10th International Symposium of Molecular Cell Biology of Macrophages  (Macrophages 2001)

"Macrophage Signaling, Apoptosis, Lectin and Trafficking"

by Tadashi Kasahara and Kouji Matsushima, Kyoritsu College of Pharmacy , 1-5-30, Shibakoen, Minato-ku, Tokyo and Tokyo University, School of Medicine, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-0033


  Macrophages 2001 was held at Tokyo, Japan on June 21 and 22 organized by Dr. Tadashi Kasahara, Kyoritsu College of Pharmacy, Tokyo, cerebrating 10th annual meeting this year.  The meeting started originally in 1991 in Kanazawa by Dr Kouji Matsushima (now at Tokyo Univ) for discussing up-to-date topics on the various aspects of macrophages.  Macrophages 2001 consisted of 4 major sessions with 9 invited speakers from overseas and 12 Japanese speakers, giving each most fascinating issues and discussion.

   In the first Session subtitled "Signal Transduction in Macrophages",  Luke O'Neill (Trinity College, Dublin) reviewed signaling pathways activated by IL-1 and Toll-like receptors (TLRs), depicting that TIR domain-containing receptors are important switch for innate immunity and inflammation. He presented that the Mal (Myd88 adapter-like) protein with TIR, which forms a heterodimer with MyD88 and selectively recruits IRAK-2, and thus Mal appears to play roles in TLR4 signaling.  Shizuo Akira (Osaka Univ) also reviewed on each TLR function with the successful generation of TLR2, 4, 9 and Myd88 knock-out mice, and indicated that TLRs recognize different microbial cell wall components.  Arturo Zychlinsky (New York Univ) talked on the cell death and innate immunity in bacterial infections, particularly secretory proteins from Shigella and Salmonella, and the role of TLRs in the signals for apoptosis. Kensuke Miyake (Tokyo Univ)  talked on the role MD molecules, which are required for the recognition of TLR4 by the LPS.  Jun Ninomiya-Tsuji (Nagoya Univ) was on the prerequisite role of TAK1 MAPKKK functions in the IL-1 signaling.


In Session 2 “Macrophages and Apoptosis”, cells undergoing apoptosis are rapidly and selectively eliminated by phagocytes, and evidence showing the importance of this event has been accumulated.  The issues to be immediately solved are the mechanism of phagocyte recognition of target apoptotic cells and the physiological role of this phenomenon.  In this session, six speakers presented their recent observations. Valerie Fadok (Denver) emphasized that a phosphatidylserine (PS) receptor she recently cloned, is essential for macrophage engulfment of apoptotic cells and demonstrated the importance of “being eaten”.  She focused on the consequences of apoptotic cell engulfment and has constantly observed the repression of proinflammatory cytokines and the release of anti-inflammatory mediators, such as TGF-b.   Giovanna. Chimini (Marseilles, France) presented the role of ABC1 transporter, the mammalian homologue of C. elegance Ced-7, in macrophage recognition of apoptotic cells.  ABC1 is presumably required for both macrophages and apoptotic cells, influencing the structure of the membrane microdomains.  Yoshinobu Nakanishi (Kanazawa Univ) presented the mechanism and role of macrophage phagocytosis of influenza virus-infected cells in a manner mediated by PS and neuraminidase presumably leading to virus clearance.  Dror Mevorach (Jerusalem, Israel) has been studying the role of complement on phagocytosis of apoptotic cells.  He presented the evidence for the involvement of complement in opsonization of apoptotic cells and pointed to the much more efficient phagocytosis by macrophages in comparison with immature DC.  Yoshiro Kobayashi (Toho Univ) also presented the macrophage responses to the ingestion of apoptotic cells. Tadashi Kasahara (Kyoritsu College Pharmacy) presented that the focal adhesion kinasehas a role of anti-apoptocic activity in various cells including macrophages.


In Session 3 subtitled “Lectin and Carbohydrate Recognition”,  five presentations were focused on regulated expression and functional relevance of lectins, carbohydrate ligands, and glycosyltransferases, implying that interaction between endogenous lectins and their carbohydrate ligands are potentially involved in antigen uptake, cell trafficking and intercellular communication in the immune system.  Sem Saeland (Schering Plough) showed molecular structures and biological functions of two lectins, a Langerhans cell-specific lectin, Langerin and newly identified DC-specific lectin asialoglycoprotein receptor (DC-ASGPR). The latter one was identical with the lectin originally defined as a macrophage lectin by Prof. Irimura’s group.  He implied a potential role of the lectin as an endocytic receptor.  Tatsuro Irimura (Tokyo Univ) reviewed recent his efforts on regulation of O-glycan formation on mucin core polypeptides, recognition of O-glycosylated mucin peptides by lectins, and gene expression, molecular diversity, and functional significance of a macrophage lectin specific for Gal/GalNAc (MGL).  Minoru Fukuda (Burnham Institute) claimed glycosyltransferases involved in construction of L-selectin ligands in the high endothelial venules. Core2 branched O-glycans with sulfation have been thought to be inevitable for synthesis of L-selectin ligands in in vitro studies.  However, lymphocyte homing in vivo was barely affected in core2 GlcNAc transferase knockout mice.  He presented a recently identified key enzyme for construction of novel L-selectin ligands (core1-beta1,3-N-acetylglucosaminyl transferase). Kenjiro Matsuno (Dokkyo Univ) has visualized recruitment of DC precursors in liver infection.  The initial step of the recruitment in liver is the interaction between DC and Kupffer cells.  He presented that GalNAc-specific lectin-carbohydrate interaction is crucial in the step.  Kazuhiko Takahara and Kayo Inaba (Kyoto Univ) identified genes encoding murine Langerin and DC-SIGN.  They are potential tools for precise classification of a number of different DC subpopulations in mice.

In the final Session, “Chemokines on the Dynamics of Leukocyte Trafficking”,  Antonio Lanzavecchia (Bellinzona, Switzerland) presented outline of T lymphocyte activation, differentiation and migration in several model system. Of note was that human memory T cells comprise two distinct populations; non-polarized CCR7+ central memory cells and polarized (Th1 or Th2), CCR7- effector memory cells.  Reinhold Forster (Univ Erlangen, Germany) presented that chemokines and their receptors are essentially involved in lymphocytes trafficking. Particularly, by generating BLR1/CXCR5-/-  and CCR7-/- mice, BLC/CXCL13 is involved in the severe impairment of B cells to secondary lymphoid organs and CCR7 is for the DC migration.  Hiromichi Ishikawa (Keio Univ) talked on the gut cryptopaches for the generation of thymus-independent IEL.  Sho Ishikawa (Tokyo Univ) talked on the B cell trafficking and autoimmunity, demonstrating that BLC was markedly enhanced in the thymus and kidney in aged BWF1 mice and suggesting that aberrant expression of BLC by myeloid DCs may breaking immune tolerance and generating autoimmunity.

10th International Symposium on Molecular Cell Biology of Macrophages 2001

Macrophages Signaling, Apoptosis, Lectin and Trafficking


June 21 (Thur)

9:15-  9:20    Opening remarks  Tadashi Kasahara (Kyoritsu College of Pharmacy)

Session 1: 9:20-12:30 Signal Transduction in Macrophages: Organizers:S.Akira and K. Miyake              

9:20- 10: 00  Luke O'Neill  (Trinity College, Dublin, Ireland):  Signaling pathways activated by IL-1 and Toll-like receptors

10:00- 10: 40  Arturo Zychlinsky   (NYU, New York, USA) : Cell death and immunity in bacterial infections

10:40-10:55                       Coffee Break

10:55-11:30   Shizuo Akira (Osaka Univ, Osaka): Toll-like receptors and their signaling pathways

11:30-12:00  Kensuke Miyake (Saga Medical College, Saga/IMS, Tokyo) : Innate recognition

of lipopolysaccharide by Toll-like receptors and associated MD molecules

12:00-12:30  Jun Ninomiya-Tsuji, Kunihiro Matsumoto (Nagoya Univ, Aichi) : TAK1 MAPKKK functions in the IL-1 signaling pathway

12:30-14:30              Lunch Break & Poster Session


Session 2:  14:30-17:40    Macrophages and Organizers:  Y. Kobayashi and Y. Nakanishi       

14:30-15:00   Valerie A Fadok (NJMRC, Denver, USA):   The role of a new phosphatidyl-serine receptor in macrophage recognition and response to apoptotic cells 

