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The leukocyte immunoglobulin-like receptor A3 (LILRA3) is a soluble protein primarily expressed by peripheral blood monocytes and is abundant in sera of healthy donors. Extracellular LILRA3 is anti-inflammatory and displays neuro-regenerative functions in vitro. However, its intracellular expression, distribution, and function(s) remain unknown. Using a combination of high-resolution confocal and super-resolution microscopy, we identified intracellular expression of native LILRA3 in the nucleus of peripheral blood monocytes and in vitro-derived macrophages. This unexpected nuclear localization of LILRA3 was confirmed in LILRA3-GFP-transfected HEK293T cells. Western blot of proteins fractionated from primary macrophages and the transfected HEK293T cells confirmed nuclear localization of the native and expressed LILRA3 proteins. Interestingly, most of the LILRA3 in the nucleus was in a monomeric form like the biologically active secreted protein, while that in the other cellular compartments was in mixed monomeric, dimeric, and oligomeric forms. The predominant presence of monomeric LILRA3 in the nucleus was independently corroborated in transfected live HEK293T cells using the number and molecular brightness (N&B) analysis method. Immunoprecipitation and mass spectrometric peptide sequencing studies revealed that nuclear LILRA3 co-immunoprecipitated with several nuclear proteins involved in host protein synthesis machinery via direct interactions to a key multifunctional RNA-binding protein, the Ewing sarcoma breakpoint region 1 protein (EWS) (data are available via ProteomeXchange with identifier PXD024602). The biological significance of the nuclear expression of LILRA3 and its interaction with these key proteins remain to be elucidated.

The leukocyte Ig-like receptor B4 (LILRB4) is an inhibitory cell surface receptor, primarily expressed on mono-myeloid cells. It contains 2 C-type Ig-like extracellular domains and a long cytoplasmic domain that contains three intracellular immunoreceptor tyrosine-based inhibitory motifs (ITIMs). Data suggest that LILRB4 suppresses Fc receptor-dependent monocyte functions via its ITIMs, but relative contributions of the three ITIMs are not characterised. To address this, tyrosine (Tyr) residues at positions 337, 389 and 419 were single, double or triple mutated to phenylalanine and stably transfected into a human monocytic cell line, THP-1. Intact Tyr 389 was sufficient to maximally inhibit FcγRI-mediated TNF-α production in THP-1 cells, but, paradoxically, Tyr 337 significantly enhanced TNF-α production. In contrast, bactericidal activity was significantly enhanced in mutants containing Tyr 419 , while Tyr 337 markedly inhibited bacteria killing. Taken together, these results indicate that LILRB4 might have dual inhibitory and activating functions, depending on the position of the functional tyrosine residues in its ITIMs and/or the nature of the stimuli.

FcγRI cross-linking on monocytes may trigger clathrin-mediated endocytosis, likely through interaction of multiple intracellular molecules that are controlled by phosphorylation and dephosphorylation events. However, the identity of phospho-proteins and their regulation are unknown. We proposed the leukocyte immunoglobulin-like receptor B4 (LILRB4) that inhibits FcγRI-mediated cytokine production via Tyr dephosphorylation of multiple kinases, may also regulate endocytosis/phagocytosis through similar mechanisms. FcγRI and/or LILRB4 were antibody-ligated on THP-1 cells, lysates immunoprecipitated using anti-pTyr antibody and peptides sequenced by mass spectrometry. Mascot Search identified 25 Tyr phosphorylated peptides with high confidence. Ingenuity Pathway Analysis revealed that the most significantly affected pathways were clathrin-mediated endocytosis and Fc-receptor dependent phagocytosis. Tyr phosphorylation of key candidate proteins in these pathways included common γ-chain of the Fc receptors, Syk, clathrin, E3 ubiquitin protein ligase Cbl, hepatocyte growth factor-regulated tyrosine kinase substrate, tripartite motif-containing 21 and heat shock protein 70. Importantly, co-ligation of LILRB4 with FcγRI caused significant dephosphorylation of these proteins and was associated with suppression of Fc receptor-dependent uptake of antibody-opsonised bacterial particles, indicating that LILRB4. These results suggest that Tyr phosphorylation may be critical in FcγRI-dependent endocytosis/phagocytosis that may be regulated by LILRB4 by triggering dephosphorylation of key signalling proteins.

The activating immunoglobulin-like receptor, subfamily A, member 2 (LILRA2) is primarily expressed on the surface of cells of the innate immunity including monocytes, macrophages, neutrophils, basophils and eosinophils but not on lymphocytes and NK cells. LILRA2 cross-linking on monocytes induces pro-inflammatory cytokines while inhibiting dendritic cell differentiation and antigen presentation. A similar activating receptor, LILRA4, has been shown to modulate functions of TLR7/9 in dendritic cells. These suggest a selective immune regulatory role for LILRAs during innate immune responses. However, whether LILRA2 has functions distinct from other receptors of the innate immunity including Toll-like receptor (TLR) 4 and FcγRI remains unknown. Moreover, the effects of LILRA2 on TLR4 and FcγRI-mediated monocyte functions are not elucidated. Here, we show activation of monocytes via LILRA2 cross-linking selectively increased GM-CSF production but failed to induce IL-12 and MCP-1 production that were strongly up-regulated by LPS, suggesting functions distinct from TLR4. Interestingly, LILRA2 cross-linking on monocytes induced similar amounts of IL-6, IL-8, G-CSF and MIP-1α but lower levels of TNFα, IL-1β, IL-10 and IFNγ compared to those stimulated with LPS. Furthermore, cross-linking of LILRA2 on monocytes significantly decreased phagocytosis of IgG-coated micro-beads and serum opsonized Escherichia coli but had limited effect on phagocytosis of non-opsonized bacteria. Simultaneous co-stimulation of monocytes through LILRA2 and LPS or sequential activation of monocytes through LILRA2 followed by LPS led lower levels of TNFα, IL-1β and IL-12 production compared to LPS alone, but had additive effect on levels of IL-10 and IFNγ but not on IL-6. Interestingly, LILRA2 cross-linking on monocytes caused significant inhibition of TLR4 mRNA and protein, suggesting LILRA2-mediated suppression of LPS responses might be partly via regulation of this receptor. Taken together, we provide evidence that LILRA2-mediated activation of monocytes is significantly different to LPS and that LILRA2 selectively modulates LPS-mediated monocyte activation and FcγRI-dependent phagocytosis.

