Cellular Immunology 271 (2011) 335–341

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Cellular Immunology

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Development of peritoneal macrophage along a dendritic cell lineage in responseto uptake of oligomannose-coated liposomes

Naoya Kojima ⇑, Chiaki Kato, Megumi Igarashi, Mariko IshiiDepartment of Applied Biochemistry, Tokai University, Hiratsuka, Kanagawa 259-1292, Japan

a r t i c l e i n f o

Article history:Received 18 March 2011Accepted 27 July 2011Available online 10 August 2011

Keywords:Dendritic cellsLiposomeOligomannosePeritoneal macrophages

0008-8749/$ - see front matter � 2011 Elsevier Inc. Adoi:10.1016/j.cellimm.2011.07.013

⇑ Corresponding author. Address: 4-1-1 Kita-kanam259-1292, Japan. Fax: +81 463 50 2012.

E-mail address: naoyaki@keyaki.cc.u-tokai.ac.jp (N

a b s t r a c t

In this study, we investigate the potential of peritoneal macrophages to differentiate into dendritic cell(DCs) in response to preferential uptake of oligomannose-coated liposomes (OMLs). About 30% of perito-neal cells (PECs) preferentially took up OMLs that were administered into the peritoneal cavity. TheOML-ingesting cells expressed CD11b and F4/80, but lacked CD11c expression, indicating that theOML-ingesting PECs with a CD11bhighCD11c� phenotype are resident peritoneal macrophages. Duringin vitro cultivation, CD11c+ cells arose among the PECs with ingested OMLs. CD11c+ cells also developedamong enriched peritoneal CD11bhighCD11� cells from OML-treated mice, and the resulting CD11c+ cellsexpressed co-stimulatory molecules and MHC class II. In addition, OML-ingesting CD11bhighCD11c+ cellswere found in spleen after the enriched peritoneal macrophages with ingested OMLs were transplantedin the peritoneal cavity of mice. These results show that a fraction of peritoneal macrophages can differ-entiate into mature DCs following uptake of OMLs.

� 2011 Elsevier Inc. All rights reserved.

1. Introduction

Dendritic cells (DC) are potent professional antigen-presentingcells (APCs) that capture antigens and migrate to T cell regions ofdraining lymph nodes, where they mature into functional DCs toinitiate primary immune responses [1–4]. Therefore, strategiesthat target DCs and modulate DC function in vivo may have signif-icant implications for vaccine design [5,6].

We have demonstrated that liposomes coated with a neoglycol-ipid constructed from mannotriose and dipalmitoylphosphatidy-lethanolamine (Man3-DPPE) (oligomannose-coated liposomes,OMLs) induce a strong T helper 1 (Th1) immune response againstencapsulated antigens in mice, with significant IFN-c productionand suppression of IL-4 production following preferential uptakeof OMLs by peritoneal CD11bhigh cells [7,8]. In addition, intraperi-toneal or subcutaneous administration of OMLs protects againstsubsequent protozoan infections and tumor inoculation when spe-cific antigens are entrapped within the OMLs [9–12]. We alsoshowed that OML uptake leads to upregulation of expression ofcostimulatory molecules and MHC class II molecules on the perito-neal CD11bhigh cells, with enhanced production of IL-12 [7], whichis essential for induction of a Th1 immune response [13]. We alsofound that the peritoneal CD11bhigh cells effectively present en-cased antigen-derived peptides via MHC class I and II molecules

ll rights reserved.

e, Hiratsuka-shi, Kanagawa

. Kojima).

and activate both CD4+ and CD8+ T cells in response to OML uptake[8]. These results indicate that OMLs can be used both as a specificantigen delivery vehicle to APCs and as an APC-activating adjuvantto induce strong cellular immunity. The peritoneal CD11bhigh cellsalso express high levels of F4/80, indicating that these cells can beclassified as resting resident peritoneal macrophages, and thesecells are the most potent target APCs in the peritoneal cavity forinduction of OML-dependent immune responses [8]. On the otherhand, it is well accepted that DCs, and not macrophages, are themost potent APCs that capture antigens at peripheral tissues,migrate to T cell regions of draining lymph nodes, and present anti-gens to initiate immune responses [1–4]. Therefore, to understandwhy OMLs induce specific immune responses after peritonealadministration requires clarification of how resting resident peri-toneal macrophages can act as professional APCs after uptake ofOMLs.