15:00-15:25   Yoshinobu Nakanishi (Kanazawa Univ,Kanazawa):  Inhibition of  influenza virus growth by apoptosis-dependent  phagocytosis of virus-infected cells by macrophages

15:25-15:55   Giovanna Chimini (INSERM-CNRS, France):   The role of ABC1and membrane lipid architecture in macrophage competence to engulf

15:55-16:10                    Coffee Break

16:10-16:35  Yoshiro Kobayashi (Toho Univ, Chiba): Macrophage responses to the ingestion of apoptotic cells

16:35-17:05  Dror Mevorach (Hebrew Univ, Israel) :  Opsonization of apoptotic cells : Implication for uptake and autoimmunity 

17:05-17:30  Tadashi Kasahara  (Kyoritsu College of Pharmacy, Tokyo): Focal adhesion kinase as an anti-apoptotic mechanism

18:00-20:00       Reception  (Heian-no-ma, Tokyo Garden Palace)


June 22 (Fri)  

Session 3:  9:20-12:30  Lectin and Carbohydrate Organizers: T. Irimura and K. Matsuno         

9:20-10:00   Sem Saeland (Schering-Plough, France): Langerin and DCasialoglycoprotein-receptor: two closely related endocytic type-II lectins with divergent functions in dendritic cells

10:00-10:40    Tatsuro Irimura (Tokyo Univ, Tokyo): C-type lectin  for the macrophage differentiation and migration

10:40-10:55                      Coffee Break

10:55-11:30 Minoru Fukuda (Burnham Inst. USA): Structural diversity of oligosaccharides that provides functional diversity

11:30-12:00     Kenjiro Matsuno (Kumamoto Univ, Kunamoto/Dokkyo Univ, Tochigi) :  Kupffer cell-mediated recruitment of dendritic cells to the liver: roles of N-acetylgalactosamine -specific sugar receptors

12:00-12:30  Kazuhiko Takahara, Kayo Inaba (Kyoto Univ, Kyoto): Identification and expresion of C-type lectins on mouse dendritic cells and possible use as a marker of dendritic cells subsets

12:30-14:00                 Lunch Break & Poster Session


Session 4:  14:00-16:45 Chemokines on the Dynamics of Leukocyte Organizers: K. Matsushima and H. Ishikawa       

14:00-14:40     Antonio Lanzavecchia (Bellinzona,  Switzerland): T lymphocyte activation, differentiation, and migration

14:40-15:15     Reinhold Forster  (Univ Clinic for Surgery, Erlangen, Germany): Homeostatic chemokines functionally organize lymphoid organs

15:15-15:30                 Coffee Break

15:30-15:55  Hiromichi Ishikawa (Keio Univ, Tokyo): Gut Cryptopaches: intestinal development and function of intraepithelial T cells

15:55-16:20   Makoto Suematsu (KeioUniv,Tokyo):  Roles of platelet-associated adhesion molecules in regulation of leukocyte adhesion to microvascular endothelium

16:20-16:45     Sho Ishikawa (Tokyo Univ, Tokyo): B cell trafficking and autoimmunity

16:45-17:00    Concluding Remarks     Kouji Matsushima (Tokyo Univ, Tokyo)


 Signalling Pathways Activated by IL-1 and Toll-Like Receptors

Luke O’Neill

Department of Biochemistry and Biotechnology Institute, Trinity College Dublin, Ireland

Interleukin-1 (IL-1) is an important cytokine which links innate and adaptive immunity.  IL-1 induces genes which encode mediators of inflammation and enhanced immune reactivity and much attention has focussed on the signalling pathways it activates.  Signals include the transcription factor NF-kappaB and the MAP kinases p38, p42/p44 and JNK.  These are triggered when IL-1 binds the Type I IL-1 receptor (IL-1RI).  IL-1RI is the founder member of a superfamily of receptors which all contain a similar sequence in their cytosolic regions termed the Toll IL-1 receptor (TIR) domain.  Toll was first described in the fruit fly where it was initially shown to regulate dorsoventral polarity in the developing embryo.  Adult flies however utilise Toll as part of their innate immune response to fungal pathogens.  There are a total of 10 human Toll-like receptors (TLRs) and all are predicted to participate in the innate response to microbial products.  A dimer of TLR-2 and TLR-6 is required for responses to products from gram positive bacteria and fungi, a  TLR-4 homodimer responds to LPS and CpG DNA signals via TLR-9.  The functions of the other TLRs is at present unknown.  During signalling, the TIR domain recruits a cytosolic TIR domain-containing protein, MyD88. This protein recruits IRAK and IRAK-2, which in turn recruit TRAF-6 leading to NF-kappaB and MAP kinase activation.  In addition we and others have found that the low molecular weight G protein Rac1 participates in TIR-dependent signalling and is involved in a pathway culminating in enhanced transactivation by NF-kappaB.  In addition, Ras and Rap are involved in p38 MAP kinase activation by IL-1, with Rap having an antagonistic role.

   We have discovered 3 additional proteins with putative TIR domains which are homologus to MyD88.  2 of these, termed A46R and A52R, are in Vaccinia virus.  They act to antagonise TIR-dependent signalling, which may one of the strategies used by the virus to manipulate host defense.  A52R specifically sequesters IRAK-2 and promotes its degradation.  The third protein is the final TIR-domain ? containing protein in the human genome and we have termed this protein Mal (MyD88 adapter-like).  Mal has an obvious TIR domain but differs from MyD88 in that it lacks a death domain.  Over-expression of Mal activates NF-kappaB, JNK and p42/p44 MAP kinase.  Mal forms a heterodimer with MyD88, and its effects can be blocked with a dominant negative form of MyD88.  Interestingly, Mal appears to selectively recruit IRAK-2 (unlike MyD88 which recruits both IRAK and IRAK-2).  Its effects are completely blocked by A52R, consistent with A52R sequestering IRAK-2.  The role of Mal is in TLR-4 signalling since a dominant negative form blocks NF-kappB activation by TLR-4 over-expression or in response to LPS in THP-1 macrophages.  It does not participate in IL-1 signalling however.  These effects are consistent with results obtained in MyD88 ? deficient mice.  Cells from these mice do not respond to IL-1 but still respond to LPS, although the LPS responses (NF-kappaB and JNK activation) are somewhat impaired.  This implies that for optimal LPS signalling, Mal and MyD88 co-operate, with the particular role of Mal being to recruit IRAK-2.
Studies into the TIR domain therefore identify it as an important switch for innate immunity and inflammation. The study of TIR domain-containing receptors has greatly improved our understanding of the molecular basis to inflammation and the host response to microbes.

Toll-Like receptors and their signaling pathways.

Shizuo Akira

Department of Host Defense, Research Institute forA@Microbial Diseases, Osaka University, Yamadaoka 3-1, Suita, Osaka 565-0871, Japan


Toll-like receptors are phylogenetically conserved receptors that are essential in the recognition ofpathogens. The TLR family harbors an extracellular  leucine-rich  repeat (LRR) domain and a cytoplasmic domain that is homologous to that of the IL-1R family. Analogous to the IL-1R, TLR recruits IRAK via adaptor MyD88, and then induces activation of TRAF6, NIK and finally NF-kB. We previously generated mice lacking TLR2, TLR4, TLR9, and MyD88. Using these mutant mice we demonstrated that TLR4 functions as the transmembrane component of the lipopolysaccharide (LPS) receptor, while TLR2 recognizes peptidoglycans  from Gram-positive bacteria, and lipoproteins. TLR9 is a receptor for CpG DNA (microbial DNA). Although various microbial cell wall components are recognized by different  receptors, all of these responses are abrogated in MyD88-deficient  cells.  These results show that different TLRs recognize different microbial cell wall components, and that MyD88 is an essential signaling molecule shared among IL-1 R/Toll family members. However, LPS activation of MAP kinases and NF-kB remains intact in MyD88-deficient  macrophages. This indicates that the LPS response is mediated by both MyD88-dependent and -independent pathways, and that the MyD88-dependent pathway is essential for the inflammatory response mediated by LPS.


Cell Death And Innate Immunity In Bacterial Infections

Arturo Zychlinsky

   Skirball Institute and Department of Microbiology,  NYU Medical School,  540 First Av., NY, NY 10016

     Acute bacterial diseases result from the activation of the host’s innate immune system and the ensuing inflammation.  Gram negative enterobacteria are good models to analyze how microbes manipulate innate immunity. Enterobacteria of different genera induce apoptosis, which is crucial in the initiation of inflammation.

