Animal Models of Systemic Lupus Erythematosus (SLE) and Ex Vivo Assay Design for Drug Discovery

Matthew M. Seavey1, Lily D. Lu1, Kristine L. Stump1

1 Worldwide Discovery Research, Cephalon, Inc., West Chester, Pennsylvania
Publication Name:  Current Protocols in Pharmacology
Unit Number:  Unit 5.60
DOI:  10.1002/0471141755.ph0560s53
Online Posting Date:  June, 2011
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Abstract

Systemic Lupus Erythematosus (SLE) is a debilitating and often fatal autoimmune disease that involves multiple organ systems. It can develop for years before being diagnosed. Current treatments for SLE usually involve the use of cytotoxic or immunosuppressive agents that can lead to infection or cancer. The design of appropriate models and assays will determine the efficiency and speed with which an investigator can test a new chemical entity (NCE) or expect results to move a drug discovery program forward. This unit describes a series of preclinical assays for the identification of new agents for the treatment of SLE. Most importantly, this unit will guide the reader through a step‐by‐step process to select appropriate models, validation drugs, and readouts, depending on the objective of the study. The reader will acquire a working knowledge of what models are available and the potential advantages and disadvantages of each, including ex vivo assays relevant to the discovery of new SLE therapeutics. Curr. Protoc. Pharmacol. 53:5.60.1‐5.60.40. © 2011 by John Wiley & Sons, Inc.

Keywords: lupus; SLE; drug discovery; animal models

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Optimization and Validation of Selected Lupus Mouse Models for Drug Screening and Proof‐of‐Concept Studies
  • Basic Protocol 2: ELISA for the Detection of Serum Anti‐Nuclear Antibodies (ANAs)
  • Alternate Protocol 1: ELISPOT for the Detection of ANA Antibody Secreting Cells (ASCs)
  • Support Protocol 1: Preparation of Chicken Chromatin for Use in ELISA and ELISPOT Assays
  • Basic Protocol 3: Measuring Serum Cytokines Using Multiplex Bead Technology
  • Alternate Protocol 2: Processing of Mouse Paws for Use in Luminex or ELISA Cytokine Assays (Lupus Arthritis Model)
  • Basic Protocol 4: Urine Analysis for Monitoring Renal Involvement
  • Basic Protocol 5: Renal Histology to Assess Lupus Nephritis
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Optimization and Validation of Selected Lupus Mouse Models for Drug Screening and Proof‐of‐Concept Studies

  Materials
  • Mice: MRL/lpr and a control non‐lupus forming strain, MRL/Mp, as a negative control for lupus formation
  • Standard‐of‐care reference compound/drug(s) (e.g., Table 5.60.3)
  • Test compound(s)
  • Additional reagents and equipment for determining pharmacokinetics (unit 7.1), blood collection (Donovan and Brown, ), and animal identification (Donovan and Brown, )

Basic Protocol 2: ELISA for the Detection of Serum Anti‐Nuclear Antibodies (ANAs)

  Materials
  • Coating antigen stock solutions (one of the following):
    • 0.5 mg/ml purified Smith antigen, native protein (GenWay, cat. no. 11‐511‐248366; http://genwaybio.com/) in borate‐buffered saline (BBS, see recipe)
    • 0.5 mg/ml dsDNA (see recipe)
    • 1.5 mg/ml purified, boiled chicken chromatin (see protocol 4)
  • Borate‐buffered saline (BBS), pH 8.4 (see recipe)
  • Borate‐buffered saline with 0.5% Tween‐20 (BBS‐T; see recipe)
  • Borate‐buffered saline with 0.5% Tween‐20 and 1.0% BSA (BBS‐TB; see recipe)
  • Serum sample(s) to be assayed for anti‐chromatin/Sm/dsDNA antibodies (can be fresh or frozen); vortex then dilute the sample appropriately for assay; avoid freeze‐thaw cycles
  • Primary antibody stock solutions (one of the following):
    • 0.2 mg/ml mouse anti–Smith antigen (clone: Y12; Abcam, cat. no. ab3138) in BBS‐TB
    • 1 mg/ml mouse anti‐dsDNA (clone: HYB331/01; Abcam, cat. no. ab27156) in BBS‐TB
    • 2 mg/ml mouse anti‐chromatin (Sigma, clone 2B1, cat. no. S2944); also, mouse anti‐chromatin standard stocks can be made directly using serum from 25‐week‐old MRL/lpr mice; as the serum concentration of anti‐chromatin IgG is extremely high at this time (titer using 2B1 Ab from Sigma), dilute the serum at least 800× to 1000× to obtain a signal below saturation
  • Detection antibody: HRP‐conjugated goat anti‐mouse IgG H&L–Fab fragment – HRP conjugated, 1mg/ml in BBS‐TB (Abcam, cat. no. ab6823)
  • ELISA peroxidase substrate premixed TMB solution (Rockland, cat. no. TMBE‐1000; http://www.rockland‐inc.com/)
  • Stop solution: 1 M H 2SO 4
  • Flat‐bottom BD Falcon 96‐well microtiter plates, clear plastic (BD Falcon, cat. no. 353379)
  • Plate washer (Stat‐Matic II Plate Washer, VWR, cat. no. 100166‐394)
  • Orbital nonrefrigerated plate shaker
  • Microtiter plate reader with automated computer interface and dual wavelength spectrophotometer
  • Additional reagents and equipment for ELISA (Hornbeck, ; also see Crowther, , as a guide to basic ELISA design and theory)