Cells of a monocyte/macrophage lineage can be directed to de-velop into potent immunostimulatory DCs in human when cul-tured in the presence of GM-CSF and IL-4 [14,15]. Randolph et al.showed that human and mouse monocytes differentiate into DCson uptake of zymosan or microspheres with reverse transendothe-lial migration [16,17], and Rezzani et al. found that resting residentperitoneal macrophages obtained from peritoneal lavage can dif-ferentiate into DCs upon treatment with GM-CSF [18,19]. A perito-neal pool of CD14+ mononuclear cells in humans can alsodifferentiate into DCs or macrophages in vitro under appropriateconditions [20]. These findings prompted us to investigate the po-tential of resident peritoneal macrophages to differentiate into a

336 N. Kojima et al. / Cellular Immunology 271 (2011) 335–341

DC lineage in response to OML uptake. In the present study, weshow that peritoneal cells that belong to a monocyte/macrophagelineage become mature DC-like cells in response to OML uptake.

2. Materials and methods

2.1. Antibodies and reagents

Fluoroscein isothiocyanate (FTIC)- or phycoerythrin (PE)-la-beled antibodies directed against mouse CD3e, CD14, CD19,CD40, CD86, CCR7, F4/80, B220, and I-A/I-E class II molecules werepurchased from eBioscience (Boston, MA, USA). A peridinin chloro-phyll protein-cyanine 5.5 (PerCP-Cy5.5)-labeled anti-CD11b mono-clonal antibody (mAb), an allophycocyanin (APC)-labeled CD11cmAb, and Fc-block (anti-mouse CD16/32) were purchased fromBD PharMingen (San Diego, CA, USA). Isotype control antibodieswere purchased from R&D Systems (Minneapolis, MN, USA).

2.2. Preparation of oligomannose-coated liposomes (OMLs)

Neoglycolipids constructed from mannotriose (Man3; Mana1-6(Mana1-3)Man) and dipalmitoylphosphatidylethanolamine(DPPE) were synthesized and purified in our laboratory and coatedliposomes were prepared, as described previously [9,10]. Briefly, a

Fig. 1. Phenotypic analysis and CD11c expression of PECs with ingested OMLs. OMLs orPECs were recovered 1 h after injection and stained with PerCP-Cy5.5-labeled anti-CD11band MHC class II. The expression of these molecules on PECs was analyzed by flow cyhematopoietic cell lineage markers in the cells with ingested OMLs. Peritoneal cells withsquare in panel A), and expression of other molecules in the gated cells was analyzed. Graantibodies, respectively. (C) Expression of CD11b and CD11c in the gated cells obtainedThe data are representative of those obtained in three separate experiments.

chloroform–methanol (2:1, v/v) solution containing 1.5 lmol ofDPPC, 1.5 lmol of cholesterol, and 0.15 lmol of neoglycolipid wereadded to a flask and evaporated to prepare a lipid film containingneoglycolipid. PBS (200 ll), 5 mg/ml of FITC-BSA containing PBS,or 5 mg/ml of ovalbumin (OVA) containing PBS was added to thedried lipid film and multilamellar vesicles were prepared by in-tense vortex dispersion. The vesicles were extruded 10 timesthrough a 1-lm pore polycarbonate membrane (Nucleopore, Pleas-anton, CA, USA). The amount of entrapped protein was measuredusing a modified Lowry protein assay kit (Pierce, Rockford, IL) inthe presence of 0.3% (w/v) sodium dodecyl sulfate, using BSA asthe standard. Molar ratios of the lipid components of the liposomeswere determined using HPLC. Particle sizes of liposomes weredetermined by dynamic light scattering using a particle size ana-lyzer (LB-550, Horiba, Kyoto, Japan).

