Activation of FcγRI on Triggers Differentiation into Immature Dendritic Cells That Induce Autoreactive T Cell Responses

This information is current as Motoyuki Tanaka, Stephan R. Krutzik, Peter A. Sieling, of September 23, 2021. Delphine J. Lee, Thomas H. Rea and Robert L. Modlin J Immunol 2009; 183:2349-2355; Prepublished online 27 July 2009; doi: 10.4049/jimmunol.0801683

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2009 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Activation of Fc␥RI on Monocytes Triggers Differentiation into Immature Dendritic Cells That Induce Autoreactive T Cell Responses1

Motoyuki Tanaka,*† Stephan R. Krutzik,* Peter A. Sieling,* Delphine J. Lee,* Thomas H. Rea,‡ and Robert L. Modlin2*†

The formation of immune complexes results in activation of the innate immune system and subsequent induction of host inflammatory responses. In particular, the binding of IgG immune complexes to Fc␥R on monocytes triggers potent inflam- matory responses leading to tissue injury in disease. We investigated whether activation of monocytes via Fc␥R induced cell differentiation, imparting specific inflammatory functions of the innate immune response. Human IgG alone induced mono- cytes to differentiate into cells with an immature (iDC) phenotype, including up-regulation of CD1b, CD80, Downloaded from -CD86, and CD206. Differentiation into CD1b؉ iDC was dependent on activation via CD64 (Fc␥RI) and induction of GM CSF. The human IgG-differentiated iDC were phenotypically different from GM-CSF-derived iDC at the same level of CD1b expression, with higher cell surface CD86, but lower MHC class II, CD32, CD206, and CD14. Finally, in comparison to GM-CSF-derived iDC, IgG-differentiated iDC were more efficient in activating T cells in both autologous and allogeneic mixed lymphocyte reactions but less efficient at presenting microbial Ag to T cells. Therefore, activation of Fc␥RI on monocytes triggers differentiation into specialized iDC with the capacity to expand autoreactive T cells that may contribute http://www.jimmunol.org/ to the pathogenesis of immune complex-mediated tissue injury. The Journal of Immunology, 2009, 183: 2349–2355.

he formation of immune complexes triggers a series of on human monocytes results in the production of several proin- inflammatory responses that contribute to the patho- flammatory cytokines, such as TNF-␣, IL-6, and GM-CSF T physiology of autoimmune diseases, including rheuma- (7–9). toid arthritis and systemic lupus erythematosus (1–3). One Cells of the lineage can be activated by various mechanism by which immune complexes trigger inflammatory receptors and cytokines to differentiate into specialized functional responses is by their ability to interact with receptors for the Fc cellular subsets of the innate immune system, including dendritic by guest on September 23, 2021 portion of the Ig. In particular, immune complexes composed of cells (DC),3 specialized APC that play a crucial rolls in the regu- ␥ IgG cross-link Fc R on cells of the monocyte/macrophage lin- lation of innate responses and in the initiation of adaptive immu- ␥ eage (4). The cross-linking of Fc R triggers phagocytosis of nity (10, 11). We have previously shown that activation of specific opsonized foreign bodies, Ab-dependent cell-mediated cytotox- TLR triggers the rapid differentiation of monocytes into two dis- icity, and production of various proinflammatory cytokines and ϩ tinct populations of cells, CD1b DC that are potent APC, and chemokines (5, 6). ϩ ϩ CD209 macrophages that are highly phagocytic (12). CD1b DC In humans, three different classes of Fc␥R have been de- accumulate at the site of infection and have been shown to corre- scribed as follows: Fc␥RI (CD64), Fc␥RII (CD32), and Fc␥RIII ␥ late with the resistance against the pathogen in leprosy (12, 13). In (CD16). These Fc R differ in cell distribution, function, and ϩ affinity for IgG isotype (4). CD64 is a high-affinity receptor addition to treatment of monocytes with TLR ligands, CD1b DC ϭ Ϫ8 differentiation is induced by low-dose treatment with GM-CSF (Kd 10 M for monomeric IgG), whereas CD32 and CD16 alone (0.2–10 U/ml) as well as a combination of GM-CSF and exhibit low affinity for monomeric IgG, with Kd for monomeric IgG ranging from 10Ϫ5 to 10Ϫ7 M (4). CD64 is exclusively IL-4. The presence of CD209 on GM-CSF/IL-4-derived DC but expressed on myeloid cells, including monocytes, macrophages, not circulating or tissue DC suggests that the derivation of Ϫ and IFN-␥-activated granulocytes. The cross-linking of CD64 CD209 DC with GM-CSF is more physiologically relevant (12). Given that Fc␥R are expressed on monocytes and given the ability ␥ *Division of Dermatology and †Department of Microbiology and Immunology David of monocytes to differentiate into DC, we hypothesized that Fc R Geffen School of Medicine, University of California, Los Angeles, CA 90095; and activation of monocytes could trigger differentiation into DC, cells ‡ Section of Dermatology, University of Southern California School of Medicine, Los of the innate immune system with the ability to instruct the adap- Angeles, CA 90033 tive immune response in a manner that could contribute to auto- Received for publication May 27, 2008. Accepted for publication June 16, 2009. immune disease. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported in part by grants from the Heiser Program for Research in Leprosy and Tuberculosis of the New York Community Trust, the Japan Antibiotics Research Association Pfizer Infectious Diseases Fund 2005, and National Institutes of 3 Abbreviations used in this paper: DC, dendritic cell; h, human; iDC, immature Health Grants AR40312 and A122553. DC; MFI, mean fluorescence intensity; MHC II, MHC class II; qPCR, quantita- 2 Address correspondence and reprint requests to Dr. Robert L. Modlin, University of tive real-time PCR. California, Division of Dermatology, 52-121, HS, 10833 Le Conte Avenue, Los An- geles, CA 90095. E-mail address: [email protected] Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00 www.jimmunol.org/cgi/doi/10.4049/jimmunol.0801683 2350 Fc␥RI-MEDIATED DC DIFFERENTIATION

