Published online before print February 24, 2006
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Instituto de Biología y Genética Molecular, Consejo Superior de Investigaciones Científicas and Universidad de Valladolid, Spain
1Correspondence: Instituto de Biología y Genética Molecular, Calle Sanz y Forés s/n, 47003-Valladolid, Spain. E-mail: mscres{at}ibgm.uva.es
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1 and C2, the MAPK cascade, and the Akt/PKB route. Linkage of C3bi to the -chain of IgG produced a decrease of the protein bands displaying tyrosine phosphorylation, whereas the MAPK cascades and the Akt/PKB route remained almost unaffected. Zymosan particles, which because of their ß-glucan content mimic the effect of fungi, produced a limited increase of tyrosine-phosphorylated protein bands, whereas treatment of zymosan under conditions adequate for C3bi coating increased its ability to induce protein tyrosine phosphorylation. Noteworthy, this was also observed under conditions where other components of serum might be bound by zymosan particles, for instance, serum IgG, thereby suggesting their potential involvement in Syk activation. The induction of cytokines showed a changing pattern consistent with the changes observed in the signaling pathways. IC induced monocyte chemoattractant protein-1 (MCP-1)/CC chemokine ligand 2 (CCL2), interleukin (IL)-1ß, and eotaxin-2/CCL24, which were not observed with C3bi-coated IC. Zymosan induced the expression of tumor necrosis factor 
(TNF-), TNF-ß, IL-10, IL-6, and MCP-2/CCL8, whereas the cytokine signature of C3bi-coated zymosan also included interferon-inducible protein 10/CXC chemokine ligand 10, platelet-derived growth factor-BB, and I-309/CCL1. Taken together, these findings indicate that C3bi targets the phagocytic cargo, and engagement or diversion of the Syk route determines the phagocyte response.
Key Words: Fc receptors • phagocytosis
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-mannose moieties. Polysaccharides composed of ß-glucan repeats can bind complement receptor 3 (CR3; membrane-activated complex-1, Mß2 integrin, CD18/CD11b) [4
] and dectin-1, a proinflammatory, nonopsonic receptor [5
6
7
]. Polymorphonuclear and mononuclear phagocytes express receptors for the Fc portion of immunoglobulin G (IgG; FcRs) antibodies, which are of prime importance in the production of immune-mediated tissue injury [8
, 9
], and for molecules such as C3bi [10
] and C5a [11
], which are generated upon activation of the complement system. In a previous study, using preformed immune complexes (IC), we have observed that covalent binding of C3bi to the Fc portion of IgG integrated into the IC network blocked the ability of IC to release arachidonic acid (AA) from human monocytes, whereas binding of C3bi to zymosan (a polysaccharide of yeast wall mainly composed of ß-glucan moieties) significantly enhanced its ability to release AA. This finding was taken as an indication that coupling of C3bi to IC formed during adaptive immune responses in the presence of complement factors might divert cell signaling events from the FcR route to a CR3-dependent pathway with reduced proinflammatory activity [12
], whereas binding of C3 by PAMP under conditions similar to those occurring during the innate immune response enhances the inflammatory response. Based on these findings, we have studied the signaling events triggered downstream from the engagement of FcR and CR3 in human monocytes, looking for possible cross-talk mechanisms and focusing on the analysis of the phosphorylation cascades coupled to these receptors and the production of cytokines. As a result of the diversity of routes potentially involved, we focused on the activation of spleen tyrosine kinase (Syk), a 72-kDa nonreceptor protein kinase enclosing tandem Src homology domains, the phosphorylation of which allows binding to the immunoreceptor tyrosine-based activation motifs (ITAM) of FcR [13
] and has also been associated with ß-integrin signaling [14