2.3. Preparation and culture of peritoneal cells (PECs)

Six- to 10-week-old female C57BL/6 mice were purchased fromShizuoka Laboratory Animal Corporation (Hamamatsu, Japan). Allanimal experiments were conducted in compliance with the ethi-cal requirements of the Animal Committee at Tokai University. APBS suspension (200 ll) of OMLs or uncoated liposomes (60 lg ofcholesterol) with or without encased FITC-labeled bovine serum

uncoated liposomes with encased FITC-BSA were injected into the peritoneal cavity.mAb and PE- or APC -labeled mAbs against mouse CD11c, CD14, CD86, CCR7, F4/80tometry. (A) Incorporation of liposomes in the peritoneal cells. (B) Expression of

incorporated liposomes were gated based on the FITC fluorescence (indicated in they solid peaks and open peaks indicate cells treated with isotype control and specific

from OML-treated mice was analyzed before and after in vitro cultivation for 1 day.

N. Kojima et al. / Cellular Immunology 271 (2011) 335–341 337

albumin (BSA) was injected into the peritoneal cavity of mice. Onehour after injection, peritoneal cells (PECs) were harvested by la-vage using 5 ml of ice-cold PBS and washed twice with PBS. ThePECs were plated at a density of 2 � 106/ml on a 12-well non-adherent culture plate (HydroCell�, CellSeed Inc., Tokyo, Japan)and cultured in RPMI1640 medium supplemented with 10% FCS,100 U/ml penicillin, 100 lg/ml streptomycin, and 5 mM 2-mercap-toethanol for the indicated time (1–3 days). After incubation, PECswere stained with a mixture of PerCP-Cy5.5-labeled anti-CD11b,APC-labeled anti-CD11c and PE-labeled anti-CD40, CD86, CCR7,or I-A/I-E mAbs after Fc block treatment, and analyzed by flowcytometry.

2.4. Enrichment of resident peritoneal macrophages

PECs obtained from the peritoneal cavity of OML-administratedmice were seeded on a 3-cm Petri dish. The cells were incubated inRPMI1640 medium supplemented with 10% FCS, 100 U/ml penicil-lin, 100 lg/ml streptomycin, and 5 mM 2-mercaptoethanol at37 �C for 2 h. After non-adherent cells were removed by extensivewashing with pre-warmed culture medium, adherent cells wereharvested by pipetting. The recovered cells were cultured on a12-well non-adherent culture plate for 1 day and stained withanti-CD11b, CD11c, CD19, or F4/80 after Fc block treatment, andanalyzed by FACS. To analyze migration of peritoneal cells, the en-

Fig. 2. Expression of CD11c in a peritoneal cell population that expressed high levels of Ccholesterol) were injected into the peritoneal cavity. PECs were recovered 1 h after injstained with PerCP-Cy5.5-labeled anti-CD11b mAb and APC-labeled anti-CD11c mAb, an

riched cells (2 � 106) with ingested FITC-BSA containing were theninjected into the peritoneal cavity of a mouse, and the single cellsuspensions of spleen and omentum was prepared 1 day afterthe injection. To investigate the initiation of immune responsesby peritoneal cells, PECs obtained from mice administrated withOVA-containing OMLs, OVA-containing uncoated liposomes, andOVA (each 10 lg of protein) were seeded on 3-cm Petri dishes,respectively, and cultured for 24 h. The adherent cells were recov-ered and the enriched cells (1 � 106) were injected into the perito-neal cavity of a mouse. The single cell suspension of spleen wasprepared 7 days after the injection, and the cytokines secretedfrom the cells were measured.

