perpendicular to the medial side of the knee. The 3-p sagittal sections were cut from the medial to lateral ... ness. The tibial growth plate values were arbitrarily set to 100% and the valuesfor each articular ..... We thank William Peterson for help with the computer ... 4. van Eden W, Holoshitz J, Nevo Z, Frendel A, Klajman.
American Journal of Pathology, Vol. 144, No. 5, May 1994 Copyright © Amenican Society for Investigative Pathology
Comparison of Mobility Changes with Histological and Biochemical Changes During Lipopolysaccharide-lnduced Arthritis in the Hamster
Ivan G. Otterness,* Marcia L. Bliven,* A. J. Milici,* and A. Robin Poolett From the Department of Immunology and Infectious Diseases,* Central Research Division, Pfizer Inc., Groton, Connecticut; and Shriners Hospitals for Crppled Children,* Joint Diseases Laboratory; and Division of Surgical Research,t Department of Surgery, McGill University, Montreal, Quebec, Canada
Arthritis refers to a heterogeneous class of diseases characterized by impairment of movement. Yet animal models of arthritis have traditionally been based on the utilization of animals housed without the capability of extendedfree movement and without adjunctive measurement of mobility. To define the determinants of mobility impairment, we have established a lipopolysaccharide (LPS)-induced arthritis model in the hamster that prominently features monitoring of mobility and compares mobility changes with histological and biochemical changes during arthritis. Intraarticular LPS induces a dose-dependent inhibition of the hamster's mobility as measured by decreased daily distance on a running wheel (normal distance 9 to 12 km/day). At low concentrations of LPS (0.1 and 1 lug/knee), daily distances returned to normal after 4 and 6 days, respectively. At higher concentrations, the mobility was still markedly suppressed after 6 days, and,
sulfate failed to correlate with either proteoglycan loss or mobility changes. Proteoglycan synthesis, which was maximally suppressed the second day after LPS, was enhanced over controls at the time of restoration of mobility, suggesting the onset of repair. These results suggest a possible association of mobility inhibition with local cytokine synthesis. This model provides an approach to define the causes of mobility impairment. (Am J Pathol 1994, 144:1098-1108)
at 100ug/knee, irreversible chondrocyte loss was observed on the femoral condylar margins. Further studies were therefore conducted using 1 jug
The term arthritis refers to a heterogeneous class of diseases characterized by impairment of movement. Numerous published papers have addressed the causes of histological and biochemical changes observed during arthritis. Yet, with the exception of orthopedic problems, no one has systematically addressed the question of which pathological changes are responsible for loss of mobility. To address this question, a new model of arthritis was necessary. Numerous models of chronic inflammatory arthritis exist. Immunological responses to molecules of articular cartilage, eg, type 11 collagen12 or proteoglycan aggrecan,3 can lead to inflammatory erosive arthritis. Similarly, exposure to molecules cross-reactive with articular cartilage, ie, mycobacterial antigens,4 leads to adjuvant arthritis. Experimental arthritis can arise from infectious agents as in Lyme arthritis5 and Mycoplasma arthritis6 or may arise because of deposition in synovial tissue of bacterial products, such as streptococcal type A peptidoglycan-polysaccharide (PG-PS),7 Lactobacillus casei cell wall,6 or lipopolysaccharide (LPS).9'10 In addition, immunization with
the time of restoration of mobility. At the time of restoration of mobility, the synovial capsule was stiU edematous and heavily infiltrated with leukocytes; proteoglycan loss from the medialfemoral condyle was stiU increasing. Plasma keratan
ARP was supported by the Shriners of North America and the Medical Research Council of Canada. Accepted for publication December 10, 1993. Address reprint requests to Dr. Ivan G. Otterness, Department of Immunology, Box 820, Pfizer Central Research, 558 Eastern Point Road, Groton, CT 06340.
