Fc- and Complement-Receptor Activation Stimulates Cell Cycle Progression of Macrophage Cells from G1 to S1
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免疫学杂志 2005年第11期
Abstract
Phagocytosis of microorganisms by macrophages is an important host defense mechanism. While studying the phagocytosis of the human pathogenic fungus Cryptococcus neoformans, we noted that macrophage-like J774 cells with ingested fungal cells had frequent mitotic figures. By analyzing the relative proportion of phagocytic cells as a function of cell cycle phase, we observed an increase in S phase cells after Fc-mediated phagocytosis of polystyrene beads, live or heat-killed C. neoformans. This result was confirmed by increased nuclear BrdU incorporation after Fc-mediated phagocytosis. The induced progression to S phase was observed after both Fc- and complement-mediated phagocytosis of live yeasts. Fc-mediated stimulation of cell division did not require ingestion, because it could be triggered by incubating cells in IgG1-coated plates. Phagocytosis-mediated stimulation of replication was confirmed in vitro using primary bone marrow macrophages and in vivo for peritoneal macrophages. We conclude that phagocytosis of microbes or inert particles can stimulate macrophages to enter S phase and commence cell division. This observation suggests a potential mechanism for increasing the number of effector cells after microbial ingestion, but can also promote the spread of infection.
Introduction
Phagocytosis of microorganisms by host phagocytic cells is an important host defense mechanism. The process of phagocytosis involves a rearrangement of the cytoskeleton and cell membrane to ingest the microbial particle (1, 2). Microbial phagocytosis can be followed by cellular activities that result in the death of the microbe, including lysosomal fusion with the phagosome and oxidative burst (2, 3). Alternatively, phagocytosis may not lead to the killing of the microbe because many pathogens have strategies to evade cellular microbicidal mechanisms. Examples of microbes that replicate inside macrophages include Mycobacterium tuberculosis (4), Listeria monocytogenes (5), and Cryptococcus neoformans (Cn)4 (6). Given that the outcome of phagocytosis is uncertain for the host cell, it is likely that immune cells have additional safeguards for the eventuality that phagocytosis does not lead to microbial killing.
Most mature macrophages, such as those in the peritoneum, originate from blood monocytes, which are derived from bone marrow. Generally, these cells are considered to be postmitotic (7). However, peritoneal exudate macrophages have been reported to form colonies when placed in suitable culture conditions in vitro (8, 9). Several studies have shown that a significant proportion of macrophages in the peritoneum and other organs can undergo mitosis (10, 11, 12, 13, 14, 15). Cell division would lead to a doubling of the phagocytic cells, an event that could conceivably benefit the host by increasing the number of effector cells at the site of infection. However, division of cells infected with a live microbe that is replicating intracellularly could also lead to a doubling of infected cells, an event that could potentially harm the host. Hence, one can posit theoretical reasons for the supposition that phagocytosis could alter cell division through programmed host cell effects or subversion of the process by microbial actions. Although there are examples of microbial toxins impacting cell division (16, 17), we could find no reports in the literature that explored direct links between phagocytosis and cell division effects.
Cn is a major cause of life-threatening infections in patients with impaired immunity (18). This microbe is a facultative intracellular pathogen that replicates inside macrophages by using an unusual strategy that includes accumulation of polysaccharide-containing vesicles and induction of phagosome leakiness (6). While studying the phagocytosis of this organism, we noted that macrophages with ingested fungal cells frequently appeared to be dividing, as indicated by mitotic figures. Interestingly, in the late 1960s, Mackaness (10) reported that peritoneal macrophages manifested numerous mitotic figures after in vivo injection of Listeria monocytogenes into the peritoneum. Hence, we hypothesized that the phagocytosis of microbes could affect macrophage cell division. This phenomenon may have been due to the stimulation, synchronization, or arrest of cell cycle. We report in this work that the phagocytic ability of macrophages is cell cycle dependent and, most interestingly, that phagocytosis of microbes or inert particles may allow macrophages to re-enter the cell cycle and start replication. We believe these observations could herald a general phenomenon with important implications for both host defense and microbial pathogenesis.
Materials and Methods
Statistics
Pairwise comparison between groups was done by t test using Excel. Value of p < 0.05 was considered significant.
