Agents and Actions vol. 5/4 (1975) Birkhguser Verlag Basel
Changes in Cellular Enzyme Levels and the Inhibition of Selective Release of Lysosomai Hydrolases from Macrophages by Indomethacin by CARLOSFINLAY*, PHILIP DAVIES** and ANTHONY C. ALLISON** Centro Nacional de Investigaeiones Cientificas (*), Apt 6990, La Habana, Cuba, and Division of Cell Pathology (**), MRC Clinical Research Centre, Watford Road, Harrow, Middx. HA1 3UJ, England
Abstract lndomethaein increases the cellular levels of several lysosomal enzymes in cultures of mouse peritoneal macrophages exposed to the drug for periods of time ranging from one day to four weeks. This increase can be blocked by puromyein, an inhibitor of protein synthesis. Pretreatment of macrophages with indomethacin inhibits the selective release of lysosomal enzymes induced by a C-mucopolysaeeharide peptidoglycan complex purified from the cell walls of Group A streptococci.
The mode of action of anti-inflammatory drugs remains obscure. The difficulties in determining their mode of action arises, at least in part, from the complex and variable nature of the chronic inflammatory diseases which they are used to treat, as well as the experimental systems used to test their activity. As we have discussed elsewhere [1,2], macrophages are always present in chronic inflammatory lesions, and evidence is accumulating that these cells can bring about tissue damage by the selective release or secretion of hydrolytic enzymes in response to inflammatory stimuli [3-11]. These enzymes fall into at least two classes, acid hydrolases and neutral proteinases. The acid hydrolases are confined within lysosomes but the subcellular localization of the neutral proteinases remains to be determined. The release of lysosomal enzymes from macrophages can be induced by a variety of inflammatory stimuli of both immunological and
1) Part of this work was carried out while Carlos Finlay was British Council/International Cell Research Organization Research Fellow at the Clinical Research Centre.
nonimmunological types . One of the ways in which anti-inflammatory drugs may exert their beneficial effects would be to influence the synthesis or release of acid hydrolases by macrophages at sites of inflammation. In this paper we show that indomethacin increases the cellular levels of lysosomal enzymes in cultured mouse peritoneal macrophages, an effect which can be blocked by inhibition of protein synthesis. We also demonstrate that pretreatment with indomethacin inhibits the release of lysosomal enzymes from macrophages exposed to a C-mucopolysaccharide peptidoglycan complex purified from the cell walls of Group A streptococci. Materials and methods Experimental animals. Swiss mice were obtained
from SACI, Brentwood, Essex, or from the mouse colony established at CNIC, Habana, Cuba. Tissue culture materials. Tissue culture grade Petri dishes were obtained from Nunc Jobling Laboratories Division, Stone, Staffs, UK; M199 from Burroughs Wellcome, Beckenham, Kent, UK, newborn calf serum from Tissue Culture Services, Slough, Bucks, UK, Swine serum from Bio Cult Laboratories Ltd, Glasgow, Scotland. Biochemical reagents. Phenolphthalein glucuronic acid leucine-2-naphthylamide, Fast Garnett GBC, bovine serum albumin, HEPES buffer powder, puromycin and glycyl phenylanine naphthylamide were from Sigma Chemical Co., Surbiton, Surrey, UK; haemoglobin substrate for proteinase assay from Worthington Biochemical Corp., Freehold, N.J., USA, pnitrophenyl-fl-D-galactopyranoside and p-nitrophenyl-2acetamido-/%D-deoxyglucopyranoside from Koch-Light Laboratories, Colnbrook, Bucks, UK, lactate dehydrogenase UV-Test Packs from Boehringer Corporation (Ltd) Ealing, Middlesex, UK, highly polymerized calf thymus DNA from British Drug House, Poole, Dorset,
Inhibition of Release of Lysosomal Hydrolases by Indomethacin
UK, diamino benzoic acid from Aldrich Biochemical, Milwaukee, Wisconsin, USA, acridine orange from G. T. Gurr, London, UK, Heparin, preservative free, from Boots, Nottingham, UK. Indomethacin was a gift from Merck, Sharp and Dohme Laboratories, Hoddesdon, Herts, UK.
