Glomerulonephritis Induced by Heterologous Anti‐GBM Globulin as a Planted Foreign Antigen

Dragana Odobasic1, Joanna R. Ghali1, Kim M. O'Sullivan2, Stephen R. Holdsworth3, A. Richard Kitching4

1 These authors contributed equally to this work, 2 Centre for Inflammatory Diseases, Monash University Department of Medicine, Clayton, 3 Department of Nephrology, Monash Health, Clayton, 4 Department of Paediatric Nephrology, Monash Health, Clayton
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 15.26
DOI:  10.1002/0471142735.im1526s106
Online Posting Date:  August, 2014
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Abstract

The glomerulonephritides are diseases characterized by immune‐mediated glomerular inflammation. Most severe and rapidly progressive forms of glomerulonephritis feature the participation of injurious leukocytes that localize to glomeruli. This unit describes classical models of rapidly progressive glomerulonephritis in mice, induced by injecting heterologous globulin (raised in sheep) that binds to the glomerular basement membrane. These models have been particularly useful in defining the participation of effector leukocytes in severe glomerular disease. In these models, injury typically occurs in two phases. In the initial, heterologous phase, injury is mediated by the globulin bound within the glomerulus acting as an antibody. The later, autologous phase of injury is mediated by the host's adaptive immunity to the heterologous globulin now functioning as a planted foreign antigen within glomeruli. As autologous phase injury is driven by immunity to sheep globulin, assessment of antigen‐specific systemic immunity to sheep globulin is critical when using this model. Curr. Protoc. Immunol. 106:15.26.1‐15.26.20. © 2014 by John Wiley & Sons, Inc.

Keywords: glomerulonephritis; cell‐mediated immunity; planted antigen; T lymphocytes

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Induction of Non‐Accelerated Autologous Phase Anti‐GBM GN
  • Basic Protocol 2: Inducing Accelerated Autologous Phase Anti‐GBM GN
  • Support Protocol 1: Preparation of a Mouse Kidney Cortex Fraction
  • Support Protocol 2: Absorption and Precipitation of a Globulin Fraction
  • Support Protocol 3: Determination of Optimal Doses of Sheep Anti‐Mouse GBM Globulin
  • Support Protocol 4: Three‐Layer Immunohistochemical Staining to Assess Intrarenal Leukocyte Accumulation
  • Support Protocol 5: DAB Black Staining with Nuclear Fast Red Counterstain to Assess Intrarenal Leukocyte Accumulation
  • Support Protocol 6: Detection of Mouse IgG or C3 Deposition in Glomeruli by Immunofluorescence
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Tables
     
 
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Materials

Basic Protocol 1: Induction of Non‐Accelerated Autologous Phase Anti‐GBM GN

  Materials
  • Sheep anti‐mouse GBM globulin (see Support Protocols protocol 31 and protocol 42)
  • Male C57BL/6 mice, aged 6 to 12 weeks
  • Paraformaldehyde‐periodate‐lysine solution (PLP, see recipe)
  • 20% sucrose solution, dissolved in PBS
  • Optimal cutting temperature (OCT) compound (Tissue‐Tek, Sakura)
  • 10% formalin
  • 70% ethanol solution in distilled water
  • Paraffin
  • Liquid nitrogen
  • 30‐, 26‐, or 23‐G needles
  • 1‐ml syringes
  • Heat lamp or heat pad
  • Mouse retaining cone (or similar apparatus to facilitate i.v. injection)
  • Metabolic cages
  • 5‐ml tubes
  • Sterile 1.5‐ and 0.5‐ml microcentrifuge tubes (Eppendorf)
  • Cryomoulds (Tissue‐Tek, Sakura)
  • Additional reagents and equipment for parenteral injections (see unit ), blood collection (see unit ), euthanasia of mice (see unit ), and immunohistochemistry (see unit )

Basic Protocol 2: Inducing Accelerated Autologous Phase Anti‐GBM GN

  Materials
  • 5 mg/ml normal sheep globulin (NSG) in PBS
  • Male C57BL/6 mice (6 to 12 weeks old)
  • Complete Freund's adjuvant (CFA; unit )
  • Sheep anti‐mouse GBM globulin (see Support Protocols protocol 31 and protocol 42)
  • Sonicator
  • 1‐ml syringes
  • 30‐, 26‐, and 23‐G needles
  • Additional reagents and equipment for production of polyclonal antibodies using complete Freund's adjuvant (see unit ), and assessing disease (see protocol 1)

Support Protocol 1: Preparation of a Mouse Kidney Cortex Fraction

  Materials
  • Normal mice of any strain
  • Phosphate buffered saline (PBS), ice cold
  • Sterile 50‐ml tubes
  • Petri dishes
  • Forceps
  • Scissors
  • Scalpel blade
  • Tissue homogenizer
  • Refrigerated centrifuge
  • Microscope slides and coverslips
  • Microscope
  • Sonicator
  • Scale
  • 5‐ml tubes
  • Additional reagents and equipment for euthanasia of mice (see unit ) and blood collection (see unit )

