony formation.35

The following reagents were obtained commercially: A23187, amiloride, 8-bromoadenosine 3’:5’-cyclic monophosphate, concanavalin A (con A) (cat. no. C5275), dexamethasone,

L-cell CM.

CSF-1.

GM-CSF.

ZL-3.

G-CSF.

CSF biouctiviv.

Other reagents.




618 HAMILTON ET AL

fibrinogen, formyl-methyl-leucyl-phenyalanine (FMLP), 3-isobutyl- 1-methyl-xanthine (IBMX), monensin, ouabain, prostaglandin E, (PGE,), phospholipase C purified from C-perfringens (cat. no. P4039), and 12-0-tetradecanoyl phorbol-13-acetate (PMA) (Sigma, St Louis, MO); cholera toxin (CT) and ionomycin (Calbiochem, La Jolla, CA); lipopolysaccharide (LPS) from Escherichia coli 01 11: B4, purified by the Westphal method (Difco, Detroit, MI); pertussis toxin (PT) (List, Campbell, CA); 1-oleoyl-2-acetylglycerol (Serdary, Ontario, Canada); RPMI-1640 and FBS (Common- wealth Serum Laboratories, Parkville, Australia); [methyl-’HI thymidine (70 to 85 Ci/mmol) and [a-”PI dATP (3,000 Ci/mmol) (Amersham, Australia). 5-N,N-dimethylamiloride was provided by E.J. Cragoe, Jr (Nacogdoches, TX). All other reagents were of analytical grade. All practical precautions for minimizing endotoxin contamination were taken. Solutions were made in pyrogen- free water (Delta West, Bentley, Western Australia) and endotoxin levels were routinely monitored by limulus lysate tests (C.S.L., Parkville, Australia) with the minimum detectable level being 0.1 nglmL.

RESULTS

Cytokine Stimulation of BMM PA Activity

Various uncharacterized lymphokine preparations stimulate the PA activity of murine macrophages, but the identity(ies) of the stimulating activity(ies) has not been established.22-25 It has been found previously that highly purified L-cell-derived CSF-1 and murine GM-CSF from endotoxin-lung CM stimulated the PA activity of murine resident peritoneal macrophages and starch-elicited peritoneal macrophages.18 Using BMM this time and with recombinant materials, we show that the lymphokines, GM-CSF, and IL-3 (Fig l) , but not G-CSF (Table l), stimulate the PA activity. G-CSF showed no effect even if the cultures were continued for 72 hours (not shown). The data using purified CSF-1 are included for comparison. Thus, GM-CSF and/or IL-3 could have been contributing to the uncharacterized lymphokine activities mentioned above in prior studies with different macrophage populations?2-z

50 2 < 40 - 1 .- 5 M 30

a x

- - 20 1 .* ... 2 10

2 n none 5 2.5 1 .5 .1 100 10 1 .1 10 1 .1 .01

CSF-1 GM-CSF IL-3

CSF (U/ml x 10

Fig 1. Stimulation of BMM PA activity by CSF-1, GM-CSF, and IL-3. Quiescent BMM, derived from C3H/HeJ mice, were treated with the indicated doses of murine placental CSF-1, rGM-CSF, or nlL-3. PA activity released into the culture medium was measured after 24 hours as described in Materials and Methods. Each point represents the mean of triplicate cultures (?SEMI.

Table 1. Lack of Effect of G-CSF on BMM PA Activity

PAActivity Additions (Ploug Wculture)

None 11.5 (1.2) CSF-1 (2,500 U/mL) 80.7 (8.6) nG-CSF (10,000 U/mL) 14.3 (1.3) nG-CSF (2,000 U/mL) 13.1 (0.5)

Quiescent C3H/HeJ BMM were treated with the indicated doses of G-CSF, CSF-1 (stage 2, L-cell CSF-1). or medium alone. PA activity released into the culture medium was measured after 24 hours as described in Materials and Methods. The values represent the mean of triplicate cultures (+SEM).