]; the mitogen-activated protein kinase (MAPK) cascade, which is a general transducer of the response to many stimuli; the serine/threonine kinase Akt/protein kinase B (PKB), in view of the central role of this route in the translational control of many proteins involved in the inflammatory response and its reported association with FcR signaling [15
16
17
]; and the pattern of cytokines induced in monocytic cells. Our findings indicate that coupling of C3bi to IgG inhibits the signaling cascade downstream Syk, whereas the MAPK and the PKB/Akt routes are maintained. The expression of cytokines, a functional readout of these signaling events, showed a net enhancement of the production of interleukin (IL)-1ß, monocyte chemoattractant protein-1 (MCP-1)/CC chemokine ligand 2 (CCL2), and eotaxin-2/CCL24 by THP-1 cells in response to IC, whereas this production was inhibited by C3bi coating of the IC. Conversely, C3bi coating of zymosan particles, which are natural ligands of dectin-1 and the parent receptor dendritic cell (DC)-specific intercellular adhesion molecule (ICAM)-grabbing nonintengrin receptor, enhanced the expression of proinflammatory proteins. Taken together, these data indicate that coupling of C3bi to IgG-containing IC inhibits signaling events dependent on Syk targets, whereas the PKB/Akt and the MAPK routes remain functional. These data underline the anti-inflammatory effect of C3bi coating of IC during adaptive immune responses and the opposite effect produced on ß-glucan particles. |
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1 (#2822), anti-human PLC2 (#3872), anti-human Akt (#9272), and anti-human p38 MAPK (#9212) antibodies were from Cell Signaling Technology, Beverly, MA). Affinity-purified anti-human stress-activated protein kinase/Jun N-terminal kinase (SAPK/JNK) rabbit antibody (AF1387) was from R&D Systems (Minneapolis, MN). Phosphospecific anti-human Syk antibody reactive to the activation loop site pY525/526 (#2711, rabbit, a marker of Syk activity), antiphospho-PLC1 (#2821, rabbit, Y-783), antiphospho-PLC2 (#3874, rabbit, Y-759), antiphospho-SAPK/JNK (#9255, mAb), antiphospho-p38 MAPK (#9216, mAb, T-180/Y-182), antiphospho-Akt substrate (#9611, rabbit, R/K-X-R/K-X-X-T/S), and antiphosho-Akt (#9271, rabbit, S-473) antibodies were from Cell Signaling Technology. Anti-ACTIVE® MAPK polyclonal antibodies were from Promega (Madison, WI).
Binding of C3 to IC
To obtain IC bound covalently to C3bi, IC were incubated with normal human serum (NHS) under conditions that allow the activation of the complement system through the alternative pathway. For this purpose, 100 µl suspension containing 100 µg IC was incubated for 15 min with 800 µl NHS depleted of Ca2+ and Mg2+ by treatment with Chelex-100 and then supplemented with 5 mM Mg2+. The reaction was stopped with 1 ml ice-cold phosphate-buffered saline (PBS), and the precipitates were washed three times with the same buffer [18
].
Preparation of IgG-coated latex beads
OVA was coupled to 1 µm carboxylate-modified latex beads by incubation with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. Coating of the OVA-coupled beads with IgG was carried out by incubation with anti-OVA IgG purified by protein G chromatography. Anti-OVA F(ab')2 was obtained by pepsin digestion of purified IgG from rabbit anti-OVA antiserum, followed by protein A chromatography to remove undigested IgG and Fc fragments. Coupling of C3bi to latex beads was carried out on beads coated with anti-OVA IgG and F(ab')2 [12
].
Cell culture
THP-1 cells were cultured in RPMI-1640 medium supplemented with 2 mM glutamine and 10% heat-inactivated fetal bovine serum. Human monocytes were isolated from peripheral blood of healthy volunteer donors by centrifugation onto Ficoll cushions and adherence to plastic dishes for 2 h. Nonadhered cells were removed by extensive washing. To carry out experiments of stimulation of adhering THP-1 cells via the ß2-integrin route with fibrinogen, tissue-culture plates were coated with 20 µg/ml fibrinogen (Sigma-Aldrich, St. Louis, MO) overnight at 4°C and then washed with PBS three times [19
]. Blocking was conducted by incubation with 3% bovine serum albumin for 1 h, followed by washing with PBS.
