|
|||||||||
|
|
|
|
|
|
|
|
|
|
|
Blood, 15 January 2006, Vol. 107, No. 2, pp. 610-618. Prepublished online as a Blood First Edition Paper on September 20, 2005; DOI 10.1182/blood-2005-06-2271.
IMMUNOBIOLOGY
Fc
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and methodsResultsDiscussionReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
TLRs interact with ligands as homodimers or, as noted for TLR2, heterodimers, and thus recruit cytosolic adaptor molecules that include myeloid differentiation protein 88 (MyD88), MyD88-adaptor like/TIR-associated protein (MAL/TIRAP), Toll-receptor–associated activator of interferon (TRIF), and Toll-receptor–associated molecule (TRAM).16,18,19 The recruitment of MyD88 by TLR2 and TLR4, the focus of the present study, results in sequential activation of protein kinase interleukin-1 (IL-1) receptor-associated kinases (IRAKs) and transforming growth factor-
–activated kinase (TAK1). TAK1, in turn, regulates activation of mitogen-activated protein (MAP) kinases and the transcription factor, nuclear factor 
B (NF-B), with ensuing production of inflammatory cytokines. TLR4 also activates a MyD88-independent pathway through TRAM and TRIF that uses interferon (IFN)–regulatory factor (IRF3) and NF-B to induce production of IFN-. In addition, TLRs engage signaling molecules that are commonly used by other types of receptors, such as phosphatidylinositol 3-kinase (PI3K), AKT, and possibly Src kinases, in addition to the MAP kinases.15 However, the contribution of these "common" signaling molecules and their downstream targets to the overall response to TLR ligands is uncertain.
IgE-dependent allergic diseases are initiated by multivalent binding of allergens to IgE that is bound to receptors with high affinity for IgE (Fc
RI) on mast cells.20 Mast cells also express functional TLRs,21 which may account for the protection conferred by mast cells against bacterial and parasitic infections in animal models.22,23 Activated mast cells release an array of potent inflammatory mediators by rapid discharge of preformed mediators in granules, the generation of inflammatory lipids from arachidonic acid, and the production of numerous Th2-type cytokines and chemokines.23 All these responses are evoked by allergens via FcRI, while stimulation of mast cells via TLR2 and TLR4 results primarily in generation of cytokines such as IL-4, IL-5, IL-6, IL-10, IL-13, and TNF
.24 There are reports that TLR2 ligands also induce degranulation and generation of leukotriene C4,22,25,26 but current models for TLR signaling do not define a clear mechanism for these responses, and such responses have not been observed in all studies.27 Although less well studied, TLR1, TLR3, TLR6, TLR7, TLR8, and TLR9 are variably expressed in mast cells of different origins.25,28-30
Stimulation of mast cells through FcR1 leads to activation of Src kinases and Syk. The ensuing phosphorylation of adaptor proteins enables recruitment of other signaling molecules and propagation of downstream signals.31 Signaling molecules so recruited include phosphatidylinositol 3-kinase (PI3K), phospholipase (PL) C
1/2, and Ras. A PLC-mediated increase in intracellular levels of free Ca2+ and activation of protein kinase C provide essential signals for degranulation,20,32 whereas the activation of the Ras/Raf/MAP kinase pathway results in production of arachidonic acid.33 These signaling pathways also regulate activation of transcription factors such as NF-B, c-Jun, c-Fos, ATF2, and NFAT, to promote cytokine-gene transcription (Hundley et al34 and citations therein). The repertoire of activated transcription factors is expanded further on co-stimulation of mast cells with antigen and Kit ligand (stem cell factor). As a consequence, the production of cytokines is markedly augmented in a synergistic manner.34
Here we show that FcRI also interacts synergistically with TLR2 and TLR4 in several rodent mast cell lines. Both types of receptor use unique as well as common signaling pathways, which in combination reinforce the activation of cytokine-related transcription factors. This results in up to 20-fold enhancement in production of cytokines, an enhancement that may contribute to the exacerbation of allergic symptoms by infectious agents.