Results
Phagocytosis is a cell cycle-dependent process
To investigate the relationship between phagocytic efficacy and cell cycle phase, we evaluated the proportion of J774 cells with ingested yeast cells relative to their cell cycle stage. The growth curve showed that J774 cells duplicated every 12 h in nutritional culture medium (Fig. 1A). The phagocytic efficacy of 3T3 cells was reported to depend on cell cycle phase, with cells in G1 phase being most efficient in their ability to phagocytose particles (21). Consistent with that report, our results showed that macrophages in G1 phase have higher phagocytic ability than those in S and G2 phase. J774 cells were synchronized to G1 phase by starvation in serum-depleted medium for 72 h (Fig. 1B). More than 80% of J774 cells were synchronized to G1 phase by this procedure, as analyzed by FACS. In the G1-enriched fractions, the percentage of phagocytosis rose to 70%. While under normal conditions, the percentage of J774 cells in G1 was from 40 to 60%, and the percentage of phagocytosis was below 50%. There was a strong correlation between the percentage of phagocytosis and the proportion of J774 cells in G1 phase (Fig. 1B). In a separate experiment, the phagocytic index of J774 cells cultured for 48 h in serum-depleted medium increased >5-fold and the percentage of phagocytosis increased 3-fold, relative to cells cultured for 5 h in fresh medium (Fig. 1, C and D). These experiments confirmed that J774 cells, as 3T3 cells, are more phagocytic in G1 phase.
Cell cycle distribution of peritoneal macrophages is altered after Fc-mediated phagocytosis in vivo
We investigated whether the phenomenon of enhanced cell cycle progression observed in the J774 cell line and in cultured primary macrophages would occur in vivo. Analysis of peritoneal macrophages from uninfected mice revealed that 90% of the cells collected expressed macrophage marker MAC-3 and all of these peritoneal macrophages were in G1 phase. After mice were infected i.p. with Ab-opsonized Cn, peritoneal macrophages were harvested and analyzed. Although phagocytosis of Ab-opsonized Cn by peritoneal macrophages in vivo was not as robust as in the optimized J774 culture assay, it nevertheless revealed that those peritoneal macrophages, which had internalized Cn, were shifted to S and G2 just as in the previous assays. This was observed despite the variability between individual mice (Table I). This experiment was done eight times, and the effect was observed in four independent experiments.
Discussion
This study originated from two observations. First, we made the serendipitous observation that cells with ingested Cn were more likely to have morphological features consistent with cell division. Second, we noted great interexperimental variation in the phagocytic index of macrophage-like cells for Cn. After considering and excluding such explanations as inadequate time for phagocytosis, opsonin quantity, and yeast cell to macrophage ratio, we combined these observations to hypothesize that the interexperimental differences reflected the state of the phagocytic cells and focused on the cell cycle phase. Our results establish that the efficiency of phagocytosis is a cell cycle-dependent process, and demonstrate for the first time that phagocytosis in turn promotes cell division. J774 cells are more efficient in phagocytosis when they reside in G1 phase of the cell cycle compared with other phases. This result is consistent with, and confirmatory of, a report that phagocytosis in 3T3 cells occurred primarily in G1 phase cells (21). Most importantly, we found that phagocytosis can drive the cell cycle of macrophages from G1 into S phase, which has numerous implications. The FACS results showed that phagocytosis changed the percentage of distribution of cells in the different cycle phases; namely, that in the nonphagocytic population, there were more cells in G1 than in S phase, and in the phagocytic population there were fewer cells in G1 and more in S phase. The conclusion that phagocytosis stimulated cell division was supported by the BrdU incorporation experiments, which revealed increased DNA synthesis in cells undergoing phagocytosis. These observations with cultured J774 cells were confirmed with bone marrow-derived macrophages and peritoneal cells in vivo, demonstrating for the first time that induction of cell cycle progression is enhanced by phagocytosis regardless of the type of ingested particles.
Cell replication is affected by external stimuli such as growth factors, cell-cell contact, and cell adhesion to the extracellular matrix. Despite the fact that both cell division and phagocytosis are basic processes of cell biology that have been exhaustively studied for decades, we could not find prior studies in the literature that directly explored connections between phagocytosis and cell division. However, certain observations hint the two processes may be connected. It was recently reported that cyclin D1 deficiency impaired the motility and guided migration of bone marrow macrophages and reduced membrane ruffles (23), in which the ability to make ruffles has been shown to be important for internalizing particles. This suggests an explanation for the result that macrophages in G1 phase have the optimal ability to carry out phagocytosis because cyclin D dominates in the G1 phase of cell cycle. Although the relationship between cytoskeletal changes and cyclins has also been explored (24, 25), a direct link between phagocytosis and cell cycle has not been made. Our findings in this study show that phagocytosis is potentially a powerful stimulus to promote macrophage replication.
