[methods in molecular biology] mouse models for drug discovery volume 602 || collagen-induced...

Chapter 11

Collagen-Induced Arthritis in Mice

Lisette Bevaart, Margriet J. Vervoordeldonk, and Paul P. Tak

Abstract

Collagen-induced arthritis (CIA) in mice is an animal model for rheumatoid arthritis (RA) and can beinduced in DBA/1 and C57BL/6 mice using different protocols. The CIA model can be used to unravelmechanisms involved in the development of arthritis and is frequently used to study the effect of newtherapeutics. The development of a CIA model in C57BL/6 mice recently enabled researchers to useknockout mice on this background for arthritis research.

In this chapter, the protocol for induction of arthritis in both mice strains is described, includingthe monitoring of clinical arthritis and paw swelling in the mice during the experiment. Furthermore,protocols for decalcification of paws and for the detection of collagen-specific antibodies in mice sera aredescribed.

Key words: Arthritis, CIA, collagen, DBA/1, C57BL/6.

1. Introduction

Rheumatoid arthritis (RA) is a debilitating disease which afflictsabout 1% of the population. It is characterized as a chronic,immune-mediated inflammatory disease with destruction of car-tilage and bone in the joints. Treatment strategies vary fromthe use of conventional disease-modifying anti-rheumatic drugs(DMARDs) to biologicals (e.g., TNF-� or CD20-specific anti-bodies). Unfortunately, not all patients respond to treatment andthere is still a need for new drug development. Collagen-inducedarthritis (CIA) is a frequently used animal model to study theeffect of new therapeutics for RA. Similarities between CIA andRA include symmetrical joint involvement, synovial hyperplasia,inflammatory cell infiltrates, and the presence of autoantibodies

G. Proetzel, M.V. Wiles (eds.), Mouse Models for Drug Discovery, Methods in Molecular Biology 602,DOI 10.1007/978-1-60761-058-8 11, © Humana Press, a part of Springer Science+Business Media, LLC 2010

181

182 Bevaart, Vervoordeldonk, and Tak

to type II collagen (1). Even production of rheumatoid factor hasbeen reported.

CIA is induced by injection of an emulsion of collagen incomplete Freund’s adjuvant (CFA). This initiates an immuneresponse directed toward collagen, involving T-and B-cell activa-tion and antibody production. Antibodies recognize collagen inthe joints and instigate the local autoimmune response in whichmonocytes, granulocytes, and T cells are attracted to the joints.This further fuels the inflammatory process resulting in produc-tion of cytokines and inflammatory mediators (2, 3).

CIA in DBA/1 mice is induced via an initial intradermal (i.d.)injection of an emulsion consisting of bovine type II collagen inCFA. Mice are intraperitoneally (i.p.) injected with bovine type IIcollagen 21 days later which boosts the immune system, resultingin chronic inflammation in both the hind and the front paws. InC57BL/6 mice, CIA can be induced by two i.d. injections withchicken type II collagen in CFA, on day 0 and day 21 (4). Com-pared to CIA in DBA/1 mice, CIA on the C57BL/6 backgroundshows a reduced severity in clinical scores and paw swelling anda decreased incidence, approximately 70–80% in C57BL/6 com-pared to 90–100% in DBA/1 mice.

The presence of a CIA model in C57BL/6 mice enablesresearchers to investigate the role of specific genes, receptors, orimmune mediators such as cytokines in knockout or transgenicmice, which often are on a C57BL/6 background. In this way,opportunities arise to study new targets for drug development.

2. Materials

2.1. CIA in DBA/1Mice

1. Male DBA/1 mice (Harlan [see Note 1]), 8–12 weeksold. All animal experiments have to be done in accor-dance with institutional and national guidelines andregulations.

2. Bovine type II collagen 2 mg/ml (Chondrex Inc., cat. no.2002-2).

3. Complete Freund’s Adjuvant, CFA (Mycobacterium tuber-culosis, heat inactivated); 4 mg/ml (Chondrex Inc.; cat. no.7001).

4. Incomplete Freund’s Adjuvant, IFA (Chondrex Inc.; cat.no. 7002).

2.2. CIA in C57BL/6Mice

1. Male or female C57BL/6 mice (C57BL/6J, Charles Riveror The Jackson Laboratory), 8–12 weeks old (see Note 2).Try to use animals of one sex. All animal experiments have

Collagen-Induced Arthritis in Mice 183

to be done in accordance with institutional and nationalguidelines and regulations. When using knockout mice, usewild-type (WT) littermates as control (see Note 3).