Shigella and Salmonella induces apoptosis in macrophages in vitro and in vivo. These two bacteria secrete and deliver into the cytoplasm the homologous proteins Invasion Plasmid Antigen (Ipa) B (Shigella) and Salmonella Invasion Protein (SipB).  IpaB and SipB are necessary to induce cell death and bind to caspase (Casp)?1, a host cysteine protease that is required for apoptosis.  Apoptosis mediated by Casp-1 is pro-inflammatory in Shigella and Salmonella infections, since Casp-1 proteolytically activates the cytokines pro-Interleukin (IL)-1s and pro-IL-18.
 A second, and independent, mechanism of induction of host cell apoptosis is the activation of the pattern recognition receptors Toll Like Receptors (TLR). In response to bacterial molecules, TLRs activate a signal sequence cascade that culminates in either activation of the transcription factor NF-kB or apoptosis.
The activation of apoptosis in the innate immune system of animals and plants are analogous. Surprisingly, many of the host players also have sequence homology, suggesting a common ancestral innate immune defense system.


Innate Recognition of Lipopolysaccharide by Toll-like Receptors and Associated MD molecules.

Kensuke Miyake

Division of Infectious Genetics, Department of Microbiology and Immunology,

The Institute of Medical Science, The University of Tokyo4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan

   The innate immune response is the first line of defense against microbial pathogens.  The principal challenge for the immune system is to recognize pathogens and mount an immediate defense response.  A wide variety of bacterial components are capable of stimulating innate immunity.  These include LPS, peptidoglycan, lipoteichoic acid, lipoarabinomannan, lipopeptides, and bacterial DNA.  LPS is a principal component of Gram-negative bacteria that activates the innate immune system, and one of the best-studied molecules.  Toll-like receptor 4 (TLR4) has been implicated in innate recognition and signaling of LPS.  The mutations of the TLR4 gene lead to hyporesponsiveness to LPS in mice and humans.  Exogenous expression of TLR4, however, does not confer LPS responsiveness on cell lines.  We recently cloned the MD-2 molecule that is associated with the extracellular domain of TLR4.  MD-2 imparts LPS responsiveness to TLR4.  LPS is therefore recognized by TLR4/MD-2, but not by TLR4 alone.  Our recent results indicate that MD-2 has a role in triggering an activation signal via TLR4 after physical contract of LPS with TLR4/MD-2. 
RP105/CD180 is the first to be described as a molecule similar to Drosophila Toll. RP105 is expressed on the B cell surface and antibody-dependent ligation leads to massive B cell proliferation in mice. RP105 is associated with MD-1, and forms the RP105/MD-1 heterodimer. Roles of RP105/MD-1 in LPS responses were addressed by making mice lacking RP105 or MD-1. The defect was most apparent in B lymphocytes. A lack of MD-1 resulted in defective expression of RP105 on the B cell surface, demonstrating an indispensable role for MD-1 in cell surface expression of RP105. The lack of RP105/MD-1 led to hyporesponsiveness to LPS in B cells. Using transfectants expressing TLR4/MD-2 and RP105/MD-1, functional cooperation between TLR4/MD-2 and RP105/MD1 were suggested. Collectively, Immune cells use the heterodimeric complexes consisting of TLR4/MD-2 and RP105/MD-1 for innate recognition of Gram-negative bacteria.

TAKI MAPKKK functions in the lL-1 signaling Pathway

Jun Ninomiya-Tsuji, Giichi Takaesu, Satosi Kishida and Kunihiro Matsumoto

Department of Molecular Biology, Graduate School of Science, Nagoya University and CREST, Japan Science and Technology Corporation


    Interleukin-1 (lL-l ) is a proinflammatory cytokine that functions in the generation of systemic and local responses to infection  and injury. After binding to the ceII surface receptor IL-lRI, TL-l activates intraceIIular signaling cascades leading to the activation of the transcription factors AP-l and NF-kB, which in turn up-regulate the expression of many proinflammatory genes in the nucleus. Recently, considerable progress has been made in delineating the IL-l signaling pathway. The first signaling event initiated by IL-1 is formation of a complex containing IL-l RI, the receptor accessory protein (IL-1AcP) and an adaptor protein MyD88, which in turn facilitates the association of the serine/threonine IL-1 receptor-associated kinase (IRAK). IRAK then interacts with TNF receptor-associated factor 6 (TRAF6), a factor required for IL-1-induced AP-1 and NF-kB activation.   We demonstrated that TAKI MAPKKK is activated by lL-l and plays essential roles to activate both AP-1 and NF-kB. We identified a novel TAK1-interacting protein, TAB2, which interacts with TRAF6, linking TAKl to the IL-l pathway. 1L-l induces formation of the TRAF6-TAB2-TAKl complex. We also found that TAB2 resides in the membrane in the absence of lL-1 and lL-l induces translocation of TAB2 into the cytosol where TRAF6 and TAKl reside. FoIIowing IL-1 stimulation, TAB2 transiently associates with IRAK concomitantly with the translocation. Furthermore, we found that TAB2 translocation does not occur in the IRAK deficient ceIIs even in the presence of IL-1. In the IRAK deficient cells, both the TRAF-6- TAB2-TAKl complex formation and the activation of TAKl are completely abolished. Therefore, IRAK regulates TAB2 translocation, which is an essential step for both the TRAF6-TAB2-TAKl complex formation and the activation of TAKl in the lL-l signaling pathway.


The Role of A New Phosphatidylserine Receptor in Macrophage Recognition and Response to Apoptotic Cells

Valerie A. Fadok, Donna L. Bratton, Peter M. Henson,

Program in Cell Biology, Dept. Pediatrics, National Jewish Medical and Research Center, D509, 1400 Jackson Street, Denver, CO 80206; fadokv@njc.org


Clearance of apoptotic cells by phagocytes is a critical feature in embryogenesis, tissue remodeling, and resolution of inflammation.  The ability of viable cells to recognize their dying neighbors has been preserved throughout phylogeny.    In mammals, although fibroblasts, epithelial cells, and endothelial cells are capable of recognizing and removing apoptotic cells, the redoubtable macrophage is the phagocyte that mediates swift and efficient clearance.  Several receptors have been described to mediate the binding and engulfment of apoptotic cells.  These include lectins, integrins (particularly avb3 on macrophages and avb5 on dendritic cells), several scavenger receptors (Class A, Class B, and other oxidized phospholipid receptors e.g. CD68, LOX-1), other pattern recognition receptors (e.g. CD14), the mer receptor, CD91, and a newly described receptor for phosphatidylserine.  In addition, there are a number of soluble proteins which appear to act as opsonins for uptake, including collectins (MBP, SPA, SPD) and C1q, other complement proteins, thrombospondin, b2GP1, and gas-6.  In particular, we have most recently discovered that the collectins on apoptotic cells bind to surface calreticulin which is itself bound to CD91.  Crosslinking this receptor appears to induce engulfment of apoptotic cells. 