Materials and Methods mixture (StemCell Technologies). Media or T cells were added. We ␥ ␣ Ligands and Ags measured IFN- , TNF- , IL-4, and IL-17 levels by ELISA (BD Bio- sciences and Invitrogen). Proliferation was measured as described pre- We purchased TLR2/1 ligand Mycobacterium tuberculosis 19-kDa li- viously (28). For MHC class II (MHC II) studies, monocytes were popeptide (EMC Microcollections). M. tuberculosis extracts were prepared differentiated and irradiated as above. Cells were cultured with MHC by probe sonication as previously described (24) and provided by Dr. J. II-restricted T cells and the GroES or GroES peptide as de- Belisle (Colorado State University, Fort Collins, CO). The GroES protein scribed previously (12). was provided by Dr. P. Brennan (Colorado State University, Fort Collins, CO) through a contract with the National Institute of Allergy and Infectious Disease (contract N01-AI-25469, “Leprosy Research Support”). The Results GroES peptide (25) was synthesized by SynPep. Fc␥R activation induces monocyte differentiation Plate-bound hIgG Signaling through Fc␥R is known to inhibit the differentiation Human (h)IgG derived from healthy donor-pooled sera (Equitech-Bio) was and maturation of cytokine-derived DC (14). In this study, the reconstituted in various concentration (0.2–125 ␮g/ml) into endotoxin-free ability of Fc␥R stimulation to directly induce monocyte differ- PBS (Invitrogen) and incubated in the plastic culture plate overnight in 4°C entiation was investigated using plate-immobilized hIgG as a before monocyte culture. Coated plastic was washed twice with PBS before ␥ monocyte culture. model for immune complex activation of Fc R on monocytes (6, 8). Human peripheral blood monocytes were cultured in Monocyte differentiation various concentrations (0.04–125 ␮g/ml) of immobilized human We obtained whole blood from healthy donors (University of California serum IgG. On day 2, cells were harvested, and the expression of Los Angeles Institutional Review Board no. 92-10-591-33) with informed the DC marker CD1b was measured by flow cytometry. Immobi- Downloaded from consent. PBMC were purified by Ficoll-Hypaque gradient centrifugation lized IgG induced CD1b expression in a dose-dependent manner (Ficoll-Paque; Pharmacia Biotech). Peripheral blood monocytes were iso- (Fig. 1a). In contrast, soluble hIgG did not induce CD1b expres- lated from PBMC by Percoll (Amersham Biosciences) density gradient centrifugation. Unless otherwise stated, monocyte cultures were performed sion on monocytes. Previously, we demonstrated that rGM-CSF at 37°C in RPMI 1640 supplemented with glutamine (2 mM), penicillin treatment of monocytes for 2 days induced differentiation into im- (100 U/ml), streptomycin (100 ␮g/ml), and 10% heat-inactivated FCS mature DC (iDC) as characterized by morphology and phenotype,

