American Journal ofPathology, Vol. 137, No. 5, November 1990 Copyright K American Association ofPathologists
Pathogenesis of Antigen-induced Arthritis in Mice Deficient in Neutrophil Elastase and Cathepsin G Roy Pettipher,* Jo Edwards,t Simon Cruwys,* Eileen Jessup,* Julian Beesley,* and Brian Henderson* From the Department of Pharmacology,* Wellcome
Research Laboratories, Beckenham, Kent; and the Department of Rheumatology Research,t University College and Middlesex School of Medicine,
London, United Kingdom
The contribution of neutrophil-derived elastase and
cathepsin G to joint pathology has been examined in immune arthritis in the mouse. Neutrophilsfrom beige mice are genetically deficient in lysosomal elastase and cathepsin G, but have normal levels of the acid hydrolases, p-glucuronidase, and N-acetyl-f3-glucosaminidase. The development of antigeninduced arthritis in normal mice has been compared with that in beige mice. The pattern of synovitis (both leukocyte accumulation and plasma leakage) were indistinguishable in normal and beige mice. Cartilage proteoglycan depletion was quantified by measuring the decrease in safranin 0 staining intensity, and this, too, was unaltered in mice lacking elastase and cathepsin G. These results suggest that neutrophil elastase and cathepsin G do not contribute to these aspects of joint pathology in antigen-induced arthritis in the mouse. (Am J Pathol 1990; 13 7:1077-1082)
Joint swelling and degradation of articular cartilage are characteristic features of inflammatory joint diseases such as rheumatoid arthritis (RA).1 It has been suggested that lysosomal proteases, such as elastase and cathepsin G, contribute to joint pathology in arthritis, as they can degrade both vascular basement membrane2 and proteoglycan3 at physiologic pH and thereby cause intraarticular edema and cartilage destruction, respectively. Consequently it is of interest to investigate the involvement of these neutrophil-derived neutral proteases in the pathogenesis of chronic arthritis. We have chosen an immunologically induced arthritis in the mouse because this
model closely resembles RA in histopathology, chronicity, and response to drug treatment.4' The development of chronic arthritis in C57 mice has been compared with that in congenic C57 bg/bg beige (Chediak-Higashi) mice, which are selectively deficient in neutral elastase and cathepsin G.7 This strain of mouse has been used by several investigators to examine the role of neutrophil-derived neutral proteases in experimental pathology. In the reversed passive Arthus reaction in the skin,e in experimental alveolitis,9 and in zymosan-induced arthritis,10 no difference in tissue damage was observed between normal and beige mice. However, in a model of autoimmune glomerulonephritis, where mice are passively immunized against glomerular basement membrane, albuminuria was almost absent in beige mice.'1 This indicates that elastase and cathepsin G may play a role in tissue damage associated with some autoimmune reactions. Consequently we have investigated the role of elastase and cathepsin G in a model of arthritis in which the mice are actively sensitized to the challenging antigen. The accumulation of polymorphonuclear and mononuclear cells in the synovial tissue was quantified histologically and increased vascular permeability measured by uptake of technetium-99 (9Tc). To assess the degree of proteoglycan depletion, longitudinal sections of mouse knees were stained with safranin 0, which is a stoichiometric stain for sulfated glycosaminoglycans, the oligosaccharide components of proteoglycans.12 The content of proteoglycan then was assessed by measuring the absorbance through the articular cartilage with a scanning and integrating microdensitometer.
Materials and Methods Animals Male normal C57 and beige C57 (bg/bg) mice were obtained from Harlan OLAC Limited, United Kingdom. Mice Accepted for publication June 14, 1990. Address reprint requests to Dr. Roy Pettipher, Department of Immunology and Infectious Diseases, Pfizer Central Research, Groton, CT 06340.
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were fed standard chow and tap water ad libitum. Animals were aged between 8 and 10 weeks at the start of the experiments.
Materials Freund's complete and incomplete adjuvants were obtained from Sigma Chemical Co., Poole, Dorset, UK. Heatkilled Mycobacteria tuberculosis was a gift from the Central Veterinary Laboratories, Weybridge. Bordetella pertussis was obtained from Wellcome Research Laboratories (Beckenham, Kent, UK). Methylated bovine serum albumin (m-BSA) was obtained from Sigma. Safranin 0 was obtained from BDH; Technetium-99 (17 MCi/mg) was obtained from Amersham. Colorimetric substrates for figlucuronidase and N-acetyl-p-glucosaminidase were obtained from Sigma. Porcine pancreatic elastase was obtained from Sigma. [3H]-casein was prepared by reacting soluble casein (Sigma) with [3H]-acetic anhydride (Amersham, 500 mCi/mmole).
