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Prostaglandin biosynthesis by lapine articular chondrocytes in culture
480
Biochimica et Biophysics 0 Elsevier/North-Holland
Acta, 663 (1981) 480-490 Biomedical Press
BBA 57743
PROSTAGLANDIN BIOSYNTHESIS CHONDROCYTES IN CULTURE
CHARLES
J. MALEMUD
a, ROLAND
BY LAPINE ARTICULAR
W. MOSKOWITZ
a and AVIV
HASSID
b
a The Cartilage Research Laboratory, Department of Medicine, Division of Rheumatology and ’ Department of Medicine, Division of Nephrology, Case Western Reserve University, Cleveland, OH 44106 (U.S.A.) (Received June 19th, 1980) (Revised manuscript received
Key words: Prostaglandin chondrocy te)
October
biosynthesis;
9th, 1980)
Prostacyclin;
Thromboxane
Bz (Rabbit
articular
Summary Secondary monolayer and spinner cultures of rabbit articular chondrocytes released into the culture medium prostaglandins the synthesis of which was inhibited by sodium meclofenamate. The prostaglandins measured by radioimmunoassay were, in order of decreasing abundance, prostaglandin E2, 6-0x0prostaglandin F,, (the stable metabolite of prostacyclin) and prostaglandin F Za. Several lines of evidence indicated that chondrocytes synthesize little if any thromboxane B2 (the stable metabolite of thromboxane A,). The presence of prostaglandins was confirmed by radiometric thin-layer chromatography of extracts of culture media incubated with [3H]arachidonic acid-labeled cells. In monolayer culture, chondrocytes synthesized immunoreactive prostaglandins in serum-free as well as serum-containing medium. Monolayer chondrocytes produced higher levels of prostaglandin E2 relative to 6-oxo-prostaglandin F,, than did spinner cells, but the latter synthesized more total prostaglandins. The identity of endogenous prostaglandins as well as those synthesized in shortterm culture by rabbit cartilage slices was compared to those produced by chondrocytes in long-term culture. Chondrocytes synthesized all of the prostaglandins found in articular cartilage. Minimal quantities of thromboxane B2 were detected in cartilage. A higher percentage of 6-oxo-prostaglandin F1, relative to other prostaglandins was found in cartilage than in either monolayer or spinner chondrocyte cultures. These results demonstrate that articular chondro-
Abbreviation:
30
:4,
arachidonic
acid.
481
cytes synthesize prostaglandins and prostacyclin. These prostaglandins may exert significant physiological effects on cartilage, since exogenous prostaglandins depress chondrocyte sulfated-proteoglycan synthesis and may even promote proteoglycan degradation,
Introduction Mounting evidence implicates prostaglandins in a variety of pathophysiological events affecting mammalian synovial joints. Synovium from patients with untreated rheumatoid arthritis secrete copious amounts of prostaglandin E2 in explant culture [ 11. A marked increase in prostaglandin E has been observed in resected synovium from patients with rheumatoid arthritis compared with osteo~thritic patients as determined by ~munofluorescent techniques [ 2 1. The effects of prostaglandins as mediators of inflammation, bone resorption, and events leading to osteolysis and hypercalcaemia as a result of metastatic tumors have also been well documented [3-51. Most convincing is evidence showing that nonsteroidal drugs such as indomethacin [6] and aspirin [ 7,8], which are effective inhibitors of pros~gl~din cyclo-oxygenase activity, also possess concomitant potent antiphlogistic effects. The effects of indomethacin and aspirin may, however, be unrelated to prostaglandins, since these agents also reduce leukocyte esterase activity [ 91. Although the relationship between joint inflammation and prostaglandins is well documented, little is known of its role in normal cartilage physiology. Bonner [lo] reported increased levels of free C,,:, in articular cartilage obtained from elderly individuals, although this finding probably accompanies the general increase in intracellular lipid content with age 1111. Prostaglandins have also been found in increased amounts in calcifying cartilage, suggesting that they may be involved in the deposition of mineral [ 121. Recently, prostaglandins have been shown to depress sulfated-proteoglycan synthesis by mammalian cartilage 1131 and chondrocytes in culture [ 14,153. Two studies have failed to document effects of prostaglandins on proteoglycan degradation [ 14,161, but loss of hexosamine from cartilage slices in the presence of prostaglandins has been reported [ 171. Despite these findings, a role for prostaglandins in cartilage metabolism presumably requires that prostaglandins enter the joint cavity by way of secretion from either inflamed synovium or invading macrophages. The chondrocytes as a potential source of prost~l~d~s have largely been ignored. The data provided herein establish that articular chondrocytes in culture synthesize prostaglandin E2 and prostaglandin Fzo, as well as prostacyclin (prostaglandin I*) and that this synthesis is inhibited by sodium meclofenamate. Moreover, the kinds of prostaglandin produced by short-term cultures of rabbit cartilage are basically similar to those measured endogenously in whole cartilage homogenates. These results indicate that chondrocytes may be a major source of prostaglandins in synovial joints. The presence of prostaglandins under normal physiological conditions and their presumed pro-inflammatory nature appears discordant. This requires further examination of the role of prostaglandins in joint pathophysiology.
482
Materials and Methods Materials
Nutrient-F12 (Ham’s), Dulbecco’s modified Eagle’s medium (high glucose for monolayer), Dulbecco’s modified Eagle’s medium for spinner culture without CaClz and MgS04 but containing MgC12 (165 mg/l), Gey’s balanced salt solution, antibiotics, mycostatin and fungizone were all purchased from Grand Island Biological Co., Grand Island, NY. Fetal bovine serum was obtained from either Associated Biomedic Systems, Buffalo, NY or Microbiological Associates, Bethesda, MD. All culture-ware was from Falcon Plastics, Oxnard, CA. 20:4 (62 Ci/mmol) was purchased from New [ 5,6,8,9,11,12,14,15-3H(N)]C England Nuclear, Boston, MA. Sodium meclofenamate was a gift from Lambert-Parke-Davis Co. (Indianapolis, IN). Methods Techniques
of culture. Articular chondrocytes were obtained from pooled articular cartilages of immature New Zealand white rabbits as previously described [ 18,191. After 7-10 days of primary monolayer culture in NutrientF12 mixture supplemented with 10% fetal bovine serum, the cells were subpassaged by brief exposure to trypsin (0.25%) and centrifugation of the suspension. In some experiments, secondary monolayer and spinner cultures were generated from the same cell pellet as previously detailed [ 201. All experiments were performed on chondrocytes after one subpassage. When experiments were performed in serum-free Dulbecco’s medium (monolayer), cells cultivated in serum containing medium were washed several times prior to addition of serum-free Dulbecco’s medium (monolayer). Radioimmunoassay. Radioimmunoassay for prostaglandin E, was performed with an antibody purchased from Pasteur Institute, Paris. Anti-prostaglandin E2 cross-reacts by 2.7% with prostaglandin El. All other prostaglandins and their metabolites do not cross-react significantly. The binding and cross-reactivity characteristics of this antibody have been published in detail elsewhere [21]. Anti-bodies against prostaglandin F,, and 6-oxo-prostaglandin F,, were prepared in our laboratory, while that against thromboxane Bz was a gift from Dr. William Campell, Houston TX. Anti-prostaglandin F,, does not distinguish between prostaglandin F1, and prostaglandin F,, . Anti-thromboxane B2 does not cross-react to a significant extent with other prostaglandins or with their metabolites. 50% inhibition of [3H]prostaglandin F,, binding is achieved with 20-40 pg prostaglandin F,, . Prostaglandin F,, , prostaglandin El, prostaglandin EZ, 6oxo-prostaglandin F1,, 15-keto-prostaglandin F1,, and 13,14dihydro15-keto-prostaglandin Fzo, cross-react by 63,0.22, 0.13, 0.17, 0.27 and 33%, respectively. Prostaglandin Bz, 15-keto-prostaglandin F,, and 13,14dihydro15-keto-prostaglandin E, cross-react by less than 0.01%. The substantial crossreaction of prostaglandin F,, presented little problem, since most biological systems synthesize relatively small amounts of monoenoic prostaglandins. 50% inhibition of 12SI-labeled histamine-6-oxo-prostaglandin F1, binding to antiSoxo-prostaglandin F1, is achieved with 20-40 pg 6-oxo-prostaglandin F,,. Prostaglandm F1,, prostaglandin El and prostaglandin Fzo, cross-react with anti6 oxo-prostaglandin F la by 22, 2.5 and 2%, respectively. Prostaglandin B2,
