Fc Gamma Receptor IIb on GM-CSF Macrophages Controls Immune Complex Mediated Inhibition of Inflammatory Signals

Background In rheumatoid arthritis (RA) macrophages play a major role in amplifying synovial inflammation. Important activating signals are those induced by Toll-like receptor (TLR) ligands and by activated T cells. The balance between activating and inhibitory Fc gamma receptors (FcγRs) on macrophages might be crucial in modulating these inflammatory responses. The purpose of this study was to determine FcγR expression on pro- and anti-inflammatory macrophages (gmMφ and mMφ, respectively) and identify functional consequences on immune complex uptake and macrophage activation. Methods Human monocytes were isolated and differentiated into gmMφ and mMφ. A full FcγR characterization of both macrophage subtypes was performed and uptake of fluorescent immune complexes (ICs) was determined. FcγRIIb isoforms were determined by qPCR. Macrophages were stimulated via different TLRs or cytokine activated T cells in the presence or absence of ICs and cytokine production was determined. Blocking studies were performed to look into the pathways involved. Results mMφ expressed high levels of the activating FcγRIIa and FcγRIII and low levels of the inhibitory FcγRIIb, while the FcγR balance on gmMφ was shifted towards the inhibitory FcγRIIb. This was accompanied by a clear increase in FcγRIIb1 mRNA expression in gmMφ. This resulted in higher IC uptake by mMφ compared to gmMφ. Furthermore, FcγR-mediated stimulation of gmMφ inhibited TLR2, 3, 4 and 7/8 mediated cytokine production via FcγRIIb and PI3K signaling. In addition, gmMφ but not mMφ produced TNFα upon co-culture with cytokine activated T cells, which was reduced by IC binding to FcγRIIb. The latter was dependent on PI3K signaling and COX2. Conclusions FcγR expression patterns on gmMφ and mMφ are significantly different, which translates in clear functional differences further substantiating FcγRIIb as an interesting target for inflammation control in RA and other autoimmune/inflammatory diseases.


Introduction
One of the major pathways underlying the pathogenesis of rheumatoid arthritis (RA) is the aberrant production of inflammatory cytokines by macrophages. In the arthritic joint, macrophages are one of the main effector cells present and their levels correlate with disease activity and joint destruction [1,2]. Their levels are mainly associated with inflammatory cytokines such as TNFa and interleukin (IL) 1b, and could be sustained by factors like granulocyte-macrophage colony-stimulating factor (GM-CSF), present in the RA synovial joint [3][4][5]. Multiple pathways are proposed to play a role in macrophage activation in RA. One mechanism inducing cytokine production by RA macrophages is the triggering of Toll-like receptors (TLRs). Many endogenous TLR ligands have been found in an arthritic joint, such as GP96 and SNAPIN, which activate cells via TLR2, small heat shock protein B8 that can activate TLR4, and self-RNA from damaged cells which is likely to stimulate macrophages via TLR3 or TLR7/8 [6][7][8][9][10]. Blocking antibodies against these TLRs reduce spontaneous cytokine production by RA synovial tissue cultures, confirming they are not only present in the arthritic joint but also contribute to the abundant cytokine production seen in RA [10][11][12].
Another pathway mediating synovial macrophage activation is by direct interaction with activated T cells. Cytokine activated T cells resemble RA synovial T cells in their contact-dependent effector function and activation phenotype [13,14]. These cells can be cultured from peripheral blood lymphocytes in the presence of IL-2, IL-6 and TNFa (cytokine activated T cells, Tck) and induce an unbalanced, inflammatory cytokine response from monocytes [14].
Another component present in many RA patients are autoantibodies. These can form immune complexes (IC) and especially when deposited in tissues they can activate macrophages. Soluble ICs can have cell activating but also inhibitory effects, as is emphasized by IVIg treatment [15]. An important deciding factor for the cellular response to ICs is the balance of activating and inhibitory Fc gamma receptors (FccRs).
The FccR system consists of the activating FccRI, FccRIIa and FccRIII that trigger cell activation via an immunoreceptor tyrosine-based activation motif (ITAM) in their cytoplasmic domain and the inhibitory FccRIIb that signals via an immunoreceptor tyrosine-based inhibition motif (ITIM) [16]. As the only inhibitory FccR, FccRIIb is an important brake on the immune system by inhibition of cell activation via the activating FccRs on a wide array of cells and inhibition of the B cell receptor. FccRIIb has two major isoforms, namely FccRIIb1 and FccRIIb2, which differ in their capabilities to mediate endocytosis and in their distribution on immune cells [17][18][19][20]. FccRIIb1 predominates in B cells, while FccRIIb2 is the major isoform in myeloid cells. We and others have previously shown that IC binding to FccRIIb can also inhibit TLR4 signaling [21,22]. In our previous report, only RA patients that could control their disease activity without the need of anti-rheumatic drugs had high FccRIIb levels on their dendritic cells (DC) and were capable of this inhibition [21]. This supports an important regulatory role for FccRIIb in controlling inflammation in RA.
Since proinflammatory macrophages are important in the pathogenic process in RA and there is no data on the expression and function of the inhibitory FccR on such macrophages we aimed to delineate the expression of FccR receptors on homeostatic M-CSF macrophages (mMQ) and inflammatory GM-CSF macrophages (gmMQ). We determined the complete FccR balance on gmMQ and mMQ and tested whether functional differences were attributed to this. We mainly focused on combined FccR triggering with macrophage activation via a range of TLRs implicated in RA pathology or activated T cells and found that FccRIIb was able to dampen both TLR and Tck induced TNFa production when this inhibitory FccRIIb was highly expressed.