(OMEGA Scientific). We stimulated monocytes with medium, immobi- including CD1b expression and function (12, 15). The frequency http://www.jimmunol.org/ lized hIgG, soluble hIgG (0.04–125 ␮g/ml), rGM-CSF (0.2–10 U/ml), or ϩ the M. tuberculosis 19-kDa lipopeptide (10 ␮g/ml) for 2 or 3 days. DC of CD1b cells was comparable for hIgG vs rGM-CSF, although were differentiated with rGM-CSF and matured with lipopeptide as de- the mean fluorescence intensity (MFI) was greater for rGM-CSF- scribed previously (26). derived cells (Fig. 1b). ϩ ϩ To study the CD16 and CD64 subsets of peripheral monocytes, we Therefore, it was logical to compare the phenotype of CD1bϩ first isolated monocytes as above, and then anti-CD16-MACS beads cells derived by Fc␥R activation vs culture with rGM-CSF. We (Miltenyi Biotec) were added for 20 min at 4°C. Flow though was desig- ϩ compared expression levels of various surface molecules in nated as the CD64 monocytes. Remaining cells were designated as the ϩ CD16ϩ cells. Both cells were enriched twice before stimulation. CD1b cells treated for 2 days with hIgG (25 ␮g/ml) or rGM-CSF (0.2 U/ml) to normalize to the level of CD1b expression (Fig. 1, c

Fc␥R, GM-CSF blocking assays by guest on September 23, 2021 and d). The expression patterns of cell surface markers for both Enriched monocytes as above were preincubated with 20 ␮g/ml 3G8 activation via Fc␥R and rGM-CSF were consistent with an iDC- (anti-CD16; BioLegend), AT10 (anti-CD32, Serotec), 10.1 (anti-CD64, like phenotype, including high levels of MHC II, CD40, CD86 ␥ Serotec), 107.3 (mIgG1k isotype control for anti-Fc R mAbs; BD Bio- (B7.2, costimulatory molecule), as well as low expression of CD16 sciences), BVD2-23B6 (anti-GM-CSF; BD Biosciences), or R35-95 (rat ␥ ␥ IgG2a isotype for anti-GM-CSF mAbs; BD Biosciences) and placed on (Fc RIII), CD64 (Fc RI), CD14 (TLR coreceptor), and CD83. ice for 20 min before starting culture in hIgG-coated culture well. Yet, there were also clear differences. The levels of CD86 were found to be higher on Fc␥R-activated CD1bϩ cells. Meanwhile, Flow cytometric analysis levels of MHC II, CD32 (Fc␥RII), CD206 (), and The following mAbs were used for flow cytometry studies: BCD1b3.1 CD14 were lower on Fc␥R-activated cells. In comparison to ma- (anti-CD1b (27), mIgG1k), 4A76 (anti-CD1b, mIgG2a; Beckman Coulter), ture DC, HLA-DR, CD80, CD86 (Fig. 1d), and CD83 (data not HI149 (anti-CD1a; BD Biosciences), AD5-8E7 (anti-CD1c; Miltenyi Bio- tec), L243 (anti-HLA-DR; BD Biosciences), MEM-233 (anti-CD80; shown) expression was lower in the hIgG- and rGM-CSF-differ- Caltag Laboratories), 2331(FUN-1) (anti-CD86; BD Biosciences), HB14 entiated cells. Taken together, these data suggest that DC derived (anti-CD40; Caltag Laboratories), GHI/61 (anti-CD163; BD Biosciences), by activation with hIgG vs rGM-CSF are immature and have dis- 19.2 (anti-CD206), DCN46 (anti-CD209; BD Biosciences), Tu¨K4 (anti- tinct phenotypes. CD14; Caltag Laboratories), 3G8 (anti-CD16; Caltag Laboratories), FLI8.26 (anti-CD32; BD Biosciences), 10.1 (anti-CD64; BD Biosciences), M5E2 (anti-CD14; BD Biosciences), HB15e (anti-CD83; BD Bio- Fc␥R activation by hIgG induces GM-CSF transcription in sciences), SCO6 (anti-CDw116; Immunotech), and appropriate isotype control (Caltag Laboratories, Sigma-Aldrich, and BD Biosciences). monocytes To assess whether GM-CSF mediates the Fc␥R induction of CD1b GM-CSF real-time PCR on monocytes, we first evaluated whether Fc␥R activation could Monocytes were stimulated with media, plate-bound hIgG (0.04–25 ␮g/ml trigger the induction of GM-CSF mRNA. Peripheral blood mono- in coating solution) or 19-kDa lipopeptide for 3 h. We isolated RNA and cytes were stimulated with hIgG, and levels of GM-CSF mRNA synthesized cDNA as described previously (25). GM-CSF primer were as follows: forward, 5Ј-GCCTCACCAAGCTCAAGGG-3Ј; and reverse, 5Ј- were determined by quantitative real-time PCR (qPCR). hIgG in- GGAGGGCAGTGCTGTTTGTAG-3Ј. Reactions used Sybr Green PCR duced GM-CSF mRNA in a dose-dependent manner and to levels Master Mix (Bio-Rad). The relative quantities of GM-CSF per sample and similar to that seen with TLR2/1L activation (50- vs 45-fold) (Fig. normalization were calculated as described previously (25). 2a). Fc␥R-stimulated monocytes also secreted the proinflamma- T cell assays tory cytokines TNF-␣, IL-6, and IL-1␤, whereas treatment of monocytes with rGM-CSF did not (data not shown). For MLR, Fc␥R-activated and rGM-CSF-induced CD1bϩ cells were stimulated as above, harvested, washed, irradiated, and plated. We ob- To determine whether GM-CSF mediated the induction of tained T cell from unmatched donors for allogeneic MLR or from iden- CD1b on Fc␥R-activated monocytes, we stimulated monocytes tical donors for autologous MLR with Rosette Sep T cell enrichment with hIgG in the presence or absence of a GM-CSF blocking Ab The Journal of Immunology 2351