483
prostaglandin Dz, thromboxane Bz, prostaglandin Ez, and C,,:, cross-react by less than 0.05%. The significant cross-reaction of prostaglandin F,, does not present any problem for the reason mentioned previously. [3H]Prostaglandin F 201 and [3H]prostaglandin E2 (100-200 Ci/mmol), used for radioimmunoassay, were purchased from New England Nuclear. 1251-labeled histamine&oxoprostaglandin F lol and ‘251-labeled histamine-thromboxane B2 were prepared in our laboratory according to the method of Sors et al. [ 221. Their specific radioactivities were not determined. Radioimmunoassay of prostaglandins was performed directly with aliquots of culture medium from monolayer and spinner cells. For determination of prostaglandin content in cartilage, tissue was obtained from pooled articular joints of three New Zealand white rabbits (3.1 ? 0.2 kg (S.E.)). After brief treatment with testicular hyaluronidase, the tissue was disrupted in a Polytron homogenizer for 2 min at 0°C followed by centrifugation at 12 000 X g. Radioimmunoassay was performed on the supematant. To assess prostaglandin synthesis by short-term cultures (24 h) of rabbit articular cartilage, radioimmunoassay was performed on cartilage after homogenization of the cells and also on the culture medium. Radiometric thin layer chromatography (TLC). Chondrocyte cultures were maintained in Dulbecco’s medium (monolayer) or Dulbecco’s medium (spinner) supplemented with 10% fetal bovine serum for up to 7 days. Culture medium was changed every 2-3 days. When monolayer cells were subconfluent (generally after 1-2 days), chondrocytes were incubated with [3H]C2,,:4 ((l10) - lo6 cpm/culture) for 48 h at 37°C. At the end of the labeling period, an aliquot of the culture medium was removed and radioactivity in the medium was measured. Uptake of [ 3H]C20: 4 was generally 85-90% of the original radioactivity in monolayer culture. The radioactivity remaining in the culture media was removed from the cell cultures by three washes with unlabelled Dulbecco’s medium (monolayer) containing 10% fetal bovine serum. Medium with or without sodium meclofenamate (20 PM) was added to the cells and incubation at 37°C was continued for an additional 24 h. Prior to extraction of acidic lipids, including prostaglandins, (l-1.5) * lo3 cpm [ “C]prostaglandin E2, [ “C]prostaglandin FZa and [‘4C]C20: 4 (New England Nuclear, Boston, MA) were added as internal controls to the culture medium. Extraction was achieved by lowering the pH gradually to 3.0-3.5 with 1 M HCl followed by extraction twice with 3 ~01s. ethyl acetate. An aliquot of the extract was spotted on silicic acid thin-layer plates (silica gel 60, E. Merck, Darmstadt, F.R.G.) together with 5-10 pg standards (6-oxo-prostaglandin F1,, prostaglandin Fzcu, prostaglandin E2, thromboxane B2, prostaglandin D2 and Czoz4) and developed twice in either of two organic solvents; ethyl acetate/acetic acid (99 : 1, v/v) to separate prostaglandin E2 from thromboxane B, or ethyl acetate/iso-octane/acetic acid/water (11 : 5 : 2 : 10, V/V) to F,, . The prostaglandins separate prostaglandin F2, from 6-oxo-prostaglandin and thromboxane standards were generously supplied by J. Pike (Upjohn Co., Kalamazoo, MI). The standards were visualized by exposing the thin-layer plates to iodine vapor. The plates were cut into 15-17 segments, each segment was transferred to a scintillation vial and the radioactivity was determined by liquid scintillation spectrometry in Aquasol (New England Nuclear Co., Boston,
484
MA). Counting was performed in a Packard Tri-Carb System (Model 3255) using dual isotopic counting. Counting efficiency for 3H was 25-32% and for r4C 65-72%. Recoveries of [14~]pros~~~dins from medium monitored from the’added 14Cstandards was about 50%. Miscellaneous assays. DNA content of monolayer and spinner culture was determined by a modification of Burton’s diphenylamine method [ 23,241. Protein content of articular cartilage homogenates was assayed by the method of Lowry et al. [25]. Results Secondary cultures of monolayer chondrocytes released prostaglandin E2, prostaglandin Fza and 6-oxo-prostaglandin F,, into the culture media when assayed after 20 h in culture. In the experiment shown in Fig. 1, cells cultured for 20 h in serum-containing monolayer medium (3 - 10’ cells/60 mm* dish) were washed three times with serum-free medium, sodium meclofenamate (20 PM) added to some cultures and medium aliquots removed at indicated time intervals. In the absence of sodium meclofenamate, increasing concentrations of prostaglandin Ez, 6~xo-prost~l~din Fi, and pros~gl~din Fzt, were seen with time. In contrast, in the presence of sodium meclofenamate, prosta~andin synthesis was inhibited by 70-80% by 3.5 h of incubation. The inhibition was greatest for prostaglandin Ez and the least for prostagalandin Fzu biosynthesis. Immunoreactive thromboxane Bz was detected in the culture medium only after 3.5 h of incubation. The qualitative dis~ibution of prostagl~d~s synthes~ed in the presence of 10% fetal bovine serum over 3 days was invariant, but the amounts of each prostaglandin varied from day 1 to day 3 of culture (Fig. 2). Prostaglandin Ez concentrations were reduced on day 3 as compared to day 1 of culture. The same was true for Ci-oxo-prostaglandin F I0 and less so for prostaglandin Fzcl concentrations. Since we did not subsequently identify prostaglandin metabolites by TLC (see below), the reduction in ~munoreactive pros~l~d~s in the medium may have arisen through uptake of prostaglandins by cells. The reduction in prostaglandin E,, 6-oxo-prostaglandin F1, synthesis and to a lesser extent that of prostaglandin Fzo,also appeared to correlate with increased cellular proliferation (between days 1 and 3 after subculture) (Fig. 2, inset). In many assays, ~munoreactive thromboxane Bz found in medium from cell cultures was below or similar to that of medium plus serum ‘blanks’ indicative of high levels of endogenous thromboxane Bz in fetal bovine serum. These results made identification of thromboxane Bz in serum-containing chondrocyte cultures by radioimmunoassay equivocal. The data, therefore, are in agreement with the very minimal levels of immunoreactive thromboxane B2 detected in serum-free cultures. 3dayold subconfluent monolayer chondrocytes converted exogenous [ 3HI C20:4 to products that were separated by silica gel TLC. In the results shown in Fig. 3, the radioactive prostaglandins as well as unconverted [3H]Czo:4 were chromatographed in two solvent systems. The radioactivity corresponding to Goxo-prostaglandin F,, and prostaglandin E2 standards (Fig. 3A) confirmed
485
3.0
;
2.5
2
2.0
T x
1.5
CULTURE
TIME
1.0
z
0.5
N
(MINI
DISTANCE
0
0
I
I
0
1
2
3
FROM
ORIGIN(CM)
DAY
Fig. 1. (Top left) Radiohnmunoassay of prostaglandins (PG) from serum-free cultures on rabbit monolayer chondrocytes. O-day-old primary cultures were subpassaged by treatment with trypsin and replated at an initial density of lo5 cells/ml in Dulbecco’s medium (monolayer) supplemented with 10% fetal bovine serum. After 20 h. the cells were washed three times with serum-free medium, and incubated as indicated in serum-free medium. One half of the cultures were treated with sodium meclofenamate (20 PM). At the indicated time points, sliquots of the medium were withdrawn and radioimmunoassay was performed as indicated. The values for prostaglandin F2, and thromboxane B2 hi this experiment (*) indicate that only one value fell within the limits of the prostaglandin Fgo and thromboxane B2 standard curves. Inhibition by meclofenamate for prostaglandin tivity for the radioimmunoassay (20 pg/ml).