Ethics statement
The study protocol was approved by the medical ethical committee of the Radboud university medical center (Nijmegen, the Netherlands) and the University Medical Center Utrecht (Utrecht, the Netherlands) and all healthy volunteers gave their written informed consent. All experiments were performed in accordance with the Helsinki Declaration.

Culture of monocyte-derived gmMQ and mMQ and Tck cells
Peripheral blood mononuclear cells were isolated from venous blood of healthy volunteers using density-gradient centrifugation over Ficoll (GE Healthcare, Uppsala, Sweden). Monocytes and CD4+ T cells were obtained using CD14 and CD4 microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany). gmMQ and mMQ were generated by culturing monocytes in the presence of GM-CSF (800 U/ml; R&D Minneapolis, Minnesota, USA) or macro-phage colony-stimulating factor (M-CSF, 25 ng/ml; R&D) for 6 days. Macrophages were cultured in 6 well plates (Corning, New York, USA) with 1,0610 6 cells per well in 2 ml medium (RPMI-1640 Dutch modification (Gibco Life Technologies, Grand Island, New York, USA)) supplemented with 10% FCS, antibioticantimycotic and L-glutamine (Gibco Life Technologies). Culture medium with the same supplements (1 ml) was added at day 3 and the cells were harvested at day 6. In parallel, autologous CD4+ T cells were cultured in complete medium with recombinant human IL-2 (25 ng/ml), IL-6 (100 ng/ml) and TNFa (25 ng/ml) at 2610 6 cells/ml for 6 days (all from R&D).

Phenotypical analysis
Using standardized flow cytometry protocols as described previously gmMQ and mMQ were phenotyped using antibodies against CD14, CD163 (both BD Biosciences, Franklin Lakes, New Jersey, USA) and MHC-II DR/DP (clone Q1514) [23]. FccR expression was determined with antibodies against FccRI (CD64, PE labeled, clone 10.1; Dako, Glostrup, Denmark), FccRIII (CD16, PE labeled, clone DJ130c; Dako), clone IV.3 which preferentially binds to FccRIIa (StemCell Technologies, Vancouver, Canada) and the FccRIIb specific antibody 2B6 (Alexa488 labeled; MacroGenics, Rockville, Maryland, USA). Expression of unlabeled markers was visualized via a FITC labeled goat-antimouse secondary antibody. Cell fluorescence was measured on a FACS Calibur (BD) and analyzed using Flowjo software for the mean fluorescence intensity (MFI) and the proportion of positive cells relative to cells stained with the appropriate IgG isotypes.

RNA isolation and qPCR
Total RNA was extracted in 0.5 ml of TRI-reagent and treated with DNase to remove genomic DNA before being reversetranscribed into cDNA. qPCR was performed on a Quantstudio 12K Flex (Life Technologies) with SYBR Select Master Mix (Life Technologies), 7.5 ng cDNA and a primer concentration of 0.5 mM in a total volume of 15 ml. qPCR signals were quantified by comparing the cycle threshold value (Ct) of the gene of interest of each sample with the Ct value of the reference gene GAPDH (DCt). Results were deployed as relative expression (2 2DCt ). The following primers were used: GAPDH forward ATGGG-GAAGGTGAAGGTCG, reverse GGGGTCATTGATGGCAA-CAATA; FccRIIb1 forward GGATTTCAGCTCTCCCAG-GAT, reverse CGGTTCTGGTCATCAGGCTC; FccRIIb2 forward AAAGCGGATTTCAGCCAATC, reverse CAAGA-CAATGGAGACTAAATACGGT.