Immunoblotting of protein tyrosine phosphorylation
Protein extracts were collected in ice-cold extraction buffer containing 50 mM Hepes, 1 mM EGTA, 10% glycerol, 10 µg/ml leupeptin, and 1 mM phenylmethylsulfonyl fluoride (PMSF), pH 7.4. The amount of protein in each cell lysate was assayed using the Bradford reagent, and 100 µg protein sample was loaded on each lane of a 10% acrylamide sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS/PAGE) gel. Nonspecific protein-binding sites in the membranes were blocked with 5% milk in Tris-buffered saline, supplemented with 0.05% Tween®-20, and incubated with antiphosphotyrosine mAb. This was followed by incubation with horseradish-peroxidase (HRP)-conjugated goat anti-mouse IgG antibody. Detection was performed using the enhanced chemiluminescence (ECL) system (Amersham, Little Chalfont, UK).
Immunoprecipitation of Syk
Cells were lysed in 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 5 mM EDTA, containing 1% Nonidet P-40, 1 mM Na3VO4, 10 µg/ml aprotinin and leupeptin, 100 µg/ml soybean trypsin inhibitor, and 1 mM PMSF and clarified by centrifugation at 15,000 revolutions per minute for 20 min. The clarified lysates were preabsorbed on protein G-Sepharose and then incubated with anti-Syk antibodies for 4 h, followed by overnight incubation with protein G-Sepharose beads at 4°C with continuous shaking. Immune complexes were washed three times in lysis buffer, once in lysis buffer with 0.5 M NaCl, and again in lysis buffer and suspended in Laemmli sample buffer and subjected to SDS/PAGE. The extent of tyrosine phosphorylation and the amount of Syk in each sample were determined by immunoblot with antiphosphotyrosine mAb and rabbit anti-Syk antibodies and quantitated by densitometric scanning of the protein bands.
Assay of proinflammatory protein release
Culture supernatants were incubated with RayBio® Human Inflammation Antibody Array III from RayBiotech Inc. (Norcross, GA) and developed according to the manufacturer’s instructions. For this purpose, membranes were incubated with conditioned medium, followed by washing, incubation with HRP-conjugated streptavidin, and detection reaction with the ECL system. Blot densities were assayed using Quantity One® software from BioRad Laboratories (Hercules, CA) and normalized with the positive controls incorporated into the membranes. Only variations on control values showing a larger-than-twofold increase on basal densities were considered positive.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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R induces protein tyrosine phosphorylation reactions, which are blunted by C3bi coating
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Figure 1. Effect of C3bi coupling on protein tyrosine phosphorylation reactions elicited by FcR cross-linking and zymosan particles. THP-1 cells were incubated with 100 µg/ml OVA/anti-OVA IC (A and C) and 1 mg/ml zymosan particles (B), coupled or not to C3bi, and at the times indicated, the cell lysates were collected and used for the detection of tyrosine-phosphorylated protein bands. The effect of OVA-coupled latex beads coated with IgG or F(ab')2 anti-OVA is shown in D. Coupling of C3bi to latex beads was carried out by incubation with 1200 µl NHS, followed by extensive washing with ice-cold PBS. The stimulation of the cells was carried out with 200 µl/ml solution of 1% latex beads. These are a representative experiment of five with an identical pattern. P-Y, Phosphotyrosine.
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55-kDa and the 116-kDa protein bands were weakly stained above the extent observed in the controls, which agrees with previous reports indicating that dectin-1 differs from FcR in its ability to activate PTK [21
], although it contains an ITAM-like domain, which has been associated with the reported activation of Src PTK by zymosan particles [22
] and might recruit Syk in murine DC [23
]. Coupling of C3bi to zymosan particles allowed the appearance of additional protein bands, i.e., 55 kDa and 70 kDa Mr protein bands (Fig. 1B
and 1C)
, thus indicating that C3bi-coated zymosan, which has the property of acting on the I domain of CR3 because of its C3bi moiety [24
] and on the lectin-like domain of CR3 or on dectin-1 because of its ß-glucan component, triggers signaling events, which are not provided by isolated ß-glucan moieties nor by C3bi coupled into another frame, for instance, the Fc portion of IgG. To further delineate the protein tyrosine phosphorylation reactions specifically triggered by Fc portions and C3bi, experiments using OVA-coupled latex beads coated with F(ab')2 and F(ab')2 coupled to C3bi were carried out. Latex beads coated with anti-OVA IgG behaved as a robust stimulus similar to preformed IC, whereas latex beads coated with anti-OVA antibodies coupled to C3bi only produced a faint staining of some protein bands (Fig. 1D)
. When the experiments were conducted with latex beads coated with F(ab')2 anti-OVA and F(ab')2-C3bi, the effect on tyrosine phosphorylation of the protein bands was rather similar to that produced by the control OVA-coupled latex beads, thus indicating the absence of significant protein tyrosine phosphorylation under these conditions.