F2oand
thromboxane
B2 were at the level of sensi-
Fig. 2. (Bottom left). Effect of culture time on prostaglandin synthesis by monolayer chondrocytes. S-day-old primary chondrocytes were subpassed by treatment with trypsin and replated at a density of 1 * lo5 cells/ml in Dulbecco’s medium (monolayer) containing 10% fetal bovine serum. Culture medium was not changed during the experiment. At 1. 2 and days after subpassage, medium was withdrawn and radioimmunoassay was performed. Prostaglandin E2 (a); 6-oxo-prostaglandln Flo (0); prostaglandin F2o (0). Cells were removed by trypsinization and DNA content measured. DNA values represent mean +S.E. of three cultures for each group. Fig. 3. (Righthand figures) Isa-octane and ethyl acetate:acetic acid TLC of products of 13HlC26:4 metabolism in monolayer chondrocytes with or without sodium meclofenamate. 6-9-day-old primary monolayer cultures were subpassaged by treatment with trypsin. Chondrocytes were replated in Dulbecco’s medium (monolayer) supplemented with 10% fetal bovine serum at initial densities of 1.9 . lo5 cells/ml (A) and 3.5 . lo5 cells/ml (B) respectively. 24 h later, [3HlC20:4 ((l-1.8) * IO6 cpmlculture) was added with fresh serum-containing medium and isotope uptake continued for 48 h at 37’C. Cultures were washed three times with medium containing 10% fetal bovine serum and unlabeled medium with or without sodium meclofenamate (20 PM) was added for 24 h. Uptake of 13H1C2u:4 was 87-91% after 48 h in both experiments. The percentage effluxed radioactivity was reduced in the presence of meclofenamate by 65-678 but cell growth was unaffected. Open areas are radioactivity corresponding to internal prostaglandin (PG) standards as indicated. as well as other unknown acidiphilic lipids, hatched areas are in the presence of sodium meclofenamate. A. Do-octane solvent. B. Ethyl acetate/acetic acid solvent.
486
the relative abundance of each as measured by radio~muno~say. The biosynthesis of prostaglandin Ez and G-oxo-prostaglandin F,, was inhibited by 9496% by sodium meclofenamate. Although the efflux of radioactivity was reduced in the meclofenamate-treated cultures (control, 9.48 * lo4 cpm; meclofenamate, 3.31 . 104), the radioactivity in the meclofenamate-treated chondrocytes migrating with CZo:4 was actually greater or only slightly diminished (Fig. 3B) when compared to untreated cultures indicating that the reduction in effluxed radioactivity in the presence of meclofenamate was a result of decreased conversion of [ 3H ]CZo: 4 to prostaglandins. In addition to those prostaglandins measured by radioimmunoassay, radiometric TLC revealed measurable, albeit minute levels of radioactive migrating with the pros~gl~d~ Dz sided as well as radioacti~ty mi~ating between lo-12 cm of the silica gel (Fig. 3A). The latter may be prostaglandin AZ converted from prostaglandin Ez nonenzymatically during the acidification step prior to extraction. That this was the case was suggested by the presence of some 14C-radioactivity associated with these regions of the silica gel presumably from the [“CJabeledlprostaglandin Ez added as internal control prior to ethyl acetate extraction. The TLC developed in ethyl acetate/acetic acid showed minimal radioactivity corn&rating with authentic thromboxane B2 (Fig. 3B). Several lines of evidence suggest that this peak may be an unidentified product rather than thromboxane Bz, First, we were unable to abolish this peak by incubating the cells with benzylimid~ole (2 and 5 @I) a potent in vitro inhibitor of ~rombox~e synthetase 1261, Second, incubation of monolayer chondrocytes with the carboxylic-type Ca” ionophore, A23187 (2.5 PM) resulted in a 60-fold increase in prostaglandin E2 levels after 30 min. Neither constitutive nor ionophore-stimulated thromboxane B2 was detected. Spinner rather than monolayer chondrocytes have been shown to mimic more faithfully the phenotypic expression of rabbit cartilage [20,27]. We therefore compared the synthesis of prostaglandins in spinner and monolayer culture after 3,6 and 7 days in culture. The results, shown in Table I, indicated that monolayer cells synthesized prostaglandin E2 (72.8%) as the predominant prostaglandin with lesser quantities of Ci-oxo-prostaglandin F,, (21.9%) and other pros~gl~d~s after 3 days in culture. Although variations in absolute levels of total prosta~~d~s were measured in various experiments, spinner chondrocytes synthesized more total prostaglandin per cell than monolayer cell in all experiments. Furthermore, prostaglandin synthesis by chondrocytes in spinner culture was almost exclusively limited to prostaglandin E2. Neither at 3 nor 7 days culture was significant quantities of 6-oxo-prostaglandin F1,, or prostaglandin Fzo, synthesized relative to prostaglandin E2. Measurements of thromboxane Bz in both culture systems were hampered by high thromboxane B2 levels in medium blanks and are not included in these results for that reason. Adding sodium meclofenamate to the cultures 24 h prior to radioimmunoassay of 3 day cultures indicated that prostaglandin Ez was inhibited to the same extent in monolayer and spinner culture. Sodium meclofenamate inhibition of the 6~xo-prostagl~d~ F1, was also detected at both 3 and 7 days of spinner culture. We also compared the qualitative distribution of prostaglandins synthesized
TABLE
I
PROSTAGLANDIN DROCYTES
SYNTHESIS
BY MONOLAYER
AND
SPINNER
CULTURES
OF RABBIT
CHON-
8-12-day-old primary monolayer cultures were subcultured by incubating the cells with trypsin (0.25% for 15-20 ruin at 37’C and pelleting the chondrocytes by centrifugation. Secondary monolayer cultures ((3.3-3.8) . lo4 cells/ml) and spinner cultures ((2-3.4) . lo4 cells/ml) were initiated in Dulbecco’s medium (monolayer or spinner). respectively, supplemented with 10% fetal bovine serum and penicilllnstreptomycin (0.1%). The cultures were maintalned at 37’C for 2. 5 or 6 days. Culture medium was changed every 48 h. On those indicated days, fresh medium with or without sodium meclofenamate was added for an additional day. RIA was performed on culture medium from 3-. 6- or ‘I-day-old cultures. DNA content of the cultures was measured according to a modification of the Burton diphenylamine assay 123,241. n.d.. not determined: PG, prostaglandin. Culture mode
Experiment
Days after subculture
Radioimmunoassay PGEg *
Monolayer Monolayer
+ meclofenamate
Monolayer Monolayer
+ meclofenamate
Monolayer Monolayer
+ meclofenamate
Spinner Spinner
+ meclofenamate
Spinner Spinner
+ meclofenamate
Spinner Spinner
+ meclofenamate
TABLE
II
PROSTAGLANDIN
CONTENT
(nglpg
DNA)
6-oxo-PGFl,
PGFgo,
Total PG
1
3 3
6.00 0.35
1.81 0.04
0.31 0.05
8.12 0.44
1
6 6
1.67 0.18
0.40 0.10
0.13 0.03
2.20 0.31
2
7 7
0.95
0.94 0.12
n.d. n.d.
-
1
3 3
8.10 0.40
0.50 <0.07
0.91 0.25
9.51 0.72
2
3 3
40.98 <0.02
2.07 0.89
1.46 0.45
44.51 1.34
2
7 7
4.59 <0.08
0.20 CO.08
n.d. n.d.