|
Materials and methods |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Reagents were obtained from the following sources: media and culture reagents from Invitrogen/GIBCO (Carlsbad, CA); recombinant mouse IL-3 from Pepro Tech (Rocky Hill, NJ); monoclonal anti–DNP IgE antibody and the antigen, dinitrophenylated human serum albumin (DNP-HSA), lipopolysaccharide (Escherichia coli 055:B5), and peptidoglycan (Staphylococcus aureus) from Sigma (St Louis, MO); Pam3Cys-Ser-(Lys)4 3HCl and MALP2 from EMC microcollections GmbH (Tuebingen, Germany); myelin basic protein and antibodies against c-Fos from Upstate (Lake Placid, NY); antibody against IRAK1 from Santa Cruz Biotechnology (Santa Cruz, CA); polyclonal antibodies that detect activating phosphorylations of Akt(Ser473), ERK-2 (Thr202/Tyr204), JNK (Thr183/Tyr185),35 p38 MAP kinase (Thr180/Tyr182),36 c-Jun (Ser63),37 ATF-2 (Thr71),38 STAT1 (Ser727), STAT1 (Tyr701), STAT3 (Ser727), STAT3 (Tyr705), STAT5 (Tyr694), STAT6 (Tyr641),39-41 and NF-B p65 (Ser536)42 as well as the proteins themselves from Cell Signaling Technology (Beverly, ME); [14C]arachidonic acid from PerkinElmer Life and Analytical Sciences (Boston, MA). LPS from other strains of E coli and P3C from other companies were found to have similar activities as those described here for the products described in this section. All other chemicals were molecular biology grade from several sources.
Culture of mast cell lines and experimental conditions
MC/9 were grown in suspension in 75-mm2 flask (3 x 105 cells/mL) in RPMI 1640 supplemented with 15% fetal calf serum, 30 ng/mL IL-3, 2 mM glutamine, 100 µM nonessential amino acids, 10 µM 2-mercaptoethanol, and 1 mM sodium pyruvate. Primary bone marrow–derived mast cells (BMMCs) were obtained from C57BL/6 mice by previously described procedures43 and grown in the same medium as MC/9 cells. A spontaneously transformed IL-3–dependent BMMC culture line (tBMMC) and RBL-2H3 cells were cultured as in previous studies.44 Cultures were sensitized to DNP-HSA by incubation at 37°C for 18 hours with 50 ng/mL DNP-IgE of FcR1 by IgE. Cultures were stimulated with concentrations of ligands that achieved maximal or near maximal responses (data not shown) except as indicated for experiments where concentrations were varied.
Measurement of degranulation and release of [14C]arachidonic acid
For these measurements, cultures were washed and medium replaced with a PIPES-buffered saline medium before stimulation.33 Degranulation was determined by measurement of release of the granule marker -hexosaminidase by use of a colorimetric assay in which release of p-nitrophenol from p-nitrophenyl-N-acetyl--D-glucosaminide is measured. Values were expressed as the percent of intracellular -hexosaminidase that was released into the medium.32 Release of radiolabeled arachidonic acid was determined in cultures that had been labeled to equilibrium by incubation overnight with [14C]arachidonic acid (0.1 µCi [0.0037 MBq]/mL medium) and then washed. The amount of radiolabel released into the medium was expressed as a percent of intracellular [14C]lipids in nonstimulated cells.33
Assay of cytokine mRNA and protein
Individual cytokines (TNF, IL-6, and IL-13) were determined by enzyme-linked immunosorbent assay (ELISA) kits (Biosource, Camarillo, CA). For these determinations, cultures were incubated for 3 hours, unless stated otherwise, with the added stimulants in complete growth medium for measurement of release of cytokines into the medium. To examine the array of cytokines produced, a mouse cytokine antibody array (Ray Biotech, Norcross, GA) was used. A reference standard, biotin-labeled IgG on each array allowed determination of the relative changes in cytokine concentration.