LPS/knee. Histological and biochemical changes were examined to determine which resolved at
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cationic molecules such as methylated bovine serum albumin (BSA),11 amidated BSA,12 and polylysine,13 which can lodge in the anionic cartilage, produces arthritis. Each of these experimental arthritides has advantages for exploring particular facets of the human disease; none are considered to completely mirror the human disease. All of these models are based on the utilization of animals housed without capability of extended free movement and without adjunctive measurements of mobility. We set out to establish an arthritis model that prominently featured clinical-like measurements of mobility and biochemical and histological measures of disease. We wanted to explore the effect of arthritis on mobility and chose to induce a local LPS arthritis. LPS is not only ubiquitous in the environment but it can be released into portal blood during conditions characterized by hepatic impairment, stress, or shock.14 Its presence has been reported in the blood of patients with arthritis.15,16 Systemic administration of LPS can lead to a transient arthritis in naive rats7,17-19 and, in rats primed with PG-PS, systemic LPS will cause arthritis to flare.19 LPS will also potentiate type 11 collagen arthritis.20 LPS can stimulate the principal cell types involved in inflammatory reactions, ie, lymphocytes, monocytes, macrophages, polymorphonuclear leukocytes, and fibroblasts, through the LPS receptor.21'22 Finally, in vitro, LPS has been shown to directly inhibit cartilage proteoglycan synthesis and to stimulate proteoglycan turnover.23 These results suggest that LPS could play a direct etiological role in some forms of infectious arthritis and a secondary, but perhaps major role, in reactivation and/or maintenance of inflammatory arthritis. In this paper the nature of LPS-induced arthritis in the hamster, its effects on the animals' mobility, and correlations of mobility changes with biochemical and histological changes in cartilage are described.
(80 to 100 mg/kg intraperitoneal) 1 to 2 hours before the start of the dark cycle (5 pm). The treatment groups received intra-articular injections of 20 pl of Salmonella minnesota R595 LPS (RIBI, Hamilton, MT) in saline, saline, or no injection in the synovial cavity of both knees using a 30-gauge needle. After recording body weight, they were returned to their cages. Revolutions of the exercise wheels were continuously recorded (see below). On termination of the experiment, animals were again anesthetized (10 to 11 am) and bled into heparinized tubes (Becton Dickinson, Mountain View, CA). Knee diameters were determined using a constant tension micrometer. The patellae were removed from right knees for biochemical analyses and left knees were taken for histological analysis (see below). Thus, day 0 samples were taken approximately 18 hours after LPS administration.
Mobility Data: Collection and Analysis To monitor hamster running activity and record revolutions per time period, wheels were fitted with magnetic reed switches. A 386/25 MHZ computer was interfaced with a 24-channel I/O board to collect real time data. Wheel turns were recorded automatically in 1-minute bins and cumulative wheel turns per day were recorded. Each animal was recorded for 5 days before intra-articular injection to determine an average normal daily distance (NDD). Fractional daily distance for all experimental days was calculated by dividing the hamster's distance on that day by its mean NDD. Daily data were usually presented as the mean of the fractional distance of all hamsters in the experimental group + SD. Because the hamster is nocturnal, timing of the 24-hour day was started at 12 pm rather than 12 am to complete a full activity cycle within a day. The day of intra-articular injection was defined as day 0.