3. Results

3.1. An increase in CD11c+ cells among OML-containing PECs afterin vitro cultivation

We examined whether mouse peritoneal cells (PECs) can differ-entiate into a DC lineage in response to uptake of OMLs. OMLs withentrapped FITC-BSA (FITC-OMLs) or uncoated liposomes were in-jected into the peritoneal cavity of mice, PECs were recovered 1 hafter the injection, and liposome uptake was evaluated based onthe fluorescence intensity of FITC-BSA encased in OMLs. As shownin Fig. 1A, approximately 30% of PECs from OML-treated mice had

D11b during in vitro cultivation of total PECs. OMLs or uncoated liposomes (60 lg ofection and then cultured for 1 day on non-adherent culture dishes. The cells wered expression of CD11c in the gated cells was analyzed.

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bright fluorescent signals from OML-encased FITC-BSA, indicatingthat these cells had ingested OMLs in vivo. In contrast, less than1% of PECs took up uncoated liposomes. Analysis of the expressionof cell markers in the FITC-BSAbright population (Fig. 1B) showedthat the majority (>90%) of these cells expressed high levels ofCD11b and F4/80, which are markers for macrophages; andCD14, a marker for monocytes. In contrast, the cells in the FITC-BSAbright population lacked expression of CD11c, a pan-markerfor DCs; CCR7, an indicator of cell maturity in murine DCs [21];and the co-stimulatory molecule CD86. In addition, only 14% ofFITC-BSAbright cells expressed MHC class II molecules. These resultsindicate that the PECs that preferentially ingested OMLs belong toa monocyte/macrophage lineage and are commonly referred to asthe resident peritoneal macrophages.

Expression of CD11c in the OML-containing PECs was thendetermined during in vitro cultivation of total PECs to evaluate dif-ferentiation of resident peritoneal macrophages into DCs. Most(>97%) of the cells with ingested OMLs had a CD11bhighCD11c�

phenotype immediately after recovery from the peritoneal cavity(day 0). However, when the PECs were cultured for 24 h in vitro,the number of CD11c+ cells increased significantly among the pop-ulation of PECs with ingested OMLs (day 1) and approximately 14%of OML-containing cells had a CD11bhighCD11c+ phenotype(Fig. 1C).

3.2. Resident peritoneal macrophages can differentiate into DCs andmature in response to OML uptake during in vitro cultivation

To confirm whether OMLs can promote differentiation of resi-dent peritoneal macrophages into a DC lineage, expression of

Fig. 3. Differentiation and maturation of resident peritoneal macrophages with ingestedcavity of mice and PECs were recovered 1 h after injection and the resident peritoneal mafor 1 day on a non-adherent culture dish and then stained with PerCP-Cy5.5-labeled anmouse CD40, CD86, CCR7, MHC class II, and F4/80. (A) Expression of CD11c in the enrichthose obtained in four similar experiments. (B) Expression of co-stimulatory molecules awere gated (R1 in panel A) and expression of other molecules in the R1 population was

CD11c was examined in the CD11bhigh population among PECs cul-tured for 1 day after recovered from mice that received OMLs oruncoated liposomes intraperitoneally. As shown in Fig. 2, approxi-mately 12% of cells in the CD11bhigh population became CD11c+

cells, while less than 1% of cells from mice treated with uncoatedliposomes became CD11c+ cells.

Next, adherent cells were enriched from PECs obtained fromOML-treated mice as the resident peritoneal macrophages. Over80% of recovered adherent cells had a CD11bhighCD11c� phenotypeand there were no CD11c+ cells in this enriched fraction (Fig. 3A,left panel). In addition, the enriched cells expressed high levels ofboth F4/80 and CD14 (data not shown), indicating that most ofthe enriched cells were resident peritoneal macrophages. Whencells in the enriched fraction were cultured for 1 day in vitro, a sig-nificant number of CD11bhighCD11c+ cells arose, as in the culture oftotal PECs (Fig. 3A). The resident peritoneal macrophage-derivedCD11c+ cells obtained from OML-treated mice (gated as R1 inFig. 3A) strongly expressed CD86, CD40, CCR7, and MHC class II,but the level of F4/80 expression on the cells decreased (Fig. 3B).These results indicate that resident peritoneal macrophages be-came mature DC-like cells in response to OML uptake during1 day of in vitro cultivation.

