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Published online before print March 30, 2006
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receptor-induced phagocytosis and Syk activation in dendritic cells versus macrophages
* Divisions of Pulmonary and Critical Care Medicine and
Infectious Diseases, University of Michigan Health System, Ann Arbor; and
Departamento de Farmacologia, Universidade do Estado do Rio de Janeiro, Brazil
1Correspondence: Division of Pulmonary and Critical Care Medicine, 6301 MSRB III, 1150 W. Medical Center Drive, University of Michigan Medical Center, Ann Arbor, MI 48109-0642. E-mail: petersm{at}umich.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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R) requires the spleen tyrosine kinase (Syk) and serves an important antimicrobial function. We have reported previously that FcR-mediated ingestion and Syk activation in MØ are amplified by and depend on the proinflammatory lipid mediator leukotriene B4 (LTB4). Although FcR-mediated ingestion is also important for antigen uptake, there is no information about LTB4 regulation of these processes in dendritic cells (DCs). In this study, we compared murine bone marrow (BM)-derived DCs to MØ from BM, peritoneum, and the pulmonary alveolar space. Neither phagocytosis nor Syk activation in DCs was influenced by exogenous LTB4. Unlike the various MØ populations, Syk activation in DCs was likewise unaffected by pharmacologic or genetic strategies to inhibit endogenous LTB4 synthesis or to block the high-affinity LTB4 receptor BLT1. DCs were refractory to regulation by LTB4 despite the fact that they expressed BLT1 and mobilized intracellular calcium in response to its ligation. This resistance to LTB4 in DCs instead reflected the fact that in contrast to MØ, Syk activation in DCs was itself entirely independent of calcium. These results identify a fundamental difference in FcR signaling between DCs and MØ, which may relate to the divergent, functional consequences of target ingestion in the two cell types.
Key Words: innate immunity • lipid mediators • cell signaling
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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R) is essential for DCs to process and subsequently present antigens to T lymphocytes in acquired immunity and for MØ to clear microbes in innate immunity.
The protein spleen tyrosine kinase (Syk) plays an essential role in various signaling cascades necessary for immune cell responses, including the ingestion of IgG-coated targets by MØ [1
] and DCs [2
]. Following FcR engagement, the two N-terminal Src homology 2 domains of Syk bind to the immunoreceptor tyrosine-based activation motifs of the -chain of FcRI and FcRIII and with the cytoplasmic domain of FcRIIA. Following this interaction, Syk becomes phosphorylated on tyrosine and is itself activated, initiating a cascade of signaling events leading to actin polymerization and phagocytosis [3
, 4
].
Leukotrienes (LTs) are lipid mediators of inflammation derived from the 5-lipoxygenase (5-LO) pathway of arachidonic acid (AA) metabolism. The enzyme 5-LO, in conjunction with its helper protein 5-LO-activating protein (FLAP), oxygenates AA to form LTA4. This intermediate can be hydrolyzed to form the potent leukocyte activator and chemoattractant LTB4 or conjugated with glutathione to form cysteinyl-LTs (cysLT; LTC4, LTD4, and LTE4), which elicit smooth muscle contraction and microvascular permeability [5 ]. Important in vivo roles for LTs have been established in acquired [6 , 7 ] and innate [8 ] immune responses.