Materials and Methods Induction of Arthritis
Proteoglycan Measurement
Female golden Syrian hamsters (Mesocricetus auratus) strain LAK.LVG(SYR) were purchased from Charles River Laboratories (Kingston, NY) at 100 to 110 g weight. They were maintained on a 10 to 14 hours light/dark cycle with food and water ad libitum and were acclimatized for at least 1 week in standard housing before studies were initiated. Each hamster was placed in a cage with free access to a 33-cm diameter wheel (Nalge, Rochester, NY) at least 3 days before manipulation. For intra-articular injection, hamsters were anesthetized with sodium pentobarbital
The distal patellar tendon was exposed and severed. The patella, separated from the adjacent muscle and loose connective tissue, was freed by cutting the proximal tendon and held at room temperature in a conical microcentrifuge tube containing 1 ml of RPMI medium supplemented with L-glutamine (2 mM), penicillin (10,000 U/ml), and streptomycin (10,000 pg/ ml) (S-RPMI) until all samples were collected. The individual patellae were removed from the tubes, blotted to remove excess media, and each was placed in a 96-well microtiter plate with the patellar
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cartilage surface facing up in 250 pl of S-RPMI containing 10 pc/ml 35SQ4. After incubation for 3 hours at 37 C in 5% C02 in air, the patellae were removed, washed six times in media, blotted, placed in 3 ml absolute ethanol, and fixed for a minimum of 12 hours. Individual patellae were placed in glass screwcapped vials and partly decalcified in 3 ml of 5% formic acid in water at 37 C for approximately 1 hour. Under a dissecting microscope, the patellar cartilage was peeled from the patella and digested in 12 x 75 mm snap-capped plastic tubes each with 500 pl papain (approximately 26 mg protein/ml, specific activity 21 U/mg of protein; Sigma, St. Louis, MO) diluted 1:100 in 50 mM phosphate buffer, pH 6.5, containing 2 mM EDTA and 2 mM N-acetyl cysteine. Cartilage proteoglycan was released during incubation in a 60 C water bath for 1 hour. Undigested material was removed by centrifugation for 15 minutes at 1000 x g at room temperature, and aliquots were taken for measurement of proteoglycan synthesis and content.
Proteoglycan Synthesis Three hundred microliter samples of the solubilized cartilage was pipetted into scintillation vials, mixed with 5 ml of Ready Safe (Beckman) liquid scintillation cocktail, and counted in a Beta Scintillation Counter set for 35S counting. Counts per minute were compared between test and control groups and the relative rate of sulfate incorporation into proteoglycan was determined. An absolute rate for proteoglycan synthesis was also determined from the specific activity of the 35S04.
Proteoglycan Content Fifty-microliter samples of papain solubilized cartilage were diluted serially in water in a 96-well microtiter plate. Chondroitin sulfate (Sigma) was used as a standard and diluted twofold from 100 to 3.12 pg/ml. The 250 pl of 1.6% 1,9 dimethylmethylene blue29 was added to each well and the absorbances were read immediately at 540 nm on a plate reader (Molecular Devices Corporation, Menlo Park, CA) connected to a Macintosh computer with a program (SOFTmax, Molecular Devices Corporation) for determining a best fit standard curve from the optical density.
radiolabeled rabbit proteoglycan (-10,000 cpm/ tube) in 100 pl of radioimmunoassay (RIA) buffer (10 mM sodium potassium phosphate buffer, pH 7, containing 0.85% NaCI, 0.5% NaN3, and 0.2% RIA grade BSA) was added to 50 pi of unlabeled standard proteoglycan or sample and 50 pl of Fab prepared as described.31 AN9P1 mouse monoclonal antibody to keratan sulfate was diluted in RIA buffer to give -50% maximal binding of 1251-labeled proteoglycan. The mixture was incubated for 1 hour at 37 C. After incubation, 50 pi of hyperimmune rabbit antiserum to mouse Fab (1:250) was added and the mixture was incubated for an additional 1 hour. Twenty-five microliters of nonimmune rabbit serum (diluted 1:16 in RIA buffer) was added, followed by 100 pl of an ammonium sulfate-concentrated preparation of pig immunoglobulin to rabbit F(ab')2.30 After overnight incubation at 4 C, 1 ml of RIA buffer was added, the mixtures centrifuged, and pellets were counted. Assays were performed in triplicate. By reference to a standard curve prepared using human adult proteoglycan aggrecan, 32 microgram equivalents of proteoglycan were determined. Samples from a single experiment were assayed together to minimize interassay variation.
Histology Scanning Electron Microscopy The hindlimbs of control and LPS-treated animals were removed and fixed overnight at 4 C in 10% neutral-buffered formalin. The joint cavity of each limb was then carefully opened to expose the femoral condyles. These were then further fixed by immersion for 1 hour in 3% formaldehyde plus 0.1% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.2, 18 C), washed in buffer (15 minutes), then postfixed in 2% OS04 in 0.1 M sodium cacodylate buffer (45 minutes 18 C). After a quick rinse in distilled water, the condyles were rapidly dehydrated with a graded series of ethanol and then chemically dried for 5 minutes with hexamethyldisalizane (Polysciences, Warrington, PA). The dried condyles were mounted on aluminum stubs, coated with a thin layer of gold palladium, and examined in a JEOL 840 scanning electron microscope.
