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Pages 659-664

1990

CHANGES

IN THE EXPRESSION DIFFERENTIATION

C. J. Ward,

J. Cracker,

OF ELASTASE

OF U937

AND

CATHEPSIN

PROMONOCYTES

S.J. Ghan, RLk

Stockley

B WITH

BY GMCSF and D. Burnett*

The General Hospital, Birmingham, East Birmingham Hospital, Departments of Immunology and Medicine, University of Birmingham, UK, and Howard Hughes Medical Institute, University of Chicago, Chicago, IL Received

January

25,

1990

The human pronionocytic cell line, U937. when treated for up to 72h with 12,0,t.etradecanoyl-phorbol-13-acetate or granulocyte-macrophage colony-stimulating factor, exhibited increased phagocytic activity and expression of the marker p150/95. There was an associated increase in the monocyte proteinase cathepsin B and its mFWA but decreased cellular levels of neutrophil elastase and elastase mRNA. Granulocyte-macrophage colony-stimulating factor therefore causes differentiation of U937 cells, Q1990 with appropriate effects on the synthesis of leukocyte proteinases. Academic

Press.

Inc.

The humnn U937 promonocytic cell line has the potential to differentiate into monocyte-like cells under the influence of several factors including phorbol esters [l], vitamin D3 [2], CAMP and retinoic acid [3]. The U937 cell line has recently been used as a model to study the effects of differentiation on the synthesis of leucocyte proteinases. This cell line has the potential to synthesise neutrophil enzymes, such as elastase and cathepsin G and monocyte proteinases such as collagenase and cathepsin B [ 1,4,5]. Granulocyte-Macrophage Colony-Stimulating Factor (GMCSF) supports the growth and differentiation of granulocytes and monocytes in culture but its effect on the differentiation of U937 cells is disputed [6-91 . The present study was designed to investigate the effects of GMCSF on the synthesis of elastase (E.C. 3.4.21.37) and cathepsin B (E.C. 4.4.22.1) by U937 cells.

l Correspondence to D.Burnett. Clinical Teaching Birmingham B4 6NH, England,U.K. Abbreviations used: GMCSF, Granulocyte-macrophage factor: TPA, 12.0,-tetracecanoyl-phorbol-13-acetate.

Block,

General

Hospital,

colony-stimulating 0006-291X/90

659

All

$1.50

Copyright 0 I990 bv Academic Press, Inc. rights of reproduction in any form reserved.