Measurement of cytosolic Ca2+
Cells were incubated with 2:M Fura-2 AM ester (Molecular Probes, Eugene, OR) for 30 minutes in 96-well black culture plates (CulturPlate-96 F, Perkin Elmer Life Sciences; 5 x 104 cells/0.1 mL/well). Fluorescence was monitored in a Wallac VICTOR2 plate reader (Perkin Elmer Life Sciences) with alternating excitation at 340 nm and 380 nm and an emission wavelength set at 510 nm. Data were corrected for background fluorescence of cells that did not contain Fura-2, and the ratio of fluorescence at 340 nm and at 380 nm was determined.
Assay of IRAK activity
Immunoprecipitated IRAK1 was incubated at 30°C for 30 minutes with 1 µg of bacterially expressed myelin basic protein (MBP) as substrate in 20 µL of a kinase buffer: 10 mM HEPES (pH7.4), 1 mM dithiothreitol, 5 mM MgCl2, and 5 µCi (0.185 MBq) [-32/P]-ATP (3000 Ci/mmol [111 Bq/mmol]). Samples were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), and the dried gels were then exposed to X-ray film.
Immunoblotting
Cells were washed twice with ice-cold phosphate-buffered saline (PBS) and then lysed (30 minutes) in 0.1 mL lysis buffer: 20 mM HEPES, pH 7.3, 1% Triton X-100, 10% glycerol, 12.5 mM sodium pyrophosphate, 10 mM sodium orthovanadate, 50 mM sodium fluoride, 1 mM phenylmethylsulfonyl fluoride, 30 µg/mL leupeptin, 30 µg/mL aprotinin, and 25 mM p-nitrophenyl phosphate. Proteins were separated by SDS-PAGE and then transferred to nitrocellulose membranes for immunoblotting with the indicated primary antibodies. Immunoblots were visualized by chemiluminescence (Amersham, Piscataway, NJ) and quantitated by densitometry (Molecular Dynamics, Piscataway, NJ).
Expression levels and DNA-binding activity of transcription factors
A nuclear extraction kit (Active Motif, Carlsbad, CA) was used to prepare nuclear extracts. Activated transcription factors were identified by use of oligonucleotide arrays (TranSignal Protein/DNA Arrays, Panomics, Redwood City, CA) according to the manufacturer's instructions. Promotor binding activity was determined for NFATc1, NF-B, and c-Fos by use of TransAM ELISA-based kits (Active Motif). In this procedure, nuclear extracts were added to multiwell plates with an immobilized oligonucleotide that contained the consensus binding site for the given transcription factor. The quantity of bound transcription factor was then determined by use of a primary antibody and a secondary antibody conjugated to horseradish peroxidase in an ELISA-type assay.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
The TLR2/TLR1 ligand, P3C, the TLR2/TLR6 ligands, PGN and MALP2, and the TLR4 ligand LPS were tested for their ability to stimulate degranulation and release of [14C]arachidonic acid. None of these ligands stimulated degranulation or release of [14C]arachidonic acid by themselves (Figure 1A,B) nor augmented such responses to antigen (Figure 1C-F) in MC/9 cells and BMMCs. Similar negative results were obtained in response to high concentrations of LPS (up to 10 µg/mL) and P3C (up to 100 µg/mL), and after prior exposure of cells to conditioned media or growth factors, namely, pokeweed mitogen-stimulated spleen-conditioned medium in the case of primary BMMCs22 and IL-3, IL-4, IFN, WEHI-conditioned media, or stem cell factor in the case of MC/9 cells (data not shown). P3C, PGN, and LPS also failed to stimulate or augment degranulation and release of radiolabeled arachidonic acid in RBL-2H3 cells and the transformed cell line, tBMMC (data not shown).