Phagocytosis-induced stimulation of cell cycle progression of J774 cells was repeatedly observed after the phagocytosis of polystyrene beads, live Cn or dead Cn. We observed the same phenomenon with bone marrow macrophages, thus extending this to another cell type and to primary macrophages. In the Cn experimental system, IgG1-mediated opsonization can trigger phagocytosis through the FcR or through complement receptor in an unusual complement-independent mechanism whereby the capsule interacts directly with CD18 (26). Because both mechanisms occur simultaneously, we conducted additional experiments to discriminate whether Fc- or complement-mediated opsonization was responsible for the observed stimulation of cell division. Furthermore, we were interested in establishing whether phagocytosis was necessary for the effect. Incubating J774 cells on a plate coated with IgG1 stimulated cell cycle progression, indicating that FcR activation could induce the phenomenon. Furthermore, we noted cell cycle progression after C3-mediated phagocytosis, indicating that the phenomenon could follow activation of the complement receptors. Hence, engaging either Fc or complement receptors can trigger cell cycle progression, and at least the Fc-mediated process does not require ingestion. However, the observation that J774 cells incubated with naked polystyrene beads stimulated cell cycle progression indicates that additional receptors may feed into this signal transduction pathway. The fact that multiple surface receptors central to the phagocytic process can stimulate cell cycle progression, combined with the observation that it occurs in both primary and continuous cell lines, suggests a basic link between two physiological pathways.
A 10–15% progression from G1 to S phase was consistently observed after the phagocytosis of polystyrene beads, live Cn or dead Cn, in 20 independent experiments. Although at first glance it may appear that only a small percentage of the cells in any one experiment progressed to S phase, this percentage is relatively large when one considers that approximately half the cells are in a resting state (40–50% for J774 cells and bone marrow macrophages). Incidentally, we obtained similar results with longer incubation time (as long as 4 h). Considering that phagocytosis is a rapid event that is largely completed by 15 min, the relatively short duration of activation of Fc/complement receptors might lead to the measured change of the cell cycle progression.
In the past two decades, the signaling cascades responsible for and resulting from phagocytosis and cell division have been elucidated. Therefore, it is possible to identify shared pathways that may explain our findings. The cell cycle is divided into G1, S, G2, and M phases in eukaryotic cells. The regulation of this cycle is primarily controlled by periodic synthesis and destruction of cyclins, which in turn bind to cyclin-dependent kinases (cdks) and activate them. To enter S phase, cells must pass a restriction point in late G1 phase. This process is conducted by cyclin D, which phosphorylates retinoblastoma tumor suppressor protein (Rb) through the binding of cyclin D with cdk4 and cdk6. Cyclin D expression is induced by mitogenic stimuli instead of oscillating during cell cycle, as the other cyclins. Phosphorylation of Rb prevents it from binding to E2F factors and thus switches E2F from a repressor to an activator of gene expression of cell cycle proteins, including cyclins E and A. Cyclin E participates in a positive feedback control of Rb by maintaining Rb in a hyperphosphorylated state, and thus drives the cell cycle through S and G2 (27). Intensive studies showed that the cell cycle is under subtle regulation by signaling molecules such as PI3K and Rho GTPases (25, 28, 29, 30). The activation of PI3K and Rho GTPases is required for the process of macrophage phagocytosis (2, 3). This leads us to posit that activation of known signaling pathways involved in phagocytosis could potentially influence cell proliferation. For instance, activation of PI3K in phagocytosis could participate not only in the molecular events in phagocytosis, but also in the cell cycle regulation such as the inhibition of p27kip1, which in turn increases the activity of cyclin E/cdk2 and thus enhances the G1 to S transition (29, 31, 32). Hence, phagocytosis could activate PI3K and stimulate cells to progress in the cell cycle progression via cyclin E.