2. Acetic acid 100% (Merck, cat. no. 1.00063.1000).3. 0.1M acetic acid. Dilute acetic acid to a concentration of

0.1M and filter through a 0.2 �m filter with use of asyringe. Keep the solution on ice and add acid slowly tothe water!

4. Chicken type II collagen, 5 mg (Sigma, cat. no. C9301).Dissolve the complete content of the vial in 2.5 ml sterilefiltered 0.1 M acetic acid. Place the vial on a rotator anddissolve the collagen overnight at 4◦C.

5. Complete Freund’s Adjuvant, CFA (M. tuberculosis, heatinactivated); 5 mg/ml (Chondrex Inc.; cat. no. 7023).

2.3. Preparation ofCFA/CollagenEmulsion

1. 14 ml round bottom tubes (Falcon, cat. no. 35-2057)2. Homogenizer (for example, IKA; model T10 basic, with

dispersing tool S10N-5G, IKA, cat. no. 3304000)

2.4. Injection of theCFA Emulsion

1. 100 �l glass syringes (Hamilton 1710 TLLX with stop;P/N 81022/01)

2. 25G needles3. Blades4. Tweezers5. Anesthetics (for example, isofluorane)

2.5. Measurement ofArthritis

1. Caliper (for example, Kroeplin Langenmesstechnik, cat.no. SO247)

2.6. Sacrificing Mice 1. Eppendorf tubes2. Tweezers and scissors3. Anesthetics4. 4% Formalin5. Ethanol (Merck, cat. no. 1.00983.2500)

2.7. Decalcifcation ofMice Paws

1. 70% alcohol2. EDTA (Titriplex II, Merck, cat. no. 1.08418)3. Sodium hydroxide (NaOH) pellets GR for analysis (Merck,

cat. no. 1.06498)4. Millipore water5. 4M HCl6. Tissue embedding and processing cassettes


7. Decalcification solution:a. Add 300 g of EDTA to 1800 ml millipore water.b. Add approximately 40 g of NaOH pellets to increase the

pH. This will help to dissolve the EDTA.c. After dissolving of the EDTA, set the pH tot 7.5 with

use of 4M HCl.8. Beaker with a grid and a stir bar

2.8. DeterminingLevels ofAnti-collagen IgG1and IgG2a Antibodies

1. High binding ELISA plates (for example, 96-wellRIA/EIA plate, high binding, flat bottom, Costar, cat. no.3590).

2. Bovine or chicken collagen, see Section 2.1.2 or 2.2.4.3. PBS.4. PBS/1%BSA, BSA used should be suitable for ELISA appli-

cation (for example, Sigma Aldrich, cat. no. A7030).5. Wash buffer: 0.05% Tween-20 in 1 × PBS. To prepare wash

buffer add 500 �l Tween-20 (Merck, cat. no. 8.22184) to1 liter of 1 × PBS.

6. Mouse anti-type II collagen IgG1 antibody solution(10 ng, Chondrex Inc., cat. no. 20306).

7. Mouse anti-type II collagen IgG2a antibody solution(10 ng, Chondrex Inc., cat. no. 20307).

8. Rat anti-mouse IgG1-HRP (1 ml, BD Pharmingen, cat.no. 559626).

9. Rat anti-mouse IgG2a-HRP (1 ml, BD Pharmingen, cat.no. 553391).

10. Sodium acetate buffer, pH 5.5. Prepare by adding 1.3 ml100% acetic acid to 198.7 ml H2O. Place the H2O on iceand slowly add the acetic acid. Be sure to add acid to water!Set pH at 5.5.

11. TMB (3,3′–5,5′-tetramethylbenzidine, Merck, cat. no.1.08622) in DMSO. Dissolve 100 mg TMB in 10 mlDMSO (dimethylsulfoxide, Merck, cat. no. 1.02950).

12. 3% H2O2

13. 1M H2SO4

3. Methods

3.1. Preparation ofBovine Type IICollagen/CFAEmulsion forInjection in DBA/1Mice

1. Per mouse a volume of 100 �l is injected. Before prepara-tion of the emulsion, calculate the total volume of emul-sion needed and multiply that by 2 to provide excess forpractical losses. A significant portion of the emulsion willbe lost due to dead needle volume and refilling of thesyringe. For example, when 40 mice need to be injected,


Fig. 11.1. Preparation of the collagen/CFA emulsion with the use of the homogenizer.