Light Microscopy Keratan Sulfate Assay Keratan sulfate in plasma was measured using a triple antibody method as described except that a monovalent Fab was used instead of IgG,30 1251-
The hindlimbs of control and LPS-treated animals were removed and fixed overnight at 4 C in 10% neutral-buffered formalin. The knees were decalcified for 72 hours in 1:1 mixture of 8 N formic acid and 1 N
Mobility Changes During LPS Arthritis 1101 144. Ao. .7 1/lPIaY 1/99.4 1)1 /4
sodium formate (Kristensen's solution), rinsed for 24 hours with cold tap water, and routinely processed and embedded in Paraplast Plus (Oxford Labware, St. Louis, MO). Great care was used in the mounting of the tissues to ensure that sectioning would begin perpendicular to the medial side of the knee. The 3-p sagittal sections were cut from the medial to lateral side of the medial condyle using a Reichert microtome. The sections were numbered and approximately matched sections from all animals were routinely stained with safranin 0 and fast green and examined in a light microscope.
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Quantitation of Proteoglycan Depletion by Image Analysis Sections were examined in an Olympus microscope using a 1Ox objective connected to an Optimas Image Analyzer System (Bioscan Inc., Seattle, WA). Densitometric measurements of the inverse log of the integrated gray value were made of the noncalcified cartilage of the medial femoral condyle and the tibial growth plate and used to determine the staining intensity per mm2. The values from the tibial growth plate were used as an internal standard to correct for variations in staining intensity due to section thickness. The tibial growth plate values were arbitrarily set to 100% and the values for each articular cartilage were expressed as the relative percentage of tibial growth plate staining. The analysis assumed that the amount of proteoglycan in the growth plate was unaffected by intra-articular injection of LPS. We tested that premise by determining that the absolute intensity of growth plate staining was not significantly different between control and LPS-treated hamsters (data not shown).
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Results Mobility Changes after Intra-articular LPS The hamster is a nocturnal animal and normally begins wheel running activity within an hour after the onset of the dark period. During the 14-hour dark period, the hamster is engaged in wheel running for 7 to 9 hours covering typically a distance of 9 to 14 km/ night. The daily distance traveled by an individual hamster is quite constant from day to day and thus provides a good background against which one can sensitively detect changes in mobility due to arthritis. We first examined whether different dosages of intra-articular LPS would suppress mobility as measured by a decrease in the normal daily distance.
Stimpson et al17 found that in the rat 10 pg of intraarticular Salmonella typhimurium LPS elicited an arthritis that resolved in approximately 7 days. To determine whether LPS induced arthritis in hamsters, we examined a dose range of 0.1 to 100 pg of S. minnesota LPS for each knee. For each hamster, a normal daily distance was determined as the average daily distance of 5 consecutive days. The averaged normal daily distance for the 24 hamsters was 1 1.3 ± 1.3 (SD) kilometers per day. For each experimental day beginning with the day of intra-articular injection, a fractional daily distance was determined by dividing the daily distance for that hamster by its normal preinjection daily distance. For each group of hamsters, a
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Figure 3. Time sequence of the proteoglycan depletion effect of 1 yg of LPS on the articular surfiace of the medialfemoral condyle. A: Control knee 96 hours after saline injection. There was an intense staining of the cartilage proteoglyvan (arrows) doun to the tidemark. B: 48 hours after LPS injection. In the articular cartilage (arrows) there was a moderate loss of proteoglyvan down to the tidemark. C: 96 houirs after LPS injection. Although the proteoglycan loss from the femoral condyle became ver extensive (arrows) at this time, there was little to no apparent loss ofproteoglycan from the tibial articular cartilage. D: 168 hoturs after LPS injection. The near total depletion of proteoglvcan from the articuilar surface (arrows) and a celluilar infiltrate within the syvnovtim can be seen at this time. Magnification: X 125 (safraniin 0 and fast green).