FIGURE 1. Fc␥R activation by immobilized hIgG induces CD1bϩ iDC differentiation. a, Monocytes were stimulated with various concen- trations of immobilized IgG (hIgG), soluble hIgG (0.04–125 ␮g/ml), or rGM-CSF (10 U/ml). Two days later, the percentage of CD1bϩ cells was determined using flow cytometry.

Data represent mean percentage of Downloaded from CD1b expression Ϯ SEM from three independent experiments. b, Mono- cytes were stimulated with hIgG (25 ␮g/ml) or rGM-CSF (10 U/ml) or left unstimulated, and levels of CD1b were measured by flow cytometry.

Dot plots are representative of three http://www.jimmunol.org/ independent experiments. c, Human monocytes were stimulated with hIgG (25 ␮g/ml), rGM-CSF (0.2 U/ml), or medium. After 2 days, ex- pression of cell surface markers were examined by flow cytometry, gating on CD1bϩ cells (for hIgG and GM- CSF stimulation). Data are shown as MFI of at least three independent ex- by guest on September 23, 2021 periments Ϯ SEM. d, Representative histograms for HLA-DR, CD80, and CD86 are shown to illustrate the dif- ferentiation stage of the DC.

or isotype control. Monocytes stimulated with hIgG in the pres- control or hIgG alone (Fig. 2b). These data demonstrate that acti- ence of the anti-GM-CSF blocking Ab expressed ϳ60% lower vation of monocytes by hIgG triggers the induction of GM-CSF levels of CD1b compared with monocytes cultured with isotype required for optimal differentiation into CD1bϩ iDC.