It has been shown previously that murine peritoneal macrophages express u-PA activity and mRNA in response to various s t i m ~ l i . ~ ’ , ~ ~ The molecular weight of the PA activity by sodium dodecyl sulfate-zymography’7 in CSF-1- stimulated BMM culture supernatants was 48 Kd, a value obtained previously for murine macrophage u-PA” (data not shown). Northern analysis showed a transient induction of u-PA mRNA by CSF-1 with stimulation seen within 1 hour, reaching maximal levels between 4 and 8 hours with a decline to lower but still elevated levels at 24 hours (Fig 2A). Comparison of the CSF-1 dose responses for the stimulation of BMM PA activity (Fig 1) and U-PA mRNA induction (Fig 2B) shows a close correlation between both parameters. The RNA synthesis inhibitor, actinomycin D, inhibited the CSF-1-stimulated increase in PA activity (Table 2). Thus, CSF-1 would appear to be elevating u-PA activity in BMM by raising its mRNA levels possibly by increasing gene transcription. GM-CSF and IL-3 also increased u-PA mRNA levels in BMM, with these levels reflecting the degree of stimulation of PA activity by these agents (Fig 3). The phorbol ester, PMA, also dramatically increased u-PA mRNA levels in BMM. This is consistent with this agent also stimulating BMM PA activity (see below). The stimulation of BMM PA activity by GM-CSF, IL-3, and PMA was also inhibited by actinomycin D (data not shown), indicating that RNA synthesis is also required for stimulation of PA activity by these agents.

Con A Stimulation of PA Activity

The potent macrophage activating agent con A stimulated BMM PA activity. When measured at 24 hours in a representative experiment, 10 pg/mL con A increased the basal level of PA activity in CBA BMM from an average (-cSEM) of 2.0 (0.4) to 21.5 (1.0) Ploug U/culture, which was comparable with the value of 23.5 (1.1) Ploug U/culture obtained in the presence of 2,500 U/mL rhCSF-1. Just as for the other agents, the stimulation of PA activity by con A was inhibited by actinomycin D (data not shown). Consis- tent with a requirement for RNA synthesis is the observation that con A also elevated the u-PA mRNA levels in BMM (Fig 3). However, at 3 hours the degree of stimulation of u-PA mRNA was somewhat lower than either GM-CSF (Fig 3) or CSF-1 (data not shown). We have found that the elevation of u-PA mRNA by con A is prolonged, showing greater relative stimulation at later





0 1 2 4 8 13 24 (h) A Table 2. Effect of Actinomycin D on CSF-l-Stimulated BMM PA Activitv

Additions PA Activity

(Ploug U/culture)

- Actinomycin D + Actinomycin D None 5.6 (0.5) 1.3 (0.1) CSF-1 (2,500 U h L ) 53.2 (6.4) 8.0 (0.3)

Quiescent CBA BMM in 12-weil dishes were pretreated with or without 2 KglmL actinomycin D as indicated before the addition of recombinant murine CSF-1 or medium alone. PA activity released into the culture medium was measured after 8 hours as described in Materials and Methods. The values represent the mean of triplicate cultures (kSEM).

- uPA

time Doints (ie, 24 hours) compared with the CSFs (data not

[CSF-11 (U/ml) B

and other agents are unlikely to be caused by endotoxin contamination because LPS (100 ng/mL) did not stimulate the PA activity in either the CBA- or C3HmeJ-derived

1 2 3 4 5

Fig 2. Stimulation of EMM u-PA mRNA levels by CSF-1. (A) Kinetics: Northem analysis of u-PA and p, microglobulin mRNA In quiescent C3H/HJ BMM. Total RNA was extracted (Materials end Methods) at the indicated times after the addkion of 20% (vollvol) L-cell CM as a source of CSF-1. The data are from the some Northern blot probed successively for U-PA and p, microglobulin mRNA. (E) CSF-1 dose response: Northern analysis of U-PA and p, microglobulin mRNA in quiescent C3H/HeJ BMM treated with the indicated doses of rhCSF-1 for 3 hours before the RNA extraction (Materials and Meth- ods). The data are from the same Northern blot probed successively for u-PA and p, microglobulin mRNA.

Fig 3. Effects of OM-CSF, IL-3, PMA, and con A on BMM U-PA mRNA levels. Northern analysis of u-PA and p, microglobulin mRNA in quiescent CEA BMM treated with the appropriate agent for 3 hours before RNA extraction (Materials and Methods). The treatments are: lane 1, medium alone; lane 2, rGM-CSF (10.000 U/mL); lane 3, rlL-3 (10.000 U/mL); lane 4, PMA (lo-’ mol/L); lane 5, con A (10 pg/mL). The data are from the same Northem blot probed successively for U-PA and p, microglobulin mRNA.




620

80 -

60 -

HAMILTON ET AL

0 alone

+PGE

+CT

+Dex

BMM (data not shown), or resident peritoneal macrophages (see Fig 8).