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DAYS Figure 4. Kinetics of proteoglyvan loss from the nonicalcified cartilage of the central area of the medialfemoral condyle. IPS ( 1 kig/knee joint) was given and 18 hours later (day 0) and on subsequent daIs knee joints were taken, fixed and stained uith safranin 0 and fast green. The relative proteoglyvan content was determinedfrom the safranin 0 absorption by a microscopic optical densitometer and section thickness normalized to the staining intensity of the grouth plate. Typically, normnal noncalcified cartilage in this region stains with approximately 75% of the intensity of the growth.
mean fractional daily distance and its standard deviation was calculated; the results are shown in Figure 1. The saline-injected control group showed a reduction in activity during recovery from anesthesia, which
is unrelated to intra-articular injection but nonetheless led to a suppression of daily distance. At the lowest dose of LPS (0.1 pg/knee), a much more substantial and protracted suppression of mobility was found than in the control group; only 10% of normal daily distance was traveled the night after LPS injection. On days 2 and 3, there was a progressive return toward the normal daily distance. By day 4, the daily distance had returned to control values. At 1 pg, the depression in daily distance was even more pronounced on day 1. Recovery to normal daily distance was slower and still incomplete on day 4. At neither 10 nor 100 pg LPS was running close to being restored to normal within the 5-day experimental period, although with 10-pg amounts there was a significant trend toward restoration of normal daily distance.
Patellar Proteoglycan Content Changes after LPS On the fifth day after LPS injection, the hamsters were killed and the patellar cartilages from the right knees were separated from the patellar bone, adjoining liga-
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1103 AJP May 1994, Vol. 144, No. 5
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Figure 5. Effect of intra-articular LPS on the articular surface of the medialfemoral condyle. The knees of LPS- or saline-treated hamsters were examined by scanning electron microscopy. A: Saline-injected joint. Tbe articular surface was relatively smooth with numerous small indentations (X 200). B: 10 pg LPS-injected joint. The articular surface nouw contains roughened and etched regions (X 200). C: and D: 100 yig LPS-injected joints. Regions can be found where the superficial layers of collagen have been worn away (arrowheads) (x55). At higher magnification (x240) (D), numerous empty chondrocyte lacunae open to the articular surface can be seen in this region. Scale bar = 100 .
ments, and soft tissue and their proteoglycan content was determined. A mean proteoglycan content of 16.4 ± 3.0 SD pg/patella measured as glycosaminoglycan was determined for control knees by the dimethyl methylene blue assay. Intra-articular LPS produced a significant loss of proteoglycan at 0.1 pg/ knee and the loss of proteoglycan was increased at both 1 and 10 pg/knee, although the differences between 1 and 10 pg did not reach significance at P = 0.05 (Figure 2). At 1 pg LPS, the maximum loss of proteoglycan was found to be 19% of the normal content.