To confirm the physiological relevance of the results obtained in THP-1 cells, additional studies were conducted in human monocytes, which when adhered overnight on plastic dishes, showed a high level of phosphotyrosine in the absence of soluble stimuli (not shown), most likely explained by the occurrence of adhesion-dependent activation. As this impeded the appraisal of agonist-induced protein tyrosine phosphorylation, further experiments were conducted after 48 h to allow down-regulation of the signals. As shown in Figure 2A
and 2B
, the pattern of phosphorylation in tyrosine was similar to that observed in THP-1 cells, although prominent tyrosine phosphorylation was also detected in the 48-kDa cluster. Moreover, IC induced the appearance of tyrosine-phosphorylated bands in the 55-kDa, the 70-kDa, and the 100-kDa clusters, which is a strong argument in favor of the physiological relevance of the results observed in THP-1 cells.
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Figure 2. Effect of C3bi coupling on protein tyrosine phosphorylation reactions elicited by IC and zymosan particles. Human monocytes were adhered to plastic dishes and after 48 h of culture at 37°C, were incubated with 100 µg/ml IC (A) and 1 mg/ml zymosan particles (B), coupled or not to C3bi. At the times indicated, cell lysates were collected and used for the detection of tyrosine-phosphorylated protein bands. This is a representative experiment of three with an identical pattern.
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Figure 3. Effect of C3bi coupling on Syk phosphorylation elicited by FcR cross-linking and zymosan particles. THP-1 cells were treated as described in the legend to Figure 1
, and at the times indicated, the cell lysates were used for the detection of phosphorylated Syk (P-Syk) using phosphospecific anti-human Syk antibodies reactive to the activation loop site pY525/526 (A–C). The load of protein in the different lanes was addressed by using anti-Syk antibodies. These are representative experiments of three with an identical pattern.
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Figure 4. Effect of C3bi coupling on Syk phosphorylation elicited by FcR cross-linking and zymosan particles in human monocytes. Adhered human monocytes were stimulated with OVA/anti-OVA IC (A) and zymosan (B) at the concentrations described in the legend to Figure 2
, and at the times indicated, the cell lysates were collected and used for the detection of P-Syk and P-ERK-2 with phosphospecific antibodies. The load of protein in the different lanes was addressed by using anti-Syk and anti-ERK-2 antibodies. These are representative experiments of three with an identical pattern. The effect of C3bi coupling on the protein tyrosine phosphorylation of Syk elicited by OVA/anti-OVA IC and zymosan was addressed by immunoprecipitation with 1.5 µg anti-Syk antibodies and detection with antiphosphotyrosine antibody in THP-1 cells and adhered human monocytes. Blots were quantitated by densitometric scanning, and results are shown after normalization for the amount of Syk loaded in each lane. In the case of monocytes, blotting with phosphospecific anti-human Syk antibodies reactive to the activation loop was also carried out (C). A.U., Arbitrary units.
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Figure 5. Effect of serum components on zymosan effects and cooperation of zymosan and peptidoglycan with CR3 signaling. THP-1 monocytes were incubated with 1 mg/ml zymosan particles treated with NHS, serum depleted of Ca2+ and Mg2+ by treatment with Chelex-100, and purified rabbit IgG to allow coating with C3bi and/or IgG. Cell lysates were used for the detection of P-Syk and P-ERK-2. This is a representative experiment of three with an identical pattern (A). THP-1 monocytes were incubated with 100 µg/ml OVA/anti-OVA IC, OVA/anti-OVA IC coupled to C3bi, 30 µg/ml S. aureus peptidoglycan, and a combination of stimuli to address the possible cooperation of these stimuli on Syk and ERK-2. This is a representative experiment of two showing an identical pattern (B). THP-1 cells were placed on tissue-culture plates coated with fibrinogen, and at the times indicated, cell lysates were collected for the detection of protein tyrosine, Syk, and ERK-2 phosphorylation (C).