Cyclic Adenosine Monophosphate (CAMP) and BMM PA Activiv

The biochemical pathways controlling the modulation of macrophage PA activity are not well understood. PGE and CT (Fig 4A), which have been shown to raise intracellular CAMP levels ([CAMP]~) in BMM’ and in other cells,x reduced both the basal and CSF-1-stimulated BMM PA activity. The results shown are for a 24-hour incubation, although the effect was observed as early as 3 hours. Similarly, the permeable CAMP analogue, 8-bromoadenosine 3’:5’-cyclic monophosphate (8BrcAMP) (1 mmolb), and the CAMP phosphodiesterase inhibitor, IBMX (1 mmolb), also completely abolished any detectable PA

401

0 625 1250 2500

CSF-1 (U/ml)

2 3 4 5 6 7 8

activity in the presence or absence of CSF-1 when measured at 7 to 8 hours (data not shown). The synthetic glucocorticoid, dexamethasone, has also been shown to inhibit u-PA induction in other cell type^?^.^ In Fig 4A it can be seen that increasing concentrations of CSF-1 were not able to override the inhibitory effects of PGE, mol/L), CT (lo-” mol/L), or of dexamethasone (lo-’ mol/L) for stimulation of PA activity. In Fig 4B we show that PG4, 8BrcAMP, and CT inhibit the CSF-1-stimulated increase in u-PA mRNA levels. These agents alone have no effect on u-PA mRNA levels. Dexamethasone also inhibited CSF-1- stimulated u-PA mRNA levels (Fig 4C). Thus, modulation of u-PA mRNA induction is likely to be involved in the inhibition of CSF-1-stimulated BMM PA activity by CAMP and dexamethasone.

The elevation of [CAMP], has also been shown to corre-

e A

- 5000

B

4 uPA Fig 4. Inhibition of CSF-latimulated BMM PA

activity, U-PA mRNA Induction, and DNA synthesis by PGE,, CT, and dexamethasone. (A) Effect of increasing doses of CSF-1 on inhibition of PA activity. Quiescent CBA BMM in 12-well plates were treated with increasing doses of rhCSF-1 in the presence or absence of PGE, (lo-’ mol/L), CT (lo-” mol/L), or dexamethasone (lo-’ mol/L). PA activity released into the culture medium was measured after 24 hours as described in Materials and Methods. (E) Effects of PGE,, 8BrcAMP. and CT on basal and CSF-l-stimulated BMM u-PA mRNA levels. Northern analysis of U-PA and p, microglobulin mRNA in quiescent CBA BMM. Total RNA was extracted (Materials and Meth- ods) at 3 hours from cells treated as follows: lane 1, medium alone; lane 2, PGE, (10.’ mol/L); lane 3, 8BrcAMP (1 mmol/L); lane 4, CT (lo-” mol/L); lane 5, rhCSF-1 (2,500 U/mL); lane 6, CSF-1 and PG+; lane 7, CSF-1 and 8BrcAMP; lane 8, CSF-1 and CT. The cells were pretreated for 5 minutes with PGE, and 8BrcAMP and for 2 hours with CT before CSF-1 addition. The data are from the same Northern blot probed successively for U-PA and p, microglobulin mRNA.




MACROPHAGE PLASMINOGEN ACTIVATOR REGULATION 62 1

1 4 3 2

0 c9

C

0 alone

+PGE

+CT

+Dex

0 625 1250 2500

CSF-1 (U/ml)

late with inhibition of CSF-l-~timulated~ and also GM- CSF- and IL-3-stimulated7 BMM DNA synthesis. Fig 4D shows that the inhibitory effect of PGE,, and to a lesser extent CT, were overcome at high CSF-1 concentrations.' Similarly, inhibition by dexamethasone was also overcome at these CSF-1 doses. Thus, we can have experimental conditions where there is no PA expression yet DNA synthesis occurs, such as at high CSF-1 doses in the presence PGE, or dexamethasone (Figs 4A and D). It has been suggested for other cell types that increased PA activity correlates with the subsequent expression of DNA synthesis." In Table 3 it can be observed that CT (lo-'' molk) can

also prevent the stimulation of BMM PA activity by the other active hematopoietic growth factors, GM-CSF and IL-3.