P3C and LPS act in synergy with antigen to augment production of cytokines
P3C and LPS stimulated release of TNF from MC/9 cells but to a lesser extent than antigen. However, the combination of P3C or LPS with antigen markedly augmented release of TNF (Figure 2). This augmentation was evident at concentrations of antigen (Figure 2A,C) or P3C (Figure 2B) that by themselves elicited minimal or no production of TNF. At concentrations where the individual stimulants caused some production of TNF, the antigen-induced response was augmented by up to 20-fold on co-stimulation with TLR ligand (data from all experiments). Therefore, antigen and the TLR ligands appeared to act in a mutually synergistic manner or even potentiate one another at threshold concentrations. The TNF was apparently newly generated, as kinetic studies revealed no detectable release of TNF 10 minutes after addition of stimulants (data not shown). Synergy or potentiation between antigen and P3C or LPS was observed also in the production of IL-6 (Figure 2D) and IL-13 (Figure 2E,F). As was true for TNF production, P3C was more effective in augmenting production of IL-6 and IL-13 than LPS. Studies with MALP2 (10 and 100 ng/mL) in MC/9 cells also indicated that this ligand acted in synergy with antigen to increase production of TNF from 3.7- to 4.4-fold and IL-13 from 3.1- to 5.3-fold over that expected from the sum of responses to the individual stimulants (data not shown).
|
and IL-6 from primary BMMC cultures derived from C57BL/6 mice (Figure 3). Production of TNF and IL-6 was significantly augmented by the combination of antigen and LPS and P3C (Figure 3A,B). The TLR2/6 ligands PGN and MALP2 also acted in synergy with antigen in stimulating cytokine production but were less active in this regard than LPS and P3C (Figure 3A,B). We note, however, that synergy was most pronounced at threshold and supramaximal concentrations of antigen, where responses to antigen are minimal, and least pronounced at optimal concentrations of antigen. This was so for MALP2 (Figure 3C), PGN, LPS, and P3C in studies conducted in BMMCs as well as MC/9 cells (data not shown). In contrast to the findings in MC/9 cells, LPS was more effective than P3C in augmenting production of cytokines in antigen-stimulated BMMCs (Figure 3A,B). LPS is reported to be less, equi-, or considerably more potent than TLR2 ligands in stimulating cytokine production, depending on the study and mast cell line,22,26,27,29,30,45 a possible reflection of the phenotypic variability of mast cells.
Studies with antibody arrays revealed that in addition to TNF, IL-6, and IL-13, antigen stimulated production of IL-12 p70, TCA-3, monocyte chemoattractant protein (MCP1), and macrophage inflammatory factor (MIP2) in MC/9 cells. In combination with P3C or LPS, production of these cytokines was consistently enhanced to levels several-fold greater than would be expected from additive responses (data not shown).
|
|
FcRI and TLR-related signaling processes were examined to identify possible mechanisms for the synergy between antigen and TLR ligands. The calcium signal was examined first because an increase in concentration of cytosolic Ca2+ is an essential signal for degranulation and for release of arachidonic acid in antigen-stimulated mast cells.20 Figure 4 shows typical results for P3C, LPS, MALP2, and PGN, individually and in combination with antigen. Antigen by itself caused an increase in concentration of cytosolic Ca2+, which reached a maximum 2 to 3 minutes after addition of antigen and then slowly declined in both MC9 cells (Figure 4A,B) and BMMCs (Figure 4C,D). None of the TLR ligands induced an increased cytosolic Ca2+ or enhanced the antigen-induced calcium signal. Indeed, P3C attenuated the calcium response to antigen in MC/9 cells and BMMCs by a mechanism yet to be determined. The absence of a calcium signal most likely accounts for the inability of the TLR ligands to influence degranulation or release of arachidonic acid in our studies.
