The role of CD11b in phagocytosis and dendritic cell development

Immunology Letters 120 (2008) 42–48

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The role of CD11b in phagocytosis and dendritic cell development Jingtao Chen a , Sahori Namiki a , Makiko Toma-Hirano b , Shoichiro Miyatake c , Koji Ishida a , Yasue Shibata a , Naoko Arai a , Ken-ichi Arai a,d , Yumiko Kamogawa-Schifter a,d,∗ a

Department of Immunobiology, SBI Biotech Co. Ltd., Ginkgo Biomedical Research Institute, Tokyo, Japan Department of Otolaryngology - Head and Neck Surgery, Graduate School of Medicine, University of Tokyo, Tokyo, Japan Cytokine Project, The Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan d Research Center for Advanced Science and Technology, University of Tokyo, Tokyo, Japan b c

a r t i c l e

i n f o

Article history: Received 6 March 2008 Received in revised form 25 June 2008 Accepted 28 June 2008 Available online 30 July 2008 Keywords: DC Phagocytosis Integrin Antigen presentation Apoptosis

a b s t r a c t Activation of resting T cells is highly dependent on dendritic cells (DCs), which take up antigens and present antigenic peptides to T cells in the context of the major histocompatibility complex (MHC). In this study, we generated a monoclonal antibody, which we call 1C4 that recognizes integrin ␣M ␤2 (CD11b/CD18) on the surface of conventional DCs (cDCs) and is internalized after binding. Addition of 1C4 inhibited the ability of immature DCs to phagocytose apoptotic cells. 1C4 treatment also partially inhibited the generation of cDCs from bone marrow in the presence of granulocyte macrophage colony-stimulating factor (GM-CSF). Our findings suggest that not only CD11b is involved in the phagocytosis of apoptotic cells, but also that mAb such as 1C4 may be a useful tool for the delivery of specific proteins into the cytoplasm of immature DCs. © 2008 Elsevier B.V. All rights reserved.

1. Introduction Dendritic cells (DCs) are derived from bone marrow. They internalize antigen and process and present antigen-derived peptide on histocompatibility complex (MHC) molecules for recognition by T cells [1]. As antigen presenting cells, DCs prime immunologically naïve T cells initiate and modulate the immune response [2]. Phagocytosis is necessary to acquire and present foreign antigens. These antigens activate the immune system to attack dangerous invaders. In contrast, engulfing apoptotic cells suppresses immune responses and induces self-tolerance. Immature DCs in particular express a wide array of phagocytic receptors including lectins, scavenger receptors and pathogen receptors [3].

Abbreviations: DC, dendritic cell; cDC, conventional dendritic cell; MHC, major histocompatibility complex; GM-CSF, granulocyte macrophage colony-stimulating factor; Flt3-L, FMS-like tyrosine kinase 3 ligand; MFI, median fluorescence intensity; FITC, fluorescein isothiocyanate; PE, phycoerythrin; PS, phosphatidylserine. ∗ Corresponding author at: Department of Immunobiology, SBI Biotech Co. Ltd., Ginkgo Biomedical Research Institute, 4-7-4-8F Shirokane-dai, Minato-ku, Tokyo 108-0071, Japan. Tel.: +81 3 5789 3200; fax: +81 3 5789 3201. E-mail address: [email protected] (Y. Kamogawa-Schifter). 0165-2478/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.imlet.2008.06.010