3.3. Resident peritoneal macrophages functionally differentiate into DCin vivo

We next examined whether the resident peritoneal macro-phages that had taken up OMLs could functionally differentiateinto DC in vivo. We have shown previously that peritoneal macro-phages accumulate in extranodal lymphoid tissues of the omen-

OMLs into DCs during in vitro cultivation OMLs were injected into the peritonealcrophages were enriched as described in Section 2. The enriched cells were culturedti-CD11b mAb, APC-labeled anti-CD11c mAb and PE- or FITC-labeled mAbs againsted cells before and after in vitro cultivation for 1 day. The data are representative ofnd the MHC class II molecule in CD11c+ cells. The CD11c+ cells in the enriched PECsanalyzed.

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tum within 1 day after OMLs are administered into the peritonealcavity [22,23]. Therefore, FITC-OMLs were injected into the perito-neal cavity and the cells in the omentum were analyzed 1 day afterthe injection. Cells with bright fluorescent signals from FITC-BSAencapsulated in OMLs appeared in the omentum (Fig. 4A, left pa-nel), suggesting that these cells are resident peritoneal macro-phage with ingested OMLs. Over 20% of the FITC-BSAbright cellsfound in the omentum expressed CD11c and high levels ofCD11b (Fig. 4A, right panel), as in the case of in vitro cultivationfor 1 day. This suggests that OMLs can promote development ofDCs from peritoneal macrophages in vivo.

The antigen-loaded DCs must move from peripheral tissues tolymphoid tissues to activate naïve T cells. Furthermore, expressionof CCR7, which is required for this trafficking [24,25], was upregu-lated in the resident peritoneal macrophage-derived CD11c+ DC-like cells in vitro (Fig. 3C). Therefore, we next investigated the traf-ficking of OML-ingested PECs into the spleen. FITC-OMLs were in-jected into the peritoneal cavity and CD11bhighCD11c� cells wereenriched from PECs obtained from the mice. The enriched perito-

Fig. 4. Differentiation of resident peritoneal macrophages into DC-like cells in responseperitoneal cavity. Eighteen hours after administration, single cell suspensions were prepaAPC-labeled anti-CD11c mAb. The cells with bright fluorescent signals from FITC wereresident peritoneal macrophages with ingested FITC-BSA-containing OMLs were injecte18 h after administration. The cells with bright fluorescent signals from FITC were gated

neal CD11bhighCD11c� cells with ingested FITC-OMLs were then in-jected into the peritoneal cavity of a mouse, and spleen cells wereprepared 18 h after the injection. Approximately 0.01% of spleencells had bright fluorescent signals from FITC-BSA (gated inFig. 4B, left panel), suggesting that these cells were derived fromthe enriched peritoneal CD11bhighCD11c� cells administered intothe peritoneal cavity. Most of the cells in the spleen that came fromthe peritoneal cavity expressed high levels of CD11b, and approx-imately 40% of the cells in the spleen expressed significant levels ofCD11c (Fig. 4B, right panel).