G Proteins and BMM PA Activity

Receptors for many hormones interact with membrane- bound guanine nucleotide-binding proteins (G-proteins), which can transduce signals leading to the generation of so-called second messengers:' Certain G-proteins are sensitive to the effects of bacterial toxins such as PT.4' In Fig 5 it can be seen that 1 hour of pretreatment with PT (20 ng/mL) had no significant effect (P > .05) on the stimulation of BMM PA activity by the CSFs. Similar results were obtained with doses of PT up to 100 ng/mL and with pretreatment periods up to 5 hours (data not shown). This PT preparation was known to be biologically active because it ADP-ribosylated a 41-Kd protein in isolated rat islet cell membranes (M. Dunlop, personal communication). Similar results for BMM PA activitywere obtained using several PT preparations obtained commercially. These PT preparations also had a pronounced morphologic effect on the BMM inducing a rounded, contracted morphology. The PT also had other effects on the cells including a partial inhibition of the basal levels of Na+, K'-ATPase activity, glucose uptake, and pinocytosis (data not shown).

D

5000

Fig 4. (Cont'd). (C) Effect of dexamethasone on CSF-l-stimulated BMM u-PA mRNA levels. Northern analysis pedonned as for (E); however, cells were pretreated with or without lo-' mol/L dexamethasone for 30 minutes before the addition of 2,500 U/mL rhCSF-1. RNA was extracted (Materials and Methods) after 4 hours of further treatment. (D) Effects of increasing doses of CSF-1 on inhibition of DNA synthesis. BMM treated as described for (A) except that DNA synthesis was measured (Materials and Methods) at 20 hours. For (A) and (D) each point represents the mean of triplicate cultures (rSEM).




622 HAMILTON ET AL

$ 80-

-2 60-

\

M

40- b '5 5 20-

2 0-

+PT

0 control

T

none CSF-1 GM-CSF

CSF (U/ml) IL-3

Flg 5. Effect of PT on CSFstimulated BMM PA amity. Quiescent C3H/Hd BMM were pretreated with or without PT (20 ng/mL) for 1 hour before the addition of murine placental CSF-1 (2,500 U/mL), rOM-CSF ( W UlmL), rlL-3 (10' U/mL), or medium alone. PA activity released into the medium was measured after 24 hours (Materials and Methods). Each point represents the average of triplicate cultures (&EM).

Na+,K+-ATPase, Na+ Fluxes, and BMM PA Activity Stimulation of Na+/H+ exchange by growth factors has

often been considered to have an important role as an early signal in the proliferative response. We have evidence, including that based on the use of inhibitors, for the involvement of the Na+/H+ antiport and Na+,K+-ATPase activity (Na+ pump) in the proliferative response of BMM to CSF-1.6,42 However, we found that ouabain (1 "om), a specific inhibitor of Na+,K+ATPase activity,% had no significant effect on the induction of PA activity by CSF-1. In a representative experiment, 2,500 U/mL of rhCSF-1 increased the PA activity in C3WHeJ BMM from an average (2SEM) of 1.2 (0.2) to 12.1 (0.4) Ploug U/culture during 8 hours of culture. In the presence of 1 mmol/L of ouabain these values were 1.2 (0.4) and 11.0 (0.7) Ploug U/culture, respectively. Unfortunately, we were not able to test the action of inhibitors of the Na+/H+ antiport activity, amiloride (1 mmol/L), and the more potent and specific analogue 5-N,N-dimethylamiloride (DMA) (0.1 mmol/L), because of their direct effects on u-PA proteolytic activity:' Therefore,

Table 3. Effect of CT on CSF-Stimulated BMM PA Activity

PA Activity Additions (Ploug U/culture)

- CT +cT None 0 (1.0) 0.5 (1.0) CSF-1 (5,000 U/mL) 45.5 (3.9) 3.0 (2.0) rGM-CSF (lOsU/mL) 40.2 (4.2) 0 (1.0) nlL-3 (lO'U/mL) 7.8 (0.4) 2.0 ( 1 )

Quiescent C3HMeJ BMM were pretreated with or without CT (lo-" mol/L) as indicated for 1 hour before the addition of murine placental CSF-1, rGM-CSF, nil-3, or medium alone. PA activity released into the culture medium was measured after 24 hours as described in Materials and Methods. The values represent the mean of triplicate cultures (SEM).