Kinetics of LPS-lnduced Histological Changes The kinetics of the changes in proteoglycan content and in inflammation were examined histologically. Groups of six hamsters were killed on days 0, 1, 3, and 6 after administration of 1 pg of LPS. A saline-injected control group was also killed on day 4. The diameter of the control knees was unchanged throughout the experiment (7.1 + 0.2 mm SD). The LPS knee went from control levels to 9.8 ± 0.5 mm on day 1 to 9.1 +
0.5 mm on day 2. On day 5 it remains essentially unchanged at 8.8 ± 0.4 mm, ie, there was no significant change in swelling from day 2 to day 5 even though running normalized over that time period. The control knees were unremarkable with no evidence of inflammation except perhaps the slightest depletion of proteoglycan at the edges of the articular cartilage (Figure 3A). Day 0 (18 hours after LPS injection) there was a slight decrease in the amount of proteoglycan from the condyles compared with controls and no femoral erosion. On day 1, there was even more proteoglycan depletion and there were clear signs of femoral erosion (Figure 3B). By day 3, articular cartilage exhibited extensive proteoglycan depletion. There was femoral erosion and the beginning of frank destruction of the femoral cartilage (Figure 3C). On day 6 after LPS there was evidence of continued proteoglycan loss. In many animals there was complete depletion of the proteoglycan of the noncalcified layer and the impression of some chondrocyte loss (Figure 3D). We also examined the loss of proteoglycan by safranin 0 staining of the medial femoral condyle quantitatively by image analysis. No changes were found in the staining of the calcified cartilage beneath the
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tide mark. Because the calcified cartilage represents approximately 55% of the hamster condylar cartilage, its inclusion would greatly decrease the sensitivity of the measurements of change and so measurements were recorded on only the noncalcified cartilage. The amount of safranin 0 staining in the noncalcified cartilage was normalized to the tibial growth plate and relative amounts of proteoglycan determined as changes in intensity of staining. A steady decrease in staining of the femoral condyle was observed (Figure 4) compared with the control non-LPS-treated group (72% of growth plate intensity). Beginning on day 1 after LPS, the loss of staining continued through day 6 when an intensity of only 38% of growth plate staining, ie, 47% loss of proteoglycan staining in the noncalcified cartilage, was seen. For comparison, we also measured the total proteoglycan in the patella (calcified and noncalcified layers) by the DMMB assay. On day 6, an 18% loss of proteoglycan in the patellar cartilage (16.6 + 1.3 pg in controls to 13.3 ± 0.3 pg in the LPS group; n = 6 hamsters/group) was measured.
Scanning Electron Microscopy on Day 5 after LPS In controls and at the two lower dosages of LPS (0.1 and 1.0 pg), a smooth articular surface was observed with indentations reflecting the presence of superficial chondrocytes (Figure 5A). At 10 pg LPS, some etching of the cartilage surface was observed (Figure 5B), whereas at the highest level of LPS (100 pg) ar-
eas of frank ulceration of the articular cartilage surface were seen and there was debris on the cartilage surface (Figure 5C). In these regions, the chondrocytes were unroofed and numerous open, probably empty, chondrocyte lacunae were seen (Figure 5D). Commonly, the most extensive changes were observed at the lateral edges of the condylar articular surface whether the changes were recorded as loss of surface congruity, deposition of superficial materials, or empty lacunae.
Kinetics of Changes in LPS-lnduced Proteoglycan Synthesis The incorporation of 35SO4 was used to estimate changes in the synthesis of proteoglycans. We estimated an overall synthetic rate of 31 + 1.2 pg/hour/g cartilage in controls. Because the rate of synthesis was determined ex vivo with a total sulfate concentration of 0.5 mM, sulfate and other factors may be limiting; therefore this should be considered only a relative estimate of the true synthetic rate. Eighteen hours after injection of 1 pg of LPS, proteoglycan synthesis was already significantly suppressed (Figure 6). A maximum suppression of 55% was observed on the second day after LPS. Thereafter, the rate of synthesis began to increase and by day 6 it was enhanced well above normal levels. This enhancement has been consistently observed in repeated experiments. There also appears to be a correlation between the depression in proteoglycan synthesis and the degree of mobility inhibition (r2 = 0.52). Thus, peak suppression of mobility was on day 0 and peak suppression of synthesis was on day 1. Both synthesis and mobility were restored to normal before the end of the experi-
mental period.
Kinetics of Plasma Keratan Sulfate
Changes Immunologically detected plasma keratan sulfate has been assumed to reflect articular cartilage proteoglycan metabolism within the arthritic joint.33 We, therefore, analyzed the levels of plasma keratan sulfate in our kinetic studies. There was a significant elevation of the plasma keratan sulfate 18 hours after intraarticular injection of 1 pg LPS (Figure 7). Levels rose from a mean of 0.37 pg/ml to 0.91 pg/mI. But by 42 hours, plasma levels had essentially returned to normal. No further significant changes in keratan sulfate were observed.