Phagocytosis and binding assay
Phagocytosis assays were performed with fluorescently labeled ICs, prepared as previously described [24]. Macrophages were incubated with FITC-labeled ICs (50 mg/ml) for 30 min at 4uC and 37uC to determine binding and uptake, respectively. Unattached ICs were washed away before determining binding and uptake by flow cytometry. To determine IC uptake extracellular attached FITC-IC was quenched by adding trypan blue (1/40 diluted in PBS, Sigma-Aldrich) to the samples just before determining the IC uptake by flow cytometry.

Stimulation of monocyte-derived macrophages
At day 6 macrophages were harvested and plated in a concentration of 0.5610 6 cells/ml in 96 well culture plates (100 ml). Immune complexes used in this study were prepared by heating human IgG (Sigma-Aldrich, St. Louis, Missouri, USA) in PBS at 63uC for 30 minutes (heat-aggregated immune complexes (IC)), as previously described [25] and were used in a concentration of 50 mg/ml. Macrophages were stimulated or not with ICs for 15-30 minutes before the addition of TLR agonists for 20 hours. The following concentrations of TLR agonists were used: Pam3CSK4 (5 mg/ml, EMC Microcollections, Tübingen, Germany), Poly(I:C) (25 mg/ml, Invivogen, San Diego, California, USA), LPS (100 ng/ml, E. coli 0111:B4, Sigma-Aldrich) and R848 (2 mg/ml, Invivogen) for TLR2/1, 3, 4 and 7/8 respectively. The LPS was double-purified to remove any contaminating proteins as described previously [26]. Macrophages were also cocultured with cytokine-activated T cells for 20 hours in a 1:5 ratio in the presence or absence of IC (50 mg/ml) prestimulation for 1 h. FccRIIb blocking was performed by 30 min incubation of mQ-1 with 10 mg/ml 2B6 antibody (MacroGenics) or an isotype control at 4uC before stimulation with ICs and LPS or Tck. In other experiments gmMQ were treated with the PI3K inhibitors Wortmannin (0.1 mM; Calbiochem, San Diego, California, USA) or LY294002 (10 mM; Calbiochem) or COX2 inhibitor I (20 mM; Calbiochem) for 1 h at 37uC before stimulation.

Measurement of cytokines in culture supernatants
Levels of IL-10 and TNFa were measured in the supernatants using commercially available kits (Millipore, Billerica, Massachusetts, USA) according to the manufacturer's instructions. Cytokine

Statistical analysis
Differences were analyzed using paired Student's t-tests. P values less than 0.05 were considered significant.