We have demonstrated previously that endogenous and exogenous LTB4 enhances FcR-induced phagocytosis in MØ [9
] and neutrophils [10
]. One mechanism by which LTB4 does so in MØ is by amplifying FcR-induced Syk activation, a process that was itself dependent on Ca2+ influx [11
]. In the present study, we sought to extend this work to DCs by evaluating the role of LTB4 in modulating FcR-induced phagocytosis and Syk activation in these professional antigen-presenting cells (APC). In contrast to our findings with MØ, we now demonstrate that FcR-induced phagocytosis in DCs is independent of LTB4 and that Syk activation in DCs is itself independent of LTB4 and Ca2+.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Generation of bone marrow (BM)-derived DCs (BM-DCs) and BM-derived MØ (BM-MØ)
BM cells were harvested from flushed marrow cavities of femurs and tibiae of mice under aseptic conditions. BM-DCs were generated from mouse BM cells (1x106 cells/ml) cultured for 4 days in culture medium [RPMI with 10% fetal calf serum (FCS)], supplemented with 20 ng/ml granulocyte MØ-colony stimulating factor (GM-CSF; PeproTech, Rocky Hill, NJ) and 20 ng/ml interleukin (IL)-4 (PeproTech), as described by Asavaroengchai et al. [12
]. The BM-DC population was enriched by collecting the low-density interface following 14.5% (by weight) metrizamide density gradient centrifugation (15 min, 4°C, 2000 revolutions per minute) and washing twice in Hanks’ balanced saline solution (HBSS). Purified BM-DCs (
90% purity) were further cultured in 60 mm dishes at 1 x 106 cells/ml for 24 h before use.
Flow cytometry analysis of cell surface antigens
To prevent nonspecific FcR-mediated staining, the cell suspension (except the one for FcR staining) was preincubated with anti-CD16/CD32 (FcRIII/II) monoclonal antibody (mAb), according to the manufacturer’s instructions (PharMingen, San Diego, CA). The following mAb were used for staining of surface antigens: anti-F4/80 (murine MØ marker), anti-CD16/CD32 (FcRIII/II), anti-CD11c (integrin 
x chain), anti-CD80 (B7-1), anti-CD86 (B7-2), anti-I-Ak [major histocompatibility complex (MHC) class II], and the appropriate isotype controls (all from PharMingen). For flow cytometry, the cells were adjusted to a concentration of 5 x 106 cells/ml in staining buffer [fluorescent assay (FA) buffer (Difco, Detroit, MI) with 0.1% sodium azide and 1% FCS]. Cells (5x105) were stained with saturating antibody concentrations for 30 min at 4°C. The samples were washed in FA buffer and fixed with 1% paraformaldehyde (Sigma Chemical Co., St. Louis, MO) in buffered saline. Stained samples were stored in the dark at 4°C until analyzed on a flow cytometer (Coulter Elite ESP, Palo Alto, CA). Flow cytometry data were analyzed by using the FlowJo 5.4.5 software (Tree Star, San Carlos, CA).
Adherent BM-DCs used in this study were considered to be immature BM-DCs, a fact confirmed by flow cytometry, demonstrating a higher expression of CD11c [mean fluorescence intensity (MFI) of 110.1 vs. 87.6] and FcR (MFI 73.1 vs. 47.1) and a lower expression of MHC class II (MFI 75.6 vs. 159.5), CD80 (MFI 88.1 vs. 200.4), and CD86 (MFI 77.7 vs. 138.7), as compared with more mature, nonadherent BM-DCs. BM-MØ (>95% pure) were obtained from mouse BM cells prepared as described above and cultured at 1 x 106 cells/ml for 6 days in medium (RPMI with 10% FCS) supplemented with 20 ng/ml GM-CSF following a modified protocol published previously [13
].
Isolation and culture of alveolar and peritoneal MØ
Resident alveolar MØ were obtained by lung lavage from rats as described previously [14
]. Resident peritoneal MØ were harvested by peritoneal lavage of mice with 5 ml Dulbecco’s modified Eagle’s medium (DMEM; Gibco, Grand Island, NY). The cell suspensions were enumerated using a hemocytometer, adhered in flat-bottom, six-well plates (Becton Dickinson, Franklin Lakes, NJ) for 1 h at 37°C in a 5% CO2 atmosphere, and nonadherent cells were removed by washing. After adherence, the cultures were composed of more than 98% MØ, as assessed by a modified Wright-Giemsa stain (Diff-Quik, American Scientific Products, McGaw Park, IL). MØ monolayers were cultured overnight in DMEM with 10% fetal bovine serum (HyClone Laboratories, Logan, UT). The cells were washed, and the medium changed to DMEM without serum 20 min before the challenge with phagocytic targets.
