SP23
The interaction of IgM with apoptotic cells and differential IgM deposition in ischaemic
kidney and liver tissue
Emily E. Hesketh1, James Richards1, Martina Bucsaiova1, Mark Dockrell2, David Kluth1 &
Jeremy Hughes1. 1MRC Centre for Inflammation Research, Queen’s Medical Research Institute,
University of Edinburgh. 2SWT Institute for Renal Research, London.
Introduction: Natural IgM antibodies are able to bind apoptotic cells (ACs) and facilitate AC
clearance. However, following ischaemic reperfusion injury (IRI), endogenous IgM can be
pathogenic by binding injured cardiac muscle, skeletal muscle and the intestine where it
activates complement and augments tissue damage. In this study we explored IgM binding to
ACs and human renal proximal tubule cells in vitro and to injured ischaemic murine kidney and
liver tissue in vivo.
Methods: ACs were prepared from dissociated murine thymocytes by overnight culture in
serum free RPMI medium. Annexin-V (AnnV) and propidium iodide (PI) staining, assessed by
flow cytometry, was used to determine the level of early (AnnV + PI-) or late apoptosis (AnnV+
PI+). Murine plasma was used as a source of IgM. Cells were incubated in 12.5% murine
plasma (diluted in PBS) and IgM binding assessed by flow cytometry. Human proximal tubule
epithelial cells (HK-2) were injured using H2O2 (2.5 and 5 mM) to induce increasing levels of
apoptosis. Following incubation in 30% human serum (diluted in PBS) IgM binding was
examined by flow cytometry. Renal or hepatic IRI was modeled by occluding either the renal
vessels of Balb/c mice (24 mins) or the left hepatic artery (50 mins) of C57BL/6 mice
respectively. IgM deposition, inferred by immunohistochemistry, and cell necrosis was
assessed in frozen or paraffin embedded kidney and liver tissue harvested 1hr or 24hrs
following IRI. Data presented as percentage of IgM+ events (flow cytometry) or mean ± SEM
(immunohistochemistry).
Results: IgM did not bind early or late ACs (both < 5% IgM+) but strongly bound small AnnV+
PI+ AC-derived particles (~60% IgM+). Imaging studies using ‘flowsight’ technology
confirmed the cellular origin of these small AnnV+ PI+ particles. Inclusion of isotype control
IgM antibodies confirmed that IgM binding was not due to non-specific intracellular trapping of
IgM within PI+ cells. Interestingly, following permeabilisation with saponin prior to incubation
with mouse plasma, IgM bound strongly to late apoptotic AnnV+ PI+ (~97% IgM+) and viable
non-apoptotic cells (~90% IgM+), which are consistently IgM negative without
permeabilisation. This indicated that IgM was not binding a neo-epitope generated by caspase
activation in ACs but was interacting with an intracellular antigen present in both healthy and
apoptotic cells. IgM binding may be restricted by accessibility of the large IgM molecule (970
kDa) to this intracellular antigen. Previous published work in the heart, skeletal muscle etc and
our in vitro data suggested that IgM deposition in ischaemic kidneys with acute tubular necrosis
(ATN) would be observed. However, minimal medullary IgM deposition was evident at 1hr
(0.9%±0.5) and 24hrs (0.7%±0.5) following renal IRI despite the presence of significant ATN
(37.9%±12.6 necrotic tubules at 1hr; 67.5%±11.1 necrotic tubules at 24hrs). Similarly, IgM
bound minimally to HK-2 cells injured with 2.5mM (3% IgM+) or 5mM (5% IgM+) H2O2. In
contrast, dramatic IgM deposition was observed 1hr (~34%) and 24hrs (~55%) following
hepatic IRI, which mapped to necrotic areas of liver tissue.
Conclusion: IgM does not bind early or late ACs but bound strongly to small AnnV+PI+ ACderived particles in vitro. Intriguingly, IgM strongly binds permeabilised non-apoptotic viable
cells suggesting that IgM binds an intracellular antigen present in normal cells and ACs. Despite
these findings, published work in other organs and dramatic deposition following hepatic IRI,
minimal IgM deposition was observed following renal IRI despite the presence of significant
ATN. Furthermore, IgM did not bind injured human renal cells. The mechanisms underlying
the differential nature of IgM binding to injured liver and kidney in vivo are unclear.