FIGURE 2. Fc␥R activation by hIgG induces GM- CSF transcription in monocytes. a, Monocytes were stimulated with hIgG (0.04–25 ␮g/ml) or TLR 2/1 li- gand (10 ␮g/ml) for 3 h. Cells were harvested, and qPCR was performed. Fold changes of GM-CSF mRNA are shown Ϯ SEM from three independent experiments. b, Monocytes were stimulated with hIgG (0.2 ␮g/ml) in the presence or absence of anti-GM-CSF mAb or rat IgG2a as an isotype control. Two days later, the percentage of CD1bϩ cells was measured by flow cy- tometry. Data are representative of two independent experiments. 2352 Fc␥RI-MEDIATED DC DIFFERENTIATION

FIGURE 3. Fc␥R expression on human peripheral monocyte subsets. a, Peripheral human monocytes were labeled with mAbs specific for CD14, CD16, and CD64. The dot plot repre- sents CD16 and CD64 expression by ϩ ϩ

CD14 monocytes. b and c, CD14 Downloaded from CD16ϩ subset and CD14ϩCD64ϩ subset were labeled with CD32, HLA-DR, or CD116. Data are shown as MFI of three independent experi- ments Ϯ SEM (left and center panels) in addition to representative plots

from a single donor. http://www.jimmunol.org/ by guest on September 23, 2021

Surface expression of Fc␥R on peripheral blood monocyte block the interaction of Fc␥R and Fc portion of IgG, were used. At populations first, the effects of anti-Fc␥R blocking mAbs on the CD1b surface Previous studies have indicated that human peripheral blood mono- expression levels were studied. Monocytes were stimulated with ϩ ␥ cytes consist of two major subsets: CD14dimCD16 CD64 monocytes hIgG in the presence or absence of anti-Fc R mAbs or isotype and CD14brightCD16ϪCD64ϩmonocytes. To evaluate which subsets control. After 2 days, hIgG stimulated monocytes cultured with of monocytes are the precursors of CD1b cells, we examined surface anti-CD64 mAb expressed 46% lower levels of CD1b while an expression level of Fc␥R, HLA-DR and GM-CSF receptor. Periph- anti-CD16 mAb or isotype control Ab did not inhibit CD1b ex- eral blood monocytes from healthy donors were labeled with CD14, pression (Fig. 4a). CD16, and CD64 concurrently with CD32, CD116 (␣-chain of GM- Second, the effect of blocking CD64 was investigated in terms CSF receptor), and various DC markers. As previously described, of GM-CSF mRNA expression. Fc␥R stimulation and CD64 CD14ϩ monocytes were separated into the CD16ϩCD64Ϫ subset, a blocking were performed as described above. After3hofculture relatively small population, and the CD16ϪCD64ϩ subset, a rela- with hIgG, monocyte GM-CSF mRNA expression was inhibited tively larger population (Fig. 3a). Both subsets expressed a compa- by both the anti-CD64 mAb (73%) and anti-CD16 mAb (32%) but rable level of CD32 and HLA-DR (Fig. 3b). Interestingly, the not the isotype control (Fig. 4b). Although the anti-CD32 mAb ϩ CD16ϪCD64ϩ subset expressed higher levels of CD116 compared also suppressed both GM-CSF mRNA and CD1b expression fol- with CD16ϩCD64Ϫ subset (Fig. 3c). CD1a and CD1b expression was lowing Fc␥R activation, the extent of blocking was less than CD64 not detected in either subset. MHC I and costimulatory molecules blocking (data not shown). (CD40, CD80, and CD86) were comparably expressed (data not To determine whether the CD16ϪCD64ϩ subset of CD14ϩ shown). Given the higher expression level of GM-CSF receptors, monocytes was the precursor population for CD1b induction, mag- these data suggest that CD16ϪCD64ϩ subset of blood monocytes are netically separated monocyte subsets were stimulated with hIgG. more likely the CD1bϩ cell precursors. Activation by either hIgG or rGM-CSF induced CD1b expression on both peripheral blood monocytes and the CD16ϪCD64ϩ sub- hIgG triggers differentiation via CD64 set, whereas induction of CD1b expression on the CD16ϩCD64Ϫ To further determine which subset of human peripheral blood was similar to the unstimulated control (Fig. 4c). In conclusion, monocytes is the likely precursor of CD1bϩ iDC, mAbs, which hIgG activates the CD64 Fc␥R on CD16ϪCD64ϩ monocytes, The Journal of Immunology 2353