AND SYNTHESIS
BY RABBIT
ARTICULAR
-
CARTILAGE
Articular cartilage from three New Zealand white rabbits (mean weight = 3.1 f 0.2 kg) was pooled from hip, knee and shoulder. Tissues were minced, washed briefly with testicular hyaluronidase (0.05%) at 24’C and homogenized in the cold with a Polytron homogenizer. Radioimmunoassay was performed on the tissue homogenate. In the case of cartilage explant, articular cartilage from a single rabbit (3.4 kg) was removed from the hip, knee and shoulder, treated with testicular byaluronidase. minced and placed in Dulbecco’s modified Eagle’s medium containing 4500 mg glucose/l but without fetal bovine serum. Sodium meclofenamate (20 PM) was added to one culture while the other was untreated. 24 h later the medium was removed. tissues were homogenized and both were assayed. PG. prostaglandin. Experiment
Radioimmunoassay PGE2
PGF2,
(ng/mg
protein)
6-oxo-PGFl
o
Thromboxane B2
Whole cartilage Homogenized
tissue
Cartilage explant Homogenized Homogenized
tissue tissue + meclofenamate
Culture medium Culture medium
+ meclofenamate
2.67
10.78
10.75
0.87
<0.4 <0.4
5.04 3.52
5.63 0.80
0.89 <0.4
9.74 <0.4
6.75 1.32
10.18 <0.4
0.86 <0.4
488
in monolayer and spinner culture to those found endogenously in rabbit articular cartilage and to those prostaglandins synthesized by short-term organexplant cultures. The relative abundance of prostaglandins found in cartilage as measured by radioimmunoassay is shown in Table II. Surprisingly, little prostaglandin Ez relative to prostaglandin F,, or 6-oxo-prostaglandin F,, was measured. However, by assessing prostaglandin synthesis in short-term explant culture, a pattern similar, but not identical to monolayer chondrocytes was found (Table II). In two separate experiments, prostaglandin Ez at the limit of detectability (under 0.4 pg/mg protein) was in the tissue homogenate of explanted cartilage; significantly more prostaglandin Ez was in the culture medium. Other prostaglandins above background levels were detected in the tissue homogenate. No explanation for the minimal prostaglandin Ez in the tissue can be offered at present. Discussion These results demonstrate the biosynthesis of significant quantities of prostaglandin Ez and prostaglandin IZ (as 6-oxo-prostaglandin F1, ) and lesser amounts of prostaglandin F,, , and prostaglandin Dz by articular chondrocytes in culture. The relative abundance of endogenous chondrocyte prostaglandins resembled that of the synthetic profile of short-term rabbit cartilage organexplants, although some differences in the amount of prostaglandin Ez among the various culture modes was observed. Major differences were shown to exist in the synthesis of 6-oxo-prostaglandin F,, relative to prostaglandin Ez by monolayer and spinnercultured chondrocytes. The former synthesized significant amounts of prostaglandin 12, while the latter cells produced very little (Table II). In this respect, monolayer chondrocytes resembled short-term organ explants of articular cartilage (Table II). Furthermore, significant amounts of Goxo-prostaglandin F,, were found in cartilage homogenates. Thus, the synthesis of 6-oxo-prostaglandin F,, by monolayer cultures appeared to be uncoupled from the relative ‘protodifferentiated’ state [ 281 of these cells. By contrast, spinner cultures of rabbit chondrocytes which fail to proliferate [ 20,291 synthesize predominantly type II collagen [ 271 and a profile of sulfated glycosaminoglycans that is indistinguishable from the parent tissue [20] elaborated little 6-oxo-prostaglandin F,, relative to prostaglandin Ez. The major product of these cells (at both 3 and 7 days after subculture) was prostaglandin Ez (Table I). Spinner cultures did, however, synthesize more total prostaglandin per pg DNA than did monolayer cells. The increase in total prostaglandin synthesized by spinner chondrocytes is consistent with the generalized hypermetabolic state of these cells in that elevated sulfated proteoglycan [20] and collagen [27] synthesis occurs as well. Furthermore, this elevated prostaglandin synthesis by chondrocytes in spinner culture occurred in medium markedly reduced in Ca2+ (0.33 mM (spinner) vs. 2.45 mM (monolayer)) [ 271. Ca2+ is though to play an important role in prostaglandin synthesis, since Caz+ ionophores greatly increase prostaglandin production in other organs [ 301. Prostaglandin biosynthesis by chondrocytes may have important physiological implications for cartilage. Copeland et al. [31] demonstrated prostaglandin
489
synthesis by isolated calf chondrocytes. They showed that at least 85% of the prostaglandins assayable by radioimmunoassay in their study was prostaglandin E. They did not examine for the presence of 6-oxo-prostaglandin F1,, prostaglandin Fzo, or thromboxane Bz. Exogenously-administered prostaglandin E 1 and E2 have been shown to affect cell proliferation minimally [14,15] and suppress protein and sulfated-glycosaminoglycan synthesis by chondrocyte cultures [14]. Prostaglandin F1, and prostaglandin F?, were generally without effect [ 141. The effect of prostaglandins on cartilage matrix catabolism is less well known [14,16,17]. The effects of exogenous prostaglandin Iz on cartilage are not known at present. In a recent study, physiologic thromboxane Bz concentrations reduced chondrocyte sulfatedglycosaminoglycan synthesis [ 151. Since we were able to show only minimal amounts of thromboxane Bz in chondrocyte cultures, cartilage, and short-term organ culture, we conclude that in all likelihood thromboxane effects on cartilage probably result from secretion of thromboxane AZ (the precursor of thromboxane B,) by inflammatory cells such as polymorphonuclear leukocytes and macrophages [ 32,331. In summary, chondrocytes must now be considered as a major source of prostaglandin production in diarthrodial joints. Because the diverse pathophysiological actions of prostaglandin are generally controlled in vivo by administration of nonsteroidal anti-inflammatory drugs, it will now be necessary to assess the actions of these agents on cartilage independent of those on synovium during acute and chronic inflammation. Some studies have indicated that long-term treatment of osteoarthritis with indomethacin has deleterious rather than positive effects on articular cartilage [34]. Similar investigations in animal models of experimentally-induced osteoarthritis [35] may provide useful information about the role of prostaglandins in the development of degenerative cartilage lesions. Finally, oxygencentered radicals are generated in the conversion of C&, to cyclic endoperoxides (prostaglandin H,) [ 361. The radicals formed, primarily believed to be hydroxyl radicals, probably involve the generation of hydroperoxides which may require superoxide radical as a precursor [37]. Oxygenderived free radicals have been shown to be capable of reducing hyaluronic acid viscosity [38,39] and degrading cartilage proteoglycan and collagen in vitro [ 401. Oxygen and hydroxyl radicals generated by chondrocytes may conceivably mediate the depolymerization of cartilage hyaluronic acid required for proteoglycan aggregation, since joint tissues have not be shown to possess hyaluronidase activity. Acknowledgements We thank Robert Papay for excellent technical assistance. This study was supported in part by National Institutes of Health (NIH) grant AG-01305-01 and grant from the Northeastern Ohio Chapter of the Arthritis Foundation. References 1 Robinson, D.R.. Tashjian. A.H., Jr. and Levine, L. (1975) J. Clin. Invest. 56,1181-1188 2 Husby, G.. Bankhurst, A.D. and Williams, R.C.. Jr. (1977) Arthritis Rheum. 20.785-791
490 3 Klein, D.C. and Raisz. L.G. (1970) Endrocrinology 86.1436-1440 4 Dietrich, J.W.. Goodson, J.M. and Raisz, L.G. (1975) Prostaglandins 10,231-240 5 Seyberth, H.W., Segre. G.V., Morgan, J.L., Sweetman, B.J., Potts, J.T., Jr. and Oates. J.A. (1975) Eng. J. Med. 293.1278-1283 6 Ferreira, S.H., Moncada. S. and Vane, J.R. (1971) Nature (New Biol.) 231. 237-239 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40
N.
