Critical Review. Complement-mediated Phagocytosis The Role of Syk. Yumi Tohyama 1,2 and Hirohei Yamamura 2,3 THE COMPLEMENT SYSTEM AND PHAGOCYTOSIS

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1 IUBMB Life, 58(5 6): , May June 2006 Critical Review Complement-mediated Phagocytosis The Role of Syk Yumi Tohyama 1,2 and Hirohei Yamamura 2,3 1 Himeji Dokkyo University, Himeji, Hyogo, Japan 2 Department of Genome Sciences, Kobe University Graduate School of Medicine, Kobe, Japan 3 Hyogo Prefectural Institute of Public Health and Environmental Sciences, Kobe, Japan Summary Phagocytosis is a central event in the innate immune responses that are triggered by the association between ligands on the surface of pathogens and receptors on the membrane of phagocytes. Particularly, complement-mediated phagocytosis is accomplished by specific recognition of bound complement components by the corresponding complement receptors on the phagocytes. The protein-tyrosine kinase, Syk, plays a central role in Fcg receptormediated phagocytosis in the adaptive immune system. From recent studies using a macrophage-like differentiated cell line and serumtreated zymosan, it was found that Syk also plays an essential role in complement-mediated phagocytosis in innate immunity. Serum-treated zymosan particles promptly attached to the cells and were subsequently engulfed via complement receptor3. During this process, Syk became tyrosine-phosphorylated and accumulated around the nascent phagosomes. The transfer of Syk-siRNA or dominant-negative Syk (DN-Syk) into macrophages resulted in impaired engulfment of pathogen. Collectively, Syk is required for the engulfment of pathogen in complement-mediated phagocytosis. IUBMB Life, 58: , 2006 Keywords Phagocytosis; complement; Syk; Fc receptor; innate immunity. INTRODUCTION Phagocytosis is a central event in the innate immune responses that are triggered by the association between ligands on the surface of pathogens and receptors on the membrane of phagocytes. Phagocytes then engulf and eliminate the pathogens. Among the phagocytic receptor types expressed on mammalian neutrophils and macrophages, Fcg receptors and complement receptor3 (CR3) have been characterized in detail, especially those concerned with Fcg receptor-mediated signaling. Following Fc-receptor engagement, the immunoreceptor Received 5 March 2006; accepted 6 March 2006 Address correspondence to: Yumi Tohyama, Himeji Dokkyo University, Himeji, Hyogo , Japan. tyrosine-based activation motifs (ITAMs) in the receptor are phosphorylated by Src-family kinases, leading to the recruitment and activation of a protein tyrosine kinase, Syk. This in turn leads to the formation of a signaling complex at the membrane, in which Syk-mediated phosphorylation of several adaptor proteins causes activation of downstream pathways which give rise to the phagocytic effect (1). While the linear Src-family kinase/itam/syk pathway of Fcg receptormediated signaling is established, the signaling pathway of the complement system remains to be clarified. In this review we overview the complement-mediated phagocytosis system together with our data about the role of Syk in this system. THE COMPLEMENT SYSTEM AND PHAGOCYTOSIS The complement system is composed of many distinct proteins that react with one another to opsonize pathogens and induce a series of inflammatory responses that support host defense. There are three distinct pathways through which the complement components are activated on the pathogen surfaces: classical pathway, mannose-binding lectin pathway and alternative pathway. Each pathway follows a sequence of reactions to generate a key protease called C3 convertase. The complement system prevents infection by three steps as follows: (1) opsonization of pathogens toward engulfment by phagocytes bearing receptors for complement (C3b, C3bi), (2) recruitment of inflammatory cells (C3a, C5a), (3) direct killing of pathogens by creating pores at the bacterial membrane (C5b, C6, C7, C8, C9). There are also several regulatory mechanisms to prevent the adverse activation of complement on host cell surfaces. In consequence, the complement system is one of the major mechanisms by which pathogen recognition is converted into an effective host defense against initial infection (2). In particular, the alternative pathway proceeds on the microbial surfaces in the absence of specific antibody and generates the distinct C3 convertase. This pathway does not depend on a pathogen-binding protein for its initiation but ISSN print/issn online Ó 2006 IUBMB DOI: /