40 mice × 100 �l × 2 = 8 ml of emulsion needs to beprepared.

2. Dilute the CFA 4 mg/ml 1:1 in IFA, which will result inCFA with a concentration of 2 mg/ml. Place the tube onice. In the example, add 2 ml of IFA to 2 ml of CFA.

3. Add bovine type II collagen drop-wise to the CFA(2 mg/ml) while homogenizing (see Fig. 11.1). Keep thetube on ice! In the example, add 4 ml of bovine type IIcollagen.

4. Homogenize the emulsion until this appears white andviscous.

5. Perform a test to determine whether the emulsion is wellprepared. Pipet 20 �l of the emulsion on water. The emul-sion must not disperse for at least 40 s but form a niceround-shaped drop.

Since the CFA 2 mg/ml is mixed 1:1 with bovine type IIcollagen 2 mg/ml, the final concentration of M. tuberculosispresent in the emulsion is 1 mg/ml (100 �g per mouse as 100�l is injected). The final concentration of bovine collagen II is1 mg/ml (100 �g per mouse as 100 �l is injected).


3.2. Preparation ofChicken Type IICollagen/CFAEmulsion forInjection in C57BL/6Mice

1. Prepare chicken type II collagen the day before injection ofmice as mentioned in Section 2.2.4.

2. Per mouse a volume of 100 �l is injected. Before prepara-tion of the emulsion, calculate the total volume of emulsionneeded and multiply that by 2 to provide excess for practi-cal losses. A significant portion of the emulsion will be lostdue to dead needle volume and refilling of the syringe. Forexample, when 40 mice need to be injected, 40 mice × 100�l × 2 = 8 ml of emulsion needs to be prepared.

3. Transfer the amount of CFA (concentration 5 mg/ml) thatis need to a 14 ml round bottom tube. In the example,pipet 4 ml CFA.

4. Add the chicken type II collagen drop-wise to the CFA(5 mg/ml) while homogenizing (see Fig. 11.1). Keep thetube on ice! In the example, add 4 ml of chicken type IIcollagen.

5. Homogenize the emulsion until this appears white andviscous.

6. Perform a test to determine whether the emulsion is wellprepared. Pipet 20 �l of the emulsion on water. The emul-sion must not disperse for at least 40 s but form a niceround-shaped drop.

Since the CFA 5 mg/ml is mixed 1:1 with chicken type II col-lagen 2 mg/ml, the end concentration of M. tuberculosis presentin the emulsion is 2.5 mg/ml (250 �g per mouse as 100 �l isinjected). The end concentration of chicken collagen is 1 mg/ml(100 �g per mouse as 100 �l is injected).

3.3. Injection of theEmulsion in Mice

1. Fill the glass syringe with the emulsion. Due to its consis-tency, this may take some time.

2. Anesthetize the mice (for example, with oxygen 2 l/min,3% isofluorane).

3. Shave the injection area at the base of the tail. Be carefulnot to damage the skin.

4. A total of 100 �l emulsion is injected at the base of the tailwith the glass syringe and 25G needles (see Note 4). Thisvolume is divided over two injection sites. When injectedproperly, a white spot should be visible at the sites of injec-tion (see Fig. 11.2).

5. Clean the sites of injection with a wipe soaked in ethanol.6. Put the mice back in their cages where they can recover

from anesthesia.Treatment with compounds or drugs can be done either pro-

phylactic or therapeutic (see Note 5). CIA is reported to be


Fig. 11.2. Intradermally injected collagen/CFA emulsion.

a good predictive model for outcome of therapy in the clini-cal situation in combination with the adjuvant arthritis model inrats (5).

3.4. Boost Injectionin DBA/1 Mice

At day 21 after the first injection, mice are injected intraperi-toneally (i.p.) with bovine type II collagen diluted in saline. A1:2 dilution of bovine collagen in saline results in a final concen-tration of 1 mg/ml. Each mouse is injected i.p. with 100 �l ofthis solution, resulting in a boost injection of 100 �g bovine typeII collagen per mouse.

3.5. Boost Injectionin C57BL/6 Mice

At day 21 after the first injection, mice are injected i.d. at the baseof the tail close to the previous injection sites with a chicken typeII collagen/CFA emulsion. This emulsion is made in exactly thesame way as described in Section 3.2 and injected as explained inSection 3.3.