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AND METHODS

The U937 cell Treatment of U937 cells with Dhorbol ester and GMCSF. line was supplied by Professor R.M. Senior, Jewish Hospital of St. Louis, USA. The cells were maintained in culture in RPM1 1640 medium (Flow Labs’ Ltd., Herts, UK.) containing 10% fetal calf serum, 2mmol/l L-glutamine, non-essential amino acids (Flow Labs) andlmmol/l sodium pyruvate (Sigma Chemical Co., Poole, UK). The cells were seeded into Linbro wells (Flow Labs) at 3 x 105 cells inlml of culture medium with lGnmol/l 12,0,tetradecanoyl-phorbol13-acetate (TPA) or 25 U/ml of recombinant human GMCSF (Genzyme Biochemicals Ltd., Maidstone UK.) for intervals up to 72h. Control wells contained culture medium only. The cells were harvested by scraping with a rubber policeman, counted, and cell lysates prepared for neutrophil elastase and cathepsin B activity or mRNA measurements as described below. Cell preparations were obtained for assay of phagocytosis and expression of p150/95 antigen after 72 h incubation. Measurement of leukocvte elastase activitv. The U937 cells harvested following culture with TPA or GMCSF were lysed with 0.05 mol/l Tris/HCl buffer, pH 8.4 containing 1% Triton X-100 and lmol/l NaCl and the supernatants collected. The intracellular activity of elastase was measured in a microtitre well assay system, as described previously [lo], using the substrate Sue-ala-ala-ala-paranitroanilide (Peptide Institute, Osaka, Japan). Calibration was with elastase purified from empyema pus. Measurement of cathewin B activitv. Cathepsin B activity was assayed in cell lysates as described previously (111, using the substrate N-CBZ-arginylarginine-4-methyl-7-coumarylamide. The assay was calibrated with pure cathepsin B (from Dr.D. Buttle, Strangeways Research. Lab., Cambridge). Measurement of mRNA for elastase and cathewin B. One set of cells from each of the experiments with TPA and GMCSF were harvested for mRNA studies. Total cellular RNA was prepared by the method of Chomczynski and Sacchi [121. Three cDNA probes were used. The neutrophil elastase probe [ 131 supplied by Dr.G.Salvesen, Duke University Medical Centre,USA. was a 5 16bp partial cDNA in a Lambda gt- 11 vector which was subcloned into the Eco-Rl site of a pBluescript KS(+/-) phagemid. The cathepsin B cDNA, coding for preprocathepsin B [ 141 was a 1190 bp EcoRl fragment in pGEM-2. The p-a&in probe [ 151, a 1.9kb cDNA in the BamHl site of puc9, was supplied by Dr.T. Ley, Jewish Hospital of St. Louis, USA. For each dotblot, 0.5 mg of total RNA in 0.1 ml of 6x SSC (1 mol/l NaCl, 0.1 mol/l sodium citrate. pH 7.0) with 7% formaldehyde was applied to Gene-screen TM (NEN Research,Boston,USA) with a dot-blot manifold (BRL. Bethesda, USA). Prehybridisation was by the method of Maniatis et al [16]. Each cDNA probe (lOOr& was radiolabelled with 50 l&i [32P] dCTP by random primed labelling using a commercial kit ( Boehringer Mannheim, Lewes, UK) to give109 dpm/mg DNA. Membranes were hybridised overnight at 43OC with1OOng of prelabelled probes, after which they were washed in accordance with the manufacturer’s protocol. The membranes were autoradiographed and dot-blots scanned with an LKB laser densitometer. The results for elastase and cathepsin B were expressed as ratios to the values obtained for p-actin mRNA. Cytocentrifuge preparations Immunohistochemical staining for D150195. of U937 were stained for p150/95 by the alkaline phosphatase/an&alkaline phosphatase complex method [ 171. The monoclonal anti-p 150/95 and other reagents were obtained from Dako Ltd., High Wycombe, UK. The percentages of cells stained with the anti-p150/95 were counted. Phaeocvtosis assav. U937 were mixed with latex beads (3pm diameter) (2x105 U937 and 7.5 x 10s beads in 0.2 ml RPM1 1640). rotated for 4 h at 660

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37W. after which they were cytocentrifuged, air dried, fixed and stained Diff-Quik (Travenol Labs Ltd., Compton, UK). At least 200 cells were analysed on each slide at 400x magnification. A phagocytosis index was calculated as (% of cells containing beads) x (mean number of beads/cell).

in

RESULTS Effects of TPA and GMCSF on elastase activitv and mRNA. Figure 1 shows the effects of lGnmol/l TPA and 25U/ml GMCSF on elastase levels in U937 cells over 72h in culture. After 24h. TPA caused a significant reduction in elastase levels, from a mean of 213ng/lOs cells (SEM 15.0). No elastase (cl5 ng/lOs cells) was detectable in the TPA-treated cells after 72h in culture. When cultured with GMCSF, no significant change was observed after 24h, but at 48h elastase levels were significantly reduced and continued .to decrease up to 72h in culture. The TPA treatment resulted in virtually undetectable mFWA for elastase after only 18h in culture (Fig.2). The mRNA levels were also reduced after 18h culture with GMCSF, but low levels were still detectable after 72h. Effects of TPA and GMCSF on cathemin B activitv and mRNA. Figure 3 shows the (effects of TPA and GMCSF on intracellular levels of cathepsin B. Cathepsin :B activity was significantly increased from a mean of 15ng/ 106 cells (SEM 1.3) after 24h in culture with TPA. The cathepsin B levels continued to increase with time, to a mean of 297 ng/ 10s cells (SEM 37) after 72 h. Culture of the cells with GMCSF also caused an increase in cathepsin 13, but after 72h the levels were lower (mean77.4 ng/lOs cells, SEM 7.8) than those of cells cultured with TPA. Cathepsin B mFUVA levels increased progressively over 72h in culture with TPA and GMCSF (Fig.4).