2 ROLE OF SYK IN PHAGOCYTOSIS 305 is initiated through the spontaneous hydrolysis of C3, as shown in Fig. 1. It is exclusively important in innate immunity. The complement-mediated phagocytosis is accomplished by specific recognition of bound complement components by the corresponding complement receptors on the phagocytes. Pathogen invasion leads to the generation of C3 convertase which yields C3b. The final product C3bi binds to three other complement receptors CR1, CR3 (CD11b/CD18, integrin amb2) and CR4 (CD11c/CD18, integrin axb2). Binding of C3bi to CR3 induces phagocytosis most strongly after phagocyte activation. To clarify the molecular mechanism of phagocytosis, it is essential to establish a reproducible experimental system of phagocytosis in vitro. As for Fcg receptor mediated phagocytosis, sheep erythrocytes or zymosan (Saccharomyces cerevisiae) are used as particles to be incorporated and pretreated with specific antibodies prior to phagocytosis assays (3, 4). In the case of complement-mediated phagocytosis, the complement activation cascade in the serum is effectively utilized. As previously described, C3b is spontaneously produced and destroyed, and when non-self microbes such as zymosan are exposed to the serum at 378C, C3b binds to zymosan covalently and becomes C3 convertase by the action of cofactor proteins. Cleavage of C3 to C3b and finally to C3bi is amplified, and zymosan is surrounded by C3bi and incorporated effectively by phagocytes which express CR3. BACKGROUND OF PROTEIN TYROSINE KINASE, SYK AND PHAGOCYTOSIS A protein-tyrosine kinase, Syk is expressed in a wide range of hematopoietic and non-hematopoietic cells and plays a key role in adaptive immune system, that is, the immunoreceptor (B cell receptor and Fc-receptors) signaling pathways including Fcg receptor-mediated phagocytosis (5, 6). On the other hand, the role of Syk in non-adaptive immune system is not well defined and the function of Syk in innate immunity has received considerable focus (7). In addition to these roles in immunoreceptor signaling, Syk is also activated on ligation with cell surface integrins, such as integrin aiibb3 in platelets and b2 integrin in neutrophils or monocytes. Studies of Syk-deficient murine macrophages indicated that Syk plays a central role in Fcg receptormediated phagocytosis but not in complement-mediated phagocytosis (3). Taking into account such a background and that the main complement receptor CR3 is integrin amb2, we have performed the present study about whether Syk is required for complement-mediated phagocytosis from a new viewpoint. ROLE OF SYK THAT IS ESSENTIAL TO COMPLEMENT-MEDIATED PHAGOCYTOSIS A human leukemia HL60 cell line induced to macrophagelike with vitamin D3 and phorbor-ester and zymosan were used as the pathogen-phagocyte system (8, 9). Zymosan particles were pretreated with the normal serum in order that C3bi in the serum bind to zymosan (C3bi-zymosan). Timelapse microscopic observation revealed that C3bi-zymosan particles were promptly attached to and sucked into macrophage-like differentiated HL60 cells (Fig. 2). Previous reports described that such a protrusion of the cell membrane was found only in the case of Fcg receptor-mediated phagocytosis and that complement-mediated phagocytosis traps pathogens Figure 1. Coordination of the complement activation (alternative pathway) and phagocyte.

