Leukocyte Immunoglobulin-like Receptor B4 Research Articles

Protein Arginine Methyltransferase 5 Promotes the Migration of AML Cells by Regulating the Expression of Leukocyte Immunoglobulin-Like Receptor B4.

Acute myeloid leukemia (AML) is the most common type of acute leukemia in adults with poor prognosis. Especially for AML-M5 type, due to the strong cell migration ability, the possibility of extramedullary invasion is large and widespread, which leads to poor therapeutic effect. Previous studies have found that protein arginine methyltransferase 5 (PRMT5) could promote the proliferation and differentiation of leukemic cells in AML, but its regulation on the invasive ability of AML cells remains unclear. This study was designed to explore the role of PRMT5 in regulating the invasion of AML cells and to investigate the mechanisms. Patient samples were collected for detection of PRMT5 expression level. AML cells were used for exploring the function of PRMT5. The results of clinical samples showed that the expression of PRMT5 was significantly increased in newly diagnosed and recurrent AML patients, and the expression of leukocyte immunoglobulin-like receptor B4 (LILRB4) was positively correlated with the level of PRMT5. In the cell experiment in vitro, we found that when PRMT5 was knocked down, the invasion, migration, and adhesion capacities of MV-4-11 cells and THP-1 cells were decreased, and the mRNA and protein levels of LILRB4 were also decreased. Moreover, we screened related signaling pathways and found that PRMT5 affected the expression of downstream LILRB4 by activating mTOR pathway, which in turn enhanced the invasive ability of AML cells. Taken together, PRMT5 plays an important role in the invasion of AML, which acts via regulating the expression of LILRB4. PRMT5 could act as a potential therapeutic candidate for AML.

The association of leukocyte immunoglobulin-like receptor subfamily B-4 expression in acute myeloid leukemia and central nervous system involvement

Central nervous system (CNS) involvement in patients with acute myeloid leukemia (AML) varies, ranging from 0.6%–46%. Leukocyte immunoglobulin-like receptor B4 (LILRB4) has been shown to be critical in orchestration of infiltration of AML cells into the CNS in animal models, however it is unknown if an association exists between LILRB4 and CNS involvement (CNS+) in human patients with AML. LILRB4 was measured by flow cytometry in a heterogeneous population of fifty-six AML patients. Patients were then followed clinically for the development of CNS + . LILRB4 was positive in 91 % of patients with CNS + compared to 38 % without CNS involvement (p < 0.002). In logistic analysis: age, BMI, serum albumin and positive LILRB4 were predictive for CNS+ [OR, 95 % CI, p-value]: 0.95, 0.92−0.99, p < 0.01; 0.85, 0.73−0.998, p < 0.05; 0.23, 0.066−0.78, p < 0.02; 16.46, 1.93–140.2, p < 0.02, respectively. This finding of the association of LILRB4 with CNS + in combination with earlier findings suggests that LILRB4 has a mechanistic role in infiltration of the CNS and may provide insight into the pathogenesis of AML seeding the CNS. Moreover, this proof of concept and the findings in the present study may lead to the development of innovative and novel therapies to improve the lives of patients with AML.

Leukocyte immunoglobulin-like receptor B4 protects against cardiac hypertrophy via SHP-2-dependent inhibition of the NF-κB pathway.

Cardiac hypertrophy is a complex pathological process, and the molecular mechanisms underlying hypertrophic remodeling have not been clearly elucidated. Leukocyte immunoglobulin-like receptor B4 (lilrb4) is an inhibitory transmembrane protein that is necessary for the regulation of various cellular signaling pathways. To investigate whether lilrb4 plays a role in cardiac hypertrophy, we performed aortic banding in lilrb4 knockout mice, lilrb4 cardiac-specific transgenic mice, and their wild-type littermates. Cardiac hypertrophy was evaluated by echocardiographic, hemodynamic, pathological, and molecular analyses. We found that lilrb4 was expressed both in myocardial tissue and on cultured cardiomyocytes under basal conditions, but the expression was obviously decreased in mouse hearts following aortic banding and in cardiomyocytes treated with angiotensin II. Lilrb4 disruption aggravated cardiac hypertrophy, fibrosis, and dysfunction in response to pressure overload. Conversely, the cardiac overexpression of lilrb4 led to the opposite effects. Moreover, lilrb4 overexpression inhibited angiotensin II-induced cardiomyocyte hypertrophy in vitro. Mechanistically, we determined that the cardioprotective effect of lilrb4 was mediated through an interaction with SHP-2, the preservation of phosphorylated SHP-2, and the inhibition of the NF-κB pathway. In addition, SHP-2 knockdown in cardiomyocytes eliminated the inhibitory effects of lilrb4 on angiotensin II-induced hypertrophy and NF-κB activation. Our results suggest that lilrb4 protects against pathological cardiac hypertrophy via the SHP-2-dependent inhibition of the NF-κB pathway and may act as a potential therapeutic target for cardiac hypertrophy. KEY MESSAGES: Lilrb4 expression is decreased by hypertrophic stimuli. Lilrb4 protects against pathological cardiac hypertrophy. Lilrb4 interacts with SHP-2 and inhibits NF-κB pathway.