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Discussion Impairment of joint function is one of the primary reawhy arthritis patients seek medical care. Yet correlations of joint function with biochemical and histological changes during arthritic disease are lacking. Animal models that could ideally allow close examination of the relationship between joint function and disease have not previously been established. We, therefore, elected to study arthritis in the hamster, a species known for its volitional wheel running, to examine the relationship between joint function (measured indirectly as mobility) and parameters of arthritis disease. A local arthritis induced by intra-articular LPS appeared to have several distinct advantages for our initial studies. Unlike adjuvant arthritis, where systemic disease would complicate interpretation of mobility changes, a local arthritis should give a more direct relationship between changes in mobility and changes in joint biochemistry and histology. In addition, at least in the rat, the LPS appeared to induce an immediate arthritis of 6 to 7 days duration.19 This seemed a reasonable time period to evaluate changes in mobility against other parameters of arthritis. A sustained suppression of daily distance was in fact observed with LPS that increased in duration and extent of suppression with increasing dose of LPS. At the higher dosages of LPS (10 and 100 pg/ knee joint), daily distance was not restored to normal over 7 days. At all dosages of LPS (0.1 to 100 pg/ml), we failed to observe a restoration of cartilage proteoglycan content in the hamster within a 7-day period, although over a several week period after 0.1 and 1.0 pg LPS proteoglycan was restored. This consons
trasts with the rat, where it was reported that at dosages of 10 and 100 pg of LPS intra-articularly the joint swelling and histology returned to normal within 6 to 7 days.19 The increased severity of arthritis in the hamster could be attributable to greater joint use in the hamster made possible by the presence of the wheel or it could be due to a species difference. Recent studies indicate that only part of the differences can be attributed to the added effect of wheel running. (K. Shay, M. L. Bliven, D. N. Scampoli, I. G. Otterness, A. J. Milici, submitted for publication) The decrease in proteoglycan content of patellar cartilage with increasing amounts of LPS was dose responsive. Proteoglycan loss was maintained for at least 6 days after administration of LPS. Despite this, at the lower dosages of LPS (0.1 and 1.0 pg) there was a full restoration of mobility to control levels on day 4. This demonstrates that proteoglycan loss cannot be the cause of inhibition of mobility. Similarly, in an interleukin-1-induced arthritis, the acute mobility inhibition is only associated with intra-articular interleukin-1 and is independent of the degree of pro-
teoglycan loss.* One of the more remarkable findings was the rapidity with which destruction of the surface layer of cartilage occurred after 100 pg of LPS. Empty lacunae were found particularly near the edges of the condyles. This suggests that either the LPS concentration or the LPS-induced destructive activity is higher at the edges. This could mean the LPS accumulates in the intercondylar and synovial lining areas or that the cartilage at the edges, which is not well covered by the meniscus, is more susceptible to damage. Because LPS itself has no enzymatic activity, this result may reflect the capacity of LPS to stimulate the production and release of proteolytic enzymes, including the metalloproteinases collagenase, stromelysin, and gelatinase. Jasin et al,23 Morales and Haskell,22 24 27 Hubbard et al,25 and Rednar et al26 have shown the ability of LPS to cause proteoglycan depletion in cartilage pieces. Our histological data suggests that in vivo exposure of articular cartilage to high amounts of LPS can be catastrophic. At lower amounts, a graded depletion of proteoglycan was found. The amounts of proteoglycan lost after LPS injection were determined by different methods in the medial femoral condyle and the patella. In the patella, the total cartilage proteoglycan content was determined biochemically 5 days after LPS. This revealed a dosedependent loss of proteoglycan. The determined Otterness IG, Bliven ML, Milici AJ: Changes in mobility of the golden hamster during an IL-1-induced arthritis. Mediators Inflamm (in press)