Activation of protein kinases
Analysis by use of cDNA arrays indicated that MC/9 cells possessed message for TLR2 and TLR4 as well as TLR3, TLR5, TLR6, and all of the associated signaling molecules such as MyD88, MAL/TIRAP, TRAM, TRIF, IRAK1, and TAK1 (data not shown). Examination of IRAK 1 revealed that P3C and LPS induced robust activation of this kinase as determined by an in vitro kinase assay of immunoprecipitated IRAK 1. Antigen, in contrast, did not stimulate IRAK 1 activity and, in fact, partially attenuated IRAK-1 activation when added in combination with TLR ligands (Figure 5A).
|
|
was dependent, to some degree, on all 3 MAP kinases (Figure 5D). For example, the JNK inhibitor SP 60012546 substantially suppressed TNF production whether cells were stimulated with TLR ligands, antigen, or the combination of stimulants. The p38 MAP kinase inhibitor, SB203580,47 completely abrogated the responses to the TLR ligands, but it had minimal or insignificant effect on the TNF response to antigen. PD 98059, an inhibitor of the MEK-1/Erk-pathway,48 partially suppressed production of TNF in response to all stimulants, individually or in combination. Induction and activation of transcription factors
ATF-2, AP-1 binding proteins (ie, c-Jun and c-Fos), and NF-B were initially investigated because these factors are activated via TLR2 or TLR4.49,50 In addition, ATF-2 and c-Jun are the downstream targets of p38 MAP kinase and JNK, respectively. We observed that ATF-2 was most strongly activated by TLR ligands, while c-Jun and c-Fos were activated primarily by antigen. NF-B was activated to the same extent by TLR ligands and antigen. Typical experimental results were as follows.
In regard to ATF-2, LPS and P3C induced robust and persistent phosphorylation of ATF-2 as compared to antigen, and in combination with antigen, this phosphorylation was augmented in an additive manner (Figure 6A). Stimulation also resulted in small increases in levels of ATF-2 protein, but these increases were relatively minor compared to the changes in phosphorylation (Figure 6A). NF-B was activated to the same extent whether activated through TLR ligands or antigen as indicated by its phosphorylation (Figure 5A) and DNA-binding activity (Figure 6B). However, these responses were not significantly augmented, either additively or synergistically, when stimulants were used in combination (Figure 6A,B).
|
We also have investigated the phosphorylation of the signal transducer and activator of transcription (STAT) family of proteins because of the potential paracrine effects of type I IFNs that might be produced as a consequence of TLR signaling through TRAM and TRIF.51 No consistent increase in phosphorylation of STAT3, STAT5, or STAT6 was observed by immunoblotting (see "Materials and methods" for list of antibodies tested) with any stimulant or combination of stimulants in MC/9 cells (data not shown). A minimal increase was observed in serine and tyrosine phosphorylation of STAT1 in response to stimulation by TLR ligands and antigen (data not shown). However, no augmentation was noted with the combination of stimulants. Use of DNA-binding arrays also indicated minimal, if any, increase in binding of STATs to DNA consensus sequences during stimulation of the cells.
