Chemically modified low density lipoproteins as inducers of enzyme release from macrophages

Volume

186, number

July 1985

FEBS 2675

2

Chemically modified low density lipoproteins as inducers of enzyme release from macrophages Hans-Peter Hartung, Rudolf G. Kladetzky* and Michael Hennerm Departments of Neurology and *Mehcme.

Umversrty of Dusseldorf. Moorenstr

5. D-4000 Dusseldorf I, FRG

Received 9 April 1985 Macrophages carry receptors on then surface for acetylated low density lipoprotein (ac-LDL) Receptormediated endocytosis of ac-LDL IS followed by intracellular cholesterol accumulation We investigated whether occupation of these binding sites evokes the release of hydrolytic enzymes from mouse peritoneal macrophages cultured for up to 48 h ac-LDL at concentrations rangmg from 25-250 c(g protem/ml was noted to promote m a dose-dependent fashion secretlon of the neutral protemase elastase (EC 3 4 21 37) and the lysosomal acid hydrolases N-acetyl$-glucosammldase (EC 3 2 1 30), j?-glucuromdase (EC 3 2 1 3 I), p-galactoadase (EC 3 2 1 23), a-mannosldase (EC 3 2 1 24) and cathepsm D (EC 3 4 23 5) This stlmulatory effect was non-cytotoxlc LDL modified by treatment with malondlaldehyde was also capable of augmenting enzyme hberatlon mto culture supernates These findmgs may have lmphcatlons for some aspects of the atherosclerotic process Macrophage receptor

1.

Acetylated

LDL

Malondraldehyde-altered

INTRODUCTION

Macrophages of various sources and species have been documented to be endowed with receptors for acetylated LDL [l-4]. Binding of the ligand is followed by receptor-mediated endocytosis. Intracellularly the lipoprotem is subject to lysosomal decomposition. Liberated cholesterol is either exported from the cell or channelled into the cholesterol ester cycle to undergo repetitive reesterificatton and hydrolysis [5,6]. We are interested m an aspect of macrophage metabolism distinct from lipoprotein processing. Macrophages are known to be a rich source of a vartety of hydrolytic enzymes that are released constttutlvely or, for the most part, upon challenge with appropriate agents which include varied pharmacological compounds, microbes, immune comcomponents, lymphocyteplexes, complement derived factors, and phagocytic stimuli [7,8]. These enzymes, when liberated into the pericellular environment, are instrumental in effecting tissue damage and medlatmg inflammatory responses

LDL

Enzyme release

A therosclerosls

[9]. Here we have adduced evidence to indicate that secretion of considerable amounts of lysosomal enzymes is a prominent sequela of macrophage stimulation elicited by chemically modified low density lipoproteins.

2. MATERIALS

AND METHODS

Bovine hemoglobin type II, /?-glycerophosphate, ,&mtrophenyl-N-acetyl-P_glucosaminide, p-nitrophenyl-,&galactostde, p-nitrophenyl-a-mannoside, phenolphthalein-&glucuromde, porcine pancreatic elastase and cycloheximide were purchased from Sigma (Munich), mcotmamide adenine dinucleotide was obtamed from Boehringer (Mannheim), tetramethoxypropane from EGA Chemie, FolinCiocalteau reagent and all other chemicals were from Merck, Darmstadt. Dulbecco’s modified minimum essential medium (DMEM) was from Gibco (Karlsruhe), and supplemented with peniclllit-r/streptomycin (100 U/ml). LDL (density 1.019-1.063 g/ml) was obtained

Published by dlsevler Science Pubbshers B V (Blomedrcal Dwrsron) 00145793/85/$3 30 0 1985 Federation of European Biochemical Socletles