Vane, J.R. (1971) Nature (New Biol.) 231.2332-2335 Smith, J.B. and Willis, A.L. (1971) Nature (New Biol.) 231.235-237 Kruze. D., Fehr. K.. Menninger. H. and Boni, A. (1976) Z. Rheumatol. 35.337-346 Banner, W.M.. Jonsson, H., Malanos, C. and Bryant, M. (1975) Arthritis Rheum. 18,461473 Collins, D.H., Ghaditiy. F.N. and Meachim. G. (1965) Ann. Rheum. Dis. 24.123-135 Northington. F.K., Ogelsby, T.D., Ishikawa. Y. and Wuthier, R.E. (1978) Calcif. Tissue Res. 26, 227236 Kleine, O.T. and Jungmann, U. (1979) Pharmacol. Res. Commun. 9.823-831 Malemud, C.J. and Sokoloff. L. (1977) ProstagIsndins 13.845-860 Kent, L., Malemud. C.J. and ,Moskowitz, R.W. (1980) Prostaglandins 19,391406 LippieIlo. L., Yamamoto, K., Robinson, D. and Mankin, H.J. (1978) Arthritis Rheum. 21.909--917 Fulkerson. J.F., Ladenbouer-Bellis, I.M. and Chrissman, O.D. (1979) Arthritis Rheum. 22, 11171121 Sokoloff. L.. Malemud. C.J. and Green. W.T., Jr. (1970) Arthritis Rheum. 13.118-124 Green, W.T.. Jr. (1971) CIin, Orthop. 75,248-260 Srivastava, V.M.L., Malemud, C.J. and Sokoloff, L. (1974) Connect. Tissue Res. 2,127-138 Dray, F., Charbonnel. B. and Maclouf, J. (1975) Eur. J. Clin. Invest. 5.311-318 Sors. H., Maclouf, J., PradeIIes, P. and Dray, F. (1977) Biochim. Biophys. Acta 486, 553-564 Burton, K. (1956) Biochem. J. 62.315-323 Corvol. M.-T., Malemud, C.J. and Sokoloff. L. (1972) Endocrinology 90,262-271 Lowry, O.H., Rosebrough. N.J.. Farr. A.L. and Randall, R.J. (1951) J. Biol. Chem. 193.265-275 Tai. H.-H. and Yuan, B. (1978) Biochem. Biophys. Res. Commun. 80.126-131 Norby, D.P.. Malemud, C.J. and Sokoloff. L. (1979) Arthritis, Rheum. 20. 709-716 Sokoloff, L.. Malemud, C.J.. Srivastava, V.M.L. and Morgan, W.D. (1973) Fed. Proc. 32, 1499-1502 Malemud, C.J. (1978) in The Human Joint in Health and Disease (Simon, W.H., ed.), PP. 43-52. University of Pennsylvania Press, Philadelphia, PA Knapp. H.R.. Oelz, 0.. Roberts, L.J.. Sweetman, B.J., O&es, J.A. and Reed, P.W. (1977) Proc. Natl. Acad. Sci. U.S.A. 74.42514255 Copeland, M.. LippieIIo, L., Guralnick, W. and Mankin, H.J. (1978) Orthop. Trans. 2.124-125 Higgs, G.A.. Bunting, S., Moncada. S. and Vane, J.R. (1976) ProstagIandins 12. 749-757 Murota, S.I., Kawamura, M. and Morita. I. (1978) Biochim. Biophys. Acta 528, 507-511 Ronningen, H. and Langeland, N. (1979) Acta Orthop. Stand. 50.169-174 Moskowitz, R.W., Davis, W., Sammarco. J., Marteus, M., Baker, J., Mayor, M., Burstein, A.H. Frankel. V.H. (1972) Arthritis Rheum. 16,397-405 Egan, R.W., Paxton, J. and Kuehl. F.A., Jr. (1976) J. Biol. Chem. 251,7329-7335 Sagone, A.L.. Jr., We&, R.M. and DeMocko, C. (2980) InfIammation 4.65-71 McCord, J.M. (1974) Science 185.529-531 Greenwald, R.A. and May. W.W. (1979) Arthritis Rheum. 23, 455-463 Greenwald. R.A., May, W.W. and Lazarus, D. (1979) Arthritis Rheum. 19. 799 (abstract)
Biochimica et Biophysics 0 Elsevier/North-Holland
Acta, 663 (1981) 480-490 Biomedical Press
BBA 57743
PROSTAGLANDIN BIOSYNTHESIS CHONDROCYTES IN CULTURE
CHARLES
J. MALEMUD
a, ROLAND
BY LAPINE ARTICULAR
W. MOSKOWITZ
a and AVIV
HASSID
b
a The Cartilage Research Laboratory, Department of Medicine, Division of Rheumatology and ’ Department of Medicine, Division of Nephrology, Case Western Reserve University, Cleveland, OH 44106 (U.S.A.) (Received June 19th, 1980) (Revised manuscript received
Key words: Prostaglandin chondrocy te)
October
biosynthesis;
9th, 1980)
Prostacyclin;
Thromboxane
Bz (Rabbit
articular
Summary Secondary monolayer and spinner cultures of rabbit articular chondrocytes released into the culture medium prostaglandins the synthesis of which was inhibited by sodium meclofenamate. The prostaglandins measured by radioimmunoassay were, in order of decreasing abundance, prostaglandin E2, 6-0x0prostaglandin F,, (the stable metabolite of prostacyclin) and prostaglandin F Za. Several lines of evidence indicated that chondrocytes synthesize little if any thromboxane B2 (the stable metabolite of thromboxane A,). The presence of prostaglandins was confirmed by radiometric thin-layer chromatography of extracts of culture media incubated with [3H]arachidonic acid-labeled cells. In monolayer culture, chondrocytes synthesized immunoreactive prostaglandins in serum-free as well as serum-containing medium. Monolayer chondrocytes produced higher levels of prostaglandin E2 relative to 6-oxo-prostaglandin F,, than did spinner cells, but the latter synthesized more total prostaglandins. The identity of endogenous prostaglandins as well as those synthesized in shortterm culture by rabbit cartilage slices was compared to those produced by chondrocytes in long-term culture. Chondrocytes synthesized all of the prostaglandins found in articular cartilage. Minimal quantities of thromboxane B2 were detected in cartilage. A higher percentage of 6-oxo-prostaglandin F1, relative to other prostaglandins was found in cartilage than in either monolayer or spinner chondrocyte cultures. These results demonstrate that articular chondro-
Abbreviation:
30
:4,
arachidonic
acid.
481
cytes synthesize prostaglandins and prostacyclin. These prostaglandins may exert significant physiological effects on cartilage, since exogenous prostaglandins depress chondrocyte sulfated-proteoglycan synthesis and may even promote proteoglycan degradation,
Introduction Mounting evidence implicates prostaglandins in a variety of pathophysiological events affecting mammalian synovial joints. Synovium from patients with untreated rheumatoid arthritis secrete copious amounts of prostaglandin E2 in explant culture [ 11. A marked increase in prostaglandin E has been observed in resected synovium from patients with rheumatoid arthritis compared with osteo~thritic patients as determined by ~munofluorescent techniques [ 2 1. The effects of prostaglandins as mediators of inflammation, bone resorption, and events leading to osteolysis and hypercalcaemia as a result of metastatic tumors have also been well documented [3-51. Most convincing is evidence showing that nonsteroidal drugs such as indomethacin [6] and aspirin [ 7,8], which are effective inhibitors of pros~gl~din cyclo-oxygenase activity, also possess concomitant potent antiphlogistic effects. The effects of indomethacin and aspirin may, however, be unrelated to prostaglandins, since these agents also reduce leukocyte esterase activity [ 91. Although the relationship between joint inflammation and prostaglandins is well documented, little is known of its role in normal cartilage physiology. Bonner [lo] reported increased levels of free C,,:, in articular cartilage obtained from elderly individuals, although this finding probably accompanies the general increase in intracellular lipid content with age 1111. Prostaglandins have also been found in increased amounts in calcifying cartilage, suggesting that they may be involved in the deposition of mineral [ 121. Recently, prostaglandins have been shown to depress sulfated-proteoglycan synthesis by mammalian cartilage 1131 and chondrocytes in culture [ 14,153. Two studies have failed to document effects of prostaglandins on proteoglycan degradation [ 14,161, but loss of hexosamine from cartilage slices in the presence of prostaglandins has been reported [ 171. Despite these findings, a role for prostaglandins in cartilage metabolism presumably requires that prostaglandins enter the joint cavity by way of secretion from either inflamed synovium or invading macrophages. The chondrocytes as a potential source of prost~l~d~s have largely been ignored. The data provided herein establish that articular chondrocytes in culture synthesize prostaglandin E2 and prostaglandin Fzo, as well as prostacyclin (prostaglandin I*) and that this synthesis is inhibited by sodium meclofenamate. Moreover, the kinds of prostaglandin produced by short-term cultures of rabbit cartilage are basically similar to those measured endogenously in whole cartilage homogenates. These results indicate that chondrocytes may be a major source of prostaglandins in synovial joints. The presence of prostaglandins under normal physiological conditions and their presumed pro-inflammatory nature appears discordant. This requires further examination of the role of prostaglandins in joint pathophysiology.