These cells are also thought to play an important role in the induction of peripheral tolerance by engulfing apoptotic cells [4,5]. Complements are activated on the surface of apoptotic cells, and it has been shown that integrins, which act as complement receptors, play an undefined role in the internalization of apoptotic cells by DCs [6–8]. Integrins were originally identified as adhesion molecules that mediate cell-to-cell interactions. They play a role in cellular trafficking, migration, co-stimulation, immunological synapse formation and phagocytosis [9]. They consist of noncovalently-associated heterodimeric chains termed ␣ and ␤ subunits. Integrin ␣M ␤2 (also known as Mac-1, CR3, or CD11b/CD18) functions as a complement receptor. It is highly expressed on DCs [10] and is suggested to be responsible for the identification of C3b/bi binding sites on the apoptotic cell corpse. In this study, we generated panels of mAbs that recognize molecules on the surface of DCs. One of them, which we termed 1C4 mAb, binds CD11c+ , CD11b+ and B220− conventional DCs (cDCs) and suppresses their ability to phagocytose apoptotic cells. It also inhibits the generation of cDCs, defined as CD11c+ and CD11b+ , from bone marrow in the presence of granulocyte macrophage colony-stimulating factor (GM-CSF). We further determined that 1C4 recognizes mouse and human integrin ␣ M ␤2 .

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2. Materials and methods

2.6. Cloning of CD11b and CD18

2.1. Reagents

Mice CD11b and CD18 cDNAs were cloned from a mouse bone marrow cDNA library [12] with PCR. The primers were designed based on the cDNA encoding a full-length CD11b and CD18 as below: 5 -TTT TTT GCT AGC ATG ACT CTT AAA GCT CTT CTG GT3 (␣M forward primer) and 5 -TTT TTT GCG GCC GCT TAC TGA GGT GGG GCG TCT TGG C-3 (␣M reverse primer); 5 -AAA ATA GAA TTC GCC ACC ATG CTG GGC CTG CGC CCC TCACT-3 (␤2 forward primer) and 5 -ATA AGA ATG CGG GCC GCT TT GAT CCT TGG CAG CAC T-3 (␤2 reverse primer). The PCR fragments were individually purified by QIAquickR gel extraction kit (Qiagen) and sub-cloned into the expression vector pcDNA3.1/Zeo(+) vector (Invitrogen) on Nhe I and Xba, or Nhe I and Xhe I sites. The entire sequence was confirmed by sequencing both strands.

Mouse recombinant GM-CSF was purchased from R&D Systems (Minneapolis, MN). PKH26 red fluorescent cell linker kit, PKH67 green fluorescent cell linker kit and Etoposide were obtained from Sigma (St. Louis, MO). Anti-CD11c, CD11b and CD18 Abs were purchased from BD Pharmingen (San Diego, CA). 2.2. Cell preparation Bone marrow-derived DCs were generated as described previously [11]. Briefly, bone marrow cells were removed from femurs and tibias of 4–6-week-old Balb/c or C57/B6J mice (Japan CLEA Tokyo, Japan). After depletion of erythrocytes, the remaining BM cells were cultured with RPMI 1640 with 10% FCS (Invitrogen Corp., Carlsbad, CA), 2 mM l-glutamine, 10 mM HEPES, 100 units/ml penicillin/streptomycin, 1 mM sodium pyruvate and 50 ␮M 2-mercaptoethanol (2-ME), supplemented with either Flt3L (10 ng/ml) or GM-CSF (1000 U/ml) for the number of days indicated. 2.3. Generation of DC-specific mAbs A 4-week-old female Wistar rat was immunized in both footpads with Flt3-derived DCs (1–2 × 106 ). The immunization protocol has been previously described [12]. Hybridoma supernatants that reacted negatively to spleen cells (<1%), but positively to Flt3L-derived BM DCs, were selected by flow cytometry. The isotypes of selected antibodies were determined with the Rat MonoAb ID/SP Kit (Zymed Laboratories, Inc., South San Francisco, CA). Purified Ab was labeled with FITC or biotin according to the instructions of manufactured kits (Pierce Biotechnology Inc., Rockford, IL) 2.4. Internalization of mAb 1C4 mAb was pre-incubated with GM-CSF-derived DCs at 4 ◦ C for 30 min. Cells were washed and then either kept on ice or incubated for 0.5, 1, 2 and 3 h at 37 ◦ C. Levels of 1C4 mAb on the cell surface were detected with PE-labeled anti-rat IgG (Pharmingen) and analyzed by flow cytometry. The median fluorescence intensity (MFI) of the 1C4-treated cells was used to calculate the percent of 1C4 mAb remaining on the cell surface, where the MFI of the cells incubated on ice for 3 h was taken to be 100%. 2.5. Phagocytosis assay Splenocytes from C57BL/6 mice were labeled with PKH67-GL (green) (Sigma), and treated with Etoposide (20 ␮g/ml) for 15 h. Apoptotic cells were examined by staining with PI and Anexin V (data not shown). GM-CSF-derived immature DCs prepared from bone marrow cells cultured in the presence of GM-CSF for 6 days were labeled with PKH26-GL (red), and purified by using antiCD11c magnetic beads. They were washed with EDTA-containing PBS, incubated with either 1C4 or rat-IgG control Abs for 30 min on ice, and then mixed with the labeled splenocytes in round-bottom polypropylene tubes in a 2:1 ratio for 2 h at 37 ◦ C in the presence of 10% mouse serum. The cells were then spun down and fixed with 2% paraformaldehyde. The number of double-positive cells (indicating that the red cDCs had engulfed the green apoptotic cells) was analyzed by flow cytometry and used to calculate the percentage of phagocytosis. Phagocytosis was taken to be 100% for the cells incubated with the rat IgG.