FIGURE 4. hIgG-triggered differentiation of CD1bϩ cells is dependent on CD64. a, Periph- eral monocytes were stimulated with hIgG (0.2 ␮g/ml) in the presence or absence of the indi- cated blocking mAbs (10 ␮g/ml) or isotype con- trol. After 2 days, cells were labeled with anti- CD1b mAb, and flow cytometry was performed (n ϭ 2). b, Monocytes were stimulated with hIgG (25 ␮g/ml) in the presence or absence of indicated blocking mAbs (10 ␮g/ml) or isotype control. RNA was collected after 3 h. Levels of GM-CSF mRNA were measured by qPCR (n ϭ 2). c, Monocytes were separated into CD16ϩ and CD16Ϫ populations using anti-CD16 mAb- conjugated magnetic beads. CD16ϩ, CD16Ϫ,or total monocytes were stimulated with hIgG (25

␮g/ml) or rGM-CSF (0.2 U/ml) for 2 days. The Downloaded from percentage of CD1bϩ cells was determined us- ing flow cytometry (n ϭ 2). http://www.jimmunol.org/ leading to the up-regulation of GM-CSF and subsequent differen- rGM-CSF were tested for their ability to present Ag to a MHC tiation into CD1bϩ iDC. II-restricted T cell clone 9-14B GroES, which recognizes a peptide from the Mycobacterium leprae 10-kDa GroES. Both CD1bϩ iDC ␥ ϩ Fc R-differentiated CD1b iDC activate T cells populations triggered T cell proliferation and cytokine production, Given the iDC-like phenotype of both Fc␥R-activated and rGM- including IFN-␥, TNF-␣, and IL-4; however, the iDC derived by CSF-induced CD1bϩ cells, we investigated the capacity of these culture with rGM-CSF were more potent for Ag presentation to an CD1bϩ cells to activate T cells. iDC derived by Fc␥R activation vs MHC II-restricted T cell clone (Fig. 5, a–c). In contrast, in the by guest on September 23, 2021

FIGURE 5. Fc␥R-differentiated CD1bϩ iDC activate T cells. For Ag presentation studies, monocytes were differentiated with either medium, hIgG, or GM-CSF as above. Cells were cultured with MHC class II-restricted T cells (1 ϫ 104) and the GroES protein or GroES peptide. We then measured T cell proliferation (a) and cytokine production (b and c). Data are reported from triplicate values and are representative of two independent experiments. d, For the MLR, hIgG- and rGM-CSF-differentiated CD1bϩ cells were obtained as above. We obtained T cells from an unmatched donor for the allogeneic MLR or from the identical donor for autologous MLR using Rosette Sep T cell enrichment mixture (StemCell Technologies). The T cell:APC ratio varied between 10:1 and 300:1. Proliferation was measured by the thymidine incorporation assay. Data are reported as triplicate values (mean Ϯ SEM) and are repre- sentative of three independent experiments. 2354 Fc␥RI-MEDIATED DC DIFFERENTIATION

