Annals ofthe Rheumatic Diseases 1990; 49:607-610

Antigen induced arthritis in beige(Chediak-Higashi) mice

Joost Schalkwijk, Leo A B Joosten, Wim B van den Berg, Levinus B A van de Putte

AbstractMice with the beige mutation, which are

known to be deficient for leucocyte elastaseand cathepsin G, were used to investigate therole of neutral proteases in a model forantigen induced arthritis. Surprisingly, it wasshown that in this model of arthritis, usingmethylated bovine serum albumin as an

antigen, C57/black/6 'beige' mice (deficientfor leucocyte neutral proteases) developed a

more severe form of arthritis than the controlmice ('black' mice), resulting in a higherdegree of tissue damage. The incidence anddegree of bone apposition and destruction ofarticular cartilage at day 21 after induction ofarthritis were significantly higher in the beigemice. These findings could not be ascribed todifferences in the cellular immune response tomethylated bovine serum albumin. Auto-radiographic detection ofradiolabelied methy-lated bovine serum albumin suggested thatmore antigen is retained in the joints of beigemice than in black mice, which might accountfor the sustained arthritis and the concomitanttissue damage. These findings do not supportthe contention that leucocyte elastase andcathepsin G contribute to the pathogenesis ofjoint destruction in this model.

Department ofRheumatology,University HospitalSt Radboud, GeertGrooteplein Zuid 8,6500 BH Nijmegen,The NetherlandsJ SchalkwijkL A B JoostenW B van den BergL B A van de PutteCorrespondence to:Dr J Schalkwijk,Department of Dermatology,University Hospital,Veegerkliniek,Javastraat 104, Nijmegen,The Netherlands.Accepted for publication21 September 1989

Recently, a mutant mouse has been describedwhich is deficient for polymorphonuclear leuco-cyte elastase and cathepsin G.' This mutation,the so-called 'beige' gene, represents a putativemodel for the Chediak-Higashi syndrome inhumans, with which it has several features incommon, such as the giant lysosomes and theneutral protease deficiency. The beige mouse istherefore a suitable model for investigating therole of neutral proteases in various inflam-matory conditions. We have recently describedthe effect of experimental arthritis, using themodel of zymosan induced arthritis, in beigemice.2 It was found that although in vitro beigepolymorphonuclear leucocytes caused lesscartilage degradation, the tissue damage result-ing from inflammation was not significantlydifferent from that in the black mice. It was

therefore concluded that polymorphonuclearleucocyte elastase and cathepsin G were not themain mediators of cartilage breakdown in thismodel. Because in a glomerulonephritis modelbased on immunological mechanisms we foundsignificant differences between beige and blackmice3 we sought to compare the effect of an

immunologicallymediated experimental arthritison beige and black mice. We therefore chose themodel of allergic arthritis which uses methy-

lated bovine serum albumin as an antigen, asdescribed before.4 We have previously shownthat part of the inflammatory reaction in thismodel, at least in the acute phase, is dependenton hydrogen peroxide generated by activatedinflammatory cells.5 As in the zymosan inducedarthritis model, the findings in this study do notsupport a role for neutral proteases.

Materials and methodsANIMALSMale, normal (black) C57B1/6 and beige C57B1/6(bg/bg) mice were obtained from Harlan Olac,UK. The animals (aged 10-16 weeks) were fed astandard diet and tap water freely.

CHEMICALSMethylated bovine serum albumin was obtainedfrom Sigma Chemicals Co, USA. Methoxy-succinyl-alanyl-alanyl-prolyl-valyl-aminomethyl-coumarin (MAAPV-AMC) was obtained fromBachem, Bubendorf, Switzerland. 1251 wasobtained from Amersham, Bucks, UK.