Preparation of Mouse Peritoneal Neutrophils Neutrophils were harvested from the peritoneal cavities of normal or beige mice 24 hours after injection of Freund's incomplete adjuvant (0.25 ml). Cells (90% neutrophils) were lysed in 0.1 % Triton X in saline at a concentration of 107/ml.
Enzyme Assays Neutral Protease Assay Cell lysates (100 ,Iu) were incubated with 250 Al 0.1 mol/l (molar) TRIS buffer, pH 7.5, containing 1 mmol/l (millimolar) CaCI2 and 200 Mug [3H]-casein. After 2 hours, the reaction was terminated with 30% trichloroacetic acid (50 ,l) and the tubes centrifuged at 1 0,000g for 10 minutes. An aliquot of the supernatant (100 Ml) was removed and counted in a liquid scintillation spectrometer. A standard curve for elastase (10 to 1000 ng/tube) was performed in the same assay. Results were expressed in terms of elastase equivalents per 107 cells.
,3-glucuronidase assay Cell lysate (100 MIu) was incubated with 0.8 mg/ml phenolphthalein glucuronide in 0.05 mol/l acetate buffer (pH 4.5) for 16 hours at 37°C. The reaction was terminated with 1 mol/l sodium hydroxide/glycine and absorbance measured at 540 nm.
N-acetyl-H-glucosaminidase Assay Cell lysate (100 ,l) was incubated with 100 ,ul 0.1 mol/ citrate-phosphate buffer (pH 4.5) containing 1.5 mg/ml p-nitrophenyl-N-acetyl-3-glucosaminide for 2 hours at 37°C. The reaction was terminated with 100 ,l 1 mol/l sodium hydroxide/glycine and the absorbance measured at 405 nm.
Induction of Arthritis Arthritis was induced by a modification of the method of Brackertz et al.4 All injections were performed under ether anaesthesia. Mice were sensitized on the first occasion by multiple intradermal injections of 0.2 ml Freund's complete adjuvant (containing 0.2 mg m-BSA and supplemented with 0.2 mg heat-killed Mycobacterium tuberculosis). On the same occasion mice also received an intraperitoneal injection of 2 X 109 Bordetella pertussis organisms in 0.2 ml saline. Two weeks later, mice received further multiple intradermal injections of 0.2 ml Freund's complete adjuvant containing 0.2 mg m-BSA. Arthritis then was induced 5 days later by the intra-articular injection of 100 jig m-BSA in 50 ,ul saline in one knee.
Measure of Joint Inflammation Leakage of plasma into knee joints was measured by a modification of the method of Kruijsen et al.'3 Mice were injected intraperitoneally with 10 MuCi 9Tc and were killed 30 minutes later by cervical dislocation. The knee joints were cut off and the radioactivity in both control and inflamed joints measured in a gamma-counter. The degree of inflammation is expressed as the percent increase in 9Tc uptake in inflamed joints compared with control.
Histologic Quantitation of Leukocyte Infiltration and Synovial Cell Proliferation Inflamed and the contralateral control knee joints were fixed in neutral buffered formalin and decalcified in Kristensen's fluid. Tissues were dehydrated in a graded series of ethanol and were double-embedded in 1% celloidin in methyl benzoate and paraffin wax. Sections (3 M) were cut and stained with hematoxylin and eosin (H&E). The sections then were randomized and assessed in coded form using a modification of the method used by Edwards et al14 for rabbit knee joints. The number of total cells, neutrophils, and lymphocytes per 250 2 of synovial tissue section were counted in 10 fields.
Elastase and Cathepsin G in Arthritis
AJP November 1990, Vol. 13 7, No. 5
with Mayer's Haematoxylin before dehydrating, clearing, and mounting in DPX. The absorbance at 550 nm through areas of staining intensity were measured with a Vickers M85a scanning and integrating microdensitometer. The mask size was chosen to cover the outer half of the articular cartilage, and 10 measurements were made on the articular cartilage of the femoral head in each section. A measurement was made through the growth plate, which served as an internal standard to control for possible variations in section thickness and staining. A measurement was also made through the bone matrix, and this was subtracted from both the value for the articular cartilage and growth plate to control for nonspecific staining.
IA 0 =
Lysosomal Enzyme Content of Neutrophils
Lysates of normal mouse peritoneal neutrophils contained high levels of neutral protease activity equivalent to more than 600 ng elastase per 1 07 cells (Figure 1). This neutral protease activity was largely absent in peritoneal neutrophils from beige mice, although the levels of the acid hydrolases, N-acetyl-,B-glucosaminidase and 3-glucuronidase, were similar in neutrophils from normal and beige mice (Figure 1).