Alternate Protocol 1: ELISPOT for the Detection of ANA Antibody Secreting Cells (ASCs)

  • Mouse anti‐IgG (H&L) antibody (MabTech; http://mabtech.com/)
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • 70% ethanol
  • Single‐cell suspension (see unit 12.1) of mouse spleen cells or other cells, such as bone marrow, peripheral blood leukocytes, lymph node cells, or hybridoma cells as a positive control, to be tested for antibody production
  • Complete RPMI‐10 medium (see recipe)
  • Third‐party antigen control: chicken ovalbumin (OVA; Sigma, cat. no. A5503)
  • Lipopolysaccharide (LPS; see recipe)
  • Goat anti‐Mouse IgG, biotinylated (MabTech; cat. no. 3825‐6‐250; http://mabtech.com/)
  • Fetal bovine serum (FBS; appendix 2A)
  • Alkaline phosphatase streptavidin (Strep‐ALP; MabTech, cat. no. 3310‐8 or Jackson ImmunoResearch, cat. no. 016‐050‐084)
  • BCIP/NBT substrate solution (Rockland, cat. no. NBT‐100; http://www.rockland‐inc.com/)
  • Millipore multiscreen 96‐well, HTS white opaque IP plates (Millipore, cat. no. MAIPSWU10)
  • Humidified chamber: Tupperware container with moistened paper towel (4°C)
  • C.T.L. Immunospot ELISPOT plate scanner with analysis software (C.T.L. Immunospot, http://www.immunospot.com); alternatively use fluorescence microscope
NOTE: As all solutions and equipment coming into contact with cells must be sterile, proper aseptic technique must be employed for this assay. Use of a BSL‐1 or BSL‐2 hood is strongly recommended to prevent contamination.NOTE:All tissue culture incubations should be performed in a humidified 37°C, 5% CO 2 incubator.NOTE: MabTech also offers a commercial kit (MabTech, cat. no. 3825‐2AW); however, making an in‐house ELISPOT is also possible and more economical.

Support Protocol 1: Preparation of Chicken Chromatin for Use in ELISA and ELISPOT Assays

  Materials
  • Chicken blood in Alsever's solution (Rockland, cat. no. R302‐0100 http://www.rockland‐inc.com/)
  • Buffer A: 0.08 M NaCl/0.02 M EDTA, pH 7.5 (0.46 g of NaCl in 100 ml of distilled H 2O plus 0.83 g EDTA in 100 ml of distilled H 2O)
  • 1.5% Triton X‐100 in buffer A
  • Gradient solutions:
    • 1.7 M sucrose in buffer A (58.2 g sucrose/100 ml of buffer A)
    • 2.25 M sucrose in buffer A (77.0 g sucrose/100 ml buffer A)
  • 50 mM Tris⋅Cl, pH 7.9 ( appendix 2A)
  • 0.1 M EDTA solution (perform a 1:5 dilution of 0.5 M EDTA stock)
  • 50‐ml centrifuge tubes
  • Tabletop centrifuge
  • 25 × 89–mm ultracentrifuge tubes (Beckman)
  • Ultracentrifuge with Beckman JA‐17 rotor (or equivalent)
  • 1000‐µl (P1000) pipet tip with tapered end cut off
  • Bath sonicator (Branson 3510 or equivalent)
  • Spectrophotometer for DNA analysis (any spectrophotometer that can perform A 240/A 260 will work for this purpose)
  • Additional reagents and equipment for determining protein concentration ( appendix 3A)