The mammalian phosphatidylserine receptor is highly homologous to genes of unknown function in Drosophila melanogaster and C. elegans.  Using flow cytometry, we have found that the phosphatidylserine receptor is expressed on the surfaces of macrophages, dendritic cells, fibroblasts, epithelial cells, endothelial cells, and even melanoma tumor cells.  Expression on macrophages is variable, with low levels on monocyte- or bone marrow-derived cells, which can be increased by exposure to apoptotic cells and some inflammatory cytokines (e.g. TNFa).  It is highly expressed on macrophages derived from inflammatory sites, including the macrophages elicited into the mouse peritoneal cavity with thioglycollate.  In contrast, circulating cells such as lymphocytes, granulocytes, monocytes, and red blood cells do not show surface expression of this receptor.  Using RT-PCR and Northern blot analysis, we have found that the mRNA is expressed ubiquitously in all human and mouse tissues tested, including the day 7 mouse embryo.  Lymphocytes, although negative for surface expression of the receptor, express mRNA for this protein.
Our data suggest that engagement of this receptor is essential for the physical process of engulfment. We and others have found that apoptosis is accompanied by loss of phospholipid asymmetry, exposure of phosphatidylserine, and recognition by phagocytes. Macrophages and other phagocytes recognize phosphatidylserine stereospecifically, and this recognition is required for engulfment but not binding to apoptotic cells. If expression of the phosphatidylserine receptor is ablated by transfection with plasmids containing anti-sense constructs, apoptotic cells are not engulfed. Triggering of the phosphatidylserine receptor by apoptotic cells, by phosphatidylserine-containing liposomes, or by a specific monoclonal antibody results in ruffling, macropinocytosis, and activation of cdc42 and Rac. In our hands, ruffling and macropinocytosis can be induced by triggering surface CD91, but not by activating CD36, CD14, scavenger receptors, or integrins, although we have not testedavb5. These data suggest to us that many receptors mediate the binding of apoptotic cells to macrophages and other phagocytes in order to increase the likelihood of engagement of a small subset of engulfment receptors.
    In our hands, one of the consequences of apoptotic cell engulfment is the repression of proinflammatory cytokines and the release of anti-inflammatory mediators. We have focused our studies on the release of TGFb, and have found that TGFb is responsible for the anti-inflammatory phenotype which occurs. TGFb release is mediated through the phosphatidylserine receptor, because the effects of apoptotic cells can be mimicked by addition of phosphatidylserine-containing liposomes or the monoclonal antibody against the receptor, and because transfection with anti-sense constructs ablates this response. It has been known for many years that phosphatidylserine inhibits macrophage production of TNFa, transcription and activation of iNOS, and killing of intracellular parasites. It is also known that engulfment of apoptotic cells by dendritic cells can tolerize the immune system, but that exposure to necrotic cells will activate it. We therefore hypothesized that engagement of the phosphatidylserine receptor on dendritic cells by phosphatidylserine-expressing apoptotic cells, phosphatidylserine-containing liposomes or the receptor-specific monoclonal antibody would inhibit their maturation and ability to induce a productive immune response. Our preliminary experiments look promising, in that dendritic cells express the phosphatidylserine receptor, that its engagement on DC inhibits upregulation of the costimulatory molecules CD80 and CD86 in response to a maturing stimulus, and that PtdSer-expressing apoptotic cells inhibit T cell IL2 production in response to antigen presentation. We therefore believe that recognition of phosphatidylserine by the phosphatidylserine receptor not only promotes engulfment of apoptotic cells but insures that inflammation and autoimmune response will not develop.



Inhibition of Influenza Virus Growth by Apoptosis-Dependent Phagocytosis of Virus-Infected Cells by Macrophages

Shiratsuchi, A.1, Watanabe, Y. 1,. Shimizu, K. 2, Takizawa, T. 3, andNakanishi, Y. 1

1Grad. Sch. Med. Sci., Kanazawa Univ., 2Sch. Med., Nihon Univ., 3Inst.Dev. Res., Aichi Human Serv. Ctr., Japan

HeLa cells infected with influenza A virus undergo Fas-mediated apoptosisaccompanied by externalization of the membrane phospholipid phosphatidylserine (PS).1~3,5,7)  Such cells were efficiently phagocytosed. We will propose here a mechanistic model of engulfment with particular attention to the role of ABC1 transporter. In our opinion, this molecule, conserved in the nematode model system, facilitates engulfment by providing  adequate membrane characteristics and we will discuss how this could reasonably account for some of the engulfment specific feature enumerated above. The contribute of ABC1 to the dynamic equilibrium of lipids across the bilayer is also crucial for the release of membrane lipids to plasmatic acceptors and thus plays a fundamental role in the maintenance of cellular cholesterol homeostasis, dramatically altered in the naturally occurring human ABC1 knock out i.e.Tangier disease.



Macrophage Responses to The Ingestion of Apoptotic Cells

Yoshiro Kobayashi

Department of Biomolecular Science, Faculty of Science, Toho University, Funabashi, Chiba 274-8510, Japan

              We have previously demonstrated that macrophages produce IL-8 or MIP-2, a mouse homologue of IL-8, following coculture with apoptotic cells (BBRC 239, 799, 1997; JI 161, 6245, 1998).  In this study we examined the response to apoptotic cells in vivo.  Injection of apoptotic cells into the peritoneal cavity of mice led to transient neutrophil infiltration and concomitant production of MIP-2.  Apoptotic cells were phagocytosed by macrophages, as revealed on two-color flow cytometric analysis and microscopic observation.  When the mice were depleted of macrophages by pretreatment with liposome-encapsulated dichloromethylene bisphosphonate, both neutrophil infiltration and MIP-2 production were significantly suppressed, suggesting that macrophages are required for MIP-2 production in this in vivo response.  In another study we recently found that whole-body X-irradiation induced apoptosis in the thymus is assosiated with transient infiltration of neutrophils (JLB 67, 780, 2000).  These results support the hypothesis that extensive apoptosis occurring rapidly may induce an inflammatory response in vivo.  
We then examined the effects of human serum on cytokine production following coculture of macrophages with apoptotic cells. We found that human serum potentiates the production of anti-inflammatory cytokines, IL-10 and TGF-b, by a PMA-treated human monocytic cell line, THP-1 cells, and human monocyte-derived macrophages in response to apoptotic cells., which results in great suppression of the production of pro-inflammatory cytokine, IL-8. Human IgG but not F(ab)' of human IgG suppressed the IL-8 production, suggesting that one of the components responsible for the suppression is a Fc portion of human IgG. When FcgRI was specifically down-modulated by a monoclonal antibody, M22, both the potentiating effects of human serum and human IgG on the anti-inflammatory cytokine production and the suppressive effects on IL-8 production were completely abolished. Thus this study reveals a hitherto unrecognized role of human IgG and FcgRI in determining the balance between the level of pro-inflammatory cytokine and that of anti-inflammatory cytokines produced by macrophage in response to apoptotic cells._



Opsonization of Apoptotic Cells: Implication for Uptake and Autoimmunity

Dror Mevorach

The Laboratory for Cellular and Molecular Immunology, Hadassah Hospital


     Apoptosis, programmed cell death, has attracted great attention in recent years.  The term was suggested by Kerr, Wyllie and Currie in 1972, and refers to the unique morphology of the condensed chromatin, contracted cytoplasma, and formation of blebs. We can divide apoptosis to two distinct sequential processes; cell killing, and the removal of the dead cells. These two processes are linked together and in vivo staining of tissues with high rate of apoptosis show us usually the morphology of an apoptotic cell within the phagocytic cell.
Although cell death by apoptosis is measured in hours, the removal of apoptotic fragments is normally so rapid that apoptotic cells are rarely seen-even in tissues such as the thymus where up to 95% of cells undergo apoptosis. It is thought that uptake of these cells by specific receptors in phagocytes results in the disposal of cellular contents without the induction of inflammation. The mechanisms whereby apoptotic cells are efficiently identified, removed, and degraded by phagocytes in mammalian cells are not well understood. Some progress has been made in the nematode C. elegans, and in human and murine systems. Interestingly, in the nematode Caenorhabditis (C.) elegans out of 12 genes involved in PCD, at least six are functioning in removal of apoptotic bodies (3). Ced?2, ced?5, ced?10, ced?1, ced?6, and ced?7, are six genes important in uptake of apoptotic bodies. Functions of the mutant genes are only partially characterized. Ced-5 encodes the mammalian homologue DOCK 180, a protein important in signaling of integrins, Ced?7 encodes a protein similar to ABC transporter and may be important in the interaction between the cell surfaces of both the dying and the engulfing cell . The protein of Ced?6 was suggested to be a adaptor molecule with a phosphotyrosine binding domain acting in signal transduction of the engulfing cell. In mammalians, studies showed several receptors and molecules as being important in the uptake of apoptotic cells . These include integrins (including receptors for the complemnt fragment iC3b), scavenger receptors, CD14, a receptor for phosphatidylserine and an ABC1 cassette transporter. As suggested by the heterogeneity of the receptors, the mechanisms underlying the recognition and uptake of apoptotic cells by macrophages is complex. The different species studied, the different nature and preparation of the phagocytes, the different source of apoptotic cells and absence of knowledge regarding the biochemical nature of the receptors in many cases, precludes a clear understanding of the role that each receptor plays in this important biological process.
As a part of innate and adoptive immunity, soluble host proteins called opsonins, which include complement ligands, initially coat microorganisms that penetrate the mammalian sterile milieu. The main purpose of opsonization is to allow efficient subsequent clearance of opsonized particles by specific receptors on the surface of leukocytes with the subsequent antigen presentation and induction of the appropriate pro-inflammatory immune response.Several proteins: thrombospondin I, complement fragments,Beta-2GPI, immunoglobulins, C-reactive protein and others, act as opsonins and have a role in uptake of apoptotic cells and bodies.
Being a part of the innate immunity, the complement system was thought to be a “complementing” host defense pro-inflammatory mechanism. We have now evidence that the complement system and additional proteins allow efficient uptake of opsonized apoptotic cells and bodies up to 8 folds, compared to non-opsonized apoptotic cells, without induction of a pro-inflammatory response.In fact, as demonstrated by ligation to the complement receptors, opsonization by complement and other proteins not only increases efficiency of uptake but also further determine signaling and down stream events leading to inhibition of a pro-inflammatory response by macrophages and dendritic cells. Importantly, inhibition of expression of DRII and costimulatory molecules following ingestion of apoptotic cells coated by homologous complement fragments may represent a mode of induction of peripheral self-tolerance. Alteration of the mechanisms of clearance of apoptotic cells may result in a pro-inflammatory response by antigen presenting cells, loss of tolerance to chromatin, self proteins and ribonucleoproteins, and the establishment of autoimmunity.