Preparation of C-mucopolysaeeharide peptidoglycan complex from Group A Streptococcal cell walls (PPG)
without acridine orange for 15 minutes and examined in
the living state by fluorescence microscopy on a Zeiss Photo microscope. Coverslip preparations for phasecontrast microscopy were fixed in 2.5% glutaraldehyde in 0.1 M phosphate buffer p H 7.5.
The sample of P P G used was that prepared and used in other studies on the release of acid hydrolases from macrophages [4, 6]. Macrophage collection and culture. Macrophages were obtained by peritoneal lavage of mice with M199 containing 100U/ml of penicillin 100, ~tg/ml streptomycin and 10 IU/ml heparin. 5 ml aliquots of the collected fluid containing 0.5-1.0 x 106 cells/ml were distributed into 50 m m Petri dishes and incubated in a humidified atmosphere of 5% carbon dioxide and air at 37 ~ for 1 to 2 hours to allow attachment of ad~herent cells. Nonadherent cells were removed by washing four times with phosphate-buffered saline. After washing, the cells were cultured in M199 containing varying amounts of newborn calf or swine serum. The serum was heated at 56 ~ for 30 minutes before use. Culture prepared in this way give a sheet of well spread cells within 24 hours. Concentrated solutions of indomethacin were prepared in dimethyl sulphoxide. Before addition to cultures the indomethacin was diluted with medium in such a way that the final concentration of D M S O was always 0.1% in all cultures. Control cultures contained 0.1% w/v dimethyl sulphoxide. Indomethacin was added to cultures after the initial washing of cells and left in contact with cells for up to four weeks. P P G was normally added after the cells had been in culture for 48 hours. P P G suspensions were sonicated in an MSE ultrasonic disintegrator for 2 minutes operating at approximately 20 kc. In all experiments quadruplicate cultures were used and biochemical data are expressed as mean ~: 1 SD. At the end of each incubation period the medium was removed and the adherent cells were washed once with phosphatebuffered saline. The cells were then released by adding saline containing 0 . 1 ~ v/v Triton x-100 and 0 . 1 ~ bovine serum albumin and scraping with sterile silicone rubber bungs. The culture medium was centrifuged at 1000 rpm for 10 minutes to pellet any detached cells; enzyme activity of the supernatant was assayed. All fractions were assayed for enzyme activity.
All assays were conducted under conditions giving linear release of product in relation to the amount of sample used and the time of incubation. Lactate dehydrogenase was assayed by determining the rate of oxidation of reduce nicotinamide adenine dinucleotide at 340 nm. fl-glucuronidase was assayed by the method of TALALAY et al. . fl-galactosidase was assayed by the method of CONCmE et al.  using p-nitrophenyl-fl-D-galactopyranoside as substrate. N-acetyl-fl-D-glucosaminidase was assayed by the method of WOOLEN et al.  using p-nitrophenyl-2-acetamido-2-fl-D-gIucopyranoside as substrate dissolved in 0.1 M acetate buffer p H 4.5 except where otherwise indicated. Leucine-2-naphthylamidase activity was measured by the method of GOLDnARG and RUTENBERG  as described by DAwEs et El. . Acid proteinase was assayed by the method of A r s o n  and the tyrosine content of the trichloracetic acid supernatant determined colorimetrically at 660 nm with Folin Cioleateau's reagent. Cathepsin C was assayed by determining the release of free naphthylamine from glycyl phenylalanine naphthylamide. 0.2 ml of enzyme sample was preincubated with 0.2 ml of 0.1 M phosphate buffer pH 6.5 containing 0.04 M cysteine and 0.04 sodium chloride for 10 minutes at 37 ~ After preincubation 0.4 ml of 0.4 m M substrate was added and incubation carried out for 5 to 240 minutes at 37~ The reaction was terminated by adding 1.4 ml of a mixture of equal volumes of (a) 10 m M chloromercuribenzoic acid and 50 m M disodium ethylenediamine tetracetic acid and (b) 0.5 mg/ml Fast Garnett GBC dissolved in 4 % w/v Tween 20. Solution (a) was prepared by dissolving 3.57G chloromercuribenzoic acid in 120 ml 0.5 M NaOH. To this 18.6G disodium ethylene diamine tetracetic acid was added and the volume brought to 950 ml with water. The pH was adjusted to 6.0 with 1 M HC1 and the volume brought to 1000 ml. The mixture of solution (a) and (b) should be prepared daily, filtered and stored at 4 ~ The coupling reaction gave a red product the optical density of which was measured at 520 nm. This assay was developed in the way suggested by BARRETT  and is based on his assay for cathepsin B1 .