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value includes the proteoglycan of both the calcified and the noncalcified cartilage layers. Kirviranta et al33 have previously shown that safranin 0 staining is proportional to the amount of proteoglycan. This allowed us to use a densitometric determination of cartilage proteoglycan loss for the femoral condyle based solely on the safranin 0 staining of the central portion of the noncalcified cartilage layer in histological section. Calculating the percentage of overall proteoglycan loss (47% loss of staining) and combining it with our measured area of the noncalcified cartilage (45% of the cartilage was noncalcified) gives a value of 21% for overall loss of proteoglycan after 1 pg LPS. This compares favorably with the overall loss calculated for the patella after 1 pg LPS, ie, 18%. Another interesting result was obtained when the kinetics of keratan sulfate release into plasma was determined. A significant elevation of keratan sulfate was found in the plasma on day 0 (18 hours after LPS administration). If keratan sulfate correlated with the kinetics of loss in the femoral condyle, a similar elevation would have been expected on days 2 through 6, with the maximum loss on day 0 and an intermediate level of loss on day 1. Keratan sulfate is found mainly on small aggregating degradation products of aggrecan in human cartilage35 but it is present in fragments of aggrecan in synovial fluid.30 These are found further reduced in size in serum.33 The timing of keratan sulfate release into the plasma may reflect the early loss of the smaller keratan sulfate-rich molecules. It is also likely that the maximum permeability/ leakage of high molecular weight materials occurs in the first 24 hours after LPS injection, and, thereafter, the joint capsule returns to a more normal level of permeability. Thus, it appears that in this model, the high early keratan sulfate levels in plasma are more likely to be determined by enhanced permeability and smaller keratan sulfate size rather than only by an enhanced degradation rate for keratan sulfate molecules. It is apparent that there are factors other than simple loss of keratan sulfate from cartilage that must be considered to account for the elevation of keratan sulfate in plasma. Proteoglycan synthesis during this period was considerably suppressed by exposure to LPS. This would be expected from in vitro studies of the effect of LPS on cartilage.23 28 Suppression was maximal at day 1 when plasma keratan sulfate levels had returned to normal and synthesis remained suppressed through day 3. By day 6, proteoglycan synthesis had risen above normal levels suggesting that a repair response had begun. We have found that under some circumstances effects on synthesis and degradation are disassociated and that proteoglycan synthesis is
restored while proteoglycan degradation continues. This would appear to be the case here. Although proteoglycan synthesis has been enhanced, within 7 days there was no restoration of proteoglycan levels in the patella or femoral condyle, though we do know that with time the proteoglycan content as measured by dimethylmethylene blue is restored (unpublished observations). It is known that interleukin-1 causes suppression of proteoglycan synthesis in vitro36,37 and we previously found that treatment with antiinterleukin-1 antibody restored synthesis of proteoglycans in mBSA arthritis,38 neither anti-tumor necrosis factor nor anti-interleukin-6 displayed similar activity. Thus, local production of IL-1 is a primary candidate for causing inhibition of proteoglycan synthesis. Matsukawa et al10 have shown using the interleukin-1 receptor antagonist in the rabbit that interleukin-1 makes a major contribution to cartilage proteoglycan loss in LPS arthritis. When we examined the relationship between decreased mobility and cartilage parameters of arthritic disease, no correlation was observed between cartilage proteoglycan depletion or plasma keratan sulfate. If pain is a major cause of limitation of mobility, this result is not surprising, because the cartilage itself is not innervated. Because the synovial lining is innervated39'40 it could provide sensory input that limits mobility. Nonetheless, at the time of restoration of mobility, the synovial lining and surrounding soft tissue were still edematous and filled with inflammatory cells, suggesting that edema and cell infiltration per se were not the primary determinants of mobility inhibition. Surprisingly, there was a relationship between loss and restoration of mobility and loss and restoration of chondrocyte proteoglycan synthetic function. This could mean that mediators such as cytokine interleukin-1, involved in suppression of proteoglycan synthesis, are temporally expressed with mediators involved in suppression of mobility. Interleukin-1 could also play a direct role in suppression of mobility. We have found interleukin-1 to suppress movement in rats housed in an activity cage with continuous recording of movement41,42 to cause limping after intra-articular injection43 and to suppress running in the hamster.* Regardless this animal model provides an approach for identification of those mediators involved in limitation of mobility.
Acknowledgments We thank William Peterson for help with the computer interface, Anne Shay and Debra Scampoli for histological analysis, and Mirela lonescu for the HA assays.
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