482
Materials and Methods Materials
Nutrient-F12 (Ham’s), Dulbecco’s modified Eagle’s medium (high glucose for monolayer), Dulbecco’s modified Eagle’s medium for spinner culture without CaClz and MgS04 but containing MgC12 (165 mg/l), Gey’s balanced salt solution, antibiotics, mycostatin and fungizone were all purchased from Grand Island Biological Co., Grand Island, NY. Fetal bovine serum was obtained from either Associated Biomedic Systems, Buffalo, NY or Microbiological Associates, Bethesda, MD. All culture-ware was from Falcon Plastics, Oxnard, CA. 20:4 (62 Ci/mmol) was purchased from New [ 5,6,8,9,11,12,14,15-3H(N)]C England Nuclear, Boston, MA. Sodium meclofenamate was a gift from Lambert-Parke-Davis Co. (Indianapolis, IN). Methods Techniques
of culture. Articular chondrocytes were obtained from pooled articular cartilages of immature New Zealand white rabbits as previously described [ 18,191. After 7-10 days of primary monolayer culture in NutrientF12 mixture supplemented with 10% fetal bovine serum, the cells were subpassaged by brief exposure to trypsin (0.25%) and centrifugation of the suspension. In some experiments, secondary monolayer and spinner cultures were generated from the same cell pellet as previously detailed [ 201. All experiments were performed on chondrocytes after one subpassage. When experiments were performed in serum-free Dulbecco’s medium (monolayer), cells cultivated in serum containing medium were washed several times prior to addition of serum-free Dulbecco’s medium (monolayer). Radioimmunoassay. Radioimmunoassay for prostaglandin E, was performed with an antibody purchased from Pasteur Institute, Paris. Anti-prostaglandin E2 cross-reacts by 2.7% with prostaglandin El. All other prostaglandins and their metabolites do not cross-react significantly. The binding and cross-reactivity characteristics of this antibody have been published in detail elsewhere [21]. Anti-bodies against prostaglandin F,, and 6-oxo-prostaglandin F,, were prepared in our laboratory, while that against thromboxane Bz was a gift from Dr. William Campell, Houston TX. Anti-prostaglandin F,, does not distinguish between prostaglandin F1, and prostaglandin F,, . Anti-thromboxane B2 does not cross-react to a significant extent with other prostaglandins or with their metabolites. 50% inhibition of [3H]prostaglandin F,, binding is achieved with 20-40 pg prostaglandin F,, . Prostaglandin F,, , prostaglandin El, prostaglandin EZ, 6oxo-prostaglandin F1,, 15-keto-prostaglandin F1,, and 13,14dihydro15-keto-prostaglandin Fzo, cross-react by 63,0.22, 0.13, 0.17, 0.27 and 33%, respectively. Prostaglandin Bz, 15-keto-prostaglandin F,, and 13,14dihydro15-keto-prostaglandin E, cross-react by less than 0.01%. The substantial crossreaction of prostaglandin F,, presented little problem, since most biological systems synthesize relatively small amounts of monoenoic prostaglandins. 50% inhibition of 12SI-labeled histamine-6-oxo-prostaglandin F1, binding to antiSoxo-prostaglandin F1, is achieved with 20-40 pg 6-oxo-prostaglandin F,,. Prostaglandm F1,, prostaglandin El and prostaglandin Fzo, cross-react with anti6 oxo-prostaglandin F la by 22, 2.5 and 2%, respectively. Prostaglandin B2,
483
prostaglandin Dz, thromboxane Bz, prostaglandin Ez, and C,,:, cross-react by less than 0.05%. The significant cross-reaction of prostaglandin F,, does not present any problem for the reason mentioned previously. [3H]Prostaglandin F 201 and [3H]prostaglandin E2 (100-200 Ci/mmol), used for radioimmunoassay, were purchased from New England Nuclear. 1251-labeled histamine&oxoprostaglandin F lol and ‘251-labeled histamine-thromboxane B2 were prepared in our laboratory according to the method of Sors et al. [ 221. Their specific radioactivities were not determined. Radioimmunoassay of prostaglandins was performed directly with aliquots of culture medium from monolayer and spinner cells. For determination of prostaglandin content in cartilage, tissue was obtained from pooled articular joints of three New Zealand white rabbits (3.1 ? 0.2 kg (S.E.)). After brief treatment with testicular hyaluronidase, the tissue was disrupted in a Polytron homogenizer for 2 min at 0°C followed by centrifugation at 12 000 X g. Radioimmunoassay was performed on the supematant. To assess prostaglandin synthesis by short-term cultures (24 h) of rabbit articular cartilage, radioimmunoassay was performed on cartilage after homogenization of the cells and also on the culture medium. Radiometric thin layer chromatography (TLC). Chondrocyte cultures were maintained in Dulbecco’s medium (monolayer) or Dulbecco’s medium (spinner) supplemented with 10% fetal bovine serum for up to 7 days. Culture medium was changed every 2-3 days. When monolayer cells were subconfluent (generally after 1-2 days), chondrocytes were incubated with [3H]C2,,:4 ((l10) - lo6 cpm/culture) for 48 h at 37°C. At the end of the labeling period, an aliquot of the culture medium was removed and radioactivity in the medium was measured. Uptake of [ 3H]C20: 4 was generally 85-90% of the original radioactivity in monolayer culture. The radioactivity remaining in the culture media was removed from the cell cultures by three washes with unlabelled Dulbecco’s medium (monolayer) containing 10% fetal bovine serum. Medium with or without sodium meclofenamate (20 PM) was added to the cells and incubation at 37°C was continued for an additional 24 h. Prior to extraction of acidic lipids, including prostaglandins, (l-1.5) * lo3 cpm [ “C]prostaglandin E2, [ “C]prostaglandin FZa and [‘4C]C20: 4 (New England Nuclear, Boston, MA) were added as internal controls to the culture medium. Extraction was achieved by lowering the pH gradually to 3.0-3.5 with 1 M HCl followed by extraction twice with 3 ~01s. ethyl acetate. An aliquot of the extract was spotted on silicic acid thin-layer plates (silica gel 60, E. Merck, Darmstadt, F.R.G.) together with 5-10 pg standards (6-oxo-prostaglandin F1,, prostaglandin Fzcu, prostaglandin E2, thromboxane B2, prostaglandin D2 and Czoz4) and developed twice in either of two organic solvents; ethyl acetate/acetic acid (99 : 1, v/v) to separate prostaglandin E2 from thromboxane B, or ethyl acetate/iso-octane/acetic acid/water (11 : 5 : 2 : 10, V/V) to F,, . The prostaglandins separate prostaglandin F2, from 6-oxo-prostaglandin and thromboxane standards were generously supplied by J. Pike (Upjohn Co., Kalamazoo, MI). The standards were visualized by exposing the thin-layer plates to iodine vapor. The plates were cut into 15-17 segments, each segment was transferred to a scintillation vial and the radioactivity was determined by liquid scintillation spectrometry in Aquasol (New England Nuclear Co., Boston,
484
MA). Counting was performed in a Packard Tri-Carb System (Model 3255) using dual isotopic counting. Counting efficiency for 3H was 25-32% and for r4C 65-72%. Recoveries of [14~]pros~~~dins from medium monitored from the’added 14Cstandards was about 50%. Miscellaneous assays. DNA content of monolayer and spinner culture was determined by a modification of Burton’s diphenylamine method [ 23,241. Protein content of articular cartilage homogenates was assayed by the method of Lowry et al. [25]. Results Secondary cultures of monolayer chondrocytes released prostaglandin E2, prostaglandin Fza and 6-oxo-prostaglandin F,, into the culture media when assayed after 20 h in culture. In the experiment shown in Fig. 1, cells cultured for 20 h in serum-containing monolayer medium (3 - 10’ cells/60 mm* dish) were washed three times with serum-free medium, sodium meclofenamate (20 PM) added to some cultures and medium aliquots removed at indicated time intervals. In the absence of sodium meclofenamate, increasing concentrations of prostaglandin Ez, 6~xo-prost~l~din Fi, and pros~gl~din Fzt, were seen with time. In contrast, in the presence of sodium meclofenamate, prosta~andin synthesis was inhibited by 70-80% by 3.5 h of incubation. The inhibition was greatest for prostaglandin Ez and the least for prostagalandin Fzu biosynthesis. Immunoreactive thromboxane Bz was detected in the culture medium only after 3.5 h of incubation. The qualitative dis~ibution of prostagl~d~s synthes~ed in the presence of 10% fetal bovine serum over 3 days was invariant, but the amounts of each prostaglandin varied from day 1 to day 3 of culture (Fig. 2). Prostaglandin Ez concentrations were reduced on day 3 as compared to day 1 of culture. The same was true for Ci-oxo-prostaglandin F I0 and less so for prostaglandin Fzcl concentrations. Since we did not subsequently identify prostaglandin metabolites by TLC (see below), the reduction in ~munoreactive pros~l~d~s in the medium may have arisen through uptake of prostaglandins by cells. The reduction in prostaglandin E,, 6-oxo-prostaglandin F1, synthesis and to a lesser extent that of prostaglandin Fzo,also appeared to correlate with increased cellular proliferation (between days 1 and 3 after subculture) (Fig. 2, inset). In many assays, ~munoreactive thromboxane Bz found in medium from cell cultures was below or similar to that of medium plus serum ‘blanks’ indicative of high levels of endogenous thromboxane Bz in fetal bovine serum. These results made identification of thromboxane Bz in serum-containing chondrocyte cultures by radioimmunoassay equivocal. The data, therefore, are in agreement with the very minimal levels of immunoreactive thromboxane B2 detected in serum-free cultures. 3dayold subconfluent monolayer chondrocytes converted exogenous [ 3HI C20:4 to products that were separated by silica gel TLC. In the results shown in Fig. 3, the radioactive prostaglandins as well as unconverted [3H]Czo:4 were chromatographed in two solvent systems. The radioactivity corresponding to Goxo-prostaglandin F,, and prostaglandin E2 standards (Fig. 3A) confirmed
485
3.0
;
2.5
2
2.0
T x
1.5
CULTURE
TIME
1.0
z
0.5
N
(MINI
DISTANCE
0
0
I
I
0
1
2
3
FROM
ORIGIN(CM)
DAY
Fig. 1. (Top left) Radiohnmunoassay of prostaglandins (PG) from serum-free cultures on rabbit monolayer chondrocytes. O-day-old primary cultures were subpassaged by treatment with trypsin and replated at an initial density of lo5 cells/ml in Dulbecco’s medium (monolayer) supplemented with 10% fetal bovine serum. After 20 h. the cells were washed three times with serum-free medium, and incubated as indicated in serum-free medium. One half of the cultures were treated with sodium meclofenamate (20 PM). At the indicated time points, sliquots of the medium were withdrawn and radioimmunoassay was performed as indicated. The values for prostaglandin F2, and thromboxane B2 hi this experiment (*) indicate that only one value fell within the limits of the prostaglandin Fgo and thromboxane B2 standard curves. Inhibition by meclofenamate for prostaglandin tivity for the radioimmunoassay (20 pg/ml).