2.7. Statistical analysis Statistical analysis of results was performed using two-tailed Student’s t-test. p-Values <0.05 were considered to be significant. The data were normally distributed. Unless otherwise indicated, results are presented as the mean ± S.D. 2.8. In vivo Ab treatment For development experiments, 300 ␮g of 1C4 mAb or control rat Abs were administered intraperitoneally to BALB/c mice at days 0 and 7. After 14 days, spleen cells were harvested and cDCs (determined as CD3− , CD19− , DX5− , B220− and CD11c+ cells) were analyzed by flowcytometry. For antigen presentation experiments, mice were injected intraperitoneally with 300 ␮g of either 1C4 or control Abs. One day later, mice were injected intraperitoneally with 1 mg OVA. Twelve hours after OVA injection, OVA-loaded DCs were then isolated by autoMACS with anti-CD11c magnetic beads. Varying levels of purified DCs were then incubated at 37 ◦ C in 96-well plates with 1 × 105 of CD4+ T cells isolated from the spleen cells of DO11.10 TCR transgenic mice. On day 4, 20 ␮l of WST-1 reagent (Roche Applied Science, Mannheim, Germany) was added to each well, and the plates were incubated for 2 h at 37 ◦ C. CD4+ T cell proliferation was measured by absorbance at 450 nm. 3. Results 3.1. Generation of 1C4 mAb specific for mouse cDC In order to study the function of mouse DCs, we immunized a 4week-old rat with whole DCs to generate DC-specific antibodies. We selected an mAb we called 1C4 for further study based on its ability to stain bone marrow-derived DCs but not primary splenocytes. Among DC subsets, this mAb stained most GM-CSF-derived cDCs, but showed less affinity for Flt3L-derived plasmacytoid DCs (Fig. 1). Positively stained cells were CD11b+ , CD11c+ and B220− , further suggesting that 1C4 mAb recognizes cDCs rather than plasmacytoid DCs. 3.2. Internalization of 1C4 mAb by mouse cDCs Macrophages and DCs have many surface proteins that deliver antigens to intracellular compartments for degradation. We incubated immature DCs (derived from day 6, GM-CSF-treated bone marrow) with labeled mAbs at 37 ◦ C for varying lengths of time and assessed whether the mAbs were internalized. Decreasing levels of PE-labeled anti-rat Ab staining indicated that 1C4 internalization occurred within 30 min (Fig. 2A, left panel). The fact that the level

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Fig. 1. 1C4 mAb recognizes cDCs. Flt3L-derived DCs or GM-CSF-derived DCs (at day 10) were stained with FITC-labeled 1C4 mAb and PE-labeled CD11c, CD11b or B220.