2 ' 0.1
Leukocyte Infiltration in the Synovial Tissue of Mice During the Development of Antigen-induced Arthritis
Figure 1. The upper panel illustrates the levels of neutral protease activity (expressed as human elastase equivalents) in lysates ofperitoneal neutrophilsfrom normal (open columns) or beige mice (hatched columns). The middle panel illustrates the levels of N-acetyl-f3-glucosaminidase in lysates ofperitoneal neutrophils from normal (open columns) or beige mice (hatched columns). The lower panel illustrates the levels of f3glucuronidase in lysates ofperitoneal neutrophilsfrom normal (open columns) or beige mice (hatched columns). Each point is the mean ± SEM ofdata from six animals. (* P < 0.01 compared with levels in neutrophilsfrom normal mice).
Histochemical Quantitation of Proteoglycan Depletion from Articular Cartilage Longitudinal sections (3 ,) of inflamed and contralateral control knees were prepared as before. The sections were dewaxed and stained for 10 minutes with 0.5% Safranin 0 in 50% ethanol. They were rinsed and counterstained
At day 1 after antigen challenge, the synovial lining was infiltrated with large numbers of neutrophils both in normal mice and in beige mice (Figure 2). The level of neutrophil accumulation declined progressively at day 7 through day 14. However at all times total cell numbers in synovial tissues were increased fourfold to fivefold compared with control levels, with an increasing proportion of large mononuclear cells (Figure 2). These are presumed to comprise a mixture of infiltrating macrophages and resident synovial cells. Surprisingly, at all times, lymphocyte numbers were low in the inflamed synovial tissues. Lymphocytes were not detectable at day 1, while at day 7 there were 3 ± 1 lymphocytes per 82 in normal mice, compared with 3 ± 1 lymphocytes per t2 in beige. This increased slightly at day 14, 10 ± 3 cells per '2 in normal mice compared with 8 ± 1 cells per #2 mice. There was no discernible difference in the histologic pattern of leukocyte infiltration during the development of antigen-induced arthritis in normal and beige mice.
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1 14 7 DURATION OF ARTHRITIS (DAYS) Figure 2. Histologic quantitation of total cells (open columns) and neutrophils (shaded columns) in the synovial tissues of normal (A) or beige (B) mice during the development ofantigeninduced arthritis. Closed circle with bars represents the total number of cells in a normal noninflamed joint. Each point is the mean ± SEM of data from eight animals.
Plasma Leakage into the Joints of Mice During the Development of Antigen-induced Arthritis At day 1 after antigen challenge, inflamed joints of normal mice displayed high plasma leakage (as measured by 99Tc uptake compared with the contralateral noninflamed joints (Figure 3). As the disease progressed to the chronic phase, plasma leakage was less apparent, which suggests that the intra-articular edema correlates with the neutrophil infiltration. The pattern of vascular permeability changes were not significantly different in beige mice.
Histochemical Analysis of Articular Proteoglycan Content in Mice During the Development of Antigen-induced Arthritis The growth plate was used as an internal standard to control for variations in section thickness and staining intensity. The absorbance through the growth plate did not vary in control or arthritic joints in normal or beige mice at any time (Table 1). The absorbance measured through the articular cartilage of noninflamed joints also did not vary significantly in normal or beige mice (Figure 4). However, the cartilage from inflamed joints showed a reduction in staining intensity in the superficial/mid-zones. The reduction in Safranin 0-staining was not statistically significant at day 1, but there were highly significant reductions at day 7 and 14 (Figure 4). Both normal and beige mice demonstrated a similar reduction in Safranin 0-staining intensity.
Lysosomal enzymes derived from neutrophils are thought to contribute to inflammation and tissue injury in arthritis.15 In particular, the proteases, elastase and cathepsin G, are active at neutral pH and can degrade vascular basement membrane and cartilage proteoglycan. This suggests that these enzymes may be responsible for the increased vascular permeability and cartilage damage that occurs in inflamed joints. To test this hypothesis, we have examined the pathogenesis of antigen-induced arthritis in mice genetically deficient in elastase and cathepsin G7. In the present study, we have confirmed that neutrophils from beige mice are selectively deficient in lysosomal neutral protease activity, but have normal levels of acid
hydrolases.78 Furthermore neutrophils from beige mice have the capacity to produce reactive oxygen species in comparable amounts to normal neutrophils.10 The present study demonstrates that the pattern of synovitis was very similar in normal and beige mice. The synovial tissue was infiltrated with high numbers of neutrophils in the first 24 hours after antigen challenge, but by 1 to 2 weeks an increase in mononuclear cell numbers was the prominent histologic feature. These mononuclear cells were predominantly macrophages and, surprisingly, very few lymphocytes were apparent in the synovial lining of either strain of mouse, in contrast to the large numbers of lymphocytes and plasma cells seen in the equivalent rabbit model.14 c)
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