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from human plasma of normolipidemic volunteers and prepared by differential ultracentrifugation in a Beckman L8-70 ultracentrifuge [lo]. LDL was acetylated by repeat additions of acetic anhydride as described [l l] and modified by treatment with malondialdehyde according to [ 121. After chemical modification, dialysis was performed against 0.15 M NaCl and 0.3 mM EDTA, pH 7.4. Acetylated and malondialdehyde-modified LDL exhibited enhanced mobility on agarose gel electrophoresis in barbital buffer, pH 8.6. Cells were collected from the peritoneal cavity of 6-week-old female NMRI mice (Zentrale Tierversuchsanlage, Universitat Dusseldorf) by rinsing with DMEM containing 5 U/ml heparin (Nordmark, Hamburg), spun down at 400 x g, resuspended at a concentration of 2 x 106/ml, and then seeded into Cluster 24 multi-well culture dishes (Costar, Cambridge, MA). Following an initial 3 h adherence period, non-adherent cells were removed by several washes with medium. Monolayers consisted of 91-95% macrophages as evidenced by staining for nonspecific esterase and latex phagocytosis [13,14]. Cells were then challenged with modified LDL and cultured for up to 48 h. Cell-free supernates and cell lysates prepared by addition of 0.1% Triton X-100 were then assayed for activity of the enzymes elastase [ 151, N-acetyl-,&glucosaminidase, P-glucuronidase, fl-galactosidase, cathepsin D, acid phosphatase (EC 3.1.3.2) and cu-mannosidase [ 161. Cell viability was assessed by determination of lactate dehydrogenase (EC 1.1.1.27) release into supernates [17]. Results were corrected for cell number by correlation with DNA content of monolayers, lo6 macrophages yielding 11 pg DNA 1181. 3. RESULTS Macrophages were exposed to various amounts of ac-LDL ranging from 12.5 to 250,ug/ml. With the notable exception of acid phosphatase, release of all enzymes tested was augmented in response to ac-LDL (table 1) although to a varying extent. A detailed dose-response curve is given in fig. 1. It is apparent that a maximal stimulatory effect on the secretion is achieved by addition of 250 pg/ml acLDL, the EDso approximating lOOpg/ml. The broad spectrum of enzymes liberated from macrophages after exposure to 250 /g/ml ac-LDL 212

July 1985 Table I

Enzyme release from macrophages LDL

challenged with modified

Enzyme

Unit

Intracellular

,&Glucuronidase

mU

2.8 3.0 2.9

f 0.4a -t Osb + 0.4’

0.2 + 1.2 + 0.8 +

0.05 0.2 0.15

rY-Mannosidase

mU

2.1 2.2 2.1

* 0.35 * 0.4 & 0.3

0.1 0.6 0.4

f k +

0.04 0.15 0.1

U

13.8 15.1 13.6

+ 1.8 * 2.1 + 1.5

0.7 4.3 3.3

+ f +

0.1 0.9 0.6

Lactate dehydrogenase

mU

52.5 56.5 53.5

* 3.9 & 4.3 * 3.7

5.0 5.1 4.9

-t + -t

0.8 0.9 0.7

Elastase

mU

39.7 48.3 45.4

+ 6.4 f 8.7 + 7.5

Acid phosphatase

mU

Cathepsin D

0.72 + 0.08 0.87 + 0.1 0.82 + 0.09

Extracellular

157.2 k 28.7 211.5 k 37.9 187.9 + 31.1 0.09 It_ 0.01 0.12 + 0.01 0.11 + 0.01

Macrophages were exposed to medium (” controls), ac-LDL LDL (250 pg/ (250 pg/ml); b or malondialdehyde-modified ml); ’ for a period of 48 h, whereafter cell lysates and supernates were examined for enzyme activities. Results are means + SD of 3 experiments

can be seen in table 1. The requirement for de novo synthesis was demonstrated by experiments in which macrophages were incubated in the simultaneous presence of ac-LDL and the protein synthesis inhibitor cycloheximide (1 pg/ml). Activity in culture supernatants of all enzymes tested decreased to almost zero after addition of cycloheximide (not shown). It should be noted that ac-LDL mediated release of macrophage enzymes was not due to increased leakage subsequent to toxic damage inflicted upon these cells by exposure to the lipoprotein. The non-cytotoxic nature of its stimulatory effect was proven by showing that lactate dehydrogenase activity in culture supernatants was virtually unchanged when compared to control

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Volume 186, number 2

all hydrolases gauged except acid phosphatase (table 1). However, equi-effective doses of this lipoprotein tended to be higher.

N-Acetyl-b Glmsamn&se LO

July 1985

t

4 DISCUSSION

I

I

I

!

50

I

100

I

I

150 mnt

cantml

I

200

250

ac-LDL

(~ghlll

Fig 1. Dose relatronshrp of ac-LDL-evoked enzyme secretion. Release of the lysosomal enzyme N-acetyl-flglucosammrdase mto supernates of adherent macrophages was determined after 48 h of mcubatton wtth indicated amounts of acetylated LDL under serum-free condrttons Results are expressed m % of total enzyme acttvtty (1 e mtra- + extracellular) and represent means f SD of 3 experiments incubates. When kinetic studres were performed sampling supernates every 8 h following challenge of macrophages with ac-LDL, it was found that httle secretion took place during the first 24 h whereafter enzyme release considerably accelerated to plateau off at about 40 h (frg.2). Malondraldehyde-modified LDL, which apparently also binds to the recogmtron site for ac-LDL [6,12], was also effective m uutratmg liberation of

“lM I

I

a

I

I

2L time

after

L8 addltlon

(hl

Fig 2 Kmettcs of enzyme secretton Macrophage cultures were challenged wrth 250pg/ml ac-LDL and Incubated for up to 48 h At timed Intervals supernates were collected and evaluated for acttvrty of the enzyme N-acetyl-,&glucosamnudase. Results are gtven per lo6 cells and represent means of quadruphcate cultures