of FITC-labeled 1C4 mAb remained constant in the cells over the same time period confirmed that this reduction in signal was likely due to internalization rather than to antibody shedding (Fig. 2A, right panel). Levels of 1C4 on the cell surface continued to decline during 3 h of incubation at 37 ◦ C (Fig. 2B).

ability of immature DCs to phagocytose apoptotic cells was hampered in a dose-dependent manner by treatment with 1C4 mAb (Ab concentration 2.5 ␮g/ml: p < 0.1; 25 ␮g/ml: p < 0.01) (Fig. 3C). Thus, 1C4 mAb partially inhibits antigen uptake by immature DCs.

3.3. 1C4 mAb inhibits uptake of apoptotic cells by immature DCs

3.4. 1C4 mAb inhibits the generation of cDCs and recognizes Mac-1

One of the most remarkable features of DCs is their ability to take up and present antigens. We used a cell-labeling system [6,13] to determine whether 1C4 mAb treatment affects cell uptake by immature DCs. It has been reported that apoptotic cells are engulfed and digested by DCs. We reasoned that this process could be followed by tracking the appearance of double-positive cells in a population of green-labeled apoptotic cells and redlabeled phagocytes (Fig. 3A). Confocal microscopy showed this to be the case (Fig. 3B). Using this technique, we found that the

Finally, we examined whether mAb treatment affects the generation of cDCs from GM-CSF-treated bone marrow cultures. Addition of 1C4 mAb to the cultures significantly inhibited the GM-CSFinduced development of CD11b+ , CD11c+ and cDCs (43.5–11.8%) (Fig. 4A) without affecting cell numbers and cell viability was detected by PI staining (data not shown). This data suggests three possibilities. First, 1C4 mAb actually suppressed the development of cDCs during the culture. Second, 1C4 mAb binds to either

Fig. 2. Internalization of 1C4 mAb. (A) GM-CSF-derived immature DCs were stained with FITC-labeled 1C4 mAb and incubated at 37 ◦ C for 0.5 and 1 h. Surface-bound 1C4 mAb was detected with PE-labeled anti-rat IgG Ab and analyzed by flow cytometry at each time point. (B) The kinetics of internalization. (Percent remaining cell surface antibodies = MFI at time point/MFI without incubation) × 100. The experiments were done three times and similar results were obtained.

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Fig. 3. 1C4 mAb inhibits phagocytosis. (A) PKH26-GL (red)-labeled immature DCs (GM-CSF-derived bone marrow cells at day 6, and purified by using anti-CD11c magnetic beads) were incubated with PKH67-GL-labeled (green) apoptotic splenocytes in the presence of either 1C4 or control mAb for 2 h at 37 ◦ C and then analyzed by flow cytometry. ((A) showed the case in the presence of control Ab.) The double-positive cells (upper right) represent apoptotic cells that have been engulfed by DCs. (B) Microscopic analysis of immature DCs engulfing apoptotic cells. Double-positive cells (right upper quadrant) were sorted and analyzed by confocal microscopy. (C) The percentage of immature DCs that engulfed apoptotic cells. Percentage of phagocytosis was taken to equal the percentage of upper right quadrant/total DCs (red cells; upper right and left quadrants). Data are representative of three experiments and the values shown represent the mean of triplicate experiments. p < 0.1; **p < 0.01.