Support Protocol 2: Absorption and Precipitation of a Globulin Fraction

  Materials
  • Sheep serum, sheep anti‐mouse GBM (and if required, non‐immune sheep serum)
  • Bottle of water (same shape and volume of serum)
  • Normal (i.e., untreated) mice (rats, if needed) of any strain
  • 3.3% (w/v) tri‐sodium citrate in distilled water
  • 0.9% (w/v) NaCl
  • Borate buffer (see recipe)
  • Saturated ammonium sulfate (SAS, see recipe)
  • PBS, ice cold
  • 37° and 60°C water baths
  • Thermometer
  • Timer
  • 5‐, 10‐, and 50‐ml tubes
  • 26‐G needles
  • 1‐ml syringes
  • Refrigerated centrifuge
  • Roller
  • Stirrer
  • 5‐liter containers
  • Spectrophotometer
  • UV cuvette
  • 100‐µm mesh

Support Protocol 3: Determination of Optimal Doses of Sheep Anti‐Mouse GBM Globulin

  Materials
  • C57BL/6 mice (male, 6 to 12 weeks old)
  • Sheep anti‐mouse GBM globulin (see Support Protocols protocol 31 and protocol 42)
  • Metabolic cages
  • 26‐ and 30‐G needles
  • 1‐ml syringes
  • Heat lamp or heat pad
  • Mouse retaining cone (or similar apparatus to facilitate i.v. injection)
  • 5‐ml tubes

Support Protocol 4: Three‐Layer Immunohistochemical Staining to Assess Intrarenal Leukocyte Accumulation

  Materials
  • PLP‐fixed OCT‐frozen kidneys (and spleen, as positive controls)
  • Blocking solution: 10% normal swine serum, 10% normal sheep serum in 5% BSA/PBS
  • Primary antibody and isotype controls diluted in 1% BSA/PBS (see Table 15.26.1)
  • PBS‐Tween 20 solution containing 0.05% (v/v) Tween 20
  • Absolute methanol
  • 30% hydrogen peroxide (H 2O 2) solution
  • Avidin‐biotin blocking kit (Vector Laboratories)
  • Secondary antibody solution: 10% normal mouse serum diluted in PBS with rabbit anti‐rat biotinylated antibody (DAKO) at 1:100
  • Tertiary antibody solution: Vectastain ELITE Standard ABC solution (Vector Laboratories)
  • 3,3′ Diaminobenzidine tetrahydrochloride (DAB) tablets (Sigma‐Aldrich)
  • Hematoxylin
  • Acid ethanol solution
  • Scott's tap water solution
  • Histosol solution
  • DPX mounting medium
  • Cryostat
  • Superfrost slides and cover slips
  • PAP pen
  • Humidity chamber
  • Coplin jars
  • Platform rocker
  • Aluminium foil
  • 0.22‐µm filter
  • 3‐ml transfer pipets
  • Oven or hair dryer
Table 5.6.1   MaterialsAntibodies and Isotype Controls Used for Renal Leukocyte Staining

Leukocyte Antibody (clone) Isotype control Antibody concentration (µg/ml)
Macrophage Rat anti‐mouse CD68 (FA‐11) a Rat IgG2a 10
CD4+ T cell Rat anti‐mouse CD4 (GK1.5) Rat IgG2b 20
Neutrophil Rat anti‐mouse Gr‐1 (RB6‐8C5) b Rat IgG2b 2–5
CD8+ T cell Rat anti‐mouse CD8 (53‐6.7) Rat IgG2a 20

 aMacrophages comprise a heterogeneous population and express a number of different markers. This marker is accepted in the literature as a reasonable marker of effector macrophages in glomeruli. Note that F4/80 does not reliably detect macrophages within glomeruli (Masaki et al., ).
 bAnti‐Gr‐1 is also known as anti‐Ly6G/C and also recognizes a subset of monocytes. An anti‐Ly6G antibody (clone 1A8) can also be used to detect neutrophils more specifically, but this antibody detects neutrophils in only some strains of mice (including C57BL/6 mice).

Support Protocol 5: DAB Black Staining with Nuclear Fast Red Counterstain to Assess Intrarenal Leukocyte Accumulation

  Additional Materials protocol 6)
  • DAB black solution (see recipe)
  • Nuclear Fast Red solution (see recipe)

Support Protocol 6: Detection of Mouse IgG or C3 Deposition in Glomeruli by Immunofluorescence

  Materials
  • Non‐fixed OCT‐frozen kidneys (and spleens) from mice with non‐accelerated or accelerated anti‐GBM GN (see Basic Protocols protocol 11 and protocol 22, respectively)
  • Acetone
  • 15% normal sheep serum in 5% BSA/PBS or 15% normal goat serum in 5% BSA/PBS
  • Sheep anti‐mouse Ig‐FITC (Silenus) or goat anti‐mouse C3‐FITC (Cappel)
  • 1% BSA/PBS
  • PBS
  • Fluorescent mounting medium (Dako)
  • Cryostat
  • Superfrost slides and coverslips
  • Coplin jars
  • PAP pen
  • Humidity chamber
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