THE IMPACT OF ISCHEMIA REPERFUSION INJURY ON THE PROTEOME OF KUPFFER CELLS

1
THE IMPACT OF ISCHEMIA REPERFUSION INJURY ON THE
PROTEOME OF KUPFFER CELLS

• Jan Hirsch, MD, Kirk C. Hansen, PhD, SooJinNa
  Choi, MD, Joonhwa Noh, MD, Ryutaro Hirose, MD,
  John P. Roberts, MD, Michael A. Matthay, MD, Alma
  L. Burlingame, PhD, Jacquelyn J. Maher and Claus
  U. Niemann, MD
• Department of Anesthesia and Perioperative Care,
  UCSF
• Cardiovascular Research Institute, UCSF
• Mass Spectrometry Facility, Department of
  Pharmaceutical Chemistry, UCSF
• Rice Liver Center Laboratory and Department of
  Medicine, UCSF
• Department of Surgery, Division of
  Transplantation, UCSF

2
Sample preparation

• Lean Zucker rats (6 per group) were randomized
• 75 min of warm ischemia
• sham operation
• Anesthesia with isoflurane
• Rats actively warmed to gt 37 C
• continuous rectal temperature monitoring
• Approx. 15 min from anesthesia induction to
  ischemia onset
• Established 70 liver ischemia model with
  reproducible results
• Rats sacrificed after 8 hrs of reperfusion

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Kupffer cell isolation

• Immediate Kupffer cell isolation after sacrifice
  
• Pronase and collagenase perfusion and digestion
  of the liver
• Accudenz density gradient centrifugation
• Suspension in 150 mM ABC
• Cell lysis by liquid homogenization (French
  press)
• Normalization of samples by protein content
• 125 mcg per sample

Maher JJ. Am J Physiol 1995269G518-523.
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Protein labeling and identification
iTRAQ labeling of N- terminal residues
Tryptic digestion into peptides and pooling of
ischemia / control samples
Fractionation (cation exchange
chromatography)

• Separation by reverse phase HPLC

ESI- qQ-TOF mass spectrometry

• Interrogation of peptide spectra (Swissprot)

5
MS-Quantification with iTRAQ
Ratio iTRAQ (heavy) / iTRAQ (light)
Liver Ischemia
Sham

• Differential labeling results in different peaks
  for same peptide
• Quantification by determination of area under
  peaks using ProteinProspector
• Result is content ratio of the peptide between
  ischemia and sham operation
• Protein ratio calculated as Mean SD of peptide
  ratios

Ross PL, et al. Mol Cell Proteomics 2004.
6
Data analysis and Statistics

• Thresholds for quantitation
• No less than four significant peptide matches per
  protein
• Protein present in gt2 sample pairs
• For each protein Mean SD of the peak intensity
  ratios from all peptides was calculated
• One-sample t-tests for significance testing
• Threshold for statistical testing
• No less than 8 peptides per comparison
• Presence in gt 2 samples
• Correction for multiple comparisons using the
  False Discovery Rate

Benjamini Y. Behav Brain Res 2001125279-284.
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Results

• Information obtained for 1559 proteins
• 185 of these proteins met the threshold criteria
  for quantitation
• Statistical comparisons performed in 161 proteins
• Testing for multiple comparisons resulted in 65
  proteins with statistically significant
  differences

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Oxidant and antioxidant proteins
9
Oxidant and antioxidant proteins

• Significant increases in Catalase, Superoxide
  dismutase mn and Transketolase
• Presence of Myeloperoxidase confirmed (Brown Am J
  Pathol. 2001)
• Changes indicate activation of the
  oxidant-antioxidant system after ischemia /
  reperfusion injury in rat Kupffer cells

10
Immunity-related proteins

• Anti-inflammatory factor-1, calgranulin B and
  complement C3 content significantly elevated
  after ischemia

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Potential Implications

• Allograft inflammatory factor -1 (AIF-1)
  modulates immune response during macrophage
  activation
• Expression up-regulated by IFN-? (Deininger, FEBS
  Letters 2002)
• Over-expressed in liver allografts with acute
  rejection in rats (Raisanen, Arterioscler Thromb
  Vasc Biol 1997)
• Whereas AIF-1 was only slightly increased in a
  cold ischemia model, we found significantly
  elevated expression 5 hrs after warm ischemia
• Elevated AIF-1 content has never been reported in
  a warm ischemia reperfusion model
• Complement factor 3 (C3) has anaphylatoxic,
  chemotactic, and histaminic actions
• Possible sources in Kupffer cells include
  production by the cell and uptake from the
  extracellular space
• Increased content in agreement with previous data
  after LPS stimulation (Ogle, Prostaglandins 1991)
• C3a was shown to increase prostaglandin D2,
  thromboxane B2 and prostaglandin F2 alpha
  formation in Kupffer cells
• (Puschel, Hepatology 1993)

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Potential implications

• Calgranulin b (S100A9) is a prominent player in
  innate immunity
• Acute allograft rejection accompanied by
  expression of calgranulin a and b
• Has been shown to induce apoptosis in colon
  cancer cell lines (Ghavami, J Leukoc Biol 2004)
• Calgranulin b has been associated with t-cell
  responses and affinity changes of the Mac-1
  integrin (Roulin, Exp Cell Res 1999)
• Calgranulin complex is linked to the arachidonic
  acid metabolism in human neutrophils
• Appears to be a mediator between calcium
  signaling and arachidonic acid effects (Kerkhoff,
  JBC 1999)

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Proteins of the arachidonic acid metabolism

• Significantly elevated content in leukotriene a-4
  hydrolase, cyclooxygenase -1 and
  glutathione-requiring prostaglandin d synthase
• Increase in Kupffer cell PGE2 production in
  response to sodium butyrate previously
  demonstrated (Perez, J Surg Res 1998)

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Conclusions

• Quantitative proteomic analysis identified
  changes in Kupffer cell protein patterns after
  ischemic liver injury
• Although some were predictable, others were
  novel, demonstrating the power of the iTRAQ
  proteomics method as a discovery tool
• gt Our results suggest that liver ischemia
  induces important alterations in protein cascades
  of the oxidant-antioxidant pathways, inflammatory
  proteins and the arachidonic acid metabolism in
  Kupffer cells