Phagocytosis by macrophages is an important process in host defense mechanisms. Microbial killing of phagocytosed microbes is beneficial to the protection of organisms from infection. Macrophage local proliferation was an efficient way to replenish the damaged macrophage after killing. Although most hemopoietic cells mature after leaving the bone marrow and cannot proliferate, local proliferation of mononuclear phagocytes has been observed (9, 33). For example, macrophages resident in spleen and peritoneum were renewed by local proliferation (14, 15). Furthermore, macrophages can replicate upon the stimulation of growth factors such as CSFs, and especially during inflammation (11). Thus, the linkage of phagocytosis and cell division could have important consequences for our understanding of microbial pathogenesis and host defense. On one hand, phagocytosis-induced cell division could increase the number of effector cells in response to infection, and also provides a novel mechanism for macrophages to cure themselves after ingesting microbes by the generation of new cells that have the potential to be noninfected. In fact, a study on persistent infection of host cells with Coxiella burnetii showed that heavily infected host cells had the ability to restrain intracellular Coxiella in one large parasite-containing vacuole, which was passaged into one of the two daughter cells of dividing host cells. Thus, a pathogen-free companion daughter cell was generated (34, 35). This would be one of the mechanisms that infected cells could cure themselves by cell replication. In contrast, many microbes are intracellular pathogens that replicate preferentially inside phagocytic cells and use intracellular residence as a mechanism to evade the immune system. Consequently, this phenomenon could also undermine host defenses by generating new infected cells in which microbes can proliferate. Clearly, the benefit or debit of this phenomenon for host defense would be dependent on the type of pathogen. The linkage of phagocytosis-induced cell division with the need for nutrients is intriguing given that phagocytosis of bacteria and other microbes is used by unicellular eukaryotes for food acquisition. Hence, phagocytosis may be linked to cell division through ancient pathways that signal the acquisition of food, which in turn would imply favorable conditions for replication.
In summary, our results indicate that phagocytosis occurs preferentially in G1 phase, and that particle ingestion stimulates the progression of cell cycle. Remarkably, this phenomenon is unknown despite the fact that both processes have been studied extensively. Cellular division following phagocytosis could have major implications for the outcome of macrophage infection. For some pathogens, we anticipate that the effect would help the host to generate additional uninfected effector cells at the site of infection. However, for other pathogens, this effect could contribute to pathogenesis. If that is the case, it is possible that future studies could identify the precise signaling pathway involved in phagocytosis-associated cell division and evaluate it as a target for antimicrobial therapy of certain infectious diseases. Furthermore, the findings suggest the need to investigate the phylogenetic origins of this phenomenon, which may have ancient roots in eukaryotic evolution.
Acknowledgments
We thank Drs. Matthew Scharff, Marshall Horwitz, and Dianne Cox for critical reading of the manuscript; Huafeng Xie and Dr. Thomas Graf for their generously providing C57BL/6 mice and primary bone marrow cells; Drs. Franco Cappoza and Michael Lisanti for generously providing BrdU and Ab to BrdU; Peng Ji in Dr. Liang Zhu’s lab for help with Western blots; and Krishanthi Subramaniam in Dr. Liise-anne Pirofski’s lab for help with labeling Cn by FITC. We also thank Clarissa Santos and Kristie Gordon from the FACS facility at the Albert Einstein College of Medicine.
Footnotes
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 This study was supported by National Institutes of Health Grants AI033142, AI033774, AI052733, and HL059842. S.C.T. is supported in part by National Institutes of Health/National Institute of Allergy and Infectious Diseases Training Grant 5T32AI07506.
2 Current address: Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Ullmann Building, Room 907, 1300 Morris Park Avenue, Bronx, NY 10461.
3 Address correspondence and reprint requests to Dr. Arturo Casadevall, Department of Medicine and Department of Microbiology and Immunology, Albert Einstein College of Medicine, Golding Building, Room 701, 1300 Morris Park Avenue, Bronx, NY 10461. E-mail address: casadeva{at}aecom.yu.edu
4 Abbreviations used in this paper: Cn, Cryptococcus neoformans; cdk, cyclin-dependent kinase; FSC, forward light scatter; Rb, retinoblastoma tumor suppressor protein.
Received for publication December 8, 2004. Accepted for publication March 16, 2005.
References
Greenberg, S.. 1999. Modular components of phagocytosis. J. Leukocyte Biol. 66: 712-717.
Underhill, D. M., A. Ozinsky. 2002. Phagocytosis of microbes: complexity in action. Annu. Rev. Immunol. 20: 825-852.
Aderem, A., D. M. Underhill. 1999. Mechanisms of phagocytosis in macrophages. Annu. Rev. Immunol. 17: 593-623.
Flynn, J. L., J. Chan. 2003. Immune evasion by Mycobacterium tuberculosis: living with the enemy. Curr. Opin. Immunol. 15: 450-455.