We then examined if the peritoneal macrophage-derived DCscould initiate immune responses. The CD11bhighCD11c� cells wereenriched from PECs obtained from mice treated with OVA-contain-ing OMLs (OVA-OML), OVA-containing uncoated liposomes (OVA-BL), and OVA alone, respectively, and the resulting enrichedCD11bhighCD11c� cells were injected into the peritoneal cavity ofa mouse. The single cell suspensions of spleen were prepared7 days after the injection, and the cytokine production from thesplenocytes was analyzed. As shown in Fig. 5, the splenocytes ob-

to OML uptake in vivo. (A) FITC-BSA-containing OMLs were administered into thered from the omentum, and stained with PerCP-Cy5.5-labeled anti-CD11b mAb and

gated (left) and the expression of CD11b and CD11c were analyzed (right). (B) Thed into peritoneal cavity and single cell suspensions were prepared from the spleen

(left) and the expression of CD11b and CD11c were analyzed (right).

Fig. 5. Initiation of immune responses by enriched peritoneal macrophages. Theresident peritoneal macrophages were enriched from mice treated with OVA-containing OMLs (OVA-OML), OVA-containing uncoated liposomes (OVA-BL) orOVA, and transplanted into the mouse peritoneal cavity. Seven days after thetransplantation, single cell suspension of spleen was prepared from individual mice,and cultured with (black bars) or without (gray bars) 10 lg/ml of OVA (gray bars)for 48 h. IFN-c in each culture supernatants was then determined by ELISA.

340 N. Kojima et al. / Cellular Immunology 271 (2011) 335–341

tained from the mouse transplanted with OVA-OML-containingCD11bhighCD11c� cells produced a significant amount of IFN-c inresponse to in vitro stimulation with OVA, while those from themice injected with OVA-BL- or OVA-treated CD11bhighCD11c� cellsdid not secreted the cytokine. On the other hand, production ofantigen-specific IL-4 and IL-5 was not observed (data not shown).These results indicate that resident peritoneal macrophages thatingested OMLs can differentiate into CD11c+ DC-like cells in vivoand migrate to secondary lymphoid tissues to activate naïve T cells,when OMLs are administrated into a peritoneal cavity of mouse.

4. Discussion

Resting resident peritoneal macrophages can be directed to de-velop into potent immunostimulatory DCs in the presence of GM-CSF alone or in combination with TNF-a or IL-4 in mice [18,19].However, it is unclear whether resident peritoneal macrophagescan differentiate into DCs under physiological conditions. We havedemonstrated that OMLs are preferentially and rapidly taken up byresident peritoneal macrophages, and that OMLs can induce anti-gen-specific Th1 immune responses and cytotoxic T lymphocytesafter administration of antigen-containing OMLs into the perito-neal cavity [7,8]. Therefore, it is possible that resting peritonealmacrophages differentiate into mature DCs in response to OML up-take, leading to initiation of specific immune responses.

In the present study, we showed for the first time that in vivouptake of OMLs by PECs of a monocyte/macrophage lineage pro-motes differentiation of the cells along a DC lineage without exog-enous cytokines. OMLs administered into the peritoneal cavity, butnot uncoated liposomes, were preferentially and rapidly ingestedby CD11bhighCD11c� peritoneal cells, which are classified as resi-dent peritoneal macrophages. A significant number of CD11bhigh

CD11c+ cells arose in the CD11bhigh population if all PECs obtainedfrom OML-treated mice were cultured in vitro for 1 day, although anegligible number of CD11c+ cells was found in the CD11bhigh pop-ulation before in vitro cultivation. The in vitro conversion of the res-ident peritoneal macrophages into DC-like cells by OML uptakewas confirmed by the appearance of CD11bhighCD11c+ cells fromCD11bhighCD11c� cells, which were enriched from PECs of OML-treated mice. The CD11bhighCD11c+ cells derived from theCD11bhighCD11c� cells expressed significant levels of co-stimula-tory molecules CD86 and CD40, a chemokine receptor CCR7 thatis required for migration of APCs from peripheral tissues to second-ary lymphoid tissues, and MHC class II molecules. Therefore, OML-induced peritoneal macrophage-derived DC-like cells displayedcharacteristics of mature APCs during in vitro cultivation. Further-more, CD11bhighCD11c+ cells containing OMLs were found in

spleen if OML-containing peritoneal CD11bhighCD11c� cells wereadministered into the peritoneal cavity of mice. This indicates thatPECs with a monocyte/macrophage lineage differentiate into DCsin vivo after uptake of OMLs in the peritoneal cavity and subse-quent trafficking from the peritoneal cavity to the spleen to initiateimmune responses.