The Association of Leukocyte Immunoglobulin-like Receptor B4 (LILRB-4) with Central Nervous System Involvement in Patients with Acute Myeloid Leukemia

Background The prevalence of central nervous system (CNS) involvement in patients with acute myeloid leukemia (AML) is variable with a reported range of 0.6% to 46%. Unfortunately, the clinical consequences of untreated CNS involvement in AML which include complete paraplegia and bilateral total blindness, can be devastating. In addition, both the diagnostic evaluation and treatment of CNS involvement are associated with potential significant morbidity. Thus, there is a need to predict CNS involvement in a noninvasive manner in patients with AML. The leukocyte immunoglobulin-like receptors are a family of receptors that regulate the activity of cancer stem cells, cancer development and relapse. These receptors are expressed on many cell types and the leukocyte immunoglobulin-like receptor B4 (LILRB-4) is expressed on monocytic myeloid cells. Purpose To investigate whether measurement of LILRB-4 is predictive of CNS involvement in patients with AML. Methods Fifty-six patients with AML and followed at the University of Texas Southwestern medical system had measurement of the LILRB-4 by flow cytometry. Demographic, laboratory, risk stratification, cytogenetics and clinical variables were acquired from electronic medical chart review. Results The study cohort of 56 patients with AML had a median age of 60 years (range: 0.83 - 92); 52% male; and racial/ethnic composition of African American 20%, Asians 4%, White Hispanic 13% and White Non-Hispanic 64%. Eleven (20%) patients were diagnosed with CNS involvement (CNS+). LILRB-4 was positive in 91% of patients with CNS+ compared to 38% without CNS (CNS-) (p Conclusions A positive LILRB-4 by flow cytometry on leukemia cells is strongly associated with CNS involvement in patients with AML. These novel findings suggest that LILRB-4+ may be useful in risk stratification for CNS+. Moreover, combined with preclinical studies, LILRB-4 may provide insight into the pathogenesis of AML seeding the CNS. Further prospective studies are needed to confirm these intriguing results.

Disrupting LILRB4/APOE Interaction by an Efficacious Humanized Antibody Reverses T-cell Suppression and Blocks AML Development.

Therapeutic strategies are urgently needed for patients with acute myeloid leukemia (AML). Leukocyte immunoglobulin-like receptor B4 (LILRB4), which suppresses T-cell activation and supports tissue infiltration of AML cells, represents an attractive drug target for anti-AML therapeutics. Here, we report the identification and development of an LILRB4-specific humanized mAb that blocks LILRB4 activation. This mAb, h128-3, showed potent activity in blocking the development of monocytic AML in various models including patient-derived xenograft mice and syngeneic immunocompetent AML mice. MAb h128-3 enhanced the anti-AML efficacy of chemotherapy treatment by stimulating mobilization of leukemia cells. Mechanistic studies revealed four concordant modes of action for the anti-AML activity of h128-3: (i) reversal of T-cell suppression, (ii) inhibition of monocytic AML cell tissue infiltration, (iii) antibody-dependent cellular cytotoxicity, and (iv) antibody-dependent cellular phagocytosis. Therefore, targeting LILRB4 with antibody represents an effective therapeutic strategy for treating monocytic AML.

Leukocyte immunoglobulin-like receptor B4 deficiency exacerbates acute lung injury via NF-κB signaling in bone marrow-derived macrophages.

Acute lung injury (ALI) is an acute inflammatory disease. Leukocyte immunoglobulin-like receptor B4 (LILRB4) is an immunoreceptor tyrosine-based inhibitory motif (ITIM)-bearing inhibitory receptor that is implicated in various pathological processes. However, the function of LILRB4 in ALI remains largely unknown. The aim of the present study was to explore the role of LILRB4 in ALI. LILRB4 knockout mice (LILRB4 KO) were used to construct a model of ALI. Bone marrow cell transplantation was used to identify the cell source of the LILRB4 deficiency-aggravated inflammatory response in ALI. The effect on ALI was analyzed by pathological and molecular analyses. Our results indicated that LILRB4 KO exacerbated ALI triggered by LPS. Additionally, LILRB4 deficiency can enhance lung inflammation. According to the results of our bone marrow transplant model, LILRB4 regulates the occurrence and development of ALI by bone marrow-derived macrophages (BMDMs) rather than by stromal cells in the lung. The observed inflammation was mainly due to BMDM-induced NF-κB signaling. In conclusion, our study demonstrates that LILRB4 deficiency plays a detrimental role in ALI-associated BMDM activation by prompting the NF-κB signal pathway.