Morphological observations of macrophages
Assay for DNA
Macrophages cultured on coverslips were observed in the living state using phase-contrast or fluorescence optics. Cultures maintained for biochemical estimations were also regularly monitored by light microscopy on an Olympus inverted-stage microscope. Unfixed coverslip preparations were incubated at 37~ with acridine orange at a concentration of 1 in 400,000 (w/v) in M199 buffered with 25 m M HEPES buffer for 5 minutes, transferred to the same medium
The D N A contents of cultures were determined by the fiuorimetric assay of KTSSANE and ROEBINS  using highly polymedsed calf thymus D N A as a standard.
Assay for protein Protein levels of macrophage cultures were determined by the method of LowRY"  using bovine serum albumin as a standard.
Inhibition of Release of Lysosomat Hydrolases by Indomethacin
Statistical tests Means and standard deviations were calculated after samples were shown to be homogeneous by calculation of coefficient of variance, Significance of difference was established by the Student t-test. Results
Morphological changes in macrophage cultures exposed to indomethacin Macrophages exposed to indomethacin in a wide dose range varying from 1 x 10-s M to 5 x 10-4 M show marked differences in their morphology compared to control cultures. These appear within 24 hours of the addition of indomethacin to the cultures. Phase-contrast microscopy shows that the cells treated with indomethacin (1 x 10-a M) appear to be better spread than controls (Fig. 1 ) w i t h more prominent 9cytoplasmic processes and ruffled membrane activity. They also show more granules, especially in the perinuclear position where lysosomes are usually located (Fig. 1 b). When cells stained
with the vital dye acridine orange are examined by fluorescence microscopy these granules are seen to be lysosomes. This identification is made on the basis of their being able to concentrate the dye and show a bright orange fluorescence against a dark cytoplasmic background and a pale green fluorescence by the nucleus . When cells are exposed to high concentrations of indomethacin (5 x 10-4 M) they show signs of toxicity. The cells round up, withdraw their cytoplasmic processes and in some instances detach from the surface of the culture vessel.
Biochemical. changes in macrophages exposed to indomethacin The morphological changes induced by indomethacin in macrophage cultures are paralleled by significant biochemical alterations. We have paid particular attention to the levels of lysosomal hydrolases in view of their potential capacity to cause tissue damage in inflammation
Phase contrast mierographs, a Control macrophage culture, b macrophage culture exposed to indomethacin 1 x 10-~ M for 72 hours in M199 + 10~ newborn calf serum ( x 560).