Microcolorimetric erythrocyte phagocytosis assay
The phagocytosis of sheep red blood cells (sRBCs) was assessed as described previously [15
]. Briefly, BM-DCs, BM-MØ, and rat alveolar MØ were plated for 24 h in 96-well, culture-treated dishes (BD Biosciences, San Jose, CA) at a density of 2 x 105 cells/well. Cells were then washed twice with warm DMEM and preincubated with the phagocytosis inhibitor cytochalasin D (Cyto D; 5 µg/ml, 45 min), LTC4 (10 nM, 2 min, Cayman Chemical, Ann Arbor, MI), or LTB4 (10 nM, 2 min, Cayman Chemical). RBCs (ICN Pharmaceuticals, Costa Mesa, CA) were opsonized with a subagglutinating concentration of polyclonal rabbit anti-sRBC IgG (Cappel Organon Teknika, Durham, NC) as described previously [16
]. Following preincubation, opsonized RBCs were added at a target:cell ratio of 50:1, and cultures were incubated for an additional 90 min at 37°C. Wells were then washed three times with phosphate-buffered saline (PBS) to remove noningested erythrocytes, and 100 µl 0.3% sodium dodecyl sulfate (SDS) in PBS was added to each well for 10 min. Serial dilutions of known amounts of RBCs were added to separate wells before the addition of the SDS solution to derive a standard curve. Lastly, 100 µl o-phenylenediamine dihydrochloride solution was added to each well as a chromogen. Following a 30-min incubation (at 22°C) in the dark, the absorbance at 450 nm was evaluated with an automated reader (VERSAmax, Molecular Devices, Sunnyvale, CA). The number of RBCs per well was derived from absorbance data at 450 nm using the standard curve made with known amounts of RBCs. Independent experiments were performed in septuplet.
Quantification of fluorescein isothiocyanate (FITC)-dextran endocytosis by flow cytometry analysis
To analyze endocytosis, cells (2x106) were incubated in RPMI-1640 medium containing 3% FCS and incubated with FITC-dextran (Sigma Chemical Co.) at a final concentration of 1 mg/ml at 37°C for 60 min, as described previously [17
]. After incubation, cells were washed with ice-cold FA buffer, then fixed in 4% paraformaldehyde, and were analyzed by flow cytometry. The FITC-dextran uptake was determined as the mean fluorescence relative to the background staining of the respective sample incubated with FITC-dextran at 4°C.
Mixed leukocyte reaction (MLR)
To assess lymphocyte proliferation in response to APC, RBC-depleted, allogeneic spleen cells were cultured in triplicate at 5 x 105 cells/well in 96-well plates together with 104 BM-DCs or BM-MØ cells as stimulators in a total volume of 200 µl RPMI-1640 medium containing 10% heat-inactivated FCS. Prior to culture, stimulator cells were irradiated (3000 rads; 137Cs source). DNA synthesis was assessed over the last 16 h of a 72-h culture by addition of 1 mCi/well 3H-thymidine (Amersham, Arlington Heights, IL). Cells were harvested onto glass fiber filters and placed in scintillation fluid (Scintiverse, Fisher Chemicals, Fair Lawn, NJ) for measurement of label incorporation in a liquid scintillation counter (Beckman Coulter, Fullerton, CA). Responses were reported as mean counts per minute (cpm) ± SEM of triplicate samples. Controls included responders or stimulators alone. Background 3H-thymidine incorporation as a result of stimulators alone was <500 cpm.
