Multinucleate cells form from mononucleate cells in a variety of physiological and pathological conditions depending on cell type. For example, skeletal muscle fibres and osteoclasts are normally multinucleate in adult mammals because of cell fusion into a syncytium. During chronic inflammation, as a result of persistent infection or foreign bodies, macrophages can form so-called multinucleated giant cells (MGCs). The function of such MGCs is unclear and they may even lack a function or be dysfunctional (Quinn and Schepetkin 2009; McNally and Anderson 2011). Multinucleate cells may in principle arise either from fusion (Zhou and Platt 2011), phagocytosis (McNally and Anderson 2005) or failure of cytokinesis (Normand and King 2010), but in general it is assumed that MGCs arise from fusion (Vignery 2008; Helming and Gordon 2009; Quinn and Schepetkin 2009; McNally and Anderson 2011).
Microglia are resident brain macrophages, which mediate innate immunity and phagocytosis of debris in the brain, but inflammatory activation of microglia is implicated in the pathology of multiple neurological diseases (Perry et al. 2010). Microglia can form MGCs when inflammatory activated in particular ways. In cultured rat microglia, MGC formation was found to be stimulated by interleukin-3 (IL-3), IL-4, interferon gamma (IFN-γ), granulocyte macrophage-colony stimulating factor (GM-CSF) and phorbol myristate acetate (PMA), while IL-1, IL-6 and tumour necrosis factor alpha (TNF-α) had no effect (Lee et al. 1993). In cultured murine microglia, single cytokines failed to induce MGC, but IL-4 and IL-13 induced microglial MGC formation in the presence of colony stimulating factors (Suzumura et al. 1999). However, in cultured pig microglia MGC formation was stimulated by mycobacteria, TNF-α (Peterson et al. 1996) and IFN-γ and inhibited by GM-CSF (Tambuyzer and Nouwen 2005). Phagocytosis of cell debris has also been found to stimulate microglia to form MGCs (Beyer et al. 2000).
Multinucleated giant cells derived from microglia have been implicated in a variety of brain pathologies. In particular, HIV-associated dementia is mediated by HIV-infected microglia which become MGCs, but how these infected multinucleate microglia cause dementia is less clear (Ghorpade et al. 2005; Nardacci et al. 2005). Microglia were also reported to form MGCs in the spinal cord of rats expressing a mutant Cu/Zn superoxide dismutase gene, modelling amyotrophic lateral sclerosis in humans (Fendrick et al. 2007). Giant-cell arteritis (GCA) is an inflammatory disease of arteries of the head, characterised by the presence of MGCs in the vessel wall. Amyloid beta (Aβ)-related angiitis, a form of GCA, is associated with an accumulation of microglia and MGCs containing intracellular deposits of Aβ (Melzer et al. 2012). Whether Aβ can induce microglia to form MGCs is unknown. MGCs derived from microglia accumulate in the brain with age (Hart et al. 2012). Thus, it is important to understand the origin and function of multinucleate microglia.
In this work, we tested whether agents known to inflammatory activate microglia could induce MGC formation by microglia. We found that lipopolysaccharide (LPS), Aβ, alpha-synuclein (α-Syn), dead PC12 cells, IFN-γ and TNF-α, but not IL-4, induced microglial multinucleation, apparently via protein kinase C (PKC). We investigated whether multinucleation was because of fusion, phagocytosis or blocked cytokinesis. We found that microglial multinucleation was because of failure of abscission, followed by reversal of the earlier stages of cytokinesis. Microglial MGCs appear normal, apart from being multinucleate and large, and have an increased phagocytic capacity.
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The proportion of multinucleate microglia varied considerably in different conditions, but was generally low (< 20%), except in the presence of an inhibitor of cytokinesis, cytochalasin D (61%). Factors that increased the proportion of multinucleate microglia included LPS, Aβ, α-Syn, IFN-γ, TNF-α, dead PC12 cells and PMA. In general these conditions inhibited the proliferation of the mononucleate cells, and the increase in multinucleation in these conditions is consistent with multinucleation being caused by an inhibition of cytokinesis, which would inhibit proliferation and cause mitotic cells to accumulate multiple nuclei. However, our results are not consistent with multinucleation being a terminally differentiated state, as multinucleation was fully reversed after withdrawal of PMA (Fig. 6b). This suggests that mononucleate and multinucleate microglia are in rapid equilibrium, and changes in the ratio may be caused by stimulations or inhibitions of the interconversion in either direction, as a result of inhibition or stimulation of cytokinesis (replication of the cell) or karyokinesis (replication of the nucleus by mitosis).