Portnoy, D. A., V. Auerbuch, I. J. Glomski. 2002. The cell biology of Listeria monocytogenes infection: the intersection of bacterial pathogenesis and cell-mediated immunity. J. Cell Biol. 158: 409-414.
Tucker, S. C., A. Casadevall. 2002. Replication of Cryptococcus neoformans in macrophages is accompanied by phagosomal permeabilization and accumulation of vesicles containing polysaccharide in the cytoplasm. Proc. Natl. Acad. Sci. USA 99: 3165-3170.
Van Furth, R., Z. A. Cohn. 1968. The origin and kinetics of mononuclear phagocytes. J. Exp. Med. 128: 415-435.
Stewart, C., H. Lin, C. Adles. 1975. Proliferation and colony-forming ability of peritoneal exudate cells in liquid culture. J. Exp. Med. 141: 1114-1132.(Yong Luo*, Stephanie C. T)
Phagocytosis of microorganisms by macrophages is an important host defense mechanism. While studying the phagocytosis of the human pathogenic fungus Cryptococcus neoformans, we noted that macrophage-like J774 cells with ingested fungal cells had frequent mitotic figures. By analyzing the relative proportion of phagocytic cells as a function of cell cycle phase, we observed an increase in S phase cells after Fc-mediated phagocytosis of polystyrene beads, live or heat-killed C. neoformans. This result was confirmed by increased nuclear BrdU incorporation after Fc-mediated phagocytosis. The induced progression to S phase was observed after both Fc- and complement-mediated phagocytosis of live yeasts. Fc-mediated stimulation of cell division did not require ingestion, because it could be triggered by incubating cells in IgG1-coated plates. Phagocytosis-mediated stimulation of replication was confirmed in vitro using primary bone marrow macrophages and in vivo for peritoneal macrophages. We conclude that phagocytosis of microbes or inert particles can stimulate macrophages to enter S phase and commence cell division. This observation suggests a potential mechanism for increasing the number of effector cells after microbial ingestion, but can also promote the spread of infection.
Introduction
Phagocytosis of microorganisms by host phagocytic cells is an important host defense mechanism. The process of phagocytosis involves a rearrangement of the cytoskeleton and cell membrane to ingest the microbial particle (1, 2). Microbial phagocytosis can be followed by cellular activities that result in the death of the microbe, including lysosomal fusion with the phagosome and oxidative burst (2, 3). Alternatively, phagocytosis may not lead to the killing of the microbe because many pathogens have strategies to evade cellular microbicidal mechanisms. Examples of microbes that replicate inside macrophages include Mycobacterium tuberculosis (4), Listeria monocytogenes (5), and Cryptococcus neoformans (Cn)4 (6). Given that the outcome of phagocytosis is uncertain for the host cell, it is likely that immune cells have additional safeguards for the eventuality that phagocytosis does not lead to microbial killing.
Most mature macrophages, such as those in the peritoneum, originate from blood monocytes, which are derived from bone marrow. Generally, these cells are considered to be postmitotic (7). However, peritoneal exudate macrophages have been reported to form colonies when placed in suitable culture conditions in vitro (8, 9). Several studies have shown that a significant proportion of macrophages in the peritoneum and other organs can undergo mitosis (10, 11, 12, 13, 14, 15). Cell division would lead to a doubling of the phagocytic cells, an event that could conceivably benefit the host by increasing the number of effector cells at the site of infection. However, division of cells infected with a live microbe that is replicating intracellularly could also lead to a doubling of infected cells, an event that could potentially harm the host. Hence, one can posit theoretical reasons for the supposition that phagocytosis could alter cell division through programmed host cell effects or subversion of the process by microbial actions. Although there are examples of microbial toxins impacting cell division (16, 17), we could find no reports in the literature that explored direct links between phagocytosis and cell division effects.
Cn is a major cause of life-threatening infections in patients with impaired immunity (18). This microbe is a facultative intracellular pathogen that replicates inside macrophages by using an unusual strategy that includes accumulation of polysaccharide-containing vesicles and induction of phagosome leakiness (6). While studying the phagocytosis of this organism, we noted that macrophages with ingested fungal cells frequently appeared to be dividing, as indicated by mitotic figures. Interestingly, in the late 1960s, Mackaness (10) reported that peritoneal macrophages manifested numerous mitotic figures after in vivo injection of Listeria monocytogenes into the peritoneum. Hence, we hypothesized that the phagocytosis of microbes could affect macrophage cell division. This phenomenon may have been due to the stimulation, synchronization, or arrest of cell cycle. We report in this work that the phagocytic ability of macrophages is cell cycle dependent and, most interestingly, that phagocytosis of microbes or inert particles may allow macrophages to re-enter the cell cycle and start replication. We believe these observations could herald a general phenomenon with important implications for both host defense and microbial pathogenesis.