Immunoprecipitation
Cell monolayers were lysed in buffer containing 1% Triton X-100 and 50 mM tris(hydroxymethyl)aminomethane (Tris; pH 8.0), 100 mM NaCl, 1 mM Na3VO4, 1 mM phenylmethylsulfonyl fluoride, 50 mM NaF, and 1 µg/mL leupeptin. Lysates were precleared with protein A-Sepharose for 30 min and incubated overnight at 4°C with anti-Syk (1:80, Santa Cruz Biotechnology, CA). Protein A-Sepharose was added to each sample and incubated for 3 h with rotation at 4°C. The beads were washed briefly three times with lysis buffer without Triton X-100 and separated on 8% SDS-polyacrylamide gel electrophoresis. The entire volume recovered after boiling the beads was loaded onto the gel; lysates are derived from equal numbers of cells, but total Syk per lane was subject to variation. The proteins were transferred to nitrocellulose membranes (Schleicher and Schuell, Keene, NH) overnight at 100 amps and for 3 h at 200 mA.
Immunoblotting
The membrane was blocked with 5% fat-free milk in Tris-buffered saline containing 0.1% Tween 20 for 1 h, washed three times, and then probed with antiphosphotyrosine (anti-PY; 1:900, PY20, Transduction Laboratories, Lexington, KY) for 1.5 h. After that, the membrane was washed and incubated with a horseradish-peroxidase (HRP)-conjugated sheep anti-mouse secondary antibody (1:15,000, Amersham Pharmacia Biotech, Piscataway, NJ). Phosphorylated bands were visualized using the enhanced chemiluminescence (ECL) system (Amersham). The membranes were then stripped, blocked, and reprobed with anti-Syk (1:800) for 1 h, followed by an incubation with HRP-conjugated donkey anti-rabbit secondary antibody (1:20,000, Amersham Pharmacia Biotech). The bands were visualized using the ECL system. Relative band densities were determined by densitometric analysis using National Institutes of Health Image software, and the ratios were calculated. The results were expressed as normalized Syk-PY/Syk, which represents the value of density obtained with the anti-PY blot divided by the value obtained with the anti-Syk blot. In all instances, density values of bands were corrected by subtraction of the background values.
Analysis of calcium mobilization
Adherent cells were harvested using the protease cocktail AccutaseTM (eBioscience, San Diego, CA) according to the manufacturer’s instructions, centrifuged for 5 min at 750 g, and then resuspended in HBSS containing 1.6 mM CaCl2. The warmed cell suspension (37°C, 107 cells/ml) was treated with 10 µM Fura-2-acetoxymethyl ester (AM) for 30 min, then washed twice with HBSS containing 1.6 mM CaCl2, and finally resuspended at a density of 107 cells/ml and transferred into the magnetically stirred cuvette of the luminescence spectrometer (Perkin Elmer LS50B, Perkin Elmer, Wellesley, MA). Calcium mobilization was monitored using excitation wavelengths of 340 and 380 nm and an emission wavelength of 510 nm. Data are presented as the ratio of fluorescence obtained from 340 and 380 (340/380) nm.
RNA extraction and reverse transcriptase-polymerase chain reaction (RT-PCR)
Total RNA was extracted using Trizol® reagent, following the manufacturer’s instructions (Invitrogen, Carlsbad, CA), and quantified by spectrophotometry. The PCR master mix was made from a Promega (Madison, WI) access RT-PCR system kit. It is a two-enzyme system, which allows an avian myloblastosis virus RT to process the RNA into cDNA in the first step and a Tfl DNA polymerase to facilitate the amplification over the number of desired cycles. The primers were designed to anneal at 55°C. The primers used for the amplification of mouse high-affinity LTB4 receptor BLT1 cDNA were (sense) 5'-GCAGTGGCCCGCCCCTTTATGTC-3' and (antisense) 5'-CACCGGGTTCACGCTGCTGCTC-3' and for BLT2, were (sense) 5'-GTAGTATGGAGCTTAGCGGGC-3' and (antisense) 5'-GGGTCTCCAGGCTCAGATG-3'. Following the amplification, the DNA product was loaded on a 1.5% agarose gel. The DNA was then transferred to a nitrocellulose membrane in NaOH (to separate the DNA strands) and probed with a 32P-labeled probe made specifically for the primer product. After hybridization, the membrane was exposed to photographic film.