3 306 TOHYAMA AND YAMAMURA Figure 2. Skillful capture of C3bi-zymosan by HL60 cells. by the caved membrane (10). However, HL60 cells in our complement-mediated phagocytosis system represented active phagocytosis by membrane protrusion. Zymosan particles which did not bind C3bi were hardly incorporated, but C3bizymosan was effectively absorbed by the cells even in IgGremoved serum. Next we analyzed the dynamics of endogenous Syk in the cells pretreated with C3bi-zymosan and found that Syk was tyrosine-phosphorylated and obviously accumulated in the region of forming phagosomes. On the contrary, Syk was neither tyrosine-phosphorylated nor accumulated in the region of forming phagosomes when C3bi-untreated zymozan was applied or when an inhibitor of C3 convertase was used. To further investigate the effect of Syk on the mechanism of complement-mediated phagocytosis, several mutant cell clones were isolated such as DN-Syk/HL (stably expressing only tandem SH2 domains of Syk kinase as a dominant-negative mutant), Syk-siRNA/HL (stably expressing Syk-siRNA), control-sirna/hl (stably expressing control sirna) and rescue-syk/syk-sirna/hl (expressing recovered amount of wild-type Syk in Syk-siRNA/HL) (Fig. 3). We quantitatively evaluated phagocytosis of the fluorescent zymosan among the mutant cell clones and found that parental macrophage-like HL60 cells incorporated only C3bi-zymosan but this phagocytosis was suppressed in DN-Syk/HL and Syk-siRNA/HL, and transfer of rescue-syk restored the phagocytic activity. From these results it became clear that Syk plays an important role in complement-mediated phagocytosis (Fig. 4). To distinguish which process of the serial phagocytosis Syk is mainly involved in, the cells were treated with a quenching reagent which discriminates fluorescent zymosan particles Figure 3. Protein structure of Syk and mutant HL60 cells. between inside and outside the cells. In the parental HL60 cells most of the attached zymosan particles were inside the cells, while few particles were ingested in the case of DN-Syk/HL or Syk-siRNA/HL cells (Fig. 5). These data indicated that Syk is required for engulfment of C3bi-zymosan but not for attachment of zymosan to CR3. MOLECULAR MECHANISM OF SYK-MEDIATED SIGNALING IN RELATION TO PHAGOSOME FORMATION AND ITS TRANSPORT To clarify the molecular mechanism how Syk affects the phagosome formation and its engulfment, the behavior of

4 ROLE OF SYK IN PHAGOCYTOSIS 307 Figure 4. Complement-dependent phagocytosis of serum-treated zymosan. Macrophage-like differentiated HL60 cells were incubated with zymosan particles which were pretreated with serum, IgG-removed serum, serum containing compstatin or the control peptide at 378C and then analyzed by flow cytometry to detect phagocytosis of fluorescent zymosan. (Left upper panel) Representative histogram patterns of the treated cells by flow cytometry are shown. M2 region includes zymosan-positive cells. (Left lower panel) The percentage of zymosan-positive cells in parental HL60 cells incubated with zymosan particles which were pretreated as indicated is presented. The mean values and SD are shown. (Right panel) The percentage of zymosan-positive cells in HL60 cells and the mutant clones treated with serum-opsonized or non-opsonized fluorescent zymosan is presented. The mean values and SD are shown. F-actin was searched microscopically. At the early stage of complement-mediated phagocytosis, marked accumulation of F-actin occurred around each phagosome and was followed by rapid depolymerization in the parental HL60 cells. In contrast, phagosomes surrounded by the accumulated actin were significantly decreased in DN-Syk/HL or Syk-siRNA/ HL cells. These results indicate that Syk promotes actin assembly around phagosomes prior to engulfment at the early stage of phagocytosis. We further searched for the signaling molecules which may affect the intracellular transport of phagosomes. CR3 is amb2, one of the integrin family, and it is known from a study of cell movement of monocytes that RhoA plays an important role in the generation of the contractile force at the downstream of integrins (11). In consideration of the possibility that a similar mechanism works after phagosome formation, we examined the activation pattern of RhoA. Treatment of the parental HL60 cells with C3bi-zymosan led to the prompt activation of RhoA, but DN-Syk/HL or SyksiRNA/HL cells revealed suppressed RhoA activation. Transfer of rescue-syk restored RhoA activation. These data indicate that Syk acts as an activator of the RhoA-pathway in C3bi-CR3-signaling, which might lead to engulfment of the particles. Next we examined the tyrosine phosphorylation of Vav, a guanine nucleotide exchange factor of RhoA and found that Vav was tyrosine-phosphorylated prior to RhoA activation in the parental HL60 cells but the phosphorylation was reduced in DN-Syk/HL or Syk-siRNA/HL cells just like the pattern of RhoA activation. Collectively Syk is a crucial upstream regulator of Vav-RhoA signaling that generates contractile force and RhoA-pathway is under the control of Syk in complement-mediated phagocytosis. CONCLUSION AND PERSPECTIVE Complement-mediated phagocytosis is essential to host defense but has been less well understood as compared with the antibody-fcg receptor-mediated phagocytosis, probably because of the difficulty of studying it in vitro. As for Syk, its role in complement-mediated phagocytosis has been denied until now, but the present study has clearly demonstrated its critical function for phagosome formation and pathogen engulfment. Recently some reports indicate that Syk is also important in Dectin-1-mediated or CD44-mediated phagocytosis (12, 13). Further investigation of cross-talk mechanism between complement-mediated signaling and other pathways important for innate immunity such as Toll-like receptor systems would shed light on the elucidation of the process from phagocytosis to achievement of adaptive immunity.