Materials and Methods
Statistics
Pairwise comparison between groups was done by t test using Excel. Value of p < 0.05 was considered significant.
Results
Phagocytosis is a cell cycle-dependent process
To investigate the relationship between phagocytic efficacy and cell cycle phase, we evaluated the proportion of J774 cells with ingested yeast cells relative to their cell cycle stage. The growth curve showed that J774 cells duplicated every 12 h in nutritional culture medium (Fig. 1A). The phagocytic efficacy of 3T3 cells was reported to depend on cell cycle phase, with cells in G1 phase being most efficient in their ability to phagocytose particles (21). Consistent with that report, our results showed that macrophages in G1 phase have higher phagocytic ability than those in S and G2 phase. J774 cells were synchronized to G1 phase by starvation in serum-depleted medium for 72 h (Fig. 1B). More than 80% of J774 cells were synchronized to G1 phase by this procedure, as analyzed by FACS. In the G1-enriched fractions, the percentage of phagocytosis rose to 70%. While under normal conditions, the percentage of J774 cells in G1 was from 40 to 60%, and the percentage of phagocytosis was below 50%. There was a strong correlation between the percentage of phagocytosis and the proportion of J774 cells in G1 phase (Fig. 1B). In a separate experiment, the phagocytic index of J774 cells cultured for 48 h in serum-depleted medium increased >5-fold and the percentage of phagocytosis increased 3-fold, relative to cells cultured for 5 h in fresh medium (Fig. 1, C and D). These experiments confirmed that J774 cells, as 3T3 cells, are more phagocytic in G1 phase.
Cell cycle distribution of peritoneal macrophages is altered after Fc-mediated phagocytosis in vivo
We investigated whether the phenomenon of enhanced cell cycle progression observed in the J774 cell line and in cultured primary macrophages would occur in vivo. Analysis of peritoneal macrophages from uninfected mice revealed that 90% of the cells collected expressed macrophage marker MAC-3 and all of these peritoneal macrophages were in G1 phase. After mice were infected i.p. with Ab-opsonized Cn, peritoneal macrophages were harvested and analyzed. Although phagocytosis of Ab-opsonized Cn by peritoneal macrophages in vivo was not as robust as in the optimized J774 culture assay, it nevertheless revealed that those peritoneal macrophages, which had internalized Cn, were shifted to S and G2 just as in the previous assays. This was observed despite the variability between individual mice (Table I). This experiment was done eight times, and the effect was observed in four independent experiments.
Discussion
This study originated from two observations. First, we made the serendipitous observation that cells with ingested Cn were more likely to have morphological features consistent with cell division. Second, we noted great interexperimental variation in the phagocytic index of macrophage-like cells for Cn. After considering and excluding such explanations as inadequate time for phagocytosis, opsonin quantity, and yeast cell to macrophage ratio, we combined these observations to hypothesize that the interexperimental differences reflected the state of the phagocytic cells and focused on the cell cycle phase. Our results establish that the efficiency of phagocytosis is a cell cycle-dependent process, and demonstrate for the first time that phagocytosis in turn promotes cell division. J774 cells are more efficient in phagocytosis when they reside in G1 phase of the cell cycle compared with other phases. This result is consistent with, and confirmatory of, a report that phagocytosis in 3T3 cells occurred primarily in G1 phase cells (21). Most importantly, we found that phagocytosis can drive the cell cycle of macrophages from G1 into S phase, which has numerous implications. The FACS results showed that phagocytosis changed the percentage of distribution of cells in the different cycle phases; namely, that in the nonphagocytic population, there were more cells in G1 than in S phase, and in the phagocytic population there were fewer cells in G1 and more in S phase. The conclusion that phagocytosis stimulated cell division was supported by the BrdU incorporation experiments, which revealed increased DNA synthesis in cells undergoing phagocytosis. These observations with cultured J774 cells were confirmed with bone marrow-derived macrophages and peritoneal cells in vivo, demonstrating for the first time that induction of cell cycle progression is enhanced by phagocytosis regardless of the type of ingested particles.
