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Title: C-reactive protein does not opsonize early apoptotic human neutrophils, but binds only
membrane-permeable late apoptotic cells and has no effect on their phagocytosis by macrophages.
Authors:
Simon P Hart ([email protected])
Karen M Alexander ([email protected])
Shonna M MacCall ([email protected])
Ian Dransfield ([email protected])
Version: 2 Date: 18 April 2005
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Title :
C-reactive protein does not opsonize early apoptotic human neutrophils,
but binds only membrane-permeable late apoptotic cells and has no effect on
their phagocytosis by macrophages.
Authors:
Simon P Hart, Karen M Alexander, Shonna M MacCall and Ian Dransfield
Reviewer 1
1. We agree with the reviewer that the apparent subpopulations of annexin V+ FITC-CRP+ cells in
figure 4a probably represent late apoptotic cells and primarily necrotic cells. This does not detract from
the principal message that most annexin V-positive cells no not bind CRP, and that all of the CRPpositive cells are permeable to PI. Subsequent analyses using IF microscopy and light microscopy
failed to distinguish two populations.
2. We have consistently used the term “late apoptotic” to describe cells that have a progressed through
apoptosis to a stage where membrane integrity has been lost. These cells exhibit all the characteristics
of apoptotic cells (internucleosomal DNA fragmentation, caspase activation, etc). This is distinct from
necrosis, which may be induced following a severe direct insult such as heating or freeze-thawing (as
demonstrated in figure 1d).
3. Fig 4b legend has been modified to include description of the nuclear stain (TO-PRO-3).
4. The phagocytosis assay described in the present manuscript has been extensively validated. We and
others have reported that inhibition or stimulation of phagocytosis by many different conditions can be
accurately and reproducibly measured using this assay. The short time course (60 mins) was chosen
because human monocyte-derived macrophages are efficient at phagocytosing apoptotic cells
(compared with THP-1 cells, for example), but time is still required for the apoptotic cell suspension to
settle onto the macrophage monolayer. We would not expect to see significant differences between
uptake of CRP-incubated or control aged neutrophils over a shorter period.
Reviewer 2
1. The present study was designed to test the hypothesis that altered FcγRIIA on apoptotic neutrophils
was reponsible for the augmented binding of CRP to apoptotic cells that has been reported by other
groups, whilst exploiting the observation that when neutrophils undergo apoptosis they remain intact
and membrane-impermeable to a greater extent than any other cell type we have examined. We have
shown that CRP fails to bind to the plasma membrane of early apoptotic neutrophils. Membraneimpermeable late apoptotic cells bound CRP principally intracellularly, so CRP may not be accessable
to a neighbouring macrophage. We have previously reported that conditions which augment
macrophage pro-inflammatory cytokine release following apoptotic cell ingestion, for example when
apoptotic neutrophils were coated with immune complexes, also profoundly influenced the rate of
phagocytosis 1. Because phagocytosis was unaffected in the present study, we believe that the bound
CRP is not influencing the mechanism used by macrophages to recognise apoptotic neutrophils, and
that measuring macrophage cytokine response would be unlikely to yield useful information.
2. We deliberately used directly conjugated CRP to avoid inherent difficulties in determining potential
binding to Fc receptors, which has caused much controversy in the past 2. However, we share the
reviewer’s concern that conjugation of CRP may alter it’s properties, so we performed stringent
analyses to confirm that it was structurally and functionally intact. Figure 1 shows that conjugated CRP
migrated as a single band in SDS-PAGE, bound in a cation-dependent manner to phosphorylcholineBSA, and bound necrotic (freeze-thawed) cells, and so fulfilled the accepted criteria for CRP binding.
Previously reported functional studies with CRP have courted controversy because of concern about
potential contamination of CRP preparations with IgG, so we are not aware of a suitable alternative
functional assay in which to look for quantitative differences between unconjugated and conjugated
CRP.
Reviewer 3
The reviewer’s question is addressed in the response to reviewer 1, question 1.
References
1. Hart SP, Alexander KM, Dransfield I. Immune complexes bind preferentially to FcgammaRIIA
(CD32) on apoptotic neutrophils, leading to augmented phagocytosis by macrophages and
release of proinflammatory cytokines. J.Immunol. 2004;172:1882-7.
2. Saeland E, van Royen A, Hendriksen K, Vile-Weekhout H, Rijkers GT, Sanders LA et al.
Human C-reactive protein does not bind to FcgammaRIIa on phagocytic cells. J.Clin.Invest
2001;107:641-3.