Finally, we examined the calcium/calcineurin-dependent activation of NFAT because of its activation and presumed role in mediating transcription of cytokine genes in antigen-stimulated mast cells.52 In contrast to antigen, LPS and P3C failed to stimulate NF-AT DNA-binding activity or to enhance this activity upon co-stimulation of MC/9 cells with antigen and LPS or P3C (Figure 7A). This result was consistent with the lack of a calcium signal in LPS or P3C-stimulated cells (Figure 4). As expected, the stimulation of NF-AT DNA-binding activity by antigen was blocked by prior treatment of cells with the calcineurin inhibitor cyclosporin A (Figure 7A, inset). Cyclosporin A also suppressed the production of TNF (Figure 7B) and IL-6 (Figure 7C) in antigen-stimulated cells, while it had minimal effect on production of these cytokines in cells stimulated with LPS or P3C. In addition, cyclosporine A markedly attenuated the augmented production of TNF and IL-6 in cells co-stimulated with antigen and LPS or P3C (Figure 7B,C). These results implied that activation of NF-AT was essential for production of TNF and IL-6 in response to antigen stimulation but not in response to the TLR ligands. However, the activation of NF-AT by antigen could act in synergy with TLR-induced signals to augment cytokine production. Presumably, the TLR ligands activate signals that can substitute for and act in synergy with NF-AT.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
RI as well as TLRs, which, respectively, participate in allergic diseases and protection against pathogens.24 Moreover, co-stimulation of BMMCs through FcR1 and TLR4 is reported to augment production of cytokines.53 Our data show that TLR2 and TLR4 ligands substantially enhance antigen-induced production of cytokines (Figures 2-3) and the associated signals, without affecting degranulation or production of arachidonic acid (Figure 1) in MC/9 cells and primary cultures of BMMCs. If this scenario is relevant to atopic disease, activation of TLRs would have the most pronounced effect at late stages of the inflammatory response to antigen that coincides with the production of inflammatory cytokines by mast cells.54,55 The pattern of signals activated correlates with the responses noted in the previous paragraph. An increase in levels of cytosolic Ca2+ is an essential signal for degranulation20 and production of arachidonic acid via phospholipase A256 in mast cells. The fact that TLR ligands elicited none of these responses in MC/9 cells, BMMCs, and other mast cell lines is consistent with this requirement for a calcium signal. This is in contrast to stem cell factor, which elicits a calcium signal and enhances antigen-induced degranulation as well as cytokine production.34
Nevertheless, TLR ligands stimulate activating phosphorylations of the MAP kinases (Figure 5), all of which appear to regulate to some degree the production of TNF (Figure 5D). Most notable was the synergistic enhancement of the phosphorylation of JNK (Figure 5C) and the additive phosphorylation of p38 MAP kinase (Figure 5B) when cells were stimulated with TLR ligands in combination with antigen. This pattern correlated with the downstream activating phosphorylations of transcription factors by MAP kinases, namely, c-Jun and ATF-2,57 as determined by use of phosphorylation-specific antibodies. The phosphorylation c-Jun (Figure 6D) and ATF-2 (Figure 6A) was most pronounced after stimulation with the combination of TLR ligands and antigen.
The transcriptional activity of c-Jun also is dependent on dimerization with itself or other bZIP proteins that permit binding to relevant promotor sites, such as the binding of the c-Jun/c-Fos dimer to AP1. Of note, antigen but not TLR ligands induced synthesis of c-Fos (Figure 6E) and binding of c-Fos to AP1 (Figure 6F), presumably as the c-Jun/c-Fos dimer. The enhancement of this binding activity by TLR ligands was likely due to the augmented phosphorylation of c-Jun when cells were co-stimulated with TLR ligands and antigen.
The net effect was that some transcription factors were activated by both types of stimulants (NF-B), while others were activated most robustly by either antigen (c-Jun/c-Fos and NFAT) or TLR ligands (ATF2). Co-stimulation with antigen and TLR ligands resulted in additive or synergistic activation of ATF-2, c-Jun, and c-Fos. Therefore, co-stimulation not only broadened the array of transcription factors activated but also reinforced the activation of some of these factors. The same results were obtained by use of DNA-binding arrays (data not shown). The array data revealed that in addition, E twenty-six (Ets) proteins were activated by all stimulants, Sp1 by antigen only, and as noted earlier, STAT proteins were minimally phosphorylated by TLR ligands and antigen.