Results
gmMQ express high levels of the inhibitory FccRIIb, while mMQ express higher levels of the activating FccRIIa and FccRIII Monocytes were cultured into pro-and anti-inflammatory gmMQ and mMQ in the presence of either GM-CSF or M-CSF. To confirm the phenotype of our gmMQ and mMQ we first analyzed their expression of CD14, CD163 and MHC-II. In line with literature the expression of CD14 and CD163 was higher on mMQ, while MHC-II was increased on gmMQ ( Figure 1A) [21]. We further evaluated the expression of activating and inhibitory FccRs. The monomeric IgG receptor FccRI was similarly expressed in gmMQ and mMQ, while FccRIII expression was highly increased on mMQ compared to gmMQ (regarding both MFI and percentage of positive cells, Figure 1B). Investigating the activating and inhibiting subtype of FccRII separately, we observed a marked difference between the gmMQ en mMQ. Whereas the activating FccRIIa is expressed higher on mMQ, expression of the inhibitory FccRIIb was increased on gmMQ ( Figure 1B). More specifically, the FccRIIb/FccRIIa ratio was 1.56 for gmMQ and 0.48 for mMQ. Thus, gmMQ display an FccR balance favored towards the inhibitory subtype whereas the opposite was found on mMQ. FccR expression was also determined on gmMQ and mMQ from some RA patients, which showed a similar FccR distribution compared to healthy controls (data not shown). In vivo in situations in which GM-CSF is produced most likely also a basal level of M-CSF will be present.
To determine the effect of the combination of both growth factors on macrophage development we also cultured macrophages with GM-CSF and M-CSF. This resulted in a phenotype similar to gmMQ, suggesting GM-CSF is dominant over M-CSF, at least regarding FccR expression (data not shown).
We further aimed to differentiate between the two major FccRIIb isoforms, FccRIIb1 and FccRIIb2. Since the extracellular domain of these isoforms is the same we used qPCR to determine the expression of these variants in mMQ and gmMQ. Using isoform specific primers we found that FccRIIb2 expression was similar in both macrophage subtypes, while FccRIIb1 expression was significantly increased in gmMQ compared to mMQ ( Figure 1C). gmMQ thus have an increased expression of the FccRIIb variant usually more predominant in B cells which is less capable of mediating endocytosis.
The capacity to take up ICs is an important function of macrophages. To evaluate the functionality of the altered aforementioned FccR balance, we investigated whether the gmMQ and mMQ display a different binding and uptake capacity of ICs. mMQ show a significantly increased potential for both binding and uptake of ICs compared to gmMQ ( Figure 1D). This is fitting with the enhanced expression of FccRIIa and FccRIII on mMQ, which have a higher affinity for most IgG isotypes compared to FccRIIb [27] and the increased expression of the non-endocytosing FccRIIb1 on gmMQ.

ICs inhibit TLR induced cytokine production by gmMQ but not by mMQ
To further evaluate the functional consequences of the differential FccR expression on gmMQ and mMQ and to test whether ICs can also inhibit TLR4 signaling in human macrophages that express high FccRIIb levels, gmMQ and mMQ were stimulated with TLR ligands in combination with ICs. mMQ and gmMQ were first stimulated with ICs alone to determine the effect of differential FccR expression on these cells on IC induced cytokine production. In mMQ ICs induced significant but low levels of TNFa and IL-10 production, while there was no clear cytokine induction observed in gmMQ (Figure 2A). Upon TLR 4 stimulation with LPS gmMQ produced high levels of TNFa and low levels of IL-10, while mMQ were marked by their relatively high IL-10 production and low production of TNFa which corroborates the literature ( Figure 2B) [5,28]. After co-stimulation with ICs, gmMQ were able to significantly attenuate TNFa production compared to those stimulated with LPS alone, while IL-10 production was relatively unaffected ( Figure 2B). In contrast, but in line with our observations on FccR expression, the addition of ICs to LPS did not result in inhibition of TLR4 mediated cytokine production in mMQ. In fact, mMQ produced significantly more IL-10 after costimulation with ICs.
To determine if this inhibitory pathway can also affect cytokine induction by other TLR ligands, similar experiments were performed with specific ligands for TLR2/1 (Pam3CSK4), TLR3 (Poly(I:C)) and TLR7/8 (R848). These experiments learned that the inhibitory effect of ICs on TLR signaling by gmMQ is not limited to TLR4, but also extends to TLR2/1, TLR3 and TLR7/ 8 ( Figure 2C), further substantiating the pivotal role of the FccR balance in the regulation of cell activation. Again, IL-10 production by gmMQ was not clearly affected by the presence of ICs and the inhibitory effect on TNFa production was not present in mMQ (data not shown). To determine if ICs could also affect TLR induced cytokine production after TLR stimulation has already occurred, we performed a time course with addition of ICs from 2 hours prior to Pam3CSK4 till 2 hours after Pam3CSK stimulation. ICs were able to significantly modulate TNFa production during the whole time range, without significantly affecting IL-10 (data not shown). ICs can thus modulate TLR induced cytokine production before and after TLR triggering.

Immune complexes can inhibit gmMQ activation by activated T cells
Another important activator of RA synovial macrophages are cytokine activated T cells. We therefore evaluated cytokine production in co-cultures of Tck with gmMQ or mMQ. Tck induced a synergistic production of TNFa when co-cultured with gmMQ ( Figure 3A), while IL-10 is almost absent, resulting in an unbalanced proinflammatory response. The TNFa production by mMQ after co-culture with Tck is much lower and not significantly different from mMQ alone ( Figure 3A). In contrast to mMQ stimulation by TLR ligands, in co-culture with Tck also IL-10 production remained low. Thus, Tck mainly stimulate gmMQ. To determine if ICs could inhibit Tck induced TNFa production when macrophages express high FccRIIb levels, gmMQ were stimulated with ICs and Tck. IC co-stimulation reduced the TNFa release by gmMQ with approximately 50% upon Tck stimulation ( Figure 3B). ICs can thus modulate both TLR and T cell induced gmMQ activation.