F2oand
thromboxane
B2 were at the level of sensi-
Fig. 2. (Bottom left). Effect of culture time on prostaglandin synthesis by monolayer chondrocytes. S-day-old primary chondrocytes were subpassed by treatment with trypsin and replated at a density of 1 * lo5 cells/ml in Dulbecco’s medium (monolayer) containing 10% fetal bovine serum. Culture medium was not changed during the experiment. At 1. 2 and days after subpassage, medium was withdrawn and radioimmunoassay was performed. Prostaglandin E2 (a); 6-oxo-prostaglandln Flo (0); prostaglandin F2o (0). Cells were removed by trypsinization and DNA content measured. DNA values represent mean +S.E. of three cultures for each group. Fig. 3. (Righthand figures) Isa-octane and ethyl acetate:acetic acid TLC of products of 13HlC26:4 metabolism in monolayer chondrocytes with or without sodium meclofenamate. 6-9-day-old primary monolayer cultures were subpassaged by treatment with trypsin. Chondrocytes were replated in Dulbecco’s medium (monolayer) supplemented with 10% fetal bovine serum at initial densities of 1.9 . lo5 cells/ml (A) and 3.5 . lo5 cells/ml (B) respectively. 24 h later, [3HlC20:4 ((l-1.8) * IO6 cpmlculture) was added with fresh serum-containing medium and isotope uptake continued for 48 h at 37’C. Cultures were washed three times with medium containing 10% fetal bovine serum and unlabeled medium with or without sodium meclofenamate (20 PM) was added for 24 h. Uptake of 13H1C2u:4 was 87-91% after 48 h in both experiments. The percentage effluxed radioactivity was reduced in the presence of meclofenamate by 65-678 but cell growth was unaffected. Open areas are radioactivity corresponding to internal prostaglandin (PG) standards as indicated. as well as other unknown acidiphilic lipids, hatched areas are in the presence of sodium meclofenamate. A. Do-octane solvent. B. Ethyl acetate/acetic acid solvent.
486
the relative abundance of each as measured by radio~muno~say. The biosynthesis of prostaglandin Ez and G-oxo-prostaglandin F,, was inhibited by 9496% by sodium meclofenamate. Although the efflux of radioactivity was reduced in the meclofenamate-treated cultures (control, 9.48 * lo4 cpm; meclofenamate, 3.31 . 104), the radioactivity in the meclofenamate-treated chondrocytes migrating with CZo:4 was actually greater or only slightly diminished (Fig. 3B) when compared to untreated cultures indicating that the reduction in effluxed radioactivity in the presence of meclofenamate was a result of decreased conversion of [ 3H ]CZo: 4 to prostaglandins. In addition to those prostaglandins measured by radioimmunoassay, radiometric TLC revealed measurable, albeit minute levels of radioactive migrating with the pros~gl~d~ Dz sided as well as radioacti~ty mi~ating between lo-12 cm of the silica gel (Fig. 3A). The latter may be prostaglandin AZ converted from prostaglandin Ez nonenzymatically during the acidification step prior to extraction. That this was the case was suggested by the presence of some 14C-radioactivity associated with these regions of the silica gel presumably from the [“CJabeledlprostaglandin Ez added as internal control prior to ethyl acetate extraction. The TLC developed in ethyl acetate/acetic acid showed minimal radioactivity corn&rating with authentic thromboxane B2 (Fig. 3B). Several lines of evidence suggest that this peak may be an unidentified product rather than thromboxane Bz, First, we were unable to abolish this peak by incubating the cells with benzylimid~ole (2 and 5 @I) a potent in vitro inhibitor of ~rombox~e synthetase 1261, Second, incubation of monolayer chondrocytes with the carboxylic-type Ca” ionophore, A23187 (2.5 PM) resulted in a 60-fold increase in prostaglandin E2 levels after 30 min. Neither constitutive nor ionophore-stimulated thromboxane B2 was detected. Spinner rather than monolayer chondrocytes have been shown to mimic more faithfully the phenotypic expression of rabbit cartilage [20,27]. We therefore compared the synthesis of prostaglandins in spinner and monolayer culture after 3,6 and 7 days in culture. The results, shown in Table I, indicated that monolayer cells synthesized prostaglandin E2 (72.8%) as the predominant prostaglandin with lesser quantities of Ci-oxo-prostaglandin F,, (21.9%) and other pros~gl~d~s after 3 days in culture. Although variations in absolute levels of total prosta~~d~s were measured in various experiments, spinner chondrocytes synthesized more total prostaglandin per cell than monolayer cell in all experiments. Furthermore, prostaglandin synthesis by chondrocytes in spinner culture was almost exclusively limited to prostaglandin E2. Neither at 3 nor 7 days culture was significant quantities of 6-oxo-prostaglandin F1,, or prostaglandin Fzo, synthesized relative to prostaglandin E2. Measurements of thromboxane Bz in both culture systems were hampered by high thromboxane B2 levels in medium blanks and are not included in these results for that reason. Adding sodium meclofenamate to the cultures 24 h prior to radioimmunoassay of 3 day cultures indicated that prostaglandin Ez was inhibited to the same extent in monolayer and spinner culture. Sodium meclofenamate inhibition of the 6~xo-prostagl~d~ F1, was also detected at both 3 and 7 days of spinner culture. We also compared the qualitative distribution of prostaglandins synthesized
TABLE
I
PROSTAGLANDIN DROCYTES
SYNTHESIS
BY MONOLAYER
AND
SPINNER
CULTURES
OF RABBIT
CHON-
8-12-day-old primary monolayer cultures were subcultured by incubating the cells with trypsin (0.25% for 15-20 ruin at 37’C and pelleting the chondrocytes by centrifugation. Secondary monolayer cultures ((3.3-3.8) . lo4 cells/ml) and spinner cultures ((2-3.4) . lo4 cells/ml) were initiated in Dulbecco’s medium (monolayer or spinner). respectively, supplemented with 10% fetal bovine serum and penicilllnstreptomycin (0.1%). The cultures were maintalned at 37’C for 2. 5 or 6 days. Culture medium was changed every 48 h. On those indicated days, fresh medium with or without sodium meclofenamate was added for an additional day. RIA was performed on culture medium from 3-. 6- or ‘I-day-old cultures. DNA content of the cultures was measured according to a modification of the Burton diphenylamine assay 123,241. n.d.. not determined: PG, prostaglandin. Culture mode
Experiment
Days after subculture
Radioimmunoassay PGEg *
Monolayer Monolayer
+ meclofenamate
Monolayer Monolayer
+ meclofenamate
Monolayer Monolayer
+ meclofenamate
Spinner Spinner
+ meclofenamate
Spinner Spinner
+ meclofenamate
Spinner Spinner
+ meclofenamate
TABLE
II
PROSTAGLANDIN
CONTENT
(nglpg
DNA)
6-oxo-PGFl,
PGFgo,
Total PG
1
3 3
6.00 0.35
1.81 0.04
0.31 0.05
8.12 0.44
1
6 6
1.67 0.18
0.40 0.10
0.13 0.03
2.20 0.31
2
7 7
0.95
0.94 0.12
n.d. n.d.