Optimal transcription of genes for cytokines such as TNF, IL-6, and IL-13 requires the coordinate actions of a repertoire of transcription factors. The identified consensus binding sites in the TNF gene loci, for example, include Ets, cyclic AMP regulatory element (CRE), AP1, AP2, Sp1, and multiple sites for NFAT and NF-B (1, 2, and 3).58-60 Of relevance to our findings, ATF-2 and c-Jun act cooperatively with NFAT family proteins on the adjacent CRE/3 sites,61 and transcriptional activity is further enhanced on co-stimulation with Sp1.58 Similar interaction occurs between the Jun (CRE) site and a nearby Ets binding site.62 The promotor region of IL-6 also contains interacting consensus sites for AP1, CRE, C/EBP, and NF-B,63,64 and cooperative interactions between NFAT and AP1 have been noted for the transcription of other cytokine genes, including IL-13.65 Although little is known about the combinatorial requirements for cytokine gene transcription in mast cells, our results suggest that optimal activation of the required repertoire of transcription factors is best achieved on co-stimulation of mast cells with antigen and the TLR ligands.
An interesting paradox is that activation of NFAT is absolutely essential for cytokine gene transcription following antigen stimulation, but this is not so for TLR stimulation. This suggests that the TLR ligands activate effective but unidentified transcription signal(s) in the absence of NFAT activation. TLR-mediated cytokine production has been linked to activation of NF-B and IFN-inducible genes, but RNase protection assays revealed no significant increase in IFN mRNA during the time course of our experiments (data not shown). Therefore, we believe it unlikely that the activation of IFN-inducible genes by TLR ligands15 was a significant factor in the synergistic interactions described here.
Although our studies encompass a variety of rodent cell lines and conditions, it should be noted that mast cells exhibit some variation in phenotype according to anatomical location, species, and culture conditions.66 Such variability might account for the expression of TLR4 in human peripheral blood–derived mast cells29 but not in human cord blood–derived mast cells.25 Nevertheless, the latter cells can be made to express TLR4 after priming with IL-4.26 Because of reports that TLR2 ligands elicit degranulation and generation of leukotriene C4 in some22,25,26 but not all27 studies, we examined the effects of various cytokines that can affect mast cell maturation and differentiation. These included stem cell factor, IL3, IL-4, and IFN.66 As noted earlier, we failed to induce degranulation and release of arachidonic acid when these cytokines were included in the growth medium, either individually or in combination. Therefore, we were unsuccessful in defining culture conditions that might explain the discrepancy in results among different laboratories.
However, if the synergy observed between antigen and TLR ligands in cultured mast cell lines has clinical relevance, there are implications in the treatment of atopic disease during infection. Apart from use of antiviral agents or the development of inhibitors of TLR activation, we note that low concentrations of glucocorticoids effectively suppress cytokine production in mast cells in response to not only antigen44 but also to TLR2 and TLR4 ligands (H.Q., unpublished data, June 2005). Therefore, glucocorticoids may serve a double purpose in the treatment of asthmatic patients during the course of airway infections.
|
Acknowledgements |
|---|
|
Footnotes |
|---|
Prepublished online as Blood First Edition Paper, September 20, 2005; DOI 10.1182/blood-2005-06-2271.
Supported in part by Intramural Research Program, National Institutes of Health, and the National Heart, Lung, and Blood Institute; and National Institutes of Health grant 1 RO1 TW006612-01 (M.V.A.).
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Michael A. Beaven, Rm 8N109/Bldg 10, National Institutes of Health, 9000 Rockville Pike, Bethesda, MD 20892-1760; e-mail: beavenm{at}nhlbi.nih.gov.