The inhibitory effect of ICs is mediated via FccRIIb and the PI3K pathway
To confirm whether the high FccRIIb expression on gmMQ was indeed responsible for the inhibitory effect of ICs on TLR and Tck induced signaling in these cells, we used a blocking antibody against FccRIIb. Blocking of FccRIIb fully abrogated the inhibitory effect of ICs on both TLR and Tck induced TNFa production ( Figure 4A and B).
In DCs our group has previously shown that the PI3K/Akt pathway is involved in the crosstalk between FccRIIb and TLR4 [21]. To determine if this pathway is also involved in the FccRIIb effect on macrophages, we blocked PI3K signaling before stimulation of gmMQ with ICs and LPS. The IC mediated inhibition of LPS induced TNFa production was abrogated in the presence of Wortmannin or LY294002, confirming the role of the PI3K pathway in FccRIIb mediated TLR4 signaling inhibition in gmMQ ( Figure 4C and data not shown). Mice studies pointed towards an additional role for prostaglandin E2 in the inhibitory actions of FccRIIb on TLR4 signaling [22]. To test this in the human setting we performed our experiments in the presence of a COX2 inhibitor. This did not affect the IC mediated inhibition of cytokine production by gmMQ upon TLR4 stimulation (Figure 4C). To test if the same mechanism was involved in IC mediated blocking of other TLRs we tested FccRIIb blocking and PI3K inhibition also for ICs in combination with TLR2/1 stimulation and this gave similar results as shown for TLR4 (data not shown).
To further determine if similar pathways are involved in inhibition of Tck induced macrophage activation we performed   the Tck experiments in the presence of Wortmannin or a COX2 inhibitor. As for TLR activation a functioning PI3K pathway was necessary for the inhibitory effect of ICs, however IC mediated blocking of gmMQ activation via Tck also appeared to be dependent on prostaglandin production, as is exemplified by the lack of IC mediated inhibition in the presence of a COX2 inhibitor ( Figure 4D). IC mediated inhibition of gmMQ TNFa production via both pathways is mediated via binding to FccRIIb and involves the PI3K pathway. Prostaglandins are necessary for the effect of IC when combined with Tck, but not for TLR mediated cell activation.

