Kaz Nagaosa

Pretaporter, a Drosophila protein serving as a ligand for Draper in the phagocytosis of apoptotic cells

Phagocytic removal of cells undergoing apoptosis is necessary for animal development and tissue homeostasis. Draper, a homologue of the Caenorhabditis elegans phagocytosis receptor CED‐1, is responsible for the phagocytosis of apoptotic cells in Drosophila, but its ligand presumably present on apoptotic cells remains unknown. An endoplasmic reticulum protein that binds to the extracellular region of Draper was isolated. Loss of this protein, which we name Pretaporter, led to a reduced level of apoptotic cell clearance in embryos, and the overexpression of pretaporter in the mutant flies rescued this defect. Results from genetic analyses suggested that Pretaporter functionally interacts with Draper and the corresponding signal mediators. Pretaporter was exposed at the cell surface after the induction of apoptosis, and cells artificially expressing Pretaporter at their surface became susceptible to Draper‐mediated phagocytosis. Finally, the incubation with Pretaporter augmented the tyrosine‐phosphorylation of Draper in phagocytic cells. These results collectively suggest that Pretaporter relocates from the endoplasmic reticulum to the cell surface during apoptosis to serve as a ligand for Draper in the phagocytosis of apoptotic cells.

Independent Recognition of Staphylococcus aureus by Two Receptors for Phagocytosis in Drosophila

Background: How multiple receptors for phagocytosis contribute to host immunity against bacterial infection has been elusive. Results: Drosophila phagocytes use two receptors, integrin and Draper, to effectively recognize and engulf Staphylococcus aureus. Conclusion: Dual recognition system exists in Drosophila for phagocytic removal of pathogenic bacteria. Significance: Study of cellular immune response is important for understanding host defense against infectious diseases. Integrin βν, one of two β subunits of Drosophila integrin, acts as a receptor in the phagocytosis of apoptotic cells. We here examined the involvement of this receptor in defense against infection by Staphylococcus aureus. Flies lacking integrin βν died earlier than control flies upon a septic but not oral infection with this bacterium. A loss of integrin βν reduced the phagocytosis of S. aureus and increased bacterial growth in flies. In contrast, the level of mRNA of an antimicrobial peptide produced upon infection was unchanged in integrin βν-lacking flies. The simultaneous loss of integrin βν and Draper, another receptor involved in the phagocytosis of S. aureus, brought about a further decrease in the level of phagocytosis and accelerated death of flies compared with the loss of either receptor alone. A strain of S. aureus lacking lipoteichoic acid, a cell wall component serving as a ligand for Draper, was susceptible to integrin βν-mediated phagocytosis. In contrast, a S. aureus mutant strain that produces small amounts of peptidoglycan was less efficiently phagocytosed by larval hemocytes, and a loss of integrin βν in hemocytes reduced a difference in the susceptibility to phagocytosis between parental and mutant strains. Furthermore, a series of experiments revealed the binding of integrin βν to peptidoglycan of S. aureus. Taken together, these results suggested that Draper and integrin βν cooperate in the phagocytic elimination of S. aureus by recognizing distinct cell wall components, and that this dual recognition system is necessary for the host organism to survive infection.

Phosphatidylserine recognition and induction of apoptotic cell clearance by Drosophila engulfment receptor Draper.

The membrane phospholipid phosphatidylserine is exposed on the cell surface during apoptosis and acts as an eat-me signal in the phagocytosis of apoptotic cells in mammals and nematodes. However, whether this is also true in insects was unclear. When milk fat globule-epidermal growth factor 8, a phosphatidylserine-binding protein of mammals, was ectopically expressed in Drosophila, the level of phagocytosis was reduced, whereas this was not the case for the same protein lacking a domain responsible for the binding to phosphatidylserine. We found that the extracellular region of Draper, an engulfment receptor of Drosophila, binds to phosphatidylserine in an enzyme-linked immunosorbent assay-like solid-phase assay and in an assay for surface plasmon resonance. A portion of Draper containing domains EMI and NIM located close to the N-terminus was required for binding to phosphatidylserine, and a Draper protein lacking this region was not active in Drosophila. Finally, the level of tyrosine-phosphorylated Draper, indicative of the activation of Draper, in a hemocyte-derived cell line was increased after treatment with phosphatidylserine-containing liposome. These results indicated that phosphatidylserine serves as an eat-me signal in the phagocytic removal of apoptotic cells in Drosophila and that Draper is a phosphatidylserine-binding receptor for phagocytosis.