We have previously shown that adherent PECs with CD11bhigh

cells obtained from mice treated with OMLs with encasedOVA were able to activate OVA-specific CD8+ (from OT-I:OVA257-264/H-2Kb-specific) and CD4+ (from OT-II: OVA323-339/H-2Ab-specific) T cells, if these cells were co-cultured for 1 day [8].We have also demonstrated that OML-containing peritonealmacrophages preferentially produce IL-12 [7], which is critical fordevelopment of Th1 cells and initiation of a cell-mediated immuneresponse [9]. In this study, we demonstrated that OML-containingperitoneal macrophages convert into mature DCs within 1 day andthat the resulting macrophage-derived DCs can present OML-encased antigens on MHC class I. Furthermore, intraperitonealplantation of the OML-ingesting peritoneal macrophages demon-strated that the resident peritoneal macrophages could differenti-ate into DC-like cells in vivo and that the macrophage-derivedDCs could migrate from the peritoneal cavity to the spleen and ini-tiates the specific immune response. Therefore, administration ofOMLs in the peritoneal cavity causes a fraction of resident perito-neal macrophages to differentiate into functional DCs in responseto OML uptake, leading to induction of characteristic immuneresponses.

OMLs but not uncoated liposomes can promote the developmentof DCs from peritoneal macrophages, suggesting that mannose-binding proteins expressed on the cells are involved in the process.DC-specific ICAM-3 grabbing nonintegrin (DC-SIGN) and DC-SIGN-related proteins, SIGNR1 and SIGNR3, expressed on resident perito-neal macrophages bind to high-mannose oligosaccharides and areinvolved in uptake of mannose-exposed particles and microbes byDCs [26–29]. We have shown that SIGNR1 and complement recep-tor 3 (CR3) expressed on resident peritoneal macrophages are phys-iological receptors for OMLs [30,31], and that SIGNR1-expressingmacrophage-like cells produce TNF-a, which is required forgeneration of DCs [32], in response to OML uptake [33]. In addition,OMLs can activate the phosphatidylinositol 3-kinase/Akt pathwaythrough phosphorylation of Src family kinases to induce activationof mitogen-activated protein kinases in macrophage-like J774A.1cells [34]. Thus, signaling triggered by mannose-binding lectin-mediated phagocytosis may modulate both DC differentiation frommacrophages and subsequent DC maturation.

The differentiation of monocytes into DCs has been tracked inintact mice after injection of fluorescent particles of 0.5–1.0 lmin diameter into mouse skin tissue, and inflammatory monocyteswith ingested particles crossed the endothelial barrier and mi-grated to lymph nodes, in which they were identified as matureDCs [16,17]. We have shown that subcutaneous administration ofOMLs with encased antigens induces strong cellular immunity tocontrol the progression of tumors, some protozoan diseases, andan allergic response [10–12]. Therefore, monocytes in skin tissuemay also be able to take up subcutaneously administered OMLs,with subsequent differentiation of these cells into mature DCs thatactivate CD4+ and CD8+ T cells to induce antigen-specific Th1 cellsand cytotoxic T cells. Additional studies are needed to investigatethe mechanisms that regulate the commitment and differentiationof cells with a monocyte/macrophage lineage to DCs after OML up-take in situ.

Acknowledgments

This work was supported by the Program for Promotion of BasicResearch Activities for Innovative Biosciences (PROBRAIN), and in

N. Kojima et al. / Cellular Immunology 271 (2011) 335–341 341

part by a Grant-in-Aid for Special Research from The Promotionand Mutual Aid Corporation for Private Schools of Japan.

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