Discussion
The present study shows that gmMQ have a relatively high expression of FccRIIb compared to the activating FccRs, while this balance is shifted towards the activating FccRs on mMQ. gmMQ secrete large amounts of TNFa upon stimulations relevant in RA, such as TLR ligands and cytokine activated T cells. Under these conditions inhibitory immune receptors, such as FccRIIb, are crucial to counter-regulate the induced inflammatory responses to prevent excessive tissue damage. We show that the switched balance towards the inhibitory FccRIIb on gmMQ is functionally relevant and can inhibit TNFa secretion from these cells induced by either TLRs or Tck in the presence of soluble ICs. This way it could function as a natural brake in an attempt to prevent excessive cytokine production and inflammation in RA.
The important regulatory role of FccRIIb is extensively shown in animal models for autoimmunity (Reviewed in [29]). In this context it was shown that the transfer of RA but not healthy control serum can induce arthritis in FccRIIb 2/2 , but not in normal B6 mice [30]. This was caused by the IgG portion supporting a pathogenic role for IgG (auto) antibodies from RA patients and an important regulatory role for FccRIIb. This model bypasses the effect of B cells because human IgG is passively transferred and it thus shows that FccRIIb expression on other effector cells, including macrophages and DCs, is crucial to prevent autoimmune inflammation.
We demonstrated for the first time that FccRIIb can inhibit cytokine induction by a wide range of TLRs, of which ligands have been found in the arthritic joint, including TLR2, TLR3, TLR4 and TLR7/8. In addition, FccRIIb can also inhibit macrophage TNFa production induced by activated T cells. This way FccRIIb can actively control two important stimulatory pathways for macrophages in RA. Inhibitor studies taught us that normal PI3K signaling is necessary for FccRIIb inhibition of both TLR and Tck induced cytokine release, while prostaglandins are only involved in the latter. Prostaglandins were postulated as an essential signaling molecule in FccRIIb mediated inhibition of TLR4 in mouse macrophages [22]. However, in our human experimental setting prostaglandins are dispensable for FccRIIb mediated inhibition of TLRs. In our cultures prestimulation of gmMQ with ICs for only 15-30 minutes or up to 2 hours after TLR stimulation was enough to get inhibition, while in mouse macrophages the dependency on prostaglandins was demonstrated after 24 hour prestimulation with ICs [22]. So prostaglandins are not necessary for the direct inhibition of TLR4 signaling by FccRIIb in humans, but might have additional inhibitory effects at later time points. This would be in line with the dependency of Tck inhibition on prostaglandin production, since the induction of TNFa in this setting is described to be much slower (peaks at 24 hrs.) compared to TLR stimulated TNFa induction (peaks at 4-8 hrs.) [31]. However, much is still unknown about the pathways involved in macrophage activation upon interaction with Tcks. CD69, CD18 and CD49d on the Tck were shown to be involved in the induction of TNFa by monocytes upon Tck co-culture [14]. On monocytes/macrophages ICAM-1 and VCAM-1 might be involved as binding partners for CD18 and CD49d, respectively. Thus far, no direct interactions are known between these molecules and FccR signaling. Hence, our work justifies more research focused at deciphering potential mechanisms involved in FccRIIb inhibition on T cell mediated macrophage activation.
Interestingly, the increased membrane FccRIIb expression on gmMQ coincides with an increased expression of FccRIIb1 on mRNA level. This suggests that FccRIIb1 expression on gmMQ could play a role in the inhibitory effects of ICs on these cells. Although the FccRIIb isoforms have been repeatedly shown to have differential endocytosis potential [17][18][19], not much is known about possible differences in inhibitory signaling. It has been described that FccRIIb1 is differently phosphorylated in B cells compared to FccRIIb2 and might have additional inhibitory functions [19,32], but this has not been repeated by another group in macrophage cell lines [33]. Whether this could have functional implications for macrophage responses towards ICs needs to be further investigated.
Some groups have tried to identify the macrophage phenotype or the FccR expression on macrophages from RA synovial tissue. FccRII overall, FccRIIb in particular and FccRIII were all increased in RA synovium and correlated with the amount of macrophages present [34,35]. Looking at in vitro markers for gmMQ and mMQ it remains difficult to fully characterize the macrophages from the synovial tissue since they express markers representing both phenotypes [36,37]. Supported by our data the expression of FccRIIb could be an additional marker for gmMQ while FccRIII marks mMQ macrophages. The ratio between these two FccRs could be a good discriminator between these macrophage subsets. The high expression of activating FccRs by mMQ, which are mainly described for their controlling/homeostatic functions, and the high expression of the inhibitory FccRIIb on the more inflammatory gmMQ might seem contradictory. However, because of the easily activated phenotype of gmMQ regulatory mechanisms including those via FccRIIb are crucial to prevent excessive inflammation and tissue damage. In addition, the capacity of mMQ to remove IC is facilitated by the high expression of FccRs, preventing accumulation of IC and thereby preventing unwanted inflammatory responses. Next to that we show that small ICs in combination with TLR stimulation mainly increase IL-10 production in mMQ and no major induction of TNFa was observed.
The in vivo situation is not as black and white as shown by this in vitro model, but knowledge about functional characteristics of these macrophage subsets combined with more detailed phenotyping of local macrophages, including differentiation between FccRIIa and FccRIIb in different diseases might give clues about the pathogenic processes going on in vivo. This could possibly give leads for therapeutic options to increase FccRIIb expression on macrophages even further to induce a more inhibitory phenotype.
Conclusions gmMQ and mMQ are characterized by a different FccR balance, with high FccRIIa and FccRIII levels on mMQ and increased FccRIIb expression on pro-inflammatory gmMQ. The relatively high FccRIIb expression on gmMQ makes these cells sensitive to IC mediated inhibition of proinflammatory cytokine release upon stimulation by TLR ligands and Tck, which can be an important feedback mechanism to prevent excessive inflammation. This shows that FccRIIb mediated cell inhibition is not restricted to ITAM containing receptors or TLR4, but can broadly regulate immune responses. Specific targeting of FccRIIb might therefore open novel therapeutic avenues for RA and other chronic immune mediated inflammatory disorders.