E s

1 * p < 0.025

1

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a9 f r .!? w

01

2000

m

40

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(hours)

02

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Figure 1. Intracellular levels of elastase in U937 cells during up to 72h with the TPA or with GMCSF. Results are shown as cultures (Mean + SEM, n=6). Significance of results tested by paired test. No elastase was detectable after 72h culture with

46 (hours)

culture for % of control Wilcoxon’s TPA.

Figure 2. Intracellular cathepsin B activity of U937 cells during culture for up to 72h with TPA or GMCSF. All results (% of control values) were statistically significant (P-z 0.025) by Wilcoxon’s test for paired data.

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Fipure 3. Elastase mRNA levels in LJ937 cells cultured for up to 72h with TPA or GMCSF. Results were calculated as the ratio to mRNA for p-actin and expressed as % of values for control cultures (mean + SEM). Figure 4. Cathepsin B mRNA levels in U937 cells cultured for up to 72h with TPA or GMCSF. Results were calculated as the ratio to mRNA for pactin and expressed as % of values for control cultures.

Phaeocvtic activitv and expression of D150/95. Treatment of U937 cells for 72h with TPA and GMCSF resulted in increased phagocytic activity and expression of p150/95 antigen (Table 1). The effects on both features were significantly greater with TPA than GMCSF.

DISCUSSION The effect of GMCSF on U937 promonocytes has been the subject of controversy. Whereas some studies have shown that GMCSF can cause U937 to differentiate into monocyte-like cells [8,9], others have suggested that it does not, unless these cells are primed first with factors that induce GMCSF receptors [6]. In the present study, we have shown that U937 can be induced, with GMCSF only, to differentiate into monocyte-like cells by the criteria of increased phagocytosis and expression of the monocyte marker p 150/95 [ 181. The differences in the reported responses of U937 to GMCSF might be due to phenotypic changes in different U937 subclones. This might also explain why the basal levels of elastase in our cells were significantly lower than those originally reported in the U937 Table 1. The effects of 72h incubation on phagocytic activity and expression Phaaocvtic Control TPA GMCSF Results

are shown

3.3 76.1 30.4 as mean

of U937 cells with TPA and GMCSF of p150/95 antigen [% positive)

Index (0.9) (13) (5.8)

with

p150/95 0.1% 45.9 % 13.6 %

SEM in brackets.

662

n = 6

exnression (0.1) (7.6) (1.3)

! 80

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from which our cells were derived 151. Elastase is a serine proteinase found in neutrophils although low levels of the protein are also present in monocytes [ 191. The mFUVA for this protein has been demonstrated in promyelocyt& but not in cells after the late neutrophil myelocytic stage or in monocytes 1201. It is not known whether promonocytes in bone marrow are able to synthesise elastase but the ability of U937 cells to produce elastase, with inhibition of synthesis on induction of differentiation with TPA, might reflect the processes occurring during cell maturation in vivo [5]. In thae present study we have shown that, following TPA-induced differentiation of U937 cells, there was virtual disappearance of elastase mFWA by :16h (l/a-life about 1Oh) and intracellular elastase activity was reduced, with a half-life of about 24h. These are similar results to those reported for cathepsin G [221. The inhibition of synthesis of cathepsin G. by TPA, was shown to be due to reduced transcription of the gene. It is likely, therefore, that the inhibition of elastase synthesis in response to differentiatjon with TPA is also due to reduced transcription but further studies will be necessary to confirm this. Cathepsin B is a cysteine proteinase found in monocytes/macrophages and intracellular levels are increased in monocytes and myelomonocytic cell lines matured or differentiated in culture [5,11.21]. The present study demonstrated that the increase in intracellular cathepsin B activity associated. with U937 differentiation was related to increased levels of mRNA. Differentiation of the U937 with GMCSF also resulted in reduced intracellular elastase activity and mRNA but increased cathepsin B. The rate and magnitude of changes due to GMCSF were, however, less than those observed with TPA-induced differentiation and were reflected in the expression of p150/95 antigen and phagocytic activity. This suggests that the rate of differentiation induced by GMCSF is slower than that induced by TPA, or tbat the concentration of GMCSF was sub-optimal. Nevertheless, the results suggest that changes in proteinase synthesis are intimately associated with the differentiation process. Human cell lines are therefore a useful model for investigating the control of synthesis of proteinases and other proteins produced by hematopoietic cells. The corollary is that granulocyte and monocyte proteinases are useful markers for following the differentiation processes of myelomonocytic cells.