we examined the effect of inhibitors of '"a+ cycle" ion transport on CSF-l-stimulated u-PA mRNA induction (Fig 6). Ouabain (1 mmol/L) did not inhibit CSF-l-stimulated u-PA mRNA induction. DMA (0.1 mmol/L) or Na' depletion also did not inhibit the increase in u-PA mRNA levels stimulated by CSF-1. These results suggest that Na+,K+- ATPase activity and/or Na+/H + exchange activity are not necessary for CSF-l-stimulated u-PA mRNA induction. Suppression of u-PA mRNA induction by amiloride (1 mmol/L) is unlikely to be related to Na+/H+ exchange inhibition because the more potent and specific inhibitor, DMA, did not have this effect. Amiloride is known to have a variety of nonspecific side effects6"' and its inhibition of u-PA mRNA levels most likely reflects this. This result highlights the danger of relating the effects of amiloride to its action as an Na+/H+ exchange inhibitor.'' We have published previously that CSF-1 rapidly stimulates BMM Na+/H+ antiport activity with the accompanying Na' influx resulting in activation of the Na+,K+ATPase activity (ie, CSF-1 stimulates an '"a+ cy~le")."~' However, attempts to determine the importance of extracellular Na+ levels for stimulation of BMM PA by CSF-1 were hindered by the cytotoxic nature of prolonged exposure (4 hours) to Na+- depleted ([Na'] 4 5 mmol/L) conditions. Under these

1 2 3 4 5 6

9'

r.

flg 6. Effects of "Na-cycie" ion transport inhibitors on CSF-1- stimulated BMM u-PA mRNA levels. Northern analysis of u-PA and 8, microglobulin mRNA in quiescent C3H/Hd BMM. Cells In HEPEW HCO; buffered medium were pretreated as described for 5 minutes before the addition of rhCSF-1 (2,500 U/mL), and the RNA was extracted 3 hours later (Materials and Methods). The treatments are: lane 1, medium alone; lane 2, CSF-1 alone; lane 3, CSF-1 and ouabain (1 mmol/L); lane 4, CSF-1 and amiloride (1 mmol/L); lane 5, CSF-1 in Na+-free buffer; lane 6, CSF-1 and DMA (0.1 mmol/L). Incubation of the cells in Na+-free buffer (using choline chloride as an osmotic replacement) was performed as described previously.u The data are from the same Northern blot probed successively for u-PA and p2 microglobulin mRNA.




MACROPHAGE PLASMINOGEN ACTIVATOR REGULATIUN 623

conditions cellular protein synthesis was inhibited by 2 80%; however, there still remained a small degree of stimulation of PA activity by CSF-1 (approximately twofold) measured at 4 hours (data not shown). Under similar conditions there appeared to be little effect on CSF-1-stimulated u-PA mRNA levels (Fig 6). The Na’/H’ exchange ionophore, monensin, results in cytoplasmic alkalinization” as well as elevating intracellular [Na’] ([Na’Ii), which in turn activates Na’,K’-ATPase acti~ity.6.~’ Monensin was not able to increase PA activity, indicating that increased pHi, elevation of [Na’], or stimulation of Na+,K+-ATPase activity alone was not sufficient to stimulate BMM PA activity (data not shown).

Protein Kinase C, Phospholipase C, Ca”, and BMM PA Activity

Other possible biochemical pathways controlling BMM PA activity were explored. Diacylglycerols and the phorbol ester, PMA, are generally considered to activate protein kinase C, leading to a number of cellular effects:’ In Table 4 it can be seen that the synthetic diacylglycerol, l-oleoyl-2- acetylglycerol (OAG), and PMA were potent at increasing BMM PA activity. Stimulation of BMM PA activity by PMA is consistent with the result in Fig 3 that shows increased u-PA mRNA levels in response to PMA. 1,2-Diacylglycer- 01s can arise intracellularly from phospholipase-mediated phospholipid hydrolysis.& We show that treatment of BMM with highly purified, nonspecific phospholipase C stimulated BMM u-PA mRNA levels (Fig 7) and PA activity (Table 4).

An increase in [Ca’’], either by Ca2’ influx or release from intracellular compartments, has been implicated as having a second messenger role in a number of cell An increased Ca2+ influx by itself would not seem to be important for BMM PA production because the Ca’’ ionophores, A23187 and ionomycin, had no significant effect; the chemotactic peptide, FMLP, which can cause Ca” release from intracellular stores via activation of the phosphatidyl inositol cycle in BMM,”was also inactive. In a representative experiment using C3H/HeJ BMM, a 24 hour treatment with A23187 (1 pnol/L), ionomycin (1 pmol/L), or FMLP (40 Fmol/L) gave an average (+SEM) of 9.4 (2.4), 27.6 (4.5), and 17.9 (4.2) Ploug U/culture, respectively.

