Immune complexes suppress IFN-γ-induced responses in monocytes by activating discrete members of the SRC kinase family.

The regulation of the innate and the adaptive immune responses are extensively intertwined and tightly regulated. Ag-driven immune responses that are modulated by immune complexes (ICs) are known to inhibit IFN-γ-dependent MHC class II expression. We have previously demonstrated that ICs dramatically inhibit IFN-γ-induced activation of human monocytes through the activation of the FcγRI signaling pathway. In the present study we further explore the mechanisms by which ICs regulate IFN-γ activation of human monocytes. We demonstrate that members of the SRC kinase family (SKF) are key mediators of IFN-γ pathway suppression: inhibitors of the SKF reverse the ability of ICs to suppress IFN-γ signaling. Small interfering RNA was used to target specific members of the SKF. The data indicate that SRC and LYN are both required for ICs to elicit their immunosuppressive activity, whereas FYN does not appear to contribute to this function. Similarly, the kinase SYK, though not a member of the SKF, is also demonstrated to be involved in this IC-mediated immunosuppression. Our data suggest a mechanism whereby ICs directly inhibit inflammatory signals by crosslinking FcγRI, resulting in the activation of the specific phosphotyrosine kinases SRC, LYN, and SYK and the concomitant suppression of the IFN-γ signaling pathway.

IgG4 can induce an M2-like phenotype in human monocyte-derived macrophages through FcγRI.

Antibodies evoke cellular responses through the binding of their Fc region to Fc receptors, most of which contain immunoreceptor tyrosine-based activation motif domains and are thus considered "activating." However, there is a growing appreciation of these receptors for their ability to deliver an inhibitory signal as well. We previously described one such phenomenon whereby interferon (IFN)γ signaling is inhibited by immune complex signaling through FcγRI. To understand the implications of this in the context of therapeutic antibodies, we assessed individual IgG subclasses to determine their ability to deliver this anti-inflammatory signal in monocyte-derived macrophages. Like IgG1, we found that IgG4 is fully capable of inhibiting IFNγ-mediated events. In addition, F(ab')2 fragments that interfere with FcγRI signaling reversed this effect. For mAbs developed with either an IgG1 or an IgG4 constant region for indications where inflammation is undesirable, further examination of a potential Fc-dependent contribution to their mechanism of action is warranted.

Immune complexes suppress IFN-gamma signaling by activation of the FcgammaRI pathway.

Antigen-driven immune responses are modulated by immune complexes (ICs), in part through their ability to inhibit IFN-gamma-dependent MHC Class II expression. We have demonstrated previously that ICs dramatically inhibit IFN-gamma-induced activation of human monocytes through the suppression of the JAK/STAT signaling pathway. In the current study, we further explore the mechanisms by which ICs regulate IFN-gamma activation of human monocytes. Consistent with previous studies in monocytes pretreated with ICs, there was a reduction in steady-state levels of RNA by real-time RT-PCR of the IFN-inducible protein 10 gene as well as the FcgammaRI gene. Pull-down assays confirm that IC pretreatment inhibits IFN-gamma-induced STAT1 phosphorylation without affecting the ability of STAT1 to bind to the STAT1-binding domain of the IFN-gamma receptor. In addition, the inhibitory function of ICs was reduced when cells from the FcR common gamma-chain knockout mice were used, supporting the role of the FcgammaRI in this inhibitory pathway. It is unexpected that ICs also require the phosphatase Src homology-2-containing tyrosine phosphatase 1 (SHP-1) to inhibit IFN-gamma induction, as demonstrated by studies with cells from the SHP-1 knockout (motheaten) mice. These data suggest a mechanism of IC-mediated inhibition of IFN-gamma signaling, which requires the ITAM-containing FcgammaRI, as well as the ITIM-dependent phosphatase SHP-1, ultimately resulting in the suppression of STAT1 phosphorylation.

Communication between NF-kappa B and Sp1 controls histone acetylation within the proximal promoter of the monocyte chemoattractant protein 1 gene.

The induction of the monocyte chemoattractant protein 1 gene (MCP-1) by TNF occurs through an NF-kappaB-dependent distal regulatory region and an Sp1-dependent proximal regulatory region that are separated by 2.2 kb of sequence. To investigate how these regions coordinate activation of MCP-1 in response to TNF, experiments were performed to examine the role of coactivators, changes in local chromatin structure, and the acetylation of histones at the MCP-1 regulatory regions. An E1a-sensitive coactivator was found to be required for expression. In vivo nuclease sensitivity assays identified changes in response to TNF at both the proximal and distal regions that were dependent on the p65 subunit of NF-kappaB and Sp1. Chromatin immunoprecipitations used to analyze factor assembly and histone acetylation at the distal and proximal regions showed that Sp1 binding to and histone acetylation of the proximal region was dependent on NF-kappaB p65. Conversely, Sp1 assembly at the proximal region was required for p65 binding to and acetylation of the distal region, suggesting communication between the two regions during gene activation. These data and the NF-kappaB p65-dependent histone acetylation of a middle region sequence suggest a potential order for the assembly, acetylation and accessibility of the MCP-1 regulatory regions in response to TNF.

Role of the intronic enhancer in tumor necrosis factor-mediated induction of manganous superoxide dismutase.

Manganous superoxide dismutase (Mn-SOD), a tumor necrosis factor (TNF)-inducible gene product, plays an important role in removing superoxide anions produced inside mitochondria. Two regulatory regions, the proximal promoter region (PPR), which is upstream from the transcription initiation site, and the TNF-responsive element (TNFRE), which is inside intron 2, are responsible for Mn-SOD expression. To understand how each of these regions contributes to the transcription of Mn-SOD, quantitative reverse transcription-PCR, chromatin immunoprecipitations, and in vivo nuclease sensitivity assays were performed on the murine Mn-SOD gene. These assays demonstrate that Sp1 and nuclear factor (NF)-kappaB p65 are required for Mn-SOD induction by TNF. Sp1 bound the PPR constitutively, whereas NF-kappaB p65 and C/EBP-beta bound the TNFRE only after TNF treatment. Binding of C/EBP-beta to the TNFRE was dependent on the presence of NF-kappaB p65. The chromatin structure within the TNFRE became more accessible to nuclease digestion after TNF treatment. This accessibility required Sp1 and NF-kappaB p65. Treatment of cells with an inhibitor of histone deacetylation, or transient transfection with coactivator-expressing plasmids, enhanced the expression of Mn-SOD. NF-kappaB p65 binding was required for acetylation of histones H3 and H4 at the PPR and the TNFRE. Together, these data suggest communication between the PPR and the TNFRE which involves chromatin remodeling and histone acetylation during the induction process of Mn-SOD in response to TNF.