5 308 TOHYAMA AND YAMAMURA Figure 5. Syk is essential for pathogen engulfment rather than for attachment via complement receptor. (Upper panel) The scheme of quenching assay with trypan blue is shown. (Lower panel) Macrophage-like differentiated HL60, DN-Syk/HL, SyksiRNA/HL or control-sirna/hl cells were treated with serum-opsonized fluorescent zymosan at 378C and observed with a fluorescent microscope before and after trypan blue staining. The cells treated with zymosan were examined by the quenching assay described above. In each culture plate, more than 100 cells and total zymosan particles (inside and outside the cell; seen before quenching), bright zymosan particles (inside the cell; seen after quenching) were counted and the average of zymosan particles/cell were calculated. The counts of outside the cell were obtained as the results of subtraction (inside and outside minus inside) in each plate. REFERENCES 1. Berton, G., Mocsai, A., and Lowell, C. A. (2005) Src and Syk kinases: key regulators of phagocytic cell activation. Trends Immunol. 26, Janeway, Jr, C. A. (Ed.) (2005) Immuno Biology, 6th edn. New York: Garland Science Publishing. 3. Kiefer, F., Brumell, J., Al-Alawi, N., Latour, S., Cheng, A., Veillette, A., Grinstein, S., and Pawson, T. (1998) The Syk protein tyrosine kinase is essential for Fcgamma receptor signaling in macrophages and neutrophils. Mol. Cell. Biol. 18, Crowley, M. T., Costello, P. S., Fitzer-Attas, C. J., Turner, M., Meng, F., Lowell, C., Tybulewicz, V. L., and DeFranco, A. L. (1997) A critical role for Syk in signal transduction and phagocytosis mediated by Fcgamma receptors on macrophages. J. Exp. Med. 186, Taniguchi, T., Kobayashi, T., Kondo, J., Takahashi, K., Nakamura, H., Suzuki, J., Nagai, K., Yamada, T., Nakamura, S., and Yamamura, H. (1991) Molecular cloning of a porcine gene syk that encodes a 72-kDa protein-tyrosine kinase showing high susceptibility to proteolysis. J. Biol. Chem. 266, Kurosaki, T., Takata, M., Yamanashi, Y., Inazu, T., Taniguchi, T., Yamamoto, T., and Yamamura, H. (1994) Syk activation by the Srcfamily tyrosine kinase in the B cell receptor signaling. J. Exp. Med. 179, Mocsai, A., Zhou, M., Meng, F., Tybulewicz, V. L., and Lowell, C. A. (2002) Syk is required for integrin signaling in neutrophils. Immunity 16, Shi, Y., Tohyama, Y., Kadono, T., He, J., Miah, S. M., Hazama, R., Tanaka, C., Tohyama, K., and Yamamura, H. (2006) Protein-tyrosine kinase, Syk is required for pathogen engulfment in complementmediated phagocytosis. Blood (in press). 9. Katagiri, K., Katagiri, T., Koyama, Y., Morikawa, M., Yamamoto, T., and Yoshida, T. (1991) Expression of src family genes during monocytic differentiation of HL-60 cells. J. Immunol. 146, Chimini, G., and Chavrier, P. (2000) Function of Rho family proteins in actin dynamics during phagocytosis and engulfment. Nat. Cell Biol. 2, Worthylake, R. A., Lemoine, S., Watson, J. M., and Burridge, K. (2001) RhoA is required for monocyte tail retraction during transendothelial migration. J. Cell Biol. 154, Rogers, N. C., Slack, E. C., Edwards, A. D., Nolte, M. A., Schulz, O., Schweighoffer, E., Williams, D. L., Gordon, S., Tybulewicz, V. L., Brown, G. D., and Reis e Sousa, C. (2005) Syk-dependent cytokine induction by Dectin-1 reveals a novel pattern recognition pathway for C type lectins. Immunity 22, Vachon, E., Martin, R., Plumb, J. D., Kwok, V., Vandivier, R. W., Glogauer, M., Kapus, A., Wang, X., Chow, C. W., Grinstein, S., and Downey, G. P. (2006) CD44 is a phagocytic receptor. Blood (in press).