Tabla 4. Effect of Protein Kinase C Activation and Phospholipase C Addfiion on BMM PA Activity

Additions PA Activiw

(Ploug Ukulture)

None CSF-1 (2,500 UImL) PMA (10.’ molIL) OAG (100 pg/mL) PL-C (1 UImL)

22.6 (3.2) 90.2 (7.3) 62.1 (6.1) 77.9 (10.0) 41.6 (1.4)

These values were not significantly different (P > .OS) to the basal PA activity of 15.2 (0.6). CSF-1 (2,500 U/ml; murine placental) increased the PA activity to 87.2 (1.5) Ploug U/culture in this experiment. These agents were also without effect on BMM u-PA mRNA induction (not shown).

Cytokine Stimulation of Resident Peritoneal Macrophage PA Activity

As mentioned, purified L-cell CSF-1 and murine GM- CSF from endotoxin-lung CM stimulated the PA activity of murine resident peritoneal macrophages when the cells were cultured on a fibrin substrate.lR We now show that purified rGM-CSF can enhance the PA activity of this type of macrophage (Fig 8). The effects of PMA, con A, and LPS are included for comparison. Purified rIL-3 (2 80 U/mL) was also stimulatoIy while purified G-CSF (I 1,000 U/mL)

Quiescent C3HIHeJ BMM were treated with the indicated concentrations of murine placental CSF-1, TPA, OAG, PL-C, or medium alone. PA activity released into the culture medium was measured after 24 hours as described in Materials and Methods. The PL-C preparation had no detectable protease activity as measured by fibrinolysis. Each value represents the mean of triplicate cultures (+SEM).

Fig 7. Effect of phospholipase C addition on BMM U-PA mRNA levels. Northern analysis of U-PA and p2 microglobulin mRNA in quiescent C3HIHe.l BMM treated as indicated with or without rhCSF-1 (2,500 U/mL) or PL-C (5 U/mL) for 3 hours before RNA extraction (Materials and Methods). The data are from the same Northern blot probed successively for u-PA and p2 microglobulin mRNA.




624 HAMILTON ET AL

25 1 T

none 5 2.5 1 .5 100 10 1 .I CSF-1 GM-CSF TPA conA LPS

(U/ml x (U/ml x I O -3)

Fig 8. Stimulation of resident peritoneal macrophage PA activity. Resident peritoneal cells were obtained from CBA mice and cultured on '"I-fibrin-coated S w e l l plates as described in Materials and Methods. The cells were cultured overnight to allow adherence of the macrophages and the nonadherent cells were removed by two washes with PD. The adherent cells were cultured in RPMI/5% ATHIFBS, with or without the indicated doses of murine placental CSF-1, rGM-CSF, TPA (lo-' mol/L), con A (10 pg/mL), LPS (100 ng/mL), or medium alone. Plasminogen- dependent fibrinolysis was measured as release of radioactivity into the medium after 24 hours- and expressed relative to total radioactivity released by trypsin. Each point represents the mean of triplicate cultures ( S E M I .

was not (data not shown). Thus, this poorly cycling macrophage population: as far as PA activity is concerned, can respond to the same agents as the BMM. With these cells, PA activity is not detected in the supernatants most likely because of the presence of PA inhibitor-2.a"9

DISCUSSION

The-above findings show that PA activity (ie, u-PA activity) joins a list of responses that the different CSFs (CSF-1, GM-CSF, and IL-3) are all able to elicit in BMM, and at similar doses, for the stimulation of other parameters, which include Na'/H' exchange and/or Na+,K+- ATPase a~tivity,"~.~' glucose uptake: c-myc and c-fos proto- oncogene expression,"*'2 pinocytosisM (Knight KR, Vairo G, Hamilton JA: submitted for publication), and DNA ~ynthesis?~' The lack of effect of G-CSF for BMM PA activity is consistent with its lack of effect on these other responses.M*'2 This result most likely reflects the lack of functional G-CSF receptors for this cell type. The actions of CSF-1, GM-CSF, and IL-3, even though they are mediated via different receptors, can therefore converge on regulat- ing u-PA activity and other BMM responses. The receptor for CSF-1 is the tyrosine kinase product of the c-fms proto-oncogene,8 while the receptors for GM-CSF and IL-3 do not appear to have tyrosine kinase activity. However, in FDC-PI cells, IL-3 can induce the appearance of several phosphotyrosine-containing proteins, suggesting that a functional tyrosine kinase may be associated with this receptor? Given the results above showing for the first time that rCSFs can increase u-PA mRNA levels (Fig 3) and given the inhibitory effects of actinomycin D (Table 2 and text), it is likely that the regulation of u-PA activity by the CSFs can occur at the level of gene expression.