Integrin βν-mediated Phagocytosis of Apoptotic Cells in Drosophila Embryos

To identify molecules that play roles in the clearance of apoptotic cells by Drosophila phagocytes, we examined a series of monoclonal antibodies raised against larval hemocytes for effects on phagocytosis in vitro. One antibody that inhibited phagocytosis recognized terribly reduced optic lobes (Trol), a core protein of the perlecan-type proteoglycan, and the level of phagocytosis in embryos of a Trol-lacking fly line was lower than in a control line. The treatment of a hemocyte cell line with a recombinant Trol protein containing the amino acid sequence RGD augmented the phosphorylation of focal adhesion kinase, a hallmark of integrin activation. A loss of integrin βν, one of the two β subunits of Drosophila integrin, brought about a reduction in the level of apoptotic cell clearance in embryos. The presence of integrin βν at the surface of embryonic hemocytes was confirmed, and forced expression of integrin βν in hemocytes of an integrin βν-lacking fly line recovered the defective phenotype of phagocytosis. Finally, the level of phagocytosis in a fly line that lacks both integrin βν and Draper, another receptor required for the phagocytosis of apoptotic cells, was lower than that in a fly line lacking either protein. We suggest that integrin βν serves as a phagocytosis receptor responsible for the clearance of apoptotic cells in Drosophila, independent of Draper.

Expression and function of class B scavenger receptor type I on both apical and basolateral sides of the plasma membrane of polarized testicular Sertoli cells of the rat

Class B scavenger receptor type I (SR‐BI), a multiligand membrane protein, exists in various organs and cell types. In the testis, SR‐BI is expressed in two somatic cell types: Leydig cells and Sertoli cells. Unlike interstitially localized Leydig cells, Sertoli cells present within the seminiferous tubules keep contact with spermatogenic cells and form the tight junction to divide the seminiferous epithelium into the basal and adluminal compartments. In this study, the expression and function of SR‐BI in rat Sertoli cells were examined with respect to dependency on the spermatogenic cycle, the plasma membrane polarity, and the pituitary hormone follicle‐stimulating hormone (FSH). When the expression of SR‐BI was histochemically examined with testis sections, both protein and mRNA were already present in Sertoli cells during the first‐round spermatogenesis and continued to be detectable thereafter. The level of SR‐BI mRNA expression in Sertoli cells was lower at spermatogenic stages I–VI than at other stages. SR‐BI was present and functional (in mediating cellular incorporation of lipids of high density lipoprotein) at both the apical and basolateral surfaces of polarized Sertoli cells. Finally, SR‐BI expression at both the protein and mRNA levels was stimulated by FSH in cultured Sertoli cells. These results indicate that SR‐BI functions on both the apical and basolateral plasma membranes of Sertoli cells, and that SR‐BI expression in Sertoli cells changes during the spermatogenic cycle and is stimulated, at least in cultures, by FSH.

Integrin αPS3/βν-mediated Phagocytosis of Apoptotic Cells and Bacteria in Drosophila

Background: Drosophila integrin βν plays a role in the phagocytosis of apoptotic cells and bacteria, but its partner α-subunit remains to be identified. Results: Of 5 α-subunits, αPS3 was physically and functionally associated with βν. Conclusion: αPS3/βν serves as a receptor for phagocytosis in Drosophila. Significance: The heterodimeric structure of Drosophila integrin has been genetically and biochemically solved. Integrins exert a variety of cellular functions as heterodimers of two transmembrane subunits named α and β. Integrin βν, a β-subunit of Drosophila integrin, is involved in the phagocytosis of apoptotic cells and bacteria. Here, we searched for an α-subunit that forms a complex and cooperates with βν. Examinations of RNAi-treated animals suggested that αPS3, but not any of four other α-subunits, is required for the effective phagocytosis of apoptotic cells in Drosophila embryos. The mutation of αPS3-encoding scb, deficiency, insertion of P-element, or alteration of nucleotide sequences, brought about a reduction in the level of phagocytosis. The defect in phagocytosis by deficiency was reverted by the forced expression of scb. Furthermore, flies in which the expression of both αPS3 and βν was inhibited by RNAi showed a level of phagocytosis almost equal to that observed in flies with RNAi for either subunit alone. A loss of αPS3 also decreased the activity of larval hemocytes in the phagocytosis of Staphylococcus aureus. Finally, a co-immunoprecipitation analysis using a Drosophila cell line treated with a chemical cross-linker suggested a physical association between αPS3 and βν. These results collectively indicated that integrin αPS3/βν serves as a receptor in the phagocytosis of apoptotic cells and bacteria by Drosophila phagocytes.