3.6. Monitoring ofArthritis

Arthritis is monitored using a clinical arthritis scoring system andpaw swelling is measured using a caliper. Both measurementsshould be obtained at least three times weekly; if the treatmentperiod is only one week before sacrificing, daily measurementsare advised to obtain a sufficient amount of data. Scoring has tobe started 5 days before the booster injection for baseline mea-surements of non-inflamed joints. Typically, signs of arthritis willstart after the boost injection; however, paw swelling and clin-ical arthritis scores are also observed shortly before the boost(see Note 3).

3.6.1. Caliper Score Hold the mice by the scruff of the neck and tail, and measurepaw thickness of both hind ankle joints (Fig. 11.3). Be sure toposition the caliper in the same way during each measurement.


Fig. 11.3. Paw swelling measurement.

Table 11.1Assignment of clinical scores in DBA/1 and C57BL/6 mice

score DBA/1 mice C57BL/6 mice

0 Normal Normal

1 Swelling of 1 joint (wrist/ankle or digit) Mild swelling2 Swelling of 2 joints or more Moderate swelling (or mild swelling

+ 1 or 2 swollen joints)

3 Swelling of all joints Swelling of all joints4 Swelling of all joints and ankylosis

(= joint stiffness)Joint distortion and/or rigidity and

dysfunction

3.6.2. Clinical Score Swelling and inflammation develop differently in the paws ofDBA/1 and C57BL/6 mice. Table 11.1 shows how clinicalscores are assigned per mouse strain. The score is determined forall four paws. A maximal score of 4 can be reached per paw, result-ing in a maximum total score of 16 per mouse (see Note 6).


3.7. Sacrificing ofMice

1. Mice are anesthetized (for example, with oxygen 2 l/min,3% isofluorane).

2. Blood is withdrawn from mice by heart puncture and col-lected in Eppendorf tubes. Typically, 500 �l of blood isobtained from diseased animals. When the blood is neededfor specific purposes EDTA tubes or heparin tubes can beused. Blood should be stored on ice if cytokines levels haveto be determined at a later time point.

3. Mice are sacrificed by cervical dislocation.4. Hind paws are cut of at the hips, as it is of great importance

to keep the knee joint intact for histological analysis. Skinis removed.

5. Paws are placed in tubes containing 4% formalin.6. Spin the Eppendorf tubes containing blood at 10,000 rpm,

4◦C for 10 min.7. Supernatant is transferred to new Eppendorf tubes after

which the tubes are centrifuged again.8. Supernatant is transferred to new, labeled Eppendorf tubes

which are stored at −80◦C. Make aliquots of serum peranimal to prevent multiple freeze–thaw cycles.

9. After 16–24 h, formalin is replaced by 70% ethanol. It isimportant to replace the formalin within this time frameto preserve the material for further immunohistochemicalanalysis (see Note 7).

Paws can be stored in 70% ethanol for years until further pro-cessing. For further processing, paws need to be decalcified (seebelow) before they are embedded in paraffin. Before decalcifica-tion, X-rays of the hind paws can be made for radiologic analysisin which demineralization and erosions of bone are scored. Forfurther details see ref. (6).

10. Depending on the research question, other organs such asspleen and lymph nodes can be removed from the mice for(immunological) analysis, fixed in 4% formalin, and subse-quently in 70% ethanol before paraffin embedding. Alter-natively, the tissue, placed in aluminum cryotubes, can besnap frozen immediately in liquid nitrogen after dissection.This procedure should be performed if RNA, DNA, or pro-tein needs to be isolated from the tissue.

Freshly isolated spleens and draining lymph nodes can also beused for ex vivo stimulation with collagen type II or other stim-uli of interest. These procedures are described in several researcharticles (7–9).

3.8. Decalcificationof Mice Paws

1. Transfer mouse hind paws to labeled tissue embedding andprocessing cassettes.


2. Put a stir bar in a 2 l beaker, place a grid within the beakerand fill this with decalcification solution. Transfer the cas-settes with the hind paws to the beaker and place it at 4◦Con a rotator so that the fluid is stirred continuously.

3. Refresh the decalcification fluid two times per week.4. Decalcification will take 4–6 weeks. Decalcification can be

monitored with use of X-rays to check whether the bonehas demineralized.

5. Once decalcification is complete, decalcification fluid canbe replaced by 70% ethanol.

6. Embed paws with paraffin and make serial 5 �m sagit-tal sections of whole hind paws. Hematoxylin and eosin(to study synovitis), safranin-O (to determine cartilageintegrity), or cytokine staining can be performed. For infor-mation and scoring, see ref. (6).