CD11b or CD11c, masking the epitopes recognized by the staining antibodies. Third, 1C4 mAb recognizes CD11b or CD11c and this binding also inhibits development of cDCs. Since fewer CD11b+ cells were observed, we examined the possibility that 1C4 mAb recognizes this epitope. We stained GM-CSF-derived cDCs with CD11b mAb in the presence of various concentrations of 1C4 mAb. We found that 1C4 mAb treatment blocked the staining of CD11b by CD11b mAb in a dose-dependent manner (Fig. 4B). To confirm that 1C4 mAb recognizes and binds to CD11b, we cloned the genes for both the CD11b and CD18, which is required for CD11b expression [14], from a bone marrow-derived cDNA library and transfected them together into COS7 cells. As expected, 1C4 mAb recognized doubly, but not singly, transfected COS7 cells (Fig. 4C). Thus, we conclude that 1C4 mAb recognizes CD11b. However, the fact that the levels of CD11c+ cells also decreased (45–26%) in the bone marrow culture in the presence of 1C4 mAb without affecting the total cell numbers at the end of the culture suggests that 1C4 mAb may suppress cDC generation in vitro. In addition, we examined the in vivo effects of 1C4 mAb on the development of cDC. Although effects were small, addition of 1C4 mAb significantly reduced the percentage of splenic cDCs on day 14 (Fig. 5A). We also examined the effects of 1C4 mAb on the presentation of foreign antigen (OVA) in vivo. In contrast to the uptake of apoptotic cell, the addition of 1C4 mAb stimulated antigen uptake and/or presentation and promoted T cell proliferation (Fig. 5B).

4. Discussion DCs are well known for their ability to take up and present antigen. Here we describe how we generated an mAb specific for CD11b, which is expressed on the surface of cDCs. CD11b is also known as ␣M ␤2 integrin. Integrins are a family of heterodimeric cell adhesion receptors that mediate wide variety of biological functions [9,15]. Integrin ␣M ␤2 , which is expressed in monocytes, granulocytes, macrophages, NK cells, and DCs, has been implicated in a variety of cellular responses, including phagocytosis, cell-mediated killing, chemotaxis and cellular activation [16]. Its activation is dependent on the binding of ligands such as fibrinogen, complement fragment iC3b, ICAM-1, blood coagulation factor X and denatured protein [17]. Treatment with 1C4 mAb affects the ability of immature DCs to phagocytose apoptotic cells in vitro. During phagocytosis, apoptotic cells are first tethered to phagocytes through an interaction between putative ligands and receptors. Many receptors, including phosphatidylserine (PS), the LDL receptor, scavenger receptors, CD14, CD68, integrin ␣v ␤5 and CD38, have been proposed to mediate the tethering and engulfment of apoptotic cells [18–23]. Moreover, apoptotic cells are capped by various soluble molecules such as C1q, milk-fat globule epidermal growth factor 8 (MFGE8), growth arrest specific gene 6 (Gas6), ␤2-glycoprotein I (␤2-GP I) and protein S, that bind to PS and tether phagocytes to apoptotic cells for sequential engulfment [24–27].

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Fig. 4. 1C4 mAb inhibits the development of cDCs (CD11c+ cells) and recognizes CD11b. (A) 1C4 mAb or control Ab (2.5 ␮g/ml) was added to cultured bone marrow in the presence of GM-CSF at days 0 and 3. At day 6, cells were stained with 0.5 ␮g/ml of FITC-labeled CD11b and PE-labeled CD11c mAb. The experiments were done three times and similar results were obtained. (B) Bone marrow cells cultured with GM-CSF for 7 days were stained with 0.5 ␮g/ml of anti-CD11b mAb in the presence of 1C4 mAb at the indicated concentrations. The percentage of CD11b-positive cells is shown. The experiments were done three times and similar results were obtained. (C) CD11b, CD18 and CD11b plus CD18 cDNA were transfected into COS7 cells and cells were stained with the mAbs indicated.

Although integrin ␣M ␤2 has been reported to play a major role in the phagocytosis of opsonized bacterial particles by macrophages [28,29], little is known about whether it is also involved in the phagocytosis of apoptotic cells. Other groups have reported that the phagocytic activity of splenic marginal zone DCs and immature DCs in mice is partially dependent on the interaction between integrin ␣M ␤2 and its ligand on the surface of apoptotic cells [13,30]. Skoberne et al. showed that integrin ␣M ␤2 , but not ␣v ␤5 , regulates the phagocytosis of apoptotic cells in humans [31]. Our results suggest that integrin ␣M ␤2 is involved in the phagocytosis of apoptotic cells by immature DCs in mice. Furthermore, we