Concomitant induction of apoptosis and expression of monocyte chemoattractant protein-1 in cultured rat luteal cells by nuclear factor-κB and oxidative stress

It has previously been shown that expression of monocyte chemoattractant protein (mcp)‐1 and apoptosis of luteal cells occur concomitantly during the estrous cycle in the rat corpus luteum; however, luteal cells containing mcp‐1 mRNA did not seem to be apoptotic. In the present study, the relationship between the induction of apoptosis and mcp‐1 expression in cultures of dispersed rat luteal cells was examined. Both apoptosis and mcp‐1 expression were spontaneously induced in cultured luteal cells in a manner inhibitable by antioxidative reagents or an inhibitor of nuclear translocation of nuclear factor‐kB. However, the cells containing mcp‐1 mRNA were distinct from those undergoing apoptosis, and the inhibition of apoptosis by the pan‐caspase inhibitor z‐VAD‐fmk did not influence the induction of mcp‐1 expression. These results collectively indicate that oxidative stress simultaneously, but independently, induces apoptosis and mcp‐1 expression in luteal cells through the activation of nuclear factor‐kB. This phenomenon might help to explain how monocytes/macrophages accumulate in regressive corpora lutea where their target apoptotic cells exist.

Immune response to bacteria in seminiferous epithelium

The luminal part of the seminiferous epithelium, a tissue compartment protected by the blood-testis barrier, has been considered a site of immune privilege. However, there are reports describing the production of anti-microbial peptides and the expression of Toll-like receptors in cells present in the seminiferous epithelium, evoking the possibility that this tissue compartment is immunologically active at least with regard to the innate immune response. To test this, we injected Escherichia coli into seminiferous tubules of live mice and examined the fate of bacteria, the production of chemokines and inflammatory cytokines, and the infiltration of neutrophils. The bacteria actively propagated and reached a maximal level in a day, but started to decrease after 5 days and completely disappeared in 2 months. The expression of macrophage inflammatory protein-2 and tumor necrosis factor-alpha became evident in macrophages present in the interstitial compartment of testes as early as 1-3 h after the inoculation of bacteria. Neutrophils first accumulated in the interstitial space at 9-12 h and entered the tubules after a day. On the other hand, impairment of spermatogenesis was observed a day after bacteria injection and seemed unrecoverable even after the bacteria were eliminated. By contrast, bacteria injected into the interstitial compartment were more rapidly cleared with no damage in the seminiferous epithelium. These results suggest the existence of immunity against invading microbes in the seminiferous epithelium although its effectiveness in maintaining tissue homeostasis remains equivocal.

Determination of Cell Type Specificity and Estrous Cycle Dependency of Monocyte Chemoattractant Protein-1 Expression in Corpora Lutea of Normally Cycling Rats in Relation to Apoptosis and Monocyte/Macrophage Accumulation

Abstract In regressive corpora lutea, apoptosis of luteal cells, expression of monocyte chemoattractant protein-1 (MCP-1), and accumulation of monocytes/macrophages occur. However, whether these three events are correlated and what cell type expresses MCP-1 have yet to be determined. To clarify these issues, we performed histochemical examinations to determine the localization and the numbers of MCP-1 mRNA-containing cells, apoptotic cells, and monocytes/macrophages in corpora lutea of normally cycling rats. We found that the Mcp-1 gene is expressed in nonapoptotic steroidogenic luteal cells. Corpora lutea that contained MCP-1 mRNA-expressing cells increased in number at estrus together with those containing apoptotic luteal cells. When individual corpora lutea at estrus were analyzed, those with many MCP-1-expressing cells contained few apoptotic cells, and vice versa. These results collectively suggest the following pathway for apoptosis- and MCP-1-dependent regression of the corpus luteum: 1) luteal cells are induced to undergo apoptosis at estrus, and the activation of Mcp-1 gene expression follows in nonapoptotic luteal cells; 2) monocytes/macrophages are chemoattracted by MCP-1 toward corpora lutea containing apoptotic luteal cells; and 3) monocytes/macrophages invade corpora lutea and eliminate apoptotic luteal cells by phagocytosis.

Phosphatidylserine‐ and integrin‐mediated phagocytosis of apoptotic luteal cells by macrophages of the rat

Corpora lutea disappear from ovaries in the absence of conception. The present study was undertaken to examine the hypothesis that disappearance of corpora lutea is accomplished through apoptosis‐dependent phagocytosis of luteal cells. When bone marrow cells expressing green fluorescence protein were transplanted into X‐ray‐irradiated mice, macrophages derived from donor mice were detected within corpora lutea, suggesting macrophage infiltration into the tissue. Dispersed rat luteal cells underwent spontaneous apoptosis during culture and were phagocytosed by luteal macrophages. Treatment with doxorubicin increased the extent of apoptosis in luteal cells, and those cells were more efficiently phagocytosed than cells left untreated. The phagocytosis was inhibited by liposomes containing phosphatidylserine or a peptide containing the integrin‐targeted sequence, and was stimulated by milk fat globule epidermal growth factor 8. These results collectively indicate that apoptotic luteal cells are phagocytosed by macrophages in a manner mediated by phosphatidylserine and integrin.