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R by IgG bound into the particles rather than by the ß-glucan and C3bi moieties; that the close proximity of C3bi and ß-glucan or the presence of an additional ligand, for instance, IgG, is necessary for optimal response; and that the effect of ß-glucan is not mimicked by peptidoglycan, another ligand of TLR-2.
Characterization of signaling routes downstream Syk phosphorylation
To address possible targets of Syk, the tyrosine phosphorylation of PLC1 and PLC2 was addressed in THP-1 cells. Tyrosine phosphorylation of PLC1 and PLC2 was observed as soon as 5 min after stimulation with IC, remained for 60 min (Fig. 6A
), and was also observed in response to OVA-coupled latex beads coated with IgG (Fig. 6B)
and zymosan-C3bi particles (Fig. 6C)
, whereas this was not observed in response to C3bi-coated IC. It is noteworthy that coupling of C3bi to IC allowed the activation of the MAPK cascade in monocytes (Fig. 4A)
. Zymosan and C3bi-coated zymosan particles exerted a clear effect on the MAPK route, as judged from the phosphorylation of ERK-2 and c-JNK (Fig. 7A
), which was maintained up to 60 min after addition of the stimulus. Unlike FcR-dependent mechanisms, the phosphorylation of the components of the MAPK route by C3bi-coated stimuli (Fig. 7A
and 7B)
is not preceded by Syk phosphorylation, thus suggesting that MAPK activation by ß-glucan, C3bi, and fibrinogen is Syk-independent and can occur in the absence of noticeable phosphorylation of Syk, PLC1, and PLC2.
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Figure 6. Effect of C3bi coupling on the protein tyrosine phosphorylation of PLC1 and PLC2. THP-1 cells were stimulated with IC (A), OVA-coupled latex beads (B), and zymosan particles (C), coupled or not to C3bi, at the concentrations described in the legend to Figure 1
. At the times indicated, cell lysates were collected to assess tyrosine phosphorylation of PLC1 and PLC2. The load of protein in the different lanes was addressed using antibodies directed against PLC1 and PLC2. This is a representative experiment of four with an identical pattern.
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Figure 7. Effect of zymosan and FcR cross-linking on the MAPK and Akt routes. THP-1 cells were stimulated as indicated and used for the detection of ERK-2, JNK, and Akt phosphorylation. These are representative experiments of three with an identical pattern.
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R cross-linking, we analyzed the activation of this kinase with anti-P-S-473 Akt and by assaying the presence of phosphorylated Akt substrates with a phosphospecific antibody for Akt substrates. As shown in the top blot in Figure 8
, cross-linking FcR did activate the Akt route, as judged from the appearance of a cluster of protein bands showing positive staining with antiphospho-Akt substrate antibodies. It is noteworthy that phosphorylation of Akt was also observed in response to C3bi-coated IC, as well as after zymosan challenge, as judged from the positive staining with anti-P-S-473-Akt antibodies (Fig. 7A)
, thus mimicking the results observed regarding the activation of the MAPK cascade and indicating that the Akt route is activated by the FcR route, the CR3 pathway, and the dectin-1/TLR-2 mechanism.
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Figure 8. Effect of C3bi coupling to IgG on the Akt route. THP-1 cells were incubated with 100 µg/ml OVA/anti-OVA IC, coupled or not to C3bi, and the cell lysates were collected and used for the detection of Akt substrates by immunoblotting with antiphospho-Akt substrate antibodies (top panel). Phosphorylated proteins were detected with phosphospecific antibodies. This is a representative experiment of three with an identical pattern.
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R cross-linking. As shown in Figure 9
and Table 1
, several proteins were detected in the supernatant of resting THP-1 cells, including the chemokines macrophage-inflammatory protein (MIP)-1ß/CCL4, IL-8/CXC chemokine ligand 8 (CXCL8), and regulated on activation, normal T cell expressed and secreted (RANTES)/CCL5, the soluble receptors IL-6sR and tumor necrosis factor (sTNF-R)II, and tissue inhibitor of metalloproteinase-2 (TIMP-2). Stimulation with IC enhanced the expression of MIP-1ß, RANTES, IL-6sR, and sTNF-RII near twofold above basal expression, but most important, it induced the production of MCP-1/CCL2, IL-1ß, and eotaxin-2/CCL24, which were not present in the conditioned medium from resting cells. Incubation with C3bi-coated IC did not produce the release of these cytokines, which indicates that MCP-1/CCL2, IL-1ß, and eotaxin-2/CCL24 constitute the cytokine signature of FcR stimulation.