MLR to measure allogeneic responses, Fc␥R-activated CD1bϩ equivalent expression level of CD1b molecule with maximum iDC were more potent than rGM-CSF-induced CD1bϩ iDC, in- Fc␥R activation (25 ␮g/ml in coating solution) was 1/5–1/50 of ducing between ϳ2- and 5-fold greater T cell proliferation across that previously used (0.2 U/ml). Similarly, the GM-CSF concen- a range of T cell/Ag-presenting cell ratios (Fig. 5d). In addition, trations observed in normal human sera or synovial fluids are very the iDC populations were evaluated for their ability to activate an low or undetectable (21). Even in synovial fluids from rheumatoid autologous MLR by adding different concentrations of T cells from arthritis patients, GM-CSF concentration measured by ELISA the same donor as the iDC. Again, the iDC derived from FcR␥ were much lower than the 0.2 U/ml (equivalent of ϳ35 ng/ml activation were more potent than those derived using rGM-CSF in GM-CSF) (20). In addition, the two iDC populations express dif- activating autologous MLR (Fig. 5d). Differences in the magnitude ferent phenotypes and, importantly, functional capacities, with the of the autologous MLR vs the allogeneic MLR may be related to Fc␥R-differentiated iDC more efficient at presentation to autolo- the difference sources of APC and T cells. These data suggest that gous and allogeneic T cells. These data indicate that although iDC derived from activation via FcR␥ vs GM-CSF are functionally Fc␥R-induced iDC differentiation is GM-CSF dependent, addi- distinct, with the FcR␥-differentiated iDC more potent in trigger- tional signaling or cytokine pathways must contribute to their dif- ing allogeneic/autologous T cell responses. ferentiation into a unique subset of iDC. The contribution of Fc␥R in the pathogenesis of autoimmune Discussion diseases has been studied in the context of reciprocal regulation of The ability of immune complexes to trigger FcR on cells of the immune responses via the pair of CD32a, which has an ITAM in innate immune system results in inflammatory responses that con- its cytoplasmic domain, and CD32b, which has an ITIM (22, 23). tribute to tissue injury in disease. In this study, we demonstrated The role of CD64 (Fc␥R) in immune complex and autoimmune Downloaded from that Fc␥R activation of human peripheral blood monocytes in- disease is less well understood, even though it has the highest duces differentiation into CD1bϩ iDC. The mechanism of iDC affinity for IgG, 10- to 100-fold higher as compared with CD32 differentiation was dependent on CD64 (FcR␥I) and up-regulation and CD16 (18). In this study, we demonstrate that CD64 is the of GM-CSF. A precursor monocyte population was identified as a major Fc␥R contributing to CD1bϩ iDC differentiation by hIgG, CD14ϩCD64ϩ subset, expressing high levels of GM-CSF recep- by finding that anti-CD64 blocking mAbs inhibited differentiation. ϩ ϩ tor, and could be separately activated by hIgG to differentiate into Moreover, the magnetically selected CD64 CD14 subpopulation http://www.jimmunol.org/ iDC. The iDC induced by Fc␥R activation vs GM-CSF were phe- of naive peripheral monocytes differentiated into CD1bϩ iDC not notypically and functionally distinct: Fc␥R-activated iDC were the CD16ϩCD14ϩ monocytes, suggesting that CD14ϩCD64ϩ more efficient at activating autoreactive and allogeneic T cells as monocytes are the precursor of CD1bϩ DC. The higher levels of compared with GM-CSF-derived iDC, whereas the latter were GM-CSF receptor in CD64ϩCD14ϩ monocytes support this idea. more efficient at presenting microbial Ags. Although the expression levels of CD32 were considerably higher The ability of the innate immune response to instruct the adap- than CD64, the effect observed by blocking CD32 was always of tive immune response is largely initiated and modulated by DC minor relevance compared with anti-CD64. The effect of Fc␥RI (16). Previous studies have focused on the regulation of DC dif- activation on differentiation may be specific to the stage of devel- ferentiation and maturation by individual and combinations of cy- opment. A recent report (14) indicated that CD64 cross-linking by guest on September 23, 2021 tokines (14). In the process of immune complex-related autoim- during monocyte stimulation by GM-CSF and IL-4 resulted in mune disease or inflammation, DC also play important roles (17, monocyte differentiation into DC with less differentiated pheno- 18). However, the direct relationships and mechanisms between types and impaired APC functions compare with the cytokine Fc␥R cross-linking and differentiation of monocytes into DC have combination alone. In conclusion, the ability of Fc␥R-activated not been well understood and has largely been investigated in naive monocytes to differentiate into CD1bϩ iDC with the poten- terms of regulating cytokine-derived DC. The present study pro- tial to promote autoreactive T cell responses provides a possible vides evidence that Fc␥R cross-linking alone triggers differentia- mechanism of immune complex-related autoimmune diseases and tion of monocytes into iDC, and these iDC stimulate autologous T inflammation. cell proliferation. Our findings suggest that the presence of im- mune complexes may be sufficient for initiating autoimmune man- Acknowledgments ifestations in immune complex disease. Further studies using im- We thank the University of California Los Angeles Flow Cytometry Core mune complexes isolated from serum are required for additional Laboratory for use of their facilities. insight into the pathogenesis of these diseases. Although the combination of GM-CSF and IL-4 result in the Disclosures generation of potent monocyte-derived DC that are useful for im- The authors have no financial conflict of interest. munotherapy, we have established a model of iDC by culture with GM-CSF alone (12, 15). The morphology, cell surface phenotype, References and enhanced Ag-presenting function of the GM-CSF-stimulated 1. Lorenz, H. M., M. Herrmann, and J. R. Kalden. 2001. The pathogenesis of au- toimmune diseases. Scand. J. Clin. Lab Invest. Suppl. 235: 16–26. monocytes indicate that these cells are consistent with iDC previ- 2. Schifferli, J. A., and R. P. Taylor. 1989. 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