found that heat-inactivated serum abrogates the ability of immature DCs to phagocytose apoptotic cells (data not shown), which suggests the involvement of complements in this process. It is possible that the existence of multiple receptors for phagocytosis of apoptotic cells is the reason why 1C4 mAb treatment has only a moderate effect on the process. Interestingly, in contrast to apoptotic cell uptake, antigen uptake and/or presentation by cDCs of foreign protein such as OVA was up-regulated in vivo by the addition of 1C4 mAb. This observation is compatible with other reports that active Mac-1 on DCs inhibits T cell activation, and that anti-Mac-1 mAb can alleviate this inhibition [32]. These

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Fig. 5. In vivo treatment of 1C4 mAb inhibited cDC development and up-regulated antigen presentation. (A) Splenic cDCs (CD3− , CD19− , DX5− , B220− and CD11c+ cells) were analyzed by flowcytometry 14 days after in vivo injection of 1C4 or control Abs. (B) 1C4 or control Abs (300 ␮g/mouse) were injected intraperitoneally into BALB/c mice 24 h prior to the injection of 1 mg of OVA. DCs from the mice were then purified and co-cultured with CD4+ T cells (1 × 105 ) from DO11.10 TCR transgenic mice splenocytes for 4 days at 37 ◦ C. Cell proliferation was measured with WST1 assay.

results suggest that CD11b act differently against self or non-self in vivo. 1C4 mAb also suppresses the GM-CSF-induced generation of cDCs without inducing cell death. Although 1C4 mAb treatment blocked the binding of anti-CD11b mAb, addition of 1C4 mAb to the bone marrow culture clearly inhibited the generation of CD11c+ cells. In addition, other anti-CD11b Ab (BD Pharmingen) also showed similar effects on the development of cDCs (data not shown). Taken together, it suggests that signaling through CD11b is required for the development of cDC in vitro. Although GM-CSF and Flt3L are commonly used to generate DCs, the microenvironment necessary for cDC development is not well-known. Despars and O’Neill reported that splenic endothelial cell lines support the development of DCs from bone marrow [33], and that this development requires both cell–cell contact and soluble factors [34,35]. We speculate that CD11b might be one of the cell surface molecules involved in this process. In conclusion, we have generated an 1C4 mAb, that modulates the phagocytic activities of cDCs. This antibody recognizes CD11b, suggesting that this cell surface molecule is involved in the uptake of apoptotic cells by immature DCs in vitro. Moreover, addition of 1C4 mAb partially inhibited the GM-CSF-induced generation of CD11c+ cDCs and in vivo development of splenic cDCs. The fact that 1C4 mAb, which also recognizes human CD11b (data not shown) are rapidly internalized after binding to cDCs suggests that they may be useful in the targeted delivery of antigens or other proteins such as DEC205 Ab that modulates DC function [36]. Acknowledgements We thank Dr. K. Conger for critical reading of this manuscript. We are grateful to A. Kotaki for technical help. References [1] Mellman I. Antigen processing and presentation by dendritic cells: cell biological mechanisms. Adv Exp Med Biol 2005;560:63–7. [2] Villadangos JA, Schnorrer P. Intrinsic and cooperative antigen-presenting functions of dendritic-cell subsets in vivo. Nat Rev Immunol 2007;7:543–55. [3] Banchereau J, Briere F, Caux C, Davoust J, Lebecque S, Liu YJ, et al. Immunobiology of dendritic cells. Annu Rev Immunol 2000;18:767–811. [4] Steinman RM, Turley S, Mellman I, Inaba K. The induction of tolerance by dendritic cells that have captured apoptotic cells. J Exp Med 2000;191:411–6. [5] Huang FP, Platt N, Wykes M, Major JR, Powell TJ, Jenkins CD, et al. A discrete subpopulation of dendritic cells transports apoptotic intestinal epithelial cells to T cell areas of mesenteric lymph nodes. J Exp Med 2000;191:435–44.

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