It has been reported that the pentraxins CRP and serum amyloid P are ligands for leukocyte Fcγ receptors . FcγRIIA becomes functionally enabled on early apoptotic human neutrophils [3, 4], but we have demonstrated that soluble CRP does not bind to classical early apoptotic human neutrophils in vitro, and we have been unable to demonstrate CRP binding to human FcγRIIA on transfected Jurkat cells. In the present study we have been careful to avoid pitfalls associated with indirect detection of ligand binding to neutrophils by using a preparation of directly labelled CRP that we have shown was essentially free of contaminating IgG, but which was structurally and functionally intact. The failure of CRP to bind FcγRIIA is consistent with the results of Hundt and colleagues who failed to find specific receptors for CRP on human leukocytes .
There have been several reports of Ca2+-dependent opsonisation of apoptotic cells by pentraxins [9, 11, 21]. The lack of CRP binding to classical early apoptotic human neutrophils, which exhibit all the biochemical and surface changes associated with apoptosis yet remain intact and membrane impermeable, raises questions about whether putative opsonins are really able to bind with high affinity to apoptotic cells. In contrast, we demonstrated very strong CRP binding to a subpopulation of aged neutrophils which displayed the characteristics of late apoptotic neutrophils previously reported by Hebert  and Ren . It has been recognised that CRP binds to necrotic cells since Kushner and Kaplan demonstrated CRP deposition in necrotic skeletal muscle fibres following typhoid vaccination in vivo . It is not always recognised that induction of apoptosis in many cell types in vitro leads to a significant proportion of membrane-permeable late apoptotic cells . The presence of leaky late apoptotic cell ghosts in a cell population may be overlooked because the lack of nuclear material means that they stain very faintly with May-Giemsa, and in the past late apoptotic cells may have been gated out as "debris" when analysed by flow cytometry. Furthermore, these cells pellet poorly in cytocentrifuge preparations, and this combined with their "invisibility" with May-Giemsa stains means that their prevalence has been underestimated. This clearly has implications for studies of apoptotic cell opsonisation, and much of the published data may reflect binding to intracellular moieties. CRP is not unique in binding to the interior of leaky apoptotic cells, and we have reported a similar phenomenon with the unrelated serum protein thrombospondin . Complement proteins, collectins, and heparin may also bind preferentially to late apoptotic cells [20, 25, 26]. The precise structures responsible for CRP binding have not been elucidated, but our data suggest that there are both Ca2+-dependent and Ca2+-independent binding sites. Binding to cell membrane phospholipids may account for the Ca2+-dependent component , whereas binding to polycations may be responsible for the heparin-inhibitable Ca2+-independent component . The relative paucity of nuclear chromatin in the late apoptotic cells and the absence of nuclear co-localisation seen with fluorescence microscopy means that chromatin binding is unlikely to be responsible .
The presence of late apoptotic cells also has implications for studies of the phagocytosis of apoptotic cells, since these cells may be recognised differently from classical early apoptotic cells [17, 23]. It is not known whether "opsonins" bound to intracellular components would be accessible for recognition by phagocyte receptors. In the present study we have shown that despite very strong CRP binding to late apoptotic neutrophils, there was no detectable effect on their clearance by macrophages. Ren and colleagues demonstrated that the efficiency of phagocytosis of early- and late apoptotic neutrophils was similar , so we think it is unlikely that an effect of CRP on uptake of late apoptotic cells has been masked by baseline uptake of early apoptotic neutrophils in the population of aged cells that we used.