Cell replication is affected by external stimuli such as growth factors, cell-cell contact, and cell adhesion to the extracellular matrix. Despite the fact that both cell division and phagocytosis are basic processes of cell biology that have been exhaustively studied for decades, we could not find prior studies in the literature that directly explored connections between phagocytosis and cell division. However, certain observations hint the two processes may be connected. It was recently reported that cyclin D1 deficiency impaired the motility and guided migration of bone marrow macrophages and reduced membrane ruffles (23), in which the ability to make ruffles has been shown to be important for internalizing particles. This suggests an explanation for the result that macrophages in G1 phase have the optimal ability to carry out phagocytosis because cyclin D dominates in the G1 phase of cell cycle. Although the relationship between cytoskeletal changes and cyclins has also been explored (24, 25), a direct link between phagocytosis and cell cycle has not been made. Our findings in this study show that phagocytosis is potentially a powerful stimulus to promote macrophage replication.
Phagocytosis-induced stimulation of cell cycle progression of J774 cells was repeatedly observed after the phagocytosis of polystyrene beads, live Cn or dead Cn. We observed the same phenomenon with bone marrow macrophages, thus extending this to another cell type and to primary macrophages. In the Cn experimental system, IgG1-mediated opsonization can trigger phagocytosis through the FcR or through complement receptor in an unusual complement-independent mechanism whereby the capsule interacts directly with CD18 (26). Because both mechanisms occur simultaneously, we conducted additional experiments to discriminate whether Fc- or complement-mediated opsonization was responsible for the observed stimulation of cell division. Furthermore, we were interested in establishing whether phagocytosis was necessary for the effect. Incubating J774 cells on a plate coated with IgG1 stimulated cell cycle progression, indicating that FcR activation could induce the phenomenon. Furthermore, we noted cell cycle progression after C3-mediated phagocytosis, indicating that the phenomenon could follow activation of the complement receptors. Hence, engaging either Fc or complement receptors can trigger cell cycle progression, and at least the Fc-mediated process does not require ingestion. However, the observation that J774 cells incubated with naked polystyrene beads stimulated cell cycle progression indicates that additional receptors may feed into this signal transduction pathway. The fact that multiple surface receptors central to the phagocytic process can stimulate cell cycle progression, combined with the observation that it occurs in both primary and continuous cell lines, suggests a basic link between two physiological pathways.
A 10–15% progression from G1 to S phase was consistently observed after the phagocytosis of polystyrene beads, live Cn or dead Cn, in 20 independent experiments. Although at first glance it may appear that only a small percentage of the cells in any one experiment progressed to S phase, this percentage is relatively large when one considers that approximately half the cells are in a resting state (40–50% for J774 cells and bone marrow macrophages). Incidentally, we obtained similar results with longer incubation time (as long as 4 h). Considering that phagocytosis is a rapid event that is largely completed by 15 min, the relatively short duration of activation of Fc/complement receptors might lead to the measured change of the cell cycle progression.
In the past two decades, the signaling cascades responsible for and resulting from phagocytosis and cell division have been elucidated. Therefore, it is possible to identify shared pathways that may explain our findings. The cell cycle is divided into G1, S, G2, and M phases in eukaryotic cells. The regulation of this cycle is primarily controlled by periodic synthesis and destruction of cyclins, which in turn bind to cyclin-dependent kinases (cdks) and activate them. To enter S phase, cells must pass a restriction point in late G1 phase. This process is conducted by cyclin D, which phosphorylates retinoblastoma tumor suppressor protein (Rb) through the binding of cyclin D with cdk4 and cdk6. Cyclin D expression is induced by mitogenic stimuli instead of oscillating during cell cycle, as the other cyclins. Phosphorylation of Rb prevents it from binding to E2F factors and thus switches E2F from a repressor to an activator of gene expression of cell cycle proteins, including cyclins E and A. Cyclin E participates in a positive feedback control of Rb by maintaining Rb in a hyperphosphorylated state, and thus drives the cell cycle through S and G2 (27). Intensive studies showed that the cell cycle is under subtle regulation by signaling molecules such as PI3K and Rho GTPases (25, 28, 29, 30). The activation of PI3K and Rho GTPases is required for the process of macrophage phagocytosis (2, 3). This leads us to posit that activation of known signaling pathways involved in phagocytosis could potentially influence cell proliferation. For instance, activation of PI3K in phagocytosis could participate not only in the molecular events in phagocytosis, but also in the cell cycle regulation such as the inhibition of p27kip1, which in turn increases the activity of cyclin E/cdk2 and thus enhances the G1 to S transition (29, 31, 32). Hence, phagocytosis could activate PI3K and stimulate cells to progress in the cell cycle progression via cyclin E.