ACKNOWLEDGMENTS

This work was supported by the Birmingham the British Lung Foundation and the General 663

Hospitals Endowment Fund, Hospital Bicentenary Fund.

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REFERENCES 1.

2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22.

Welgus,H.G., Connolly,N.L. and Senior,R,M. (1986) J Clin Invest. 77, 1675-1681. Olsson,I., Gullberg,U., Ivhed.1. and NilssonK. (1983) Cancer Res. 43, 5862-5867. Gavison,R., Matzner.Y. and Fibach,E. (1988) Isr J Med Sci 24, 697701. Senior.R.M., Campbel1,E.J.. Iandis.J.A., CoxF.R.. Kuhn,C. and Koven,H.S. (1982) J Clin Invest. 69, 384-393. Senior.R.M., Connolly,N.L., Campbel1.E. J., We1gus.H.G. and Bumett,D. (1987) In Pulmonary Emphysema and Proteolysis: 1986 (Tay1or.J.C. and Mittman,C, Eds.) pp. 219-226. Academic Press, Orlando,FL. Zuckerman,S.H., Surprenant.Y.M. and Tang,J. (1988) Blood. 71, 619624. Park,L.S., Friend,D., Gillis,S. and Urda1,D.L. (1986) J Exp Med 164, 251-262. Miller,L. J., Schwarting.R. and Springer,T. (1986) J Immunol. 137, 289 l-2900. Lindemann,A.. Riedel,D., Oster,W., Merte1mann.R. and Herrmann,F. (1988) Eur J Immunol. 18, 369-374. Bumett,D., Chamba,A., Hil1,S.L. and Stockley,R.A. (1989) Clin Sci. 77, 35-41. Bumett,D.. Cr0cker.J.. Afford,S.C., Bunce,C.M.. Brown,G. and Stock1ey.R.A. (1986) Biochim Biophys Acta 887, 283-290. Chomczynski,P. and Sacchi,N. (1987) Anal Biochem. 162, 156-159. Farley,D.. SaIvesenG. and Travis.J. (1988) Biol Chem Hoppe Seyler. 369 (Supplement), 3-7. Chan.S.J.. Segund0,B.S.. McCormick,M.B. and Steiner,D.F. (1986) Proc NatI Acad Sci USA. 83, 7721-7725. Ponte,P., Ng,S.-Y., Engel,J., Gunning,P. and Kedes,L. (1984) Nucl Acids Res. 12, 1687-1696. Maniatis,T., Fritsch,E.F. and Sambrook,J. (1982) Molecular Cloning. A Laboratory Manual. p. 387. Cold Spring Harbor Laboratory. CordeII,J.L., FaIini,B., Erber,W.N., Gosh,A.K., Abdulazig,Z., MacDonaldS., Putford,K.A., Stein,H. and Mason,D.Y. (1984) J Histochem Cytochem. 32, 219-229. Hogg,N., Takacs,L., PaImer,D.G., SaIvendranY. and AI1en.C. (1986) Eur J Immunol. 16, 240-248. Campbell,E.J., Si1vermanE.K. and Campbel1,M.A. (1989) J Immunol, 143, 2961-2968. F0uret.P.. DuI3ois.R.M.. Bemaudin,J.-F., Takahashi,H., Ferrans,V.J. and CrystaI,R.G. (1989) J Exp Med. 169, 833-846. Mor1and.B. (1985) Stand J Immunol. 22, 9-16. Hanson.R.D., Connolly,N.L., Bumett.D., Campbell,E.J., Senior,R.M. and Ley,T.J. (1989) J Biol Chem In press.

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Changes in the expression of elastase and cathepsin B with differentiation of U937 promonocytes by GMCSF.

The human promonocytic cell line, U937, when treated for up to 72h with 12,O,tetradecanoyl-phorbol-13-acetate or granulocyte-macrophage colony-stimula...
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