ELASTASE ASSAYElastase was used as a marker enzyme tocompare beige and black polymorphonuclearleucocytes. This enzyme was assayed aspreviously described using the fluorogenic sub-strate MAAPV-AMC.6

INDUCTION OF ARTHRITISjoint inflammation was induced as describedpreviously.4 Briefly, mice were immunised withmethylated bovine serum albumin in completeFreund's adjuvant, and arthritis was induced byinjection of 60 ,tg of the antigen in the rightknee joint three weeks after the primaryimmunisation. This type of inflammation isdependent onT lymphocytes and is characterisedby an infiltrate rich in polymorphonuclearleucocytes in the acute phase (first week) and amononuclear infiltrate in the chronic phase (upto four weeks). The immune status of theanimals (delayed type hypersensitivity againstmethylated bovineserumalbumin) wasmeasuredby skin testing. Antigen (10 ,ug) was injected inthe right ear and the swelling after 24 and 48hours was measured with a caliper.4

MEASUREMENT OF ARTHRITISJoint inflammation was measured at days 2, 5,7, and 21 by the technetium-99m method.7

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Schalkwnik,Jloosten, van den Berg, van de Putte

Briefly, the accumulation of 99nTc is measuredin the right (R) and left (L) control knee jointand expressed as a ratio (R/L). This ratio istaken as a measure for joint swelling. Ratiosabove 1-1 were considered to indicate inflam-mation of the right knee joint.

RETENTION OF RADIOLABELLED METHYLATEDBOVINE SERUM ALBUMINMethylated bovine serum albumin was radio-labelled by the chloramine T method.8 Toinvestigate the effect of arthritis on the clearanceof antigen from the inflamed joint in the beigeand normal mice radiolabelled antigen (37 kBq)was injected, and the retained antigen wasmonitored by external gammacounting or byautoradiography. Previous studies have indi-cated that this method does follow the fate ofthe injected antigen and that the measuredradiolabel actually represents immunoreactivemethylated bovine serum albumin.8

HISTOLOGY AND AUTORADIOGRAPHYMouse knee joints (six to eight in each group ateach timepoint) were processed for histologyand autoradiography.9 A histological examin-ation was made on days 2, 5, and 21. Arthritiswas scored on serial sections (haematoxylin andeosin staining). Infiltration, cartilage damage,and bone apposition were scored semiquanti-tatively.'° Antigen retention was visualised byautoradiography of 1251 labelled methylatedbovine serum albumin, and was scored on a fourpoint scale. All histological examinations wereperformed 'blindly' by two observers.

ResultsMEASUREMENT OF EXPERIMENTAL ARTHRITIS INBEIGE AND BLACK MICEBlack and beige mice were immunised withmethylated bovine serum albumin in completeFreund's adjuvant. No differences in delayedtype hypersensitivity (skin testing) were foundbetween the two strains (data not shown).Arthritis was induced by the intra-articularinjection of 60 [ig of antigen in saline. Table 1shows the time course of arthritis as assessed by99'Tc uptake, which is taken as a measure ofjoint oedema. At days 7 and 21 9'Tc uptakewas significantly higher in the beige (neutralprotease deficient) mice than in the black mice,indicating that vascular effects of inflammationwere prominent in the beige mice. For all

Table 1: Time course of arthritis in beige and black mice.Values are means (SD)

Day Ratio of 'Tc uptake

Beige mice Black mice

2 2-16 (0 30) 2-37 (0-35) NS5 1-84 (0-19) 1-58 (0-28) NS7 1-80 (0-11) 1-38 (0-13)*

21 1-27 (0-10) 1-03 (0-10)*

Seven to eight mice a group were injected intra-articularly with60 KAg of methylated bovine serum albumin in the right knee jointat day 0. Arthritis was measured at several intervals by thetechnetium uptake method (ratio of right and left knee joint).*p<0-001, Wilcoxon rank sum test.

experiments the elastase content of poly-morphonuclear leucocytes from both strainswas measured2 as a routine check of the neutralproteinase deficiency. For the groups of miceused in these studies the elastase content ofbeige mice was always found to be less than 10%of that of the black mice (data not shown).