Focal Adhesion Kinase as an Anti-Apoptotic Mechanism

Tadashi Kasahara and Yoshiko Sonoda

   Kyoritsu College of Pharmacy, Shibakoen 1-5-30, Minato-ku, Tokyo 105-8512


   Focal adhesion kinase (FAK) has an anti-apoptotic role in anchorage-dependent cells via an unknown mechanism.  In order to elucidate the role of FAK in anti-apoptosis, we have established several FAKcDNA-transfected HL-60 cell lines and examined whether FAK-transfected cells have resistance to apoptotic stimuli.  FAK-transfected HL-60 (HL-60/FAK) cells were highly resistant to hydrogen peroxide (1 mM), etoposide (50 mg/ml) or irradiation-induced apoptosis compared to the parental HL-60 cells or vector-transfected cells. Since no proteolytic cleavage of pro-caspase-3 to mature caspase-3 fragment was observed in HL-60/FAK cells, FAK was presumed to inhibit an upstream signal pathway leading to the activation of caspase-3. HL-60/FAK activated PI3-kinase-Akt survival pathway and exhibited significant activation of NF-kB with marked induction of IAPs (cIAP-1, cIAP-2, XIAP).  Elevated basal NF-kB activation appeared to be critical for the maintenance of anti-apoptotic signals.  Mutagenesis of FAKcDNA revealed that Y397F and Y925F, both of which are involved in the tyrosine-phosphorylation sites, were prerequisite for the anti-apoptotic activity as well as induction of IAPs, and K454R which is involved in the kinase-activity, was also but less required.  Above results were also confirmed by the transient expression of FAK in the adenovector in the human glioblastoma cell lines, i.e., transfection of wild-type FAK markedly protected the cells from the apoptosis, while Y397F-mutated FAK did not protect from apoptosis but rather induced apoptosis byitself.
   Prental HL-60 cells in unstimulated condition, did not express endogenous FAK, while macrophage-like differentiated cells by PMA exhibited significant level of FAK. Since PMA-differentiated cells were relatively resistant to the hydrogen peroxide-induced apoptosis compared to parental HL-60 cells, suggesting that intrinsic FAK has also an anti-apoptotic role in the macrophages.
We conclude that FAK activates PI3-kinase-Akt survival pathway with the concomitant activation of NF-kB and induction of IAPs, which ultimately inhibit apoptosis by inhibiting caspase-3 cascade.  In accordance with these results,A@interruption of the survival pathway by the mutated FAK (Y397F) downregulated XIAP expression and NF-kB activation, leading to the apoptosis.



Langerin and the DC Asialoglycoprotein-Receptor: Two Closely Related Endocytic Type-Ii Lectins with Divergent Functions in Dendritic Cells

Sem Saeland

Schering-Plough Laboratory for Immunological Research, 69571 Dardilly, France


Immature dendritic cells (DC) are highly specialized in antigen capture, notably via a number of endocytic receptors. These include type-I lectins, such as the mannose-receptor and DEC205, with multiple Ca++-dependent (C-type) carbohydrate recognition domains (CRD). The important role of type-II lectins with a single CRD in DC endocytosis has been more recently realized.
  Langerin is a mannose-binding type-II lectin that we originally cloned from DC supplemented with TGF_. Langerin is expressed on Langerhans cells of the epidermis and epithelia. Strikingly, Langerin routes extracellular ligand into Birbeck granules (BG), the unique organelles of Langerhans-type cells. This process is paralelled by an active role of Langerin in the formation of BG by membrane superimposition and zippering when expressed in cell lines. Of note, the intracellular portion of Langerin contains a proline-rich (WPREPPP) domain but lacks the tyrosine-based internalization motif used by a number of other endocytic receptors. Mouse Langerin shares key features of its human counterpart, including a similar genomic organization. Langerin is most closely related to the rodent Kuppfer cell receptors, constituting a subfamily of C-type lectins characterized by ana-helical coil-coiled stalk between the transmembrane domain and the CRD. Studies with deleted and mutant forms of Langerin have shown that the CRD is essential for BG formation. Also, mouse Langerin in which a single conserved residue was modified within the CRD did not give rise to BG but to a different type of superimposed membranes similar to the cored tubules described in mouse Langerhans cells.
  We have also identified several isoforms of an asialoglycoprotein receptor (DC-ASGPR) on human DC. These represent splice-variants of the macrophage lectin HML, a galactose-specific type-II Ca++-dependent lectin highly related to Langerin. However, in contrast to Langerin, the DC-ASGPR / HML is expressed by interstitial DC but not by Langerhans cells. The DC-ASGPR, which features an intracellular tyrosine-motif, internalizes extracellular ligand into DC with similar rapid kinetics as Langerin. However, the DC-ASGPR localizes to DC early endosomes indicative of a recycling receptor, and its expression in cell lines does not result in BG formation. Also, whereas Langerin does not intersect with the routing of MHC-II molecules, antigen targeted to the DC-ASGPR reaches the MHC-II pathway resulting in highly efficient presentation to T cells.
   Our data demonstrate that closely-related endocytic receptors can have highly divergent functions in DC. Furthermore, as illustrated by Langerin, we speculate that a given receptor could have considerable plasticity in the routing of extracellular ligands, by generating distinct subcellular compartments upon endocytosis. Recent availability of mice with disrupted Langerin genes should be important to further understand the functional consequences of the diversity of type-II lectin endocytic receptors in DC.




Molecular and Cellular Recognition by MGL in the Immunobiology of Macrophages

Tatsuro Irimura, Nobuaki Higashi, Makoto Tsuiji, Kaori Denda-Nagai, Hideyuki Takeuchi, Kentaro Kato, Akiko Morikawa, and Kouki Fuioka

Graduate School of Pharmaceutical Sciences, The University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113-0033,


                   Glycosylation is the most prominent post-translational protein modification with regard to its variation.  The range of structural variations of glycans exceeds that of proteins.  Therefore, the number of endogenous lectins within the genomic repertoires is much less than the number of glycans.  This seems to represent the major obstacle to the understanding of the biology of carbohydrates and lectins.  Although it is widely  accepted that deciphering the code written in carbohydrates will open new vistas in biology, little is known how the "secret codes" are formulated, and how they are recognized and translated into biological signals.  Our endeavors in the last several years aim to develop novel approaches to overcome these fundamental problems in glycobiology and to answer specific biological questions in the following four mutually related areas.  These are: (a) regulation of O-glycan formation on mucin core polypeptides and recognition of O-glycosylated mucin peptides by lectins (1-4), (b) immunobiology of the cells expressing a C-type lectin specific for galactose and/or N-acetylgalactosamine as a monosaccharide (MGL) (5-15), (c) bioengineering of plant lectins aiming to develop tools to identify different cells through the identification of different glycans (16), and (d) carbohydrate-mediated cellular interactions in cancer metastasis (17-21). 
   The immunobiology of MGL includes genetic and genomic aspects (5, 9), distribution (6), carbohydrate specificity in the recognition (1), its role in contact hypersensitivity (8, 11, 13), and its involvement in the formation of lymph node, lung, and liver metastasis (7, 12, 21).  A peptide with consecutive threonine residues, PTTTPITTTTK and PTTTPLK, representing MUC2 core peptide was glycosylated in a highly strict manner (1-4). Peptides with unique arrangement had high affinity with lectins such as MGL (1, 4). For example, the affinity of recombinant human MGL for immobilized glycopeptides increased, as revealed by the surface plasmon resonance and fluorescence polarization, in parallel with the number of attached GalNAc. The density of GalNAc on these peptides were shown to be determined by the peptide backbone and the combination of the pp-GalNAc-Ts. Preferential binding of densely glycosylated O-linked glycopeptides was shown to be due to the trimer formation of MGL (1).