Statistical analysis
The data are reported as a representative blot from two or three different experiments. Graphs represent the mean ± SEM from two or three different experiments. The means from different treatments were compared by ANOVA. When significant differences were identified, individual comparisons were subsequently made with the Bonferroni test for unpaired values. Statistical significance was set at a P value less than 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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R expression, although BM-DCs expressed slightly higher levels than BM-MØ. BM-DCs expressed high levels of CD11c and CD80 and also exhibited slightly higher levels of costimulatory molecules CD86 and MHC class II antigen compared with BM-MØ. In summary, the results confirm that BM-MØ and BM-DCs are phenotypically distinct populations based on their surface antigen expression patterns.
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Antigen-presenting ability of BM-DCs
MLR was assessed to examine the differential antigen-presenting activity of BM-MØ and BM-DCs (Fig. 2
). As expected, BM-DCs significantly stimulated allogeneic splenocyte proliferation, whereas BM-MØ provoked a slight proliferative response, which did not reach statistical significance.
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R-mediated phagocytosis (Fig. 3
). Higher concentrations of the compounds (up to 100 nM) also failed to alter phagocytosis (not shown). Confirming that the effects of exogenous LTs on alveolar MØ are true for other MØ cell types, we found that LTC4 and LTB4 stimulated FcR-mediated ingestion by BM-MØ (Fig. 3)
. The concentrations of LTs used in these studies were shown to enhance FcR-mediated phagocytosis by alveolar MØ in parallel experiments (data not shown).
|
R-mediated Syk activation is not modulated by LTB4 or by other 5-LO-derived productsR by IgG is known to elicit activation (phosphorylation) of Syk in MØ [3
] and DCs [19
]. In MØ, this process is amplified by LTB4 [11
]. We used pharmacological and genetic approaches to investigate whether Syk activation is likewise modulated by LTB4 in BM-DCs. As can be seen in Figure 4A
, challenge of BM-DCs with IgG-opsonized sRBCs, but not unopsonized sRBCs, elicited Syk phosphorylation. However, treatment of BM-DCs with exogenous LTB4 (100 nM) failed to amplify FcR-induced Syk activation. Moreover, Syk activation was not influenced by endogenously produced LTB4, as revealed by the use of a LTB4 receptor antagonist (LY 292476; Fig. 4B
), agents that inhibit LT synthesis by inhibition of 5-LO (zileuton) or of 5-LO-activating protein (MK 886; Fig. 4C
), and cells from 5-LO–/– mice (Fig. 5
). As noted in Figure 5
, there is no difference in Syk phosphorylation between BM-DCs obtained from WT mice versus 5-LO–/– mice upon stimulation with several doses of IgG-coated RBCs.
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Next, we examined calcium influx in BM-DCs in response to LTB4 challenge, as it is well known that calcium mobilization occurs immediately after LTB4 interaction with Gq-coupled BLTs [20
]. Stimulation of BM-DCs with LTB4 evoked a substantial increase in intracellular Ca2+ concentration, as can be observed in Figure 6
, suggesting the presence of a functional receptor in BM-DCs. Confirming the presence of BLT1 on BM-DCs, the use of a BLT1 receptor antagonist (CP 105,696; 1 µM) prior to LTB4 administration impaired Ca2+ mobilization (data not shown).