HISTOLOGY OF ARTHRITISHistological examination of the joints showedthat the amount of cellular infiltrate in the beigemice was higher throughout the inflammatoryresponse, though it only reached statisticalsignificance at day 5 (table 2). Figure 1 showsthat the infiltrate and exudate were predomi-

Table 2: Histological examination of arthritis in beige andblack mice. Values are means (SD)

Day Mice Infiltrate Cartilage Bonedamage apposition

2 beige 1-2 (04) ND NDblack 1-3 (0-5) ND ND

5 beige 3-0 (0-1) ND NDblack 1-9 (0-5)* ND ND

21 beige 1-4(05) 2-6(08) 2-4(08)black 1-1 (0-7) 1-3 (1-0)t 10 (1 O)f

Seven to eight mice a group were injected with 60 ,ug of methy-lated bovine serum albumin at day 0. At several intervals animalswere killed for histological examination. The amount of cellularinfiltrate in the synovium and periosteum was scored semi-quantitatively on serial sections at days 2, 5, and 21. Cartilagedamage and bone apposition was only present in the chronicphase of arthritis (day 21). The histological grading is given on afour point scale in proportion to severity.*p<0)005; tp<001; fp<0-02, Wilcoxon rank sum test.

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Figure 1: Arthritic kneejoint ofC57B1/6 beige mice at day 2after induction ofinflammatio. Note the exudate in thejointspace and the cellular infiltration ofthe synoin (mainlypolymorphonuclearleucocytes). P=patella; F=femur;S=synovium. (Haematoxylin and eosin staining.) Bar= 100ym.

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Antigen induced arthritis in beige mice

nantly composed of polymorphonuclicytes in the acute phase. At dayinflammation was waning, a significaence in irreversible end stage joint dwas seen between the two strains (tabbeige mice then showed consideracartilage destruction and appositionformed bone than the black mice. Figi3 show the extent of joint deformationfrom this type of inflammation in

Figure 2:Joint ofaC57Bll6 beige mouse at day21 after induction ofarthritis. Extensive cartilagedamage is visible in patellaandfemur. Large areas aredevoid ofintact chondrocytes(arrows). P=patella;F=femur; (Haematoxylinand eosin staining.)Bar= 100 Msm.

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mice: total destruction of articular cartilage andenormous humps of newly formed bone.

le 2). The ANTIGEN RETENTION IN BEIGE AND BLACK MICEbly more The data described above are contrary to whatof newly would be expected if neutral proteases had aares 2 and role in joint destruction during inflammation.resulting Surprisingly, cartilage destruction was eventhe beige more severe in the beige mice. A possible

explanation for this phenomenon might be thatpolymorphonuclear leucocyte neutral proteases

J ti actually assist with the elimination of the@ inflammatory stimulus-for example, by

*3^ degradation of immune complexes, and therebycontribute to attenuation of the inflammatoryresponse rather than promoting it. To investi-gate this possibility we measured the clearanceof antigen from the inflamed joint by following

,5 > the amount of radiolabelled antigen during thecourse of arthritis, using external gamma-counting.5 It was found that the black mice

,,; . eliminated the antigen factor faster than thet2 ' beige mice, though the difference never reached*E"ff'. significance (data not shown). As external, gammacounting of radiolabelled antigen is a

rather crude method for detecting the antigen-S (as it measures antigen in the entire knee joint,

including some of the periarticular tissues) wealso studied the fate of the injected antigen in

> * situ, using autoradiography at day 8 after injec-tion of radiolabelled antigen. Previous studieshave shown that radiolabelled material detected

*>̂ ,by autoradiography can be equated with8immnoractve antigen. When the amount of

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Figure 3:Joint ofaC57BI16 beige mouse at day21 after induction ofarthritis. Formation ofnewbone is prominent at thelateral sides ofthefemur.F=femur; N=new bone.(Haematoxylin and eosinstaining.) Bar= 100 ,m.Arrows=old bone margin.