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Figure 9. Effect of different stimuli on induction of the inflammatory proteins. THP-1 monocytes were incubated overnight with 100 µg/ml OVA/anti-OVA IC, 100 µg/ml OVA/anti-OVA IC coated with C3bi, 1 mg/ml zymosan, and 1 mg/ml C3bi-coated zymosan. The conditioned medium from these incubations was used for the assay of proinflammatory proteins with RayBio® Human Inflammation Antibody Array III. The presence of the dots corresponding to the proteins is marked by circles or by arrows. PDGF-BB, Platelet-derived growth factor-BB.
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Table 1. Inflammatory Proteins Produced by THP-1 Monocytes upon Stimulation with Different Agonists
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, TNF-ß, IL-10, IL-6, IL-12 p40, and MCP-2/CCL8, whereas the cytokine signature of C3bi-coated zymosan included IP-10/CXCL10, PDGF-BB, and I-309/CCL1, in addition to the cytokines induced by zymosan. |
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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1, and PLC2 as a distinct feature of FcR cross-linking in THP-1 monocytes, whereas other stimuli such as fibrinogen and C3bi, which bind to the I-domain of CR3, failed to do so. Moreover, we did not observe any evidence of the activation of Syk by archetypical stimuli acting on PRR such as zymosan particles and peptidoglycan, although the productive binding of these stimuli was confirmed by their ability to activate several modules of the MAPK route and the induction of cytokine expression. In contrast, C3bi-coated zymosan induced Syk phosphorylation between 15 and 30 min after its addition, which represents a significant difference from the pattern of early Syk activation produced by FcR engagement and might suggest a mechanism of activation other than that related to binding to the tyrosine-phosphorylated ITAM motifs of FcR. Nevertheless, as zymosan particles, similar to other microbial-derived products, can bind IgG [28
], it cannot be ruled out that a portion of zymosan effects might involve FcR-mediated events or another opsonin. Taken collectively, these findings underline the distinct signaling pathways that can be activated, depending on the availability of antibodies, PAMP, and complement factors, all of which can produce direct signaling and cross-talk with other stimuli.
Syk has been associated with integrin signaling in platelets [29
], polymorphonuclear leukocytes (PMN) [30
, 31
], and monocytes [14
, 32
, 33
], as well as with selectins via the actin-linking proteins moesin and ezrin, which interact directly with Syk [34
]. However, the functional consequences of this association might differ according to the type of ligands interacting with ß1 and ß2 integrins and the presence of further signals. In fact, experiments directed to compare activation of PMN by physiological ligands of ß2 integrins with the stimulation produced by immobilized anti-integrin antibodies have disclosed the need for costimulation provided by TNF-Rs or by FcR to trigger the respiratory burst [35
]. Our data agree with this notion by indicating the need of additional signals for the activation of Syk in response to the engagement of CR3 by C3bi, as judged from the results obtained with C3bi-coated zymosan particles, although it is not fully clear as yet whether dectin-1 or the lectin-like domain of CR3 might be involved in the generation of the costimulatory signals. Moreover, the complexity of potential ligands enclosed in zymosan particles can be more intricate than previously suspected, as binding of IgG could provide free Fc for interaction with FcR, thus mimicking the costimulation of integrin signaling by anti-integrin antibodies, as judged from the effect of zymosan particles coated with serum in the absence of Ca2+ and Mg2+ and IgG-coated zymosan particles. We have not observed blatant differences regarding activation of MAPK and Akt/PKB by the FcR route, the CR3 pathway, and the TLR-2 system, which suggests a wide convergence of receptors on the activation of these routes.