                  The expression of MGL during the differentiation of monocytes into macrophages was investigated with human peripheral blood monocytes and mouse bone marrow cells (13, 14).  The cell surface expression of human MGL during the in vitro differentiation of monocytes into macrophages was examined by immunostaining and Western blotting with a specific mAb.  MGL was detected on cells at an intermediate stage of differentiation.  These cells were round, slightly larger in size than monocytes, and expressed macrophage markers CD68 and CD14.  The highest levels of expression occurred between 2 and 4 days of culture.  The distribution of MGL+ cells in normal human skin was then examined by immunostaining. MGL+ cells were found mainly in the upper dermis distant from vascular structures, whereas CD14+ cells localized in perivascular areas.  These data indicate that human MGL is a unique marker for cells at an intermediate stage of macrophage differentiation (13).  All five splicing variants seemed to be expressed in the same cells throughout this differentiation stages.  Bone marrow-derived DCs were examined for the expression of the murine MGL (14).  Flowcytometric analysis after double staining for MHC class II and MGL indicated that MGL was expressed only on immature DCs with low levels of MHC class II.  Immature DCs were also able to bind a-N-acetylgalactosaminides conjugated to soluble polyacrylamide (an artificial mucin), while mature DCs and DC precursor cells could not.  The mucin like complex was taken up upon incubation of the cells at 37°C.  Thus, MGL, a phagocytic receptor for mucins, appears to be transiently expressed on DCs during their differentiation, maturation, migration, and activation.


(1)     Iida S et al, J Biol Chem, 274: 16, 10697-10705, 1999.  (2) Iida S et al, FEBS Lett, 449: 230-234, 1999.  (3) Iida S et al, Biochem J, 347: 535-542, 2000.  (4) Takeuchi H et al, Submitted.  (5)Suzuki N et al, J Immunol, 56: 128-135, 1996.  (6) Mizuochi S et al, Glycobiology, 7: 137-146, 1997.  (7) Ichii S et al, J Leukocyte Biol, 62: 761-770. 1997.  (8) Sato K et al, J Immunol, 161: 6835-6844, 1998. (9) Hosoi T et al, Glycobiology, 8: 791-798, 1998.  (10)Tsuiji M et al, Immunogenetics, 50: 67-70, 1999.  (11) Chun K-H et al, J Leukocyte Biol, 68: 471-478, 2000.  (12) Ichii S et al, Cancer Immunol Immunother, 49: 1-9, 2000.  (13) Chun K-H etr al, Int Immunol, 12: 1695-1703, 2000.  (14) Higashi N et al, submitted.  (15) Denda-Nagai et al, submitted.  (16)Yim M et al,  Proc Nat Acad Sci USA, 98: 2222-2225, 2001.  (17) Nakamori S et al, J Clin Oncol, 15: 816-825, 1997.  (18) Nemoto Y et al, Clin Exp Metastasis, 16: 569-576, 1998.  (19)Fujita K et al, Brit J Cancer, 80, 301-308, 1999.  (20) Ota M et al, Cancer Res, 60: 5261-5268, 2000. (21) Higashi et al, submitted.


Kupffer Cell-Mediated Recruitment of Dendritic Cells to the Liver: Roles of N-Acetylgalactosamine-Specific Sugar Receptors.

Kenjiro MATSUNO1, Ryosuke UWATOKU2, Makoto SUEMATSU3 and Taichi EZAKI2 1Dept. Anatomy I, Dokkyo University, School of Medicine, Mibu, Tochigi 3210293, 2Department of Anatomy II, Kumamoto University, School of Medicine, 2-2-1, Honjo, Kumamoto 860-0811, 3Department of Biochemistry, Keio University, School of Medicine, Tokyo


       Liver is an essential organ for a host defense. It is armed not only with a powerful macrophage system but also is constantly surveyed by a heavy traffic of dendritic cells (DCs) and lymphocytes. In case of emergency, such as infection and inflammation, DC traffic in the liver is accelerated. Putative DC precursors are recruited to the liver, capture antigens and soon translocate to hepatic lymph. Even lymph DCs at the antigen-transporting stage can be recruited to the liver and translocate to lymph after i.v. transfer into normal rats. However, cellular and molecular mechanisms for DC recruitment to the liver are yet to be defined. In this study, purified rat DCs were injected into circulation and their traffics were analyzed in normal and Kupffer cell-depleted rats by intravital confocal microscopy and immunohistology. Affinities of DCs to sinusoidal cells were examined by in vitro cell binding assay. DC precursor recruitment was induced by i.v. injection of latex particulates.
      Both DC precursors and lymph DCs could be recruited to the liver and their majority initially showed a selective binding to Kupffer cells. In the Kupffer cell-depleted rats, DCs could neither be recruited to the liver nor adhere to sinusoidal walls. Pretreatment with varied monosaccharides in vitro showed that sugar residues consisting of N-acetylgalactosamine(GalNAc) were necessary for this binding. The binding was calcium-dependent but not inhibited by mAbs to rat selectins or selectin ligands, implying involvement of C-type lectins other than selectins. The majority of lymph DCs possessed rat GalNAc/ galactose-specific lectin-related molecules as detected by immunostaining and RT-PCR, supporting this possibility. Furthermore, approximately 40 % of these cells could endocytose GalNAc polymer in vitro in a receptor-specific manner. Lymph DCs could also ingest virus particles in vitro, although inhibition with GalNAc was not determined.
     In conclusion, the DC-Kupffer cell binding through GalNAc-specific C-type lectin-like receptors is crucial for DC recruitment to the liver. DCs possess these receptors which recognize Kupffer cells and also function as a receptor for the endocytosis of galactosylated antigens, possibly including virus particles. Kupffer cell lectins might be also responsible for this binding. Kupffer cells may elaborate chemokines to attract and trap the recruited DC via selective adhesion, leading to DC extravasation. The accelerated traffic and the presence of blood-lymph translocation would induce rapid and efficient immune responses and thus contribute to the local defense to antigens within liver tissues as well as systemic defense to blood-borne antigens.




Identification and Expression of C-type Lectins on mouse Dendritic Cells and Possible Use as a Marker of Dendritic Cells Subsets


Kazuhiko Takahara, Yoshiki Omatsu, Yusuke Yashima, Tomonori Iyoda and Kayo Inaba

Laboratory of Immunobiology, Department of Animal Development and Physiology, Division of Systemic Life Science, Graduate School of Biostudies, Kyoto University, Kitashirakawa-Oiwakecho, Sakyo-ku, Kyoto 606-8502, Japan


    Dendritic cells (DCs) develop from bone marrow-derived progenitor cells and locate in periphery as a sentinel, move to regional lymphoid organs, and selectively stimulate antigen-specific T cells.  In this process, DCs require antigen-nonspecific interactions with various cells via many different types of molecules, including C-type lectins.  For example, MMR, DEC-205 and Langerin are thought to play a role in antigen uptake, and DC-SIGN/DC-SIGNR, known as a receptor of HIV gp120, interacts with ICAM-2 on endothelial cells during migration and with ICAM-3 on naive T cells to scan specific TCR.  Here we show results of the identification of mouse homologues of human (h) Langerin and DC-SIGN.
   Langerin is a Birbeck Granule-associated molecule in epidermal Langerhans cells (LCs), and its cDNA has been cloned and mapped in human. Mouse (m) Langerin gene, cloned based on sequence of hLangerin lectin domain, was physically mapped to mouse chromosome 6D1-D2 and to rat chromosome 4q33 distal - q34.1 proximal, syntenic to chromosome 2p13 where hLangerin gene is located. While hLangerin has been shown to be maily expressed in LCs and LC-related cells in the lung and tonsil, mLangerin mRNA was detected in various tissues, including liver, heart, lung, spleen and lymph nodes as well as epidermis, but not small intestine and Peyer’s patches. None of purified T and B cells and macrophages were positive. As to homologue of hDC-SIGN, five genes were identified by database analysis, and their amino acid sequence similarities of lectin domain between hDC-SIGN were more than 65%. Analysis of high throughput genomic sequences (HTGS) showed that one of the genes, named mDC-SIGN, was located adjacent to CD23. The other 4 genes (mDC-SIGNR1-4) were found to form a gene cluster in mouse chromosome 8A1.2-1.3, syntenic to hDC-SIGN and CD23 loci (19p13). mDC-SIGN transcript was largely detected in CD11c+ spleen DCs and BM-DCs, especially in immature cells. As mDC-SIGN, mDC-SIGNRs were also expressed in immature DCs, but some of them were also in other cell types. When expression patterns of mLangerin and mDC-SIGN/mDC-SIGNs were examined, both of them decreased upon activation of DCs. Moreover, we found that their expressions were different in DC subsets defined by CD8 or CD11b expression. Collectively, these results indicate that mLangerin and mDC-SIGN/mDC-SIGNRs are possibly a useful marker to distinguish DC subsets in relation to their functions.