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Figure 4: Autoradiograph of 125I labelled methylated bovineserum albumin retained in a kneejoint at day 8 after injectionofthe antigen. Note that most ofthe silvergrains areconcentrated at the cartilage surfaces. P=patella; F=femur;C=cartilage. (Haematoxylin and eosin staining.) Bar= 100Am-

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Schalkwijk, loosten, van den Berg, van de Putte

retained antigen was assessed semiquantitativelywith a four point scale on serial sections (asshown in fig 4) a small, but significant differ-ence was found between beige and black mice.Black mice: mean (SD) 1 9 (0-3) (n=9) andbeige mice: 2-6 (0-7) (n=6), p<O05, Wilcoxonrank sum test. This indicates that at least at day8 more antigen was present in the joints of beigemice than in those of black mice.

DiscussionBeige mice, deficient for polymorphonuclearleucocyte elastase and cathepsin G, and theirnormal C57/black/6 counterparts were used inthe methylated bovine serum albumin inducedarthritis model. The hypothesis that the de-ficiency of polymorphonuclear leucocyte neutralproteases would decrease tissue damage or chro-nicity of the inflammatory response was provedwrong. It was shown that beige mice developeda more serious and more chronic lesion thanblack mice, resulting in a more pronouncedirreversible damage to articular tissues.

Neutral proteases derived from polymor-phonuclear leucocytes have been strongly impli-cated in the degradation of articular cartilage invarious arthritic conditions. In septic arthritis,gout, and in active rheumatoid arthritis massiveinfiltration and exudation of polymorphonuclearleucocytes lead to the release of lysosomalenzymes in the joint space. Several in vitrostudies have shown that elastase and cathepsinG can degrade articular cartilage proteo-glycans,"1 12 and elastase was shown to bepresent in rheumatoid cartilage.'3 Few in vivostudies are available, however, that unequi-vocally show the part played by polymorpho-nuclear leucocyte neutral proteases in tissuedamage. Recently, we have studied two modelsof inflammation in beige and black mice. It wasfound that in experimental arthritis inducedwith zymosan there was no significant differencein inflammation or tissue damage between thetwo strains.2 In a model for experimentalglomerulonephritis, however, we showed asignificant difference in proteinuria betweenbeige and black mice, suggesting that poly-morphonuclear leucocyte neutral proteasescaused damage to the glomerular basal mem-brane.3 In this paper we have investigated theantigen induced arthritis model using beige andblack mice. This arthritis model is dependenton T lymphocytes, though polymorphonuclearleucocytes are probably the effector cells in theacute phase as we have previously shown thathydrogen peroxide contributes to the acutevascular effects in this model.5 Surprisingly, itwas found that the inflammatory response andthe amount of tissue damage in the beige micewere even higher than in the black mice. In thechronic phase beige mice show considerablymore bone apposition and destruction ofarticular cartilage. The main difference betweenthe two strains (as known so far) is the amountof elastase and cathepsin G. No differences incollagenase or acid protease content could beshown.' We have also shown that there are no

significant differences in the production ofsuperoxide.2 As the neutral proteases might,

alternatively, assist in the removal of theinflammatory stimulus (proteolysis of antigen orimmune complexes) we studied the clearancerate of labelled antigen from the joint. Ourstudies suggest a slight difference in antigenclearance between the two strains. The possiblerole of elastase and cathepsin G in the degrada-tion of antigenic material is currently beinginvestigated. In this model neutral proteasesmight eventually represent an off-switchmechanism for inflammation rather than apotentiating mechanism.The tissue damage found in the beige mice

might also be explained by the action of otherinflammatory mediators that are thought to beoperating in joint inflammation, such as oxygenmetabolites' 14 and cytokines.'5 When thesemediators are produced in excess the absence ofneutral proteases would not make much differ-ence to the resulting tissue damage. The relativecontribution of the various mediators is atpresent not known and is a subject of furtherinvestigation.