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R-mediated Syk activation is not Ca2+-dependent in BM-DCsR-mediated Syk activation is not a Ca2+-dependent process in BM-DCs, which were pretreated with the extracellular Ca2+ chelator EGTA and/or the intracellular chelator 1,2-bis(O-aminophenyl-ethane-ethane)-N,N,N',N'-tetraacetic acid (BAPTA)-AM for 30 min before IgG-RBC challenge. As observed in Figure 7A
, FcR-mediated Syk activation in BM-DCs was not inhibited by treatment with EGTA, BAPTA-AM, or the combination. By contrast, pretreatment with BAPTA-AM inhibited FcR-mediated Syk activation in rat alveolar MØ (Fig. 7B)
, in BM-MØ (Fig. 7C)
, and in rat peritoneal MØ (Fig. 7D)
. The ability of LTB4 treatment to amplify FcR-mediated Syk activation in BM-MØ and in rat peritoneal MØ is also illustrated in Figure 7C
and 7D
, respectively. As an alternative approach to comparing the participation of Ca2+ in Syk activation evoked by FcR engagement in BM-DCs versus BM-MØ, we examined the effect of the addition of the calcium ionophore A23187 on FcR-mediated Syk activation. As observed in Figure 8A
, the treatment of BM-DCs with A23187 immediately before the addition of IgG-coated RBCs did not change Syk phosphorylation. In contrast, the same procedure performed in BM-MØ amplified FcR-mediated Syk activation (Fig. 8B)
. Together, these results indicate that the process of Syk activation in BM-DCs is Ca2+-independent, in contrast to that in MØ.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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R-induced phagocytosis as well as Syk activation in DCs. We examined the effects of exogenously supplied LTs and also used genetic and pharmacologic approaches to assess the role of endogenously generated LTs in these processes. Our data demonstrate two substantial differences between murine BM-DCs and MØ. First, neither FcR-mediated phagocytosis nor Syk activation in DCs was influenced by exogenous or endogenous LTs. Second, FcR-mediated Syk activation in DCs is not Ca2+-regulated, as neither Ca2+ chelators nor Ca2+ ionophore treatment exerted any effect. As our previous report indicated that the ability of LTB4 in particular to enhance FcR-induced phagocytosis and Syk activation in MØ was dependent on receptor-mediated increases in intracellular Ca2+ [11
], it is likely that the Ca2+ independence of Syk activation in DCs accounts for the lack of modulation by LTs.
We first verified that the two cell populations of interest generated in vitro from BM-derived cells exhibited the flow cytometric characteristics of BM-MØ and BM-DC phenotypes (Fig. 1A)
. We used adherence to yield a population of immature DCs known to actively capture and process antigens and migrate to the draining lymph nodes, wherein they increase their ability to stimulate resting T cells, thereby initiating various immune responses [21
22
23
]. This fact was confirmed by flow cytometric analysis, in which the adherent cell population showed lower expression of MHC class II and higher expression of CD11c and FcR compared with the nonadherent cell population. Also as expected, BM-MØ and BM-DCs exhibited comparable capacity for mannose-dependent ingestion of dextran, demonstrating the efficiency of the phagocytic machinery for both cell types (Fig. 1B)
.
BM-DCs have been reported to produce LTB4 and cysLTs upon stimulation with lipopolysaccharide or antigen [24
, 25
] and to express the biosynthetic proteins necessary to initiate LT synthesis from AA, namely 5-LO and FLAP [25
]. Furthermore, DCs have been shown to express receptors for cysLTs [25
, 26
], which have been implicated in DC migration to [26
] and from [6
] tissue sites of antigen challenge. Although LTB4 has been reported to stimulate production of IL-6 as well as BM-DC generation when added to BM cultures [21
], the receptors for LTB4 have not been examined previously in DCs. Certainly, the inability of endogenous or exogenous LTB4 to modulate FcR-induced phagocytosis and Syk activation in BM-DCs could be the consequence of lack of expression of the high-affinity receptor BLT1. However, RT-PCR and immunoblot analyses revealed expression of BLT1 in BM-DCs. It has previously been reported that in contrast to epidermal MØ or peripheral blood monocytes, epidermal DCs from humans fail to mobilize intracellular Ca2+ in response to a variety of proinflammatory stimuli, including interleukin-1, bradykinin, and formyl-Met-Leu-Phe [27
]. This raised the possibility that LTB4 ligation of BLT1 might be incapable of triggering an intracellular Ca2+ flux in the BM-DCs under investigation here. It is important, however, that the functional competence of this receptor was established by demonstrating intact LTB4-induced Ca2+ mobilization, which was inhibitable by a BLT1-selective antagonist. Thus, the refractoriness of BM-DCs to LTB4 cannot be explained by the absence of its high-affinity receptor BLT1 or its inability to signal appropriately via Ca2+ mobilization.