Structural Diversity of Oligosaccharides that Provides Functional Diversity

Minoru Fukuda


Glycobiology Program, Cancer Research Center, The Burnham Institute, La Jolla, CA 92037

   Our laboratory has been interested in identifying oligosaccharide structures that are expressed in a cell-specific manner and elucidating the roles of these oligosaccharides. We have been particularly interested in sialyl Lewis x and related oligosaccharides, polysialic acid, and HNK-1 glycan.

   Sialyl Lewis x was originally discovered by us as human neutrophil-specific oligosaccharides (1). Following our discovery, E- and P-selectin, which bind to human neutrophils, were identified as carbohydrate-binding proteins. By using cDNA encoding a1,3-fucosyltransferase, Lowe and his colleagues elegantly demonstrated that E- and P-selectin bind to sialyl Lewis x expressed in transfected cells (2). In neutrophils, these sialyl Lewis x capping structures are present on core2 branched O-glycans. Indeed, knockout of core2 b1,6-N-acetylglucosaminyltransferase results in the loss of E-, P-, and L-selectin ligands on leukocytes and mutant mice exhibit a severely impaired response to inflammation (3). On the other hand, L-selectin on lymphocytes contribute to lymphocyte homing by recognizing L-selectin ligands expressed on high endothelial venule (HEV) cells.
  Our recent studies identified a sulfotransferase (LSST) that forms 6-sulfo sialyl Lewis x on core2 branched O-glycans, which functions as an L-selectin ligand. The expression of mouse LSST is highly restricted to HEV and, moreover, is upregulated in HEV-like structures formed in hyperplastic thymus of AKR mice (4). In core2 GlcNAc-T knockout mice, however, lymphocyte homing was barely affected and the expression of MECA-79 antigen, which specifically decorates HEV, persisted (3). Our most recent studies demonstrate that a novel 6-sulfo extended core1 structure is present in HEV of core2 GlcNAcT knockout mice and this oligosaccharide functions as an L-selectin ligand as demonstrated by expressing the newly cloned core1 extension enzyme, core1-b3GlcNAcT. Moreover, we found that the same oligosaccharide is the minimum epitope for MECA-79 (5).
  Sialyl Lewis x is also enriched in carcinomas and leukemias. To determine the roles of sialyl Lewis x in tumor metastasis, mouse B16 and human MeWo melanoma cells were transfected witha1,3-fucosyltransferase III. As expected, the expression of sialyl Lewis x leads to significantly increased formation of tumor foci. Surprisingly though, those transfected cells expressing the highest amount of sialyl Lewis x did not produce lung tumor foci. Using various immunodeficient mice, we discovered that sialyl Lewis x overexpressing cells produce large numbers of tumor foci in beige mice, which have defective natural killer cell function (6). Most recent studies demonstrate that the extent of NK cell-mediated cytolysis in vitro is in proportion to the amount of sialyl Lewis x expressed on the tumor cells, and NK cell-mediated cytolysis can be inhibited by prior treatment of tumor cells with anti-sialyl Lewis x antibody or sialyl Lewis x mimicking peptide (7).

   These results taken together indicate that the diversity of oligosaccharide structure and the amount of those oligosaccharides provide for the enormous diversity in functionality of cell-type specific oligosaccharides. (supported by CA33000, CA33895, CA48737, and CA71932)


(1)    Fukuda M et al., J Biol Chem 259:10925-10935, 1984.  (2) Lowe JB et al., Cell 63:475-484, 1990.  (3) Ellies LG et al., Immunity 9:881-890, 1998.  (4) Hiraoka N et al., Immunity 11:79-89, 1999.  (5) Yeh et al., Cell, submitted.  (6) Ohyama C et al., EMBO J 18:1516-1525, 1999.  (7) Fukuda MN et al., Cancer Res 60:450-456, 2000.



Homeostatic Chemokines Functionally Organize Lymphoid Organs

Reinhold Forster

 University Clinic for Surgery, Section of Experimental Surgery and Immunology; University of Erlangen, Germany. Email: rfoerste@molmed.uni-erlangen.de


Following antigen-independent maturation in primary lymphoid organs B and T cells are released to the blood stream and continuously re-circulate through almost all compartments of the body in order to bring the entire range of antigen-specific immune cells into close contact with invading pathogens. For this purpose lymphocytes extravasate from blood vessels, migrate to lymphoid and non-lymphoid tissue and back into the blood stream. Several lines of evidence suggests that chemokines and their receptors are essentially involved in lymphocyte trafficking.

Chemokines represent a family of small basic chemotactic proteins which mediate their effect by binding to seven transmembrane-spanning G protein coupled receptors expressed on target cells. Chemokines have been identified originally by their properties to direct extravasation of inflammatory cells. However recent data identified several chemokines which are expressed constitutively in lymphoid tissues, indicating that these chemokines might have homeostatic functions by regulating lymphocyte trafficking to or within lymphoid organs. Based on two conserved cystein residues, chemokines can be allocated into four sub-families, named CC, CXC, CX3C and XC chemokines. The residues are adjacent in CC chemokines, while they are separated by one ore three amino acid in CXC and CX3C, respectively.
By generating mice deficient for the chemokine receptor Burkitt's lymphoma receptor 1 (BLR1, CXCR5), we could provide essential evidence that the chemokine system is indeed involved in homeostatic lymphocyte trafficking to secondary lymphoid organs. CXCR5-deficient mice possess none or only few aberrantly developed PP and show severe impairment of B cell migration to B cell follicles of spleen, lymph node and PP. In addition, we could recently demonstrate that the chemokine receptor CCR7 is indispensable for the migration of naive T cells through the HEV. In addition B cells exploit CCR7 to efficiently enter lymph nodes and Peyer’s patches. Once B cells are within lymphoid organs, they use CXCR5 to follow a follicular chemokine gradient of B lymphocyte chemoattractant (BLC), which guides them to the B cell-rich follicles. On T cells CCR7 is highly expressed on the majority of naive T cells and on a subpopulation of memory T cells, now known as “central memory T cells”.
Since the ligands for CCR7, ELC/CCL19 and SLC/CCL21 are both expressed on HEV, it was likely that the interaction of CCR7 with its’ ligands is required for effective trans-endothelial migration of CCR7-positive lymphocytes into secondary lymphoid organs. To test the function of CCR7, we generated mice in which the CCR7-locus has been disrupted by gene targeting. LN of CCR7-deficient mice are devoid of naive T cells and DC, whereas the T cell population is heavily expanded in the blood, the red pulp of the spleen, and in the bone marrow. CCR7-deficient mice show severely delayed kinetics regarding the antibody response and lack contact sensitivity and delayed type hypersensitivity reactions. Adoptive transfer experiments to wild-type recipients demonstrated that the migration of CCR7-deficient B cells and T cells into LN and PP, and of T cells into the splenic periarteriolar lymphoid sheath (PALS) was severely hampered. When compared to wild-type B cells, CCR7-deficient B cells rapidly left the outer PALS after being transferred into wild-type recipients, indicating that expression of CCR7 keeps B cells for a defined period of time in close contact with T cells to allow effective T cell - B cell interactions.

We have now revealed the role of CCR7 during the process of activation and mobilization of dendritic cells (DC). We show that CCR7 is required at least at three different steps of the migratory process of DC. i) Langerhans cells need this receptor to migrate within the dermis towards the lymphatic vessels. ii) once these cells have been arrived within the marginal sinus of the draining lymph nodes they exploit CCR7 to make their way into the T cell-rich areas. iii) finally, both lymphoid- and myeloid-derived DC require the interaction of CCR7 with it’s ligands to stay within their characteristic microenvironments within lymphoid organs. Within lymphoid organs CCR7-deficient DCs are dislocated to distinct areas where other homeostatic chemokines are highly expressed. Thus, our data also suggest that a hierarchy of chemotactic gradients within lymphoid organs controls the dynamic reorganization of lymphoid organs during an adoptive immune response.