The leukocyte immunoglobulin-like receptor (LILR) B4 belongs to a family of cell surface receptors that possesses cytoplasmic immunoreceptor tyrosine-based inhibitory motifs (ITIMs). LILRB4 is believed to down-regulate activation signals mediated by non-receptor tyrosine kinase cascades through the recruitment of SHP-1. However, the exact mechanisms of LILRB4-mediated inhibition are not fully elucidated. In this study, we demonstrate high level surface expression of LILRB4 on THP-1 cells and primary peripheral blood monocytes, which profoundly inhibited production of a key pro-inflammatory cytokine (TNFalpha) induced by FcgammaRI (CD64). We also report that LILRB4 aggregated to sites of activation upon co-ligation with CD64 and that this may enhance its inhibitory effects. Cross-linking of CD64 on THP-1 cells markedly increased phosphorylation of multiple proteins including tyrosine kinases and signaling molecules (Lck, Syk, LAT, and Erk), an adaptor protein that targets protein-tyrosine kinases for degradation (c-Cbl) and a protein involved in the formation of actin cytoskeletal rearrangement (alpha-actinin-4). Co-ligation of LILRB4 considerably reduced CD64-mediated phosphorylation of Lck, Syk, LAT, Erk, and c-Cbl but not alpha-actinin-4, suggesting selective inhibition of signaling molecules. Treatment of cells with a broad-spectrum phosphatase inhibitor, sodium pervanadate (SP), significantly reversed LILRB4-mediated inhibition of TNFalpha production and protein tyrosine phosphorylation. In comparison, treatment with an SHP-1 specific inhibitor, sodium stibogluconate (SS) has no effects indicating involvement of phosphatase(s) other than SHP-1 in LILRB4 signaling. Collectively, our data show LILRB4 is a potent inhibitor of monocytes activation. This may provide a new potential therapeutic strategy for inflammatory conditions characterized by excessive TNFalpha production.

S100A12 is elevated in the circulation in patients with chronic inflammatory diseases and recent studies indicate pleiotropic functions. Serum amyloid A induces monocyte cytokines and tissue factor. S100A12 did not stimulate IL-6, IL-8, IL-1β or TNF-α production by human peripheral blood mononuclear cells but low amounts consistently reduced cytokine mRNA and protein levels induced by serum amyloid A, by ∼49% and ∼46%, respectively. However, S100A12 did not affect serum amyloid A-induced monocyte tissue factor. In marked contrast, LPS-induced cytokines or tissue factor were not suppressed by S100A12. S100A12 did not alter cytokine mRNA stability or the cytokine secretory pathway. S100A12 and serum amyloid A did not appear to form complexes and although they may have common receptors, suppression was unlikely via receptor competition. Serum amyloid A induces cytokines via activation of NF-κB and the MAPK pathways. S100A12 reduced serum amyloid A-, but not LPS-induced ERK1/2 phosphorylation to baseline. It did not affect JNK or p38 phosphorylation or the NF-κB pathway. Reduction in ERK1/2 phosphorylation by S100A12 was unlikely due to changes in intracellular reactive oxygen species, Ca2+ flux or to recruitment of phosphatases. We suggest that S100A12 may modulate sterile inflammation by blunting pro-inflammatory properties of lipid-poor serum amyloid A deposited in chronic lesions where both proteins are elevated as a consequence of macrophage activation. [ABSTRACT FROM AUTHOR]

Lack of a simple, high throughput antibody-dependent cellular phagocytosis (ADCP) assay has limited our understanding of its potential role of in hepatitis C (HCV) infection. Here, we optimised a flow-cytometry based ADCP assay using HCV envelope (E2)-protein coated microbeads that were opsonised with anti-E2 monoclonal IgG antibody (αE2 mAb) and the THP-1 monocyte cell line as effector cells. We found 1.5 × 109/ml microbeads opsonised with 5 μg/ml αE2 mAb and 1.6 × 106/ml THP-1 cells were optimal conditions to distinguish between healthy controls and patients with HCV. This optimised assay was then used to investigate ADCP in plasma obtained from 72 patients with chronic HCV infection and 15 healthy controls. We found that 75% of patients with genotype 1 and 87% of patients with genotype 3 HCV infection had significantly higher levels of ADCP compared to healthy controls. In patients, there was a significant correlation between increase in ADCP and higher concentrations of anti-E2 IgG antibodies in the plasma. Taken together, we established a simple, quick and high throughput ADCP assay for HCV infection that can readily be used for screening of large cohorts of patients and investigation of the role of ADCP in the pathogenesis or protection from this disease. • Development of flow cytometric antibody-dependent phagocytosis (ADCP) assay • Higher signal to noise ADCP at low concentration of monoclonal antibody (5 μg/mL) • ADCP assay is compatible with native blood plasma antibodies without purification. • ADCP assay is adaptable to different antigen and antibody combination. • ADCP results are reproducible using primary blood phagocytes as effector cells. [ABSTRACT FROM AUTHOR]