-
1
3 3
8.10 0.40
0.50 <0.07
0.91 0.25
9.51 0.72
2
3 3
40.98 <0.02
2.07 0.89
1.46 0.45
44.51 1.34
2
7 7
4.59 <0.08
0.20 CO.08
n.d. n.d.
AND SYNTHESIS
BY RABBIT
ARTICULAR
-
CARTILAGE
Articular cartilage from three New Zealand white rabbits (mean weight = 3.1 f 0.2 kg) was pooled from hip, knee and shoulder. Tissues were minced, washed briefly with testicular hyaluronidase (0.05%) at 24’C and homogenized in the cold with a Polytron homogenizer. Radioimmunoassay was performed on the tissue homogenate. In the case of cartilage explant, articular cartilage from a single rabbit (3.4 kg) was removed from the hip, knee and shoulder, treated with testicular byaluronidase. minced and placed in Dulbecco’s modified Eagle’s medium containing 4500 mg glucose/l but without fetal bovine serum. Sodium meclofenamate (20 PM) was added to one culture while the other was untreated. 24 h later the medium was removed. tissues were homogenized and both were assayed. PG. prostaglandin. Experiment
Radioimmunoassay PGE2
PGF2,
(ng/mg
protein)
6-oxo-PGFl
o
Thromboxane B2
Whole cartilage Homogenized
tissue
Cartilage explant Homogenized Homogenized
tissue tissue + meclofenamate
Culture medium Culture medium
+ meclofenamate
2.67
10.78
10.75
0.87
<0.4 <0.4
5.04 3.52
5.63 0.80
0.89 <0.4
9.74 <0.4
6.75 1.32
10.18 <0.4
0.86 <0.4
488
in monolayer and spinner culture to those found endogenously in rabbit articular cartilage and to those prostaglandins synthesized by short-term organexplant cultures. The relative abundance of prostaglandins found in cartilage as measured by radioimmunoassay is shown in Table II. Surprisingly, little prostaglandin Ez relative to prostaglandin F,, or 6-oxo-prostaglandin F,, was measured. However, by assessing prostaglandin synthesis in short-term explant culture, a pattern similar, but not identical to monolayer chondrocytes was found (Table II). In two separate experiments, prostaglandin Ez at the limit of detectability (under 0.4 pg/mg protein) was in the tissue homogenate of explanted cartilage; significantly more prostaglandin Ez was in the culture medium. Other prostaglandins above background levels were detected in the tissue homogenate. No explanation for the minimal prostaglandin Ez in the tissue can be offered at present. Discussion These results demonstrate the biosynthesis of significant quantities of prostaglandin Ez and prostaglandin IZ (as 6-oxo-prostaglandin F1, ) and lesser amounts of prostaglandin F,, , and prostaglandin Dz by articular chondrocytes in culture. The relative abundance of endogenous chondrocyte prostaglandins resembled that of the synthetic profile of short-term rabbit cartilage organexplants, although some differences in the amount of prostaglandin Ez among the various culture modes was observed. Major differences were shown to exist in the synthesis of 6-oxo-prostaglandin F,, relative to prostaglandin Ez by monolayer and spinnercultured chondrocytes. The former synthesized significant amounts of prostaglandin 12, while the latter cells produced very little (Table II). In this respect, monolayer chondrocytes resembled short-term organ explants of articular cartilage (Table II). Furthermore, significant amounts of Goxo-prostaglandin F,, were found in cartilage homogenates. Thus, the synthesis of 6-oxo-prostaglandin F,, by monolayer cultures appeared to be uncoupled from the relative ‘protodifferentiated’ state [ 281 of these cells. By contrast, spinner cultures of rabbit chondrocytes which fail to proliferate [ 20,291 synthesize predominantly type II collagen [ 271 and a profile of sulfated glycosaminoglycans that is indistinguishable from the parent tissue [20] elaborated little 6-oxo-prostaglandin F,, relative to prostaglandin Ez. The major product of these cells (at both 3 and 7 days after subculture) was prostaglandin Ez (Table I). Spinner cultures did, however, synthesize more total prostaglandin per pg DNA than did monolayer cells. The increase in total prostaglandin synthesized by spinner chondrocytes is consistent with the generalized hypermetabolic state of these cells in that elevated sulfated proteoglycan [20] and collagen [27] synthesis occurs as well. Furthermore, this elevated prostaglandin synthesis by chondrocytes in spinner culture occurred in medium markedly reduced in Ca2+ (0.33 mM (spinner) vs. 2.45 mM (monolayer)) [ 271. Ca2+ is though to play an important role in prostaglandin synthesis, since Caz+ ionophores greatly increase prostaglandin production in other organs [ 301. Prostaglandin biosynthesis by chondrocytes may have important physiological implications for cartilage. Copeland et al. [31] demonstrated prostaglandin
489
synthesis by isolated calf chondrocytes. They showed that at least 85% of the prostaglandins assayable by radioimmunoassay in their study was prostaglandin E. They did not examine for the presence of 6-oxo-prostaglandin F1,, prostaglandin Fzo, or thromboxane Bz. Exogenously-administered prostaglandin E 1 and E2 have been shown to affect cell proliferation minimally [14,15] and suppress protein and sulfated-glycosaminoglycan synthesis by chondrocyte cultures [14]. Prostaglandin F1, and prostaglandin F?, were generally without effect [ 141. The effect of prostaglandins on cartilage matrix catabolism is less well known [14,16,17]. The effects of exogenous prostaglandin Iz on cartilage are not known at present. In a recent study, physiologic thromboxane Bz concentrations reduced chondrocyte sulfatedglycosaminoglycan synthesis [ 151. Since we were able to show only minimal amounts of thromboxane Bz in chondrocyte cultures, cartilage, and short-term organ culture, we conclude that in all likelihood thromboxane effects on cartilage probably result from secretion of thromboxane AZ (the precursor of thromboxane B,) by inflammatory cells such as polymorphonuclear leukocytes and macrophages [ 32,331. In summary, chondrocytes must now be considered as a major source of prostaglandin production in diarthrodial joints. Because the diverse pathophysiological actions of prostaglandin are generally controlled in vivo by administration of nonsteroidal anti-inflammatory drugs, it will now be necessary to assess the actions of these agents on cartilage independent of those on synovium during acute and chronic inflammation. Some studies have indicated that long-term treatment of osteoarthritis with indomethacin has deleterious rather than positive effects on articular cartilage [34]. Similar investigations in animal models of experimentally-induced osteoarthritis [35] may provide useful information about the role of prostaglandins in the development of degenerative cartilage lesions. Finally, oxygencentered radicals are generated in the conversion of C&, to cyclic endoperoxides (prostaglandin H,) [ 361. The radicals formed, primarily believed to be hydroxyl radicals, probably involve the generation of hydroperoxides which may require superoxide radical as a precursor [37]. Oxygenderived free radicals have been shown to be capable of reducing hyaluronic acid viscosity [38,39] and degrading cartilage proteoglycan and collagen in vitro [ 401. Oxygen and hydroxyl radicals generated by chondrocytes may conceivably mediate the depolymerization of cartilage hyaluronic acid required for proteoglycan aggregation, since joint tissues have not be shown to possess hyaluronidase activity. Acknowledgements We thank Robert Papay for excellent technical assistance. This study was supported in part by National Institutes of Health (NIH) grant AG-01305-01 and grant from the Northeastern Ohio Chapter of the Arthritis Foundation. References 1 Robinson, D.R.. Tashjian. A.H., Jr. and Levine, L. (1975) J. Clin. Invest. 56,1181-1188 2 Husby, G.. Bankhurst, A.D. and Williams, R.C.. Jr. (1977) Arthritis Rheum. 20.785-791
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