The origin of MGCs (i.e. large cells of monocyte/macrophage origin with multiple nuclei that appear under inflammatory conditions) has almost invariably been attributed to fusion of cells. There is extensive evidence for such fusion occurring, particularly in the presence of IL-4 or foreign bodies (large structures such as catheters that cannot be phagocytosed) (Anderson et al. 2008; Helming and Gordon 2009; McNally and Anderson 2011). However, there is also extensive evidence that multinucleation can alternatively arise from inhibition of cytokinesis, i.e. inhibition of cell division after nuclear division has been completed, and cytokinesis can stop at various stages and then reverse to form a binucleate cell (Normand and King 2010). Known causes of multinucleation by this means include: non-disjunction of chromosomes (i.e. failure to separate) during meiosis (Shi and King 2005) and endomitosis during megakaryocyte differentiation (Geddis et al. 2007). We observed no fusion between red and green stained microglia, and the multinucleate cells induced by LPS or Aβ were never a homogenous mixture of red and green, despite the fact that we could easily observe such cells when fusion was induced by PEG. We also observed no cases of multinucleation occurring by fusion when examined by time-lapse microscopy. In contrast, all cases of multinucleation observed by video microscopy arose from a single cell undergoing mitosis (nuclear division), starting cytokinesis, forming a cleavage furrow and in some cases a long cytoplasmic bridge, but then reversing cytokinesis to form a multinucleate cell. We were surprised to find that these cytoplasmic bridges could be quite long (several cell widths), with the two pseudo-daughter cells behaving independently for some time before retraction of the cytoplasmic bridge. This raises the intriguing possibility that clonal microglia (and other cells) may be linked by cytoplasmic bridges in vivo, but we know of no evidence that this is the case. Spermatids and some other cells are known to be connected by cytoplasmic bridges as a result of failure of abscission (Normand and King 2010).
Various PKCs are known to be activated in microglia by LPS (Shen et al. 2005; Wen et al. 2011), Aβ (Combs et al. 1999; Nakai et al. 2001) and INF-γ (Shen et al. 2005), and foreign-body induced multinucleation of macrophages is known to be mediated by PKCs (McNally et al. 2008). Thus, inflammatory agents might cause multinucleation of microglia via stimulation of PKCs. Consistent with this we found that the PKC activator PMA increased microglial multinucleation, and that a PKC inhibitor Gö6976 prevented LPS and Aβ-induced multinucleation. Gö6976 specifically inhibits PKCs α, β and μ (PKC μ is also known as PKD), suggesting that one or more of these PKC/PKD isoforms are involved in microglial multinucleation. PKCα and PKCδ may inhibit cytokinesis (Akakura et al. 2010), while PKCε may be required for completion of cytokinesis (Saurin et al. 2008). However, such effects may be indirect as PKCs regulate multiple processes, and multiple processes regulate cytokinesis and its last stage, abscission (Normand and King 2010; Schiel and Prekeris 2010). PKCs can activate NADPH oxidases, which are implicated in multinucleation, although via increasing fusion (Quinn and Schepetkin 2009). The PKC activator PMA has been shown to induce multinucleation of mesenchymal stem cells because of inhibition of cytokinesis rather than induction of fusion (Yoshida et al. 2007), consistent with our results.
The function, dysfunction or lack of function of MGCs is unclear (McNally and Anderson 2011). We found that multinucleate microglia behaved similarly to mononucleate microglia as far as we could discern (similar levels of cell death, Ki-67 expression and IL-6 expression), but were larger and had a higher capacity to phagocytose large beads and cells. It has previously been suggested that osteoclasts and MGCs are large to enable phagocytosis of larger structures (McNally and Anderson 2011), and this appears to be true of multinucleate microglia, enabling them to phagocytose cell-sized beads and cells that are at least as large as a mononucleate microglia. The mononucleate microglia phagocytosed 5 μm beads (0.11 ± 0.01 beads/cell/hour) at about 1/90 of the rate for 1 μm beads (9.9 ± 3.0 beads/cell/hour), presumably reflecting the difficulty of phagocytosing something approaching the size of the phagocyte. While the multinucleate microglia had a marginally higher capacity to phagocytose the smaller beads (proportionate to their larger size), these larger cells had a disproportionately higher capacity to phagocytose the larger beads, suggesting that the larger microglia could phagocytose larger structures. However, it could be that these multinucleate cells have changes (not measured by us), other than their size, that enable them to phagocytose larger structures. Large, multinucleate, cells might be less mobile, but if so this did not prevent them from having a higher phagocytic capacity.
The multinucleate microglia had a substantially increased ability to phagocytose dead and live PC12 cells (relative to mononucleate microglia), which may reflect their increased ability to phagocytose large structures, possibly as a result of their larger size. Both mononucleate and multinucleate microglia preferred to phagocytose dead rather than live PC12 cells, but the multinucleate microglia were less discriminating between dead and live cells (Fig. 7c and d). We have previously shown that activated microglia can phagocytose live neurons and thereby kill them – a form of cell death we have called ‘phagoptosis’ (Neher et al. 2011; Brown and Neher 2012). It would appear from the research reported here that a small subset of microglia, the multinucleate, is particularly active in the phagocytosis of cells, and this multinucleate subset is increased by inflammation. Stimulating multinucleation could be beneficial during neurodegenerative disease, facilitating clearance of dead and dying cells without increasing microglial activation and inflammation, or perhaps during viral infection by promoting removal of live virus-infected cells. However, in the healthy brain, multinucleation would appear to be detrimental and might result in additional neuronal loss through phagoptosis.