Following the pioneering work of Goldstem and Brown (review [6]) it has been amply documented that cells of the monocyte-macrophage lineage are an important site of lipoprotein metabolism. Expression on the surface of these cells of so-called scavenger receptors recognizing modified LDL has so far been related only to then function in promoting clearance and intracellular processmg of hpoprotems. Scavenger receptors are considered to be instrumental m the accumulatron of cholesteryl esters m macrophages. Here we have focused our interest on a different aspect of macrophage metabolism and put forth evidence that strmulatton of the scavenger receptor results m the secretion of a number of lysosomal enzymes. Kinetics and requirement for de novo synthesis of these novel actions displayed by both ac-LDL and malondialdehyde-LDL are in accord with results from previous studies examining other inducers of enzyme release from macrophages [7,8,19-211. How occupancy of the bmdmg sttes for modified LDL results m the generation and propagation of the signal to evoke lysosomal enzyme syntheses and hberatron is yet to be elucidated. Very early m the atherosclerotrc process monocyte-macrophages enter the arterial wall at sites of altered endothehal permeability [22] We suggest that our findings, estabhshmg lipoproteins as activators of macrophage enzyme release, may be stgmfrcant to the pathogenests of atherosclerosis. Interaction of these cells with hpoprotems, which leak through the damaged lummal vessel hnmg with ensuing release of tissue and cell degrading hydrolases, might conceivably initiate, accentuate or perpetuate endothehal injury and thereby accelerate development of atherosclerotrc plaque lesions.

ACKNOWLEDGEMENTS This study was supported m part by grants from Deutsche Forschungsgemeinschaft, SFB 200, D2 and Min. f Wissenschaft und Forschung, NRW. 213

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REFERENCES [1] Goldstein, J L., Ho, Y.K , Basu, S K and Brown, M.S (1979) Proc Nat1 Acad. SC] USA 76, 333-337 [2] Brown, M S , Basu, S K , Falk, J R., HO, Y K and Goldstein, J.L (1980) J Supramol. Struct 13, 67-81 [3] Traber, M.G. and Kayden, H J. (1980) Proc. Nat1 Acad Sa USA 77, 5466-5470. [4] Traber, M G , Defendi, V and Kayden, H J (1981) J Exp Med. 154, 1852-1867 [S] Brown, M.S., Ho, Y.K. and Goldstem, J L (1980) J Blol. Chem 255, 9344-9352. [6] Brown, M.S and Goldstem, J L. (1983) Annu. Rev. Blochem. 52, 223-261 [7] Schnyder, J. and Bagglohm, M (1978) J Exp Med 148, 435-450 [8] Davies, P and Bonney, R J (1980) m. The Cell Biology of Inflammation (Weissmann, G ed.) pp 497-542, Elsevler, Amsterdam, New York [9] Bagglohnl, M (1982) Adv. Inflamm Res 3, 313-326 [lo] Brown, M.S , Dana, SE and Goldstem, J L (1974) J Blol Chem 249, 789-796 [ll] Basu, S K , Goldstein, J L , Anderson, R G.W and Brown, M S (1976) Proc Nat1 Acad Se USA 73, 3178-3182

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[12] Fogelmann, A M , Shechter, 1 , Seager, J , Hokom, M., Child, J.S and Edwards, P.A (1980) Proc Nat1 Acad SCI USA 77, 2214-2218 1131 Hartung, H.P , Haddmg, U , Bitter-Suerm~n, D and Gemsa, D. (1983) J Immunol 130, 2861-2865 [14] Hartung, H P., Bitter-Suerman and Haddmg, U. (1983) J. Immunol. 130, 1345-1349 [15] Huebner, P F (1976) Anal. Bzochem 74,419-429 [16] Harnson, E H and Bowers, W E (1981) m* Methods for Studying Mononuclear Phagocytes (Adams, D.O. et al. eds) pp.433-448, Academic Press, New York [17] Wroblewskl, F and LaDue, J S. (1955) Proc Sot Exp Btol Med. 90, 210-212 1181 Ztmmer, B , Hartung, H P , Scharfenberger, G , Bitter-Suermann, D and Haddmg, U (1982) Eur J Immunol 12, 426-430 [19] Schorlemmer, H U., Davies, P and Allison, A C (1976) Nature 261, 48-49 [20] McCarthy, K., Musson, R.S and Henson, P.M (1982) J Retsuloendothel. Sot. 31, 131-144 [Zl] Werb, Z and Gordon, S. (1975) J. Exp Med 142, 361-377 [22] Gernty, R.G., Nalto, H.K , RIchardson, M and Schwartz, C J (1979) Am J Path01 95, 775-786