Inhibition of Release of Lysosomal Hydrolases by Indom~
Exposure o f macrophages to indomethacin in doses o f 5 x 10-6 and 5 x 10-5 M for 24 hours results in appreciable increases in two lysosomal enzymes fl-glucuronidase and N-acetyl-fl-D-glucosaminidase (Table 1). The increases in the levels o f these lysosomal enzymes are more apparent after 48 and 72 hours o f culture (Table 1). However, the cellular levels o f another lysosomal enzyme, /~-galactosidase, did not show any significant elevation at any time or at any concentration o f indomethacin used. These are also increases in the levels o f leucine-2-naphthylamidase, an enzyme associated with membranes and present in only low a m o u n t s in lysosomes, after exposure o f macrophages to indomethacin in doses o f 5 x 10-6 and 5 x 10-5 M (Table 1). Increases in the levels o f cellular protein in these cultures were also seen, but no s~gnificant change in the levels o f lactate dehydrogenase, a marker o f cytoplasmic enzyme activity, was detected in the presence o f these doses o f indomethacin. Cells exposed to high doses o f i n d o m e t h a c i n (5 x 10-4M) show biochemical changes that confirm cytotoxic changes seen by morphological observation (Table 1). In all instances examined the cellular levels o f lysosomal and nonlysosomal enzymes are seen to fall significantly in the presence o f 5 x 10-4 M indomethacin (Table 1). The cellular levels o f lactate dehydrogenase and protein also show significant decreases. The most effective means o f increasing cellular levels o f acid hydrolases in mouse peritoneal macrophages is to maintain them in a
culture m e d i u m containing high concentrz o f n e w b o r n calf serum . Table 2 illus~ that indomethacin potentiates the marke creases in acid hydrolase levels o f macrop cultures maintained for 72 hours in the p r e o f 50% newborn calf serum. These increase especially prominent for fl-glucuronidase N-acetyl-fl-D-glucosaminidase. On the o t h e r protein levels increase by less than 100%. clear from Table 2 that even under these cup conditions where lysosomal enzyme levels a r showing rapid rises indomethacin is able t cause further increases in lysosomal enzyrrc levels. A further examination of the effect o f a wide range o f concentrations o f indomethacin on macrophages grown in medium containing 10% swine serum was carried out. Table 3 shows that after 72 hours doses as low as 5 x 10-s M indomethacin result in significant increases in the levels o f acid hydrolases. Notably, fl-galactosidase levels did not show a regular significant increase in indomethacin treated cells. The highest concentration o f indomethacin use (1 x 10-4 M ) caused lesser increases in e n z y n , levels than lower concentrations in every instance, presumably indicating the onset o f toxic effects. The influence of cell number on the response of macrophages to indomethacin W h e n macrophages were plated at cell concentrations ranging f r o m 1.25 x 106 to 5.0
The time-dependent effect of indomethacin treatment on cellular enzyme levels in macrophage cultures. Enzyme
Concentration Indomethacin (M)
Time in hours 24
0 5 x 10 ~ 5 x 10-5 5 x 10.4
43.0 A= 54.94-452.34-429.1 4-
48.2 468.6:k 61.2=1= 25.5 :k
0 5 x 10-6 5 x 10-5 5 x 10-a
79.7 • 28.0 124.3 -t- 12.8 113.34- 3.4 59.9 4- 15.7
0 5 x 10-6 5 x 10-5 5 x 10.4
139 217 166 127
3.4 2.32) 2.31) 3.42)
• 2.9 =c39.41) • ) ~k28.7
72 4.5 1.03) 2.21) 2.23)
46.I • 67.3 471.7 417.7 4-
3.0 3.23) 1.0~) 1.03)
81.9 ~ 10.9 144.1 :t: 14.92) 129.8~: 8.92) 38.3 4- 5.22)
87.4 4- 4.8 173.8 • 11.03) 173.84-42.01 ) 30.8 4- 6.68)
160 262 202 103
172 • 7.3 324 • ) 302 zk29.03) 72.0• 7.33)
• 4- 21.62) 4-13.11 ) 4-17.62 )
~) p < 0.05 ; 2) p < 0.01 ; 3) p < 0.001. The activities of each enzyme is expressed as nmole product formed by substrate hydrolysis/plate/hour. Cells were cultured in M199 + 10~oonewborn calf serum.