|
References |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
RI signaling and the allergic response. Curr Opin Immunol. 2002;14: 688-693.[CrossRef][Medline]
[Order article via Infotrieve] and release of arachidonic acid in mast cells: indications of communication between p38 and p42 MAP kinases. J Biol Chem. 1997;272: 13397-13402.RI mediate unique and convergent signals for release of inflammatory mediators from human mast cells. Blood. 2004;104: 2410-2417.B-independent control of NF-B activity by modulatory phosphorylations. Trends Biochem Sci. 2001;26: 186-190.[CrossRef][Medline]
[Order article via Infotrieve]1-dependent pathway of FcRI-mediated mast cell activation is regulated independently of phosphatidylinositol 3-kinase. J Biol Chem. 2003;278: 48474-48484. synthesis in human macrophages by interfering with IRF3 activation. J Biol Chem. 2004;279: 28781-28788.B, and nuclear factor of activated T cells in IgE receptor–mediated cytokine expression in mature human mast cells. J Allergy Clin Immunol. 2003;111: 1062-1068.[CrossRef][Medline]
[Order article via Infotrieve] gene expression in astrocytes. J Biol Chem. 1996;271: 22383-22390.B, AP-1, and C/EBP. J Immunol. 2003;170: 3099-3108.
CiteULike 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
This article has been cited by other articles:
|
|
 |
|
  E. Ronnberg, B. Guss, and G. Pejler Infection of Mast Cells with Live Streptococci Causes a Toll-Like Receptor 2- and Cell-Cell Contact-Dependent Cytokine and Chemokine Response Infect. Immun., February 1, 2010; 78(2): 854 - 864. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Brenzovich, M. Macey, J. Fernando, H. J. Chong, B. Barnstein, P. Mirmonsef, J. K. Morales, A. Kimura, T. D. Cruz, and J. J. Ryan IgE signaling suppresses Fc{epsilon}RI{beta} expression J. Leukoc. Biol., December 1, 2009; 86(6): 1351 - 1358. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Mansson and L.-O. Cardell Role of atopic status in Toll-like receptor (TLR)7- and TLR9-mediated activation of human eosinophils J. Leukoc. Biol., April 1, 2009; 85(4): 719 - 727. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Selander, C. Engblom, G. Nilsson, A. Scheynius, and C. L. Andersson TLR2/MyD88-Dependent and -Independent Activation of Mast Cell IgE Responses by the Skin Commensal Yeast Malassezia sympodialis J. Immunol., April 1, 2009; 182(7): 4208 - 4216. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. R. Holdsworth and S. A. Summers Role of Mast Cells in Progressive Renal Diseases J. Am. Soc. Nephrol., December 1, 2008; 19(12): 2254 - 2261. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Ryzhov, R. Zaynagetdinov, A. E. Goldstein, S. V. Novitskiy, M. M. Dikov, M. R. Blackburn, I. Biaggioni, and I. Feoktistov Effect of A2B Adenosine Receptor Gene Ablation on Proinflammatory Adenosine Signaling in Mast Cells J. Immunol., June 1, 2008; 180(11): 7212 - 7220. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Fukuoka, H.-Z. Xia, L. B. Sanchez-Munoz, A. L. Dellinger, L. Escribano, and L. B. Schwartz Generation of Anaphylatoxins by Human {beta}-Tryptase from C3, C4, and C5 J. Immunol., May 1, 2008; 180(9): 6307 - 6316. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. M. Jensen, M. A. Beaven, S. Iwaki, D. D. Metcalfe, and A. M. Gilfillan Concurrent Inhibition of Kit- and Fc{epsilon}RI-Mediated Signaling: Coordinated Suppression of Mast Cell Activation J. Pharmacol. Exp. Ther., January 1, 2008; 324(1): 128 - 138. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. Kikawada, J. V. Bonventre, and J. P. Arm Group V secretory PLA2 regulates TLR2-dependent eicosanoid generation in mouse mast cells through amplification of ERK and cPLA2{alpha} activation Blood, July 15, 2007; 110(2): 561 - 567. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
 |
 |
|||||||
 |
 |
 |
 |
 |
 |
 |
 |
||
| Copyright © 2006 by American Society of Hematology Online ISSN: 1528-0020 | |||||||||
R1 and toll-like receptors mediate synergistic signals to markedly augment production of inflammatory cytokines in murine mast cells