This work was supported by the Dutch League AgainstRheumatism.

1 Takeuchi K, Wood H, Swank R T. Lysosomal elastase andcathepsin G in beige mice. Neutrophils of beige (Chediak-Higashi) mice selectively lack lysosomal elastase andcathepsin G. J Exp Med 1986; 163: 665-77.

2 Schalkwijk J, Joosten LAB, van den Berg W B, van de PutteL B A. Experimental arthritis in C57black/6 normal andbeige (Chediak-Higashi) mice: in vivo and in vitro observa-tions on cartilage degradation. Ann Rheum Dis 1988; 47:940-6.

3 Schrijver G, Schalkwijk J, Robben J C M, Assmann K J M,Koene R A P. Antiglomerular basement membranenephritis in beige mice. J Exp Med 1989; 169: 1435-48.

4 van den Berg W B, van de Putte L B A, Zwarts W A, JoostenL A B. Electrical charge of the antigen determinesintraarticular antigen handling and chronicity of arthritis inmice. J Clin Invest 1984; 74: 1850-9.

5 Schalkwijk J, van den Berg W B, van de Putte L B A, JoostenL A B, van den Bersselaar L. Cationization of catalase,peroxidase and superoxide dismutase. Effect of improvedintraarticular retention on experimental arthritis in mice.Jf Clin Invest 1985; 76: 198-205.

6 Lammers A M, van de Kerkhof P C M, Schalkwijk J, MierP D. Elastase, a marker for neutrophils in skin infiltrates.Br J7 Dermatol 1986; 115: 181-6.

7 Kruijsen M W M, van den Berg W B, van de Putte L B A,van den Broek W J M. Detection and quantification ofexperimental joint inflammation in mice by measurementof 99"Tc-pertechnetate uptake. Agents Actions 1981; 11:640-2.

8 van den Berg W B, van Beusekom H J, van de Putte L B A,Zwarts W A, van der Sluis M. Antigen handling in antigen-induced arthritis in mice: an autoradiographic andimmunofluorescence study using whole joint sections. AmJ Pathol 1982; 108: 9-16.

9 van den Berg W B, Kruiijsen M W M, van de Putte L B A,van Beusekom H J, van der Sluis M, Zwarts WA. Antigen-induced arthritis in mice: studies on in vivo cartilageproteoglycan synthesis and chondrocyte death. Br J7 ExpPathol 1981; 62: 308-15.

10 Schalkwijk J, van den BergW B, van de Putte L B A, JoostenL A B, van der Sluis M. Effects of experimental jointinflammation on bone marrow and periarticular bone. Astudy of two types of arthritis using variable degrees ofinflammation. Br J Exp Pathol 1985; 66: 435-44.

11 Janoff A. Feinstein G, Malemud C J, Elias J M. Degradationof cartilage proteoglycan by human leukocyte granuleneutral proteases. A model of joint injury. J Clin Invest1976; 57: 615-24.

12 Schalkwijk J, van den Berg, van de Putte L B A, JoostenL A B. Elastase secreted by activated polymorphonuclearleucocytes causes damage and matrix degradation in intactarticular cartilage: escape from inactivation by alpha-l-proteinase inhibitor. Br J Exp Pathol 1987; 68: 81-8.

13 Velvart M, Baici A, Sommermeyer G, et al. Degradation invivo of articular cartilage in rheumatoid arthritis byleucocyte elastase from polymorphonuclear leucocytes.Rheumnatol Int 1981; 1: 121-30.

14 Blake D R, Hall N D, Bacon P A, Dieppe P A, Halliwell B,Gutteridge J M C. Effect of a specific chelating agent onanimal models of inflammation. Ann Rheum Dis 1983; 42:89-93.

15 Pettipher E R, Higgs G A, Henderson B. Interleukin-Iinduces leucocyte infiltration and cartilage proteoglycandegradation in the synovial joint. Proc Natl Acad Sci USA1986; 83: 8749-53.

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