FcR cross-linking produces a significant induction of a functionally important set of cytokines such as IL-1ß, MCP-1/CCL2, MIP-1ß/CCL4, and eotaxin-2/CCL24, whereas this is not observed with C3bi-coated IC and agrees with previous studies about AA release by human monocytes, where C3bi-coated IC elicited a reduced release of AA as compared with native IC [12
]. The pattern of expression of chemokine proteins induced by FcR cross-linking is consistent with previous studies addressing the induction of their mRNAs using RNase protection assays [36
]. In fact, the expression of RANTES/CCL5 was observed in resting cells, although its expression level increased after stimulation with a more delayed pattern than that observed for the mRNA expression of MCP-1/CCL2, MIP-1/CCL3, and MIP-1ß/CCL4. Analysis of the pattern of activation of several transcription factors, selected on the basis of the binding sites displayed in their promoters, showed the activation of activated protein-1, nuclear factor (NF)-
B, and CCAAT/enhancer-binding protein (C/EBP)ß [37
, 38
], thereby pointing to an effect exerted at the transcriptional level of expression regulation. Of note, NF-B and C/EBPß are crucial transcription factors involved in the transcriptional regulation of IL-1ß [38
], a central cytokine of the FcR signature in THP-1 cells.
The effect of ß-glucan on cytokine production by THP-1 cells seems to be enhanced by CR3 signaling, as judged from the effect of C3bi coating. This allows the depiction of the pattern of cytokine production in the transition of innate to adaptive immunity as a series of changes initiated by microbial invasion, which involves the generation of a large list of proinflammatory cytokines and chemokines, reaching its maximal intensity when a concomitant activation of the complement system takes place. When the microbial invasion is followed by the appearance of antibodies, this targets the antigen burden to the FcR route, thereby allowing a focalization of the inflammatory response around IL-1ß and chemokines involved in the recruitment of monocytes (MCP-1/CCL2 and MIP-1ß/CCL4) and a net reduction of the production of other cytokines, which is decreased further when there is a significant formation of adducts between the IgG -chain and C3bi.
Syk function seems dispensable for the innate-immune response and for the outside-in signaling of ß2-integrins elicited by C3bi. Unlike other PTK, Syk is also dispensable for the cytokinergic response in rodent mast cells [39
], whereas the activation of PLC1 and PLC2 in a Syk-dependent manner is a unique route providing 1,2-diacylglycerol and inositol 1,4,5-trisphosphate from phosphatidylinositol 4,5-bisphosphate hydrolysis, thus allowing the activation of PKC and the mobilization of Ca2+, which provides optimal conditions for the activation of the AA cascade [40
].
Received December 1, 2005; revised January 9, 2006; accepted January 18, 2006.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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RIII (CD16)-deficient mice Immunity 5,181-188[CrossRef][Medline]R and the complement receptor 3 pathways J. Biol. Chem. 278,52179-52187R-mediated phagocytosis J. Cell Biol. 161,1151-1161 receptor-mediated phagocytosis in murine macrophages through the activation of p70S6 kinase J. Biol. Chem. 279,54416-54425R signaling in primary human microglia: differential roles of PI-3K and Ras/ERK MAPK pathways in phagocytosis and chemokine induction J. Leukoc. Biol. 75,1147-1155IIbß3 EMBO J. 16,6414-6425[CrossRef][Medline]Rs: full activation requires both integrin and nonintegrin signals J. Immunol. 173,2068-2077 receptors induces the expression of macrophage-inflammatory protein (MIP)-1, MIP-1ß, and RANTES J. Immunol. 169,3321-3328R receptors induces monocyte chemoattractant protein-1 in the human monocytic cell line THP-1 by a mechanism involving IB- degradation and formation of p50/p65 NF-B/Rel complexes Int. Immunol. 12,547-554B-and C/EBPß-driven interleukin-1ß gene expression and PAK1-mediated caspase 1 activation play essential roles in interleukin-1ß release from Helicobacter pylori lipopolysaccharide (LPS)-stimulated macrophages J. Biol. Chem. 280,4279-4288[Medline]
receptor I signals for receptor up-regulation and internalization from degranulation, cytokine production, and survival J. Immunol. 173,4317-4323 reigns supreme in arthritis and bone resorption Trends Immunol. 25,116-119[CrossRef][Medline]This article has been cited by other articles:
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