Phagocytosis by macrophages is an important process in host defense mechanisms. Microbial killing of phagocytosed microbes is beneficial to the protection of organisms from infection. Macrophage local proliferation was an efficient way to replenish the damaged macrophage after killing. Although most hemopoietic cells mature after leaving the bone marrow and cannot proliferate, local proliferation of mononuclear phagocytes has been observed (9, 33). For example, macrophages resident in spleen and peritoneum were renewed by local proliferation (14, 15). Furthermore, macrophages can replicate upon the stimulation of growth factors such as CSFs, and especially during inflammation (11). Thus, the linkage of phagocytosis and cell division could have important consequences for our understanding of microbial pathogenesis and host defense. On one hand, phagocytosis-induced cell division could increase the number of effector cells in response to infection, and also provides a novel mechanism for macrophages to cure themselves after ingesting microbes by the generation of new cells that have the potential to be noninfected. In fact, a study on persistent infection of host cells with Coxiella burnetii showed that heavily infected host cells had the ability to restrain intracellular Coxiella in one large parasite-containing vacuole, which was passaged into one of the two daughter cells of dividing host cells. Thus, a pathogen-free companion daughter cell was generated (34, 35). This would be one of the mechanisms that infected cells could cure themselves by cell replication. In contrast, many microbes are intracellular pathogens that replicate preferentially inside phagocytic cells and use intracellular residence as a mechanism to evade the immune system. Consequently, this phenomenon could also undermine host defenses by generating new infected cells in which microbes can proliferate. Clearly, the benefit or debit of this phenomenon for host defense would be dependent on the type of pathogen. The linkage of phagocytosis-induced cell division with the need for nutrients is intriguing given that phagocytosis of bacteria and other microbes is used by unicellular eukaryotes for food acquisition. Hence, phagocytosis may be linked to cell division through ancient pathways that signal the acquisition of food, which in turn would imply favorable conditions for replication.
In summary, our results indicate that phagocytosis occurs preferentially in G1 phase, and that particle ingestion stimulates the progression of cell cycle. Remarkably, this phenomenon is unknown despite the fact that both processes have been studied extensively. Cellular division following phagocytosis could have major implications for the outcome of macrophage infection. For some pathogens, we anticipate that the effect would help the host to generate additional uninfected effector cells at the site of infection. However, for other pathogens, this effect could contribute to pathogenesis. If that is the case, it is possible that future studies could identify the precise signaling pathway involved in phagocytosis-associated cell division and evaluate it as a target for antimicrobial therapy of certain infectious diseases. Furthermore, the findings suggest the need to investigate the phylogenetic origins of this phenomenon, which may have ancient roots in eukaryotic evolution.
Acknowledgments
We thank Drs. Matthew Scharff, Marshall Horwitz, and Dianne Cox for critical reading of the manuscript; Huafeng Xie and Dr. Thomas Graf for their generously providing C57BL/6 mice and primary bone marrow cells; Drs. Franco Cappoza and Michael Lisanti for generously providing BrdU and Ab to BrdU; Peng Ji in Dr. Liang Zhu’s lab for help with Western blots; and Krishanthi Subramaniam in Dr. Liise-anne Pirofski’s lab for help with labeling Cn by FITC. We also thank Clarissa Santos and Kristie Gordon from the FACS facility at the Albert Einstein College of Medicine.
Footnotes
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 This study was supported by National Institutes of Health Grants AI033142, AI033774, AI052733, and HL059842. S.C.T. is supported in part by National Institutes of Health/National Institute of Allergy and Infectious Diseases Training Grant 5T32AI07506.
2 Current address: Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Ullmann Building, Room 907, 1300 Morris Park Avenue, Bronx, NY 10461.
3 Address correspondence and reprint requests to Dr. Arturo Casadevall, Department of Medicine and Department of Microbiology and Immunology, Albert Einstein College of Medicine, Golding Building, Room 701, 1300 Morris Park Avenue, Bronx, NY 10461. E-mail address: casadeva{at}aecom.yu.edu
4 Abbreviations used in this paper: Cn, Cryptococcus neoformans; cdk, cyclin-dependent kinase; FSC, forward light scatter; Rb, retinoblastoma tumor suppressor protein.
Received for publication December 8, 2004. Accepted for publication March 16, 2005.
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