:,ion of Release of Lysosomal Hydrolases by Indomethacin
,, cells per plate their response to 1 x 10-s M :-nethacin over a p e r i o d o f 72 h o u r s did n o t ,~(Fig. 2). A consistent increase in the levels ~-glucuronidase, N-acetyl-fl-D-glucosamini~,'and l e u c i n e - 2 - n a p h t h y l a m i d a s e is seen while t e d e h y d r o g e n a s e levels are n o t influenced n d o m e t h a c i n at any o f the cell n u m b e r s
?he effect o f chronic exposure o f macrophages '~ indomethacin Since m a c r o p h a g e s are k n o w n to persist in certain types o f ch r o n i c i n f l a m m a t o r y lesions for long p er i o d s  we e x a m i n e d the effect o f p r o l o n g e d exposure to i n d o m e t h a c i n on m a c r o -
phages. Cells were exposed to the drugs for up to 4 weeks, their culture m e d i u m being ch an g ed every 3 days. T h e cultures were e x a m i n e d carefully for the presence o f fibroblasts as c o n t a m i nating cells, n o n e being detected up to 4 weeks o f culture. It is clear f r o m Tab l e 4 t h a t the exp o s u r e o f m a c r o p h a g e cultures to 1 x 10-6 or 1 x 10-5 M i n d o m e t h a c i n for one to f o u r weeks causes highly significant increases in their c o n t e n t o f t wo acid hydrolases, N-acetyl-fl-D-glucosaminidase an d cathepsin C c o m p a r e d with cultures m a i n t a i n e d in the absence o f i n d o m e t h a c i n . Cultures exposed to i n d o m e t h a c i n for 4 weeks retained the same kind o f m o r p h o l o g i c a l changes c o m p a r e d to controls as seen in the cultures s h o wn in F i g u r e 1 b.
The effect of indomethacin on the levels of protein and lysosomal enzymes in cultures of macrophages maintained in medium containing 50% newborn calf serum. Concentration Indomethacin (M)
1 x 10 7 1 x 10-6 1 x 10 5 Concentration lndomethacin (M)
Protein Culture time in hours 0 72
fl-glucuronidase Culture time in hours 0 72
97.9 • 13.1
63.6 • 3.0
134.0 • 3.5 128.2 ~ 11.4 154.0 • 9.11) 156.6 • 16.4
158.0 s 9.1 167.6 ~ 14.0 184.0 ~ 10.61) 225.5 tz 19.72) N-Acetyl-fl-D-glucosaminidase Culture time in hours 0 72 47.5 d- 5.6 229.3 = 23.3 249.9 Jc 31.6 281.8 ~c 13.21) 282.4 • 21.51)
fl-galactosidase Culture time in hours 0 72 68.9 ~ 4.0 94.7 ~ 4.3 97.0 • 9.3 119.9=t= 5.61) 118.6 :tz 8.01)
0 1 x 10-7 1 x 10-6 1 x 10-s
1) p < 0.05; 2)p < 0.01. Enzyme activities are expressed as nmol product formed by substrate hydrolysis/plate/hour. Table 3
Lysosomal enzyme levels in macrophages exposed to various concentrations of indomethacin for 72 hours. Concentration Indomethacin
0 1 x 10-8 5 x 10-s 1 • 10-7 5 • 10-7 1 • 10-6 5 • 10-6 1 • 10-5 5 x 10-5 1 • 10-4
fl-glucuronidase (nmole phenolphthalein/ plate/hour)
N-Acetyl-fi-Dglucosaminidase (nmole paranitrophenol/plate/hour)
(nmole tyrosine/ plate/hour)
(nmole p-nitrophenol/ plate/hour)
84.0 i 2.9 84.4 :~ 1.0 91.3 ~ 3.01) 94.5 :t= 2.91) 102.3 ~= 1.8 ~) 95.8 -E 5.41) 102.3 ~ 3.02) 102.9 ~ 7.91) 108.8 ~ 2.6 ~) 95.3 • 1.82)
456 • 49 490 • 47 562 • 281) 575 • 221) 612 • 232) 568 =~ 361) 624 • 22 z) 653 • 24 ~) 667 • 322) 523 • 13.0
313 • 331 • 332 • 353 • 342 • 326 • 359 • 444 • 460 • 293 •
162 t 7.5 165 ~_ 13.0 173 4- 6.0 175 ~ 7.8 179 • 1.51) 167 + 8.4 173 4- 3.3 173 • 7.8 162 • 4.1