Basic Protocol 3: Measuring Serum Cytokines Using Multiplex Bead Technology

  Materials
  • Mouse Cytokine 10‐plex bead kit (Invitrogen, cat. no. LMC0001)
  • Sera to be tested (store at –80°C)
  • 96‐well cell strainers (Millipore, cat. no. MANMN6010)
  • Multiplex Multiscreen Filter IP plates (Millipore, cat. no. MAIPSWU10)
  • Opaque 96‐well plate cover (Invitrogen, cat. no. PC10)
  • Orbital shaker (small‐diameter rotation recommended)
  • Filtration vacuum manifold for bead washing (Pall, cat. no. 5017)
  • Luminex xMAP system with data acquisition and analysis software (Invitrogen, cat. no. MAP0200)
NOTE: Described below is a modified manufacturer's protocol for the measurement of serum cytokines from NZM and MRL/lpr mouse models. This protocol can also be used for paw and spleen extracts (see protocol 6) from the same models. The manufacturer's manual should be consulted when optimizing assay conditions.

Alternate Protocol 2: Processing of Mouse Paws for Use in Luminex or ELISA Cytokine Assays (Lupus Arthritis Model)

  Materials
  • Arthritic paws from mice
  • Liquid N 2
  • Tissue extraction reagent I (Invitrogen, cat. no. FNN0071)
  • Protease inhibitor cocktail (Calbiochem, cat. no. 539136)
  • 100× Halt phosphatase inhibitor cocktail (Thermo Scientific, cat. no. 78420)
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • BCA Protein Assay Reagent (Pierce, cat. no. 23228; use according to manufacturer's recommendations)
  • Polypropylene round‐bottom tubes (BD Falcon, cat. no. 352059)
  • Polytron Homogenizer PT 10‐35 GT (VWR, cat. no. 97036‐082) with small probe
  • Refrigerated centrifuge
  • Microtiter plate reader with automated computer interface and dual wavelength spectrophotometer
  • Additional reagents and equipment for Luminex assay ( protocol 5)

Basic Protocol 4: Urine Analysis for Monitoring Renal Involvement

  Materials
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • Standard protein solution: prepared from normal mouse sera and used as a standard for mouse urinary protein assay by turbidity—prepare by collecting or purchasing normal mouse sera (Jackson Immunoresearch, cat. no. 015‐000‐120), generating a 1 mg/ml serum standard in PBS (based on manufacturer‐reported values), then filter‐sterilizing
  • Thawed urine sample
  • 0.1 N HCl
  • 3% sulfosalicylic acid (Chondrex, cat. no. 90404; http://www.chondrex.com)
  • Uristix‐4 test strips (Siemens, cat. no. 2166; can be ordered from VWR, cat. no. AM2166)
  • BD Falcon 96‐well flat bottom tissue culture treated clean plates (BD Falcon, cat. no. 353379)
  • ELISA reader
NOTE: It is recommended that the standard and samples be run in duplicate.

Basic Protocol 5: Renal Histology to Assess Lupus Nephritis

  Materials
  • Mice
  • OCT freezing medium (Fisher Scientific, cat. no. 14‐373‐65)
  • 4% (w/v) paraformaldehyde (PFA) in PBS, pH 7.4
  • Phosphate‐buffered saline (PBS), pH 7.4 ( appendix 2A)
  • Blocking buffer: PBS with 2 µg/ml of Fc block [anti‐mouse CD16/CD32 (BD Biosciences, cat. no. 14‐0161‐81)]
  • PBST (see recipe)
  • FITC Goat anti‐mouse IgG antibody (Abcam, cat. no. ab6785‐1)
  • FITC Goat anti‐mouse IgG antibody isotype control
  • FITC Goat anti‐mouse C3 antibody (Cappel, MP Biomedicals, cat. no. 55500)
  • FITC Goat anti‐mouse C3 antibody isotype control
  • Appropriate serum for primary isotype, both rat and mouse serum (Jackson ImmunoResearch Laboratories)
  • Secondary antibody (optional)
  • Anti‐fading gel mounting medium for slides (Biomeda, cat. no. M01)
  • Fine forceps
  • Surgical scissors
  • Base molds (Fisher Scientific, cat. no. 22‐038217)
  • Cryostat with necessary tools (see appendix 3E)
  • Staining trays or holder for humidified chamber for staining (self‐assemble)
  • Humidified dark chamber: opaque plastic box containing a moist paper towel
  • Fluorescent microscope with necessary tools
  • Superfrost Plus slides for sectioning (VWR, cat. no. 48311‐703)
  • Additional reagents and equipment for cryostat sectioning ( appendix 3E)
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Figures

Videos

Literature Cited

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