T Lymphocyte Activation, Differentiation, and Migration

Antonio Lanzavecchia

Institute for Research in Biomedicine, Bellinzona, Switzerland


The relationship between T lymphocyte activation, differentiation, and migration has been studied in mouse and human systems. By stimulating mouse naive T lymphocytes under defined in vitro conditions we found that T cells that receive a short TCR stimulation in the absence of polarizing cytokines proliferate and give rise to cells that lack immediate effector function (non-polarized) and that retain the constitutive expression of the lymph node homing chemokine receptor CCR7. When transferred to syngeneic mice these cells home to the T cell areas of lymph nodes and spleen and, in response to a secondary antigenic stimulation, proliferate and differentiate to effector cells, even in the absence of adjuvants. In contrast mouse T cells that receive a prolonged TCR stimulation in the presence of IL-12 or IL-4 proliferate and differentiate to Th1 or Th2 effector cells and lose CCR7 expression, while acquiring the capacity to home to inflamed peripheral tissues. We have previously shown that in human peripheral blood CD4+ and CD8+ memory cells comprise two distinct populations: i) non-polarized, CCR7+ “central memory” cells and ii) polarized (Th1 or Th2), CCR7- “effector memory” cells. Memory T cells specific for recall antigens are present in both subsets several years after priming. Using TCR heteroduplex analysis on antigen-specific CD4+ and CD8+ T cells isolated from the central memory and effector memory subsets we now show that the same clonotype can be found in different memory T cell pools. Furthermore, using TSST as a model antigen and dendritic cells as APC we could define conditions that favour the generation of non-polarized versus polarized T cells. Altogether these studies reveal that in the T cell differentiation program effector function and migratory capacities are coordinately regulated. I will discuss a model of progressive T cell differentiation and the regulation of chemokine receptors on naive, activated and memory T cells.




Gut Cryptopaches: Intestinal Development and Function of Intraepithelial T Cells

Hiromichi Ishikawa

Department of Microbiology and Immunology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan

   The alimentary tract is an essential structure for the ingesting of nutrients from the outside, and even most primitive animals have a straight alimentary tract that runs from the mouth to the anus.    Simultaneously, the fact that 60-70% of peripheral lymphocytes are congregating in the gastrointestinal tract supports the notion that the enteric mucous membrane is the locale exposed to the greatest danger.   It is here that numerous food-derived external antigens including allergenic and toxic substances and literally infinite amounts of normal intestinal flora intermingled from time to time with life-threatening microbes continually enter the body.   Surprisingly, approximately one out of the five cells in the intestinal epithelium are lymphocytes, most of which are yet unacknowledged T cells (IEL) having unusual but distinctive characteristics and situated apparently so close to external antigens in the entire body.  In the past two decades, extrathymic (thymus-independent; TI) development of mouse IEL expressing CD8aa homodimer has been established.  Our recent studies have corroborated that lympho-hemopoietic progenitors residing in the newly identified mouse gut-associated lymhoid tissues “cryptopatches (CP)” generate TI-IEL.  Thus, the developmental pathway of TI-IEL in the mouse gut mucosa is BM-derived IEL progenitor cells _ c-kithighTCR- CP cells __TCR- IEL _ TCR+ IEL.  Furthermore, a comparative study of TCR- IEL from athymic common cytokine receptorg-chain-deficient mice not possessing CP and those from athymic SCID mice possessing CP illuminated the key role of gut CP in the early maturation of CD8aa+ IEL precursors, including cell surface expression ofaEb7 integrin, CD3e gene transcription and TCR gene rearrangements.  This paper deals with the information that has recently been accumulated concerning gut CP as well as the possible function of TI-IEL in the anatomical front of the intestine.



Roles Of Platelet-Associated Adhesion Molecules In Regulation Of Leukocyte Adhesion To Microvascular Endothelium

Makoto Suematsu, Makoto Handa, Yasuo Ikeda

Department of Biochemistry and Integrative Medical Biology, and Department of Internal Medicine, School of Medicine, Keio University, Tokyo 160-8582, Japan


Multistep mechanisms for leukocyte adhesion in vivo have been established by observation through an intravital microscope.  However, roles of circulating platelets in regulation of leukocyte-endothelial cell interactions have not fully been investigated because of several technical difficulties to visualize their behavior in vivo.  To overcome these difficulties, we have established a novel method to stain platelets with the fluorophore and to visualize their individual traffics with a resolution sufficient enough to determine site-specific velocity and density in a single vessel of organ microcirculatory system.  High-speed intensified video microscopy allowed us to visualize fast-moving platelets at videorates of 300-1000 frames/sec, and normospeed replay of the video images at 30 frames/sec demonstrated that behavior of individual platelets in microvessels differ greatly between the cells flowing at centerline region and those flowing in the periendothelial space.  Our results demonstrate that, under high shear rates greater than 600 sec-1, platelets tend to form microaggregates and to flow along the periendothelial space, exhibiting transient skipping and rolling on the endothelium.  They utilize GPIb-alpha on their surface for these shear-dependent interactions with the surface of the endothelium through its affinity to vWF.  GPIb-alpha-mediated platelet adhesion appeared to help P-selectin-mediated leukocyte adhesion to microvascular endothelium at least in part, since its immunoneutralization caused a significant suppression of the stimulus-elicited microvascular adhesion of leukocytes.  Although the roles of shear-dependent redistribution of platelets in regulation of the tissue delivery of inflammatory cells are largely unknown at present, these results suggest a possible role of platelets as a determinant of inflammatory impacts.


B Cell Trafficking and Autoimmunity

Sho Ishikawa

Dept. of Molecular Preventive Medicine, School of Medicine, The University of Tokyo


We found that the expression of B lymphocyte chemokine (BLC/CXCL13) was markedly enhanced in the thymus and kidney in aged BWF1 mice developing lupus nephritis, but not in similarly aged NZB and NZW mice. BLC positive cells were present in the cellular infiltrates in the target organs with a reticular pattern of staining. CD11b+CD11c+ dendritic cells were increased in the thymus and spleen in aged BWF1 mice and identified as the major cell source for BLC. CD4+ T cells as well as B cells were dramatically increased in the thymus in aged BWF1 mice while no increase was observed in aged NZB and NZW mice. B1/B2 ratio in the thymus was significantly higher than those in the spleen and peripheral blood in aged BWF1 mice. Interestingly, BLC showed preferential chemotactic activity for B1 cells derived from several mouse strains including non-autoimmune mice. Cell surface CXCR5 expression on B1 cells was significantly higher than that on B2 cells. Thus, aberrant high expression of BLC by myeloid DCs in the target organs in aged BWF1 mice may play a pivotal role in breaking immune tolerance in the thymus and in recruiting autoantibody-producing B cells in the development of murine lupus.by macrophages prepared from the peritoneal cavities of thioglycollate-treated mice in a manner inhibitable by PS-containing liposomes. The efficacy of phagocytosis significantly decreased when cells were infected with virus having inactive neuraminidase, that exists on the surface of virus-infected cells. Moreover, virus release into the culture medium ceased upon the addition of macrophages.  The above results collectively suggested that PS- and/or neuraminidase-mediated phagocytosis of influenza virus-infected cells by macrophages contributes to virus exclusion from the organism.



The Role of Abc1 and Membrane Lipid Architecture in Macrophage Competence to Engulf.

Olivier Chambenoit, Yannick Hamon, Didier Marguet, Giovanna Chimini.

Centre d’Immunologie INSERM/CNRS de Marseille Luminy

13288 Marseille Cedex 09 France.


Engulfment is the specialized form of phagocytosis dedicated to the safe and swift clearance of corpses generated by the apoptotic program.

It plays a crucial role in preserving homeostasis during development and in physiological and pathological situations. Exclusive features of this specialized phagocytic event can  be pinpointed  at various levels. Teleologically safety is its key word. Indeed, as opposed to Fc receptor-mediated phagocytosis which triggers immune responses, engulfment both passively prevents and actively down regulates inflammatory reactions. Engulfment, similarly to Fc receptor mediated phagocytosis is usually the task of professional phagocytes but , if required, amateurs can act as surrogate body snatchers. From the pharmacological standpoint , engulfment is uniquely sensitve to drugs affecting phospholipid mobility across the bilayer. Finally a complex array of membrane receptors are implicated in the initiation of engulfment again as opposed to the unicity of surface molecules triggering anti-invader phagocytic events. These mammalian receptors are actually familiar and possess functional records anterior and unrelated to their association with engulfment.