Several possible signaling cascades were examined above to explore in more detail how the CSFs might be acting and

how the u-PA activity might be regulated in macrophages. We and others have found previously no evidence for the phospholipase C-mediated hydrolysis of phosphatidylinositol-4, 5-bisphosphate (PIP,) in BMM treated with CSF-1, GM-CSF, or IL-3."." Therefore, this pathway, leading to the formation of 1,2 diacylglycerol and inositol phosphates, would not seem to be involved in the action of these CSFs including, of course, the regulation of BMM PA activity. However, the stimulatory action of nonspecific phospholipase C (Table 4), which presumably can increase levels of intracellular diacylglycerol, suggests that this product(s) of phospholipid breakdown may be important to the modulation of u-PA activity and other responses in macrophages. Although treatment of BMM with nonspecific phospholipase C does not stimulate PIP, hydrolysis," such treatment has been found to stimulate BMM DNA synthesis and c-fos and c-myc expression.lz The stimulatory action of exoge- nous OAG (and PMA, see below) for macrophage PA activivs1 (Table 4) and other responses45 also supports this proposal. It could be that the CSFs studied here elevate the levels of diacylglycerol in macrophages (as has recently been found in human monocytessz) and this possibility is currently being explored. It is also possible that intracellular diacyglycerol may be involved in the control of u-PA activity in other cell types.

Diacylglycerol is the endogenous second messenger associated with activation of protein kinase C.45 The phorbol ester is a relatively weak mitogen for BMM but can synergize quite dramatically with a low concentration of CSF-1, GM-CSF, and IL-3 to potentiate their mitogenic action: The results reported here show that PA activity is another response in BMM that PMA shares with the different CSFs, the other responses including enhanced "Na' cycle" activity: glucose uptake: proto-oncogene expression,'2 pinocytosisSo (Knight KR, Vairo G, Hamilton





JA: submitted for publication) and, as mentioned, DNA synthesis? In other words, protein kinase C activation and whatever pathways are used by the CSFs can both stimulate u-PA expression, presumably at the gene level.

The data above suggest that CSF-mediated stimulation of BMM u-PA mRNA levels and/or activity does not involve PT-sensitive G proteins (Fig 5 ) nor activation of Na+/H+ exchange or Na',Kt ATPase activity (Fig 6) to any significant extent. In contrast, PT-sensitive G proteins have been implicated in the modulation of human monocyte Na+,K+-ATPase activity and DNA synthesis by CSF-1:3 although in murine BMM, PT had only a minor effect on CSF-1-stimulated cell g r o ~ t h . 5 ~ Also, Na' and Cazt influxes by themselves do not seem to be capable of stimulating BMM u-PA activity (see Results). Ca2+ has been proposed as an important mediator of macrophage a~t iva t ion .~~

The inhibition of CSF-stimulated u-PA mRNA levels and activity in BMM by agents that raise [CAMP], in BMM7 and other cell types? such as 8BrcAMP, CT, PGE,, and IBMX, suggests that elevation of [CAMP], is inhibitory to CSF-stimulated u-PA activity. We have found previously, when measuring the inhibition by these agents of CSF- stimulated DNA synthesis in BMM, that the effect was reversible, nontoxic, and without any effect on protein synthesis or other early CSF-1 responses.' u-PA activity is the one early response in BMM to the CSFs so observed thus far that is inhibited by the CAMP-elevating agent^.^ The inhibition by agents that raise [CAMP], would not appear to result from interference with the binding of at least CSF-1, GM-CSF, IL-3, and PMA with their receptors because we have reported that other biochemical changes induced by these agents, such as c-myc expression, Na'/H' exchange, Na',K'-ATPase, and protein synthesis, were not affected by them7 (Vairo G, Hamilton J A unpublished observations); in addition, PGE, has been reported not to inhibit binding of CSF-1 to its receptor on BMM:6 Agents that elevate CAMP, and also dexamethasone, have been shown to lower the PA activity and the basal transcription rate of u-PA gene expression in murine thioglycollate- elicited peritoneal ma~rophage,'~ given the reduction in the mRNA levels, it is likely that the inhibitory effects on u-PA activity are due to such a mechanism in the CSF-stimulated BMM.