3.9. Anti-collagenAntibodies ELISA

1. Coat an ELISA plate overnight with 5 �g/ml bovine(DBA/1 mice) or chicken collagen (dissolved accordingto Section 2.2.4) (C57BL/6 mice) diluted in PBS, 50�l/well. Make sure to prepare different plates to detecteither IgG1 or IgG2a collagen-specific antibodies. Coat 2wells for each sample and standard, including two negativecontrols (also see Note 8).

2. Incubate overnight at 4◦C, in the dark.3. Wash the plate three times with 0.05% Tween-20 in PBS

(PBS-Tween), 100 �l/well.4. Block for 1 h with PBS/1% BSA, 100 �l/well.5. Remove the PBS/1%BSA.6. Prepare standards and serum dilutions.

6.1. Use a standard curve of anti-collagen IgG1 or IgG2a,with a high concentration of 10 ng/ml and a 1:2 serialdilution in PBS/1% BSA (seven steps in total). Incu-bate two wells per plate with PBS/1% BSA only as anegative control.

6.2. Concentrations of anti-collagen antibodies can varybetween mice, so it is important to test a range of con-centrations of serum from each mouse in one ELISA.A starting dilution of 1:50 is recommended, withseven 4-fold dilution steps.

6.3. Pipet all standards and samples in duplicate, add 50�l/well.

7. Incubate standard and samples for 1 h at room tempera-ture.

8. Wash the plate three times with PBS-Tween, 100 �l/well.


9. Add the detection antibody, 50 �l/well, diluted in PBS/1%BSA. Use either rat anti-mouse IgG1-HRP 1:1000 dilutionor rat anti-mouse IgG2a-HRP 1:1000 dilution.

10. Incubate 1 h at room temperature.11. Wash the plate three times with PBS-Tween, 100 �l/well.12. Make substrate: add 5.5 ml sodium–acetate buffer, 5.5 �l

3% H2O2, and 55 �l TMB DMSO together. Prepare thesubstrate just prior to usage and be aware that substrate islight sensitive!

13. Add 50 �l substrate solution/well and incubate for 20 minat RT, in the dark!

14. Switch on the ELISA reader and prepare a sample template.15. Add 25 �l/well 1 M H2SO4 to stop the reaction.16. Read the plate at 450/540 nm. Make a 4PL curve fit

and generate a standard curve with both x-axis and y-axislinearly. Determine concentration of collagen-specific anti-bodies based on the standard curve.

4. Notes

1. The authors of the article only have experience with miceobtained from Harlan. However, other groups also usedDBA/1 obtained from the Jackson Laboratory.

2. For the CIA model in C57BL/6, mice between 12 and 16weeks of age can also be used although this might not beoptimal.

3. Mice can develop less arthritis in a clean(er) animal facil-ity. Other factors, like stress, may also influence the devel-opment of arthritis. When moving to a new facility, firsttest whether the model still works before conducting newexperiments.

4. Be sure to inject the collagen/CFA emulsion intradermally,as depth of injection (e.g., subcutaneous vs. intradermal)can influence the immune response.

5. Treatment tested in a so-called prophylactic setting is usu-ally started on days 16–21, whereas therapeutic treatmentis often started at day 33.

6. The clinical score better reflects the development of CIA inC57BL/6 mice than the caliper score.


7. Fix paws in formalin for no more than 24 h. This is to pre-vent loss of (cytokine-) epitopes and to ensure good carti-lage integrity.

8. In the collagen antibody-ELISA, normal mouse serum canbe included as an additional negative control in the samedilution range as sera from mice with CIA. This serumshould not react in this ELISA.

Acknowledgments

The authors would like to thank Dr. Cristina Lebre for providingFig. 11.2.

References

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4. Campbell, I. K., Hamilton, J. A. and Wicks,I. P. (2000) Collagen-induced arthritis inC57BL/6 (H-2b) mice: new insights intoan important disease model of rheumatoidarthritis. Eur J Immunol 30, 1568–1575.

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9. Young, D. A., Hegen, M., Ma, H. L.,Whitters, M. J., Albert, L. M., Lowe, L.,et al. (2007) Blockade of the interleukin-21/interleukin-21 receptor pathway amelio-rates disease in animal models of rheumatoidarthritis. Arthritis Rheum 56, 1152–1163.

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