Like BLT1, the high-affinity receptor for cysLTs (cysLT1) is also a Gq-coupled receptor. That ligation of these two discrete Ca2+-coupled LT receptors in BM-DCs failed to modulate FcR-mediated phagocytosis and Syk activation suggested the alternative possibility that such processes were independent of Ca2+ in these cell types. Experiments using Ca2+ chelators and ionophores demonstrated that this was indeed the case for Syk activation in BM-DCs. This contrasts with parallel findings in murine BM-MØ as well as rat alveolar and peritoneal MØ, in which Syk activation was clearly modulated by changes in intracellular Ca2+. As other DC functions are well-recognized to be regulated by Ca2+ [28
], the Ca2+ independence of Syk regulation reflects a selective property of murine BM-DCs.
Little is known about how increased intracellular Ca2+ enhances Syk activation in MØ. Nevertheless, this mode of regulation might be expected to provide a means by which many proinflammatory ligands for Gq-coupled receptors could amplify FcR-mediated phagocytosis essential for antimicrobial defense. The fact that this mechanism is not operative in DCs, at least in the immature murine BM-DCs under investigation here, reveals cell-specific differences in the means by which these two types of monocyte-derived phagocytic cells are regulated. Although the teleologic explanation for distinct regulatory mechanisms in DCs is at this point unknown, one can speculate that it may reflect differences in the milieu in which these cells encounter IgG-coated targets or in the functional significance of the phagocytic process. Certainly, the functional consequences of FcR-mediated ingestion are widely divergent between MØ (microbial killing) and DCs (antigen processing for its eventual presentation), and these downstream responses may conceivably be differentially regulated by Ca2+. Consistent with this possibility is the fact that in contrast to monocytes or MØ, DCs fail to mobilize Ca2+ [27
] or generate reactive oxygen intermediates [19
] in response to FcR ligation. It remains to be determined whether the differences we have identified in the regulation of Syk activation contribute to divergent, functional consequences of FcR ligation in the two cell types.
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ACKNOWLEDGEMENTS |
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Received July 9, 2005; revised August 18, 2005; accepted February 22, 2006.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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receptor on macrophages J. Exp. Med. 186,1027-1039 RI (CD64) expressed on human blood dendritic cells J. Immunol. 158,3090-3098[Abstract]R and fMLP in functionally distinct antigen-presenting cell subsets: ultraviolet-induced epidermal macrophages versus Langerhans cells J. Invest. Dermatol. 105,383-387[Medline]This article has been cited by other articles:
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  D. M. Aronoff, Y. Hao, J. Chung, N. Coleman, C. Lewis, C. M. Peres, C. H. Serezani, G.-H. Chen, N. Flamand, T. G. Brock, et al. Misoprostol Impairs Female Reproductive Tract Innate Immunity against Clostridium sordellii J. Immunol., June 15, 2008; 180(12): 8222 - 8230. [Abstract] [Full Text] [PDF]
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C. Lau, X. Wang, L. Song, M. North, S. Wiehler, D. Proud, and C.-W. Chow Syk Associates with Clathrin and Mediates Phosphatidylinositol 3-Kinase Activation during Human Rhinovirus Internalization J. Immunol., January 15, 2008; 180(2): 870 - 880. [Abstract] [Full Text] [PDF]
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C. M. Peres, D. M. Aronoff, C. H. Serezani, N. Flamand, L. H. Faccioli, and M. Peters-Golden Specific Leukotriene Receptors Couple to Distinct G Proteins to Effect Stimulation of Alveolar Macrophage Host Defense Functions J. Immunol., October 15, 2007; 179(8): 5454 - 5461. [Abstract] [Full Text] [PDF]
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