It has been proposed that increases in PA activity in response to mitogenic agents may be important for the subsequent onset of DNA synthesis in CSF-1-treated BMM17 and in other cell types.15 The stimulation reported above of the u-PA activity of BMM by the mitogenic agents, CSF-1, GM-CSF, IL-3, PMA, and phospholipase C would appear to support this concept, as would the observation that CAMP-elevating agents and dexamethasone inhibit mitogen-induced u-PA activity and can also inhibit DNA synthesis in BMM. However, there are several observations with macrophages that provide evidence against a relationship between stimulation of PA activity and DNA synthesis: (1) at high CSF-1 doses, PGE, and dexamethasone completely abolish CSF-1-stimulated BMM PA activity with no significant effect on CSF-1-stimulated DNA synthesis (Fig 4A and D); (2) DMA and ouabain both inhibit CSF-1-

stimulated BMM DNA ~ y n t h e s i s ~ . ~ ~ but have little effect on CSF-1-stimulated u-PA mRNA levels and/or u-PA activity (Fig 6); (3) con A stimulates u-PA activity in BMM but is not mi t~genic~ ,~ ; (4) CSF-1 stimulates the PA activity of murine resident peritoneal macrophages (Fig 8), with this cell type showing a poor proliferative response to the growth factor6; (5) untreated thioglycollate-elicited murine peritoneal macrophages express high levels of u-PA activity ( -52% of the cells express PA activiv' yet do not cycle unless treated with a growth stimulus57; (6) for CSF-1- treated BMM, the induction of PA activity is not a signal for DNA synthesis because an additional macromolecular serum component is required for CSF-1-stimulated DNA synthesis but not PA acti~ity. '~

Interestingly, con A, which potently stimulates BMM u-PA activity, has no effect on cfos or c-myc mRNA levels in these cells." This result indicates that, for at least con A as a stimulus, expression of these proto-oncogenes is not a prerequisite for that of u-PA.

Elevated macrophage PA activity has been correlated with the degree of cell-mediated immunity in murine models" and uncharacterized lymphokine-containing samples have been found to stimulate the PA activity of murine peritoneal macrophage populations.22-z5 Interferon-y has been found to increase u-PA gene transcription in murine peritoneal exudate macrophages." The fact that purified recombinant GM-CSF and IL-3 could enhance the PA activities of BMM and resident peritoneal macrophages suggests that one or both of these cytokines could have been responsible for the previous findings with the ill-defined lymphokine preparations It is also possible that CSF-1 may have contributed to the stimulatory activities in these previous reports because this CSF can be produced by monocytes and unfractionated mononuclear cells were usually used for the preparation of the lymphokine-containing samples. Because PA has been implicated in inflammatory reactions, tissue remodeling, and cell migra t i~n , '~ . '~ .~ GM-CSF and IL-3 (as well as CSF-1) may therefore have a role in these processes via their action on macrophages. Such enhanced u-PA production (and therefore plasmin generation) by the macrophages in GM-CSF transgenic mice may contribute to the necrotic lesions of the eye and striated muscle, as well as to the chronic bleeding associated with the unusually large numbers of peritoneal macrophages?' These points have been made elsewhere where it was reported that GM-CSF and IL-3 can stimulate in an analogous manner the u-PA activity of human monocyte^^^^^; thus our observations have a parallel with human cells.

ACKNOWLEDGMENT

We thank A.-M. Bordun, Dr T. Leizer, and S. Argyriou for assistance with the Northern analyses and PA assays; Dr D. Metcalf for CSF bioassays; and M. Luciani for typing the manu- script. The following are acknowledged for samples: Drs E.R. Stanley and T.R. Bradley (placental CSF-1); Dr P. Ralph (rhCSF- 1); Dr A. Burgess (GM-CSF); N. Nicola (IL-3 and G-CSF); and Dr E.J. Cragoe, Jr (DMA).




626 HAMILTON ET AL

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1991 77: 616-627

JA Hamilton, G Vairo, KR Knight and BG Cocks factors and other agentsurokinase-type plasminogen activator activity by colony-stimulatingBiochemical signals controlling the regulation of macrophage Activation and proliferation signals in murine macrophages.

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activation and proliferation signals in murine macrophages

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