LILRB4 expression in CMML and MDS based on response to hypomethylating agents.

e18550 Background: Leukocyte immunoglobulin-like receptor B4 (LILRB4) is expressed on M4 and M5 monocytic acute myeloid leukemia (AML) cells and leads to negative regulation of immune cell activation via T cell suppression. Its expression and role in chronic myelomonocytic leukemia (CMML) and myelodysplastic syndrome (MDS) are unknown. We investigated LILRB4 expression in patients with CMML and MDS and correlated it with response to hypomethylating agent (HMA) therapy. Methods: We evaluated bone marrow samples from 19 patients with CMML and 27 patients with MDS from 2008-2014. Transcriptomic and splicing analysis by RNA sequencing were performed on CD34-positive cells in CMML and MDS patient samples in addition to AML or MDS cell lines (n = 6). Gene set enrichment analysis was used to identify pathways associated with increased LILRB4 expression. Results: By the WHO 2016 classification for CMML, there were 8 CMML-0, 5 CMML-1, and 6 CMML-2 patients, and there were 2 low-risk, 8 intermediate-1-risk, 12 intermediate-2-risk, and 5 high-risk MDS patients by IPSS. A total of 12 CMML patients and 27 MDS patients received HMA, with 6 and 12 showing response, respectively. RNA expression of LILRB4 was increased in CMML patients when compared to MDS patients and healthy controls (q &lt; 0.1) with no difference in expression based on risk groups. Pathway analysis revealed upregulation of PD-1 signaling (p = 0.004), CTLA-4 signaling (p = 0.004), and inflammatory response (p = 0.004), and gene correlates revealed a positive association with CTLA-4 expression (p = 0.05). LILRB4 expression was slightly increased in CMML patients who responded to HMAs (q &gt; 0.1). In cell lines, LILRB4 was expressed in MOLM13 (AML M5), SKM1 (AML M5), and U937 (histiocytic lymphoma), with an increase in HMA-sensitive MOLM13 cells compared to HMA-resistant MOLM13 cells (q &lt; 0.01) and no difference based on HMAs seen in other cell lines. Conclusions: LILRB4 expression is increased in CMML, with further increase in those exhibiting response to HMA therapy. It is positively correlated with immune checkpoint signaling pathways, particularly CTLA-4 expression. This may set the basis for exploring the possibility of anti-LILRB4 and anti-CTLA-4 combinations.

Gp49B is a pathogenic marker for auto-antibody-producing plasma cells in lupus-prone BXSB/Yaa mice.

AbstractImmune homeostasis is critically regulated by the balance between activating and inhibitory receptors expressed on various immune cells such as T and B lymphocytes, and myeloid cells. The inhibitory receptors play a fundamental role in the immune checkpoint pathway, thus maintaining peripheral tolerance. We recently found that expression of leukocyte immunoglobulin-like receptor (LILR)B4, an inhibitory member of the human LILR family, is augmented in auto-antibody-producing plasmablasts/plasma cells of systemic lupus erythematosus (SLE) patients. However, the mechanism behind the 'paradoxical' up-regulation of this inhibitory receptor upon pathogenic antibody-secreting cells is yet to be known. To this end, in this study, we examined if glycoprotein 49B (gp49B), the murine counterpart of human LILRB4, is also elevated in auto-antibody-producing cells in several SLE mouse models, and tried to clarify the underlying mechanism. We found that gp49B is expressed on plasma cells of lupus-prone models but not of healthy C57BL/6 mice, and the level was positively correlated to the anti-double-stranded DNA IgG titer in serum. Gp49B genetic deletion, however, did not abolish the serum auto-antibodies or fully ameliorate the lethal glomerulonephritis, indicating that gp49B is not the sole regulator of lupus but a pathogenic element in the disease. We conclude that the elevated expression of this inhibitory receptor on pathogenic plasma cells was also relevant upon the murine SLE model. The mechanism of gp49B underlying the disease progression in lupus-prone mice has been discussed.

Leukocyte immunoglobulin-like receptor B4 (LILRB4) negatively mediates the pathological cardiac hypertrophy by suppressing fibrosis, inflammation and apoptosis via the activation of NF-κB signaling

Pathological cardiac hypertrophy is a leading cause of morbidity and mortality in the world. However, it is still unclear the molecular mechanism revealing the progression of the disease. In the study, we illustrated that the expression of leukocyte immunoglobulin-like receptor B4 (LILRB4), associated with the pathological development of various inflammatory diseases, was down-regulated in pressure overload-induced hearts of patients and mice. LILRB4-knockout mice developed cardiac hypertrophy and heart failure by promoting cardiac dysfunction, fibrosis, inflammation and apoptosis. Mechanistically, transforming growth factor β1 (TGF-β1) expression was significantly promoted by LILRB4 deficiency in hearts of mice after aortic banding (AB) surgery. AB-induced inflammation in cardiac tissues was accelerated by LILRB4 deletion through elevating nuclear factor κB (NF-κB) signaling pathway. Furthermore, apoptosis triggered by AB operation in heart tissues was markedly enhanced in LILRB4-KO mice through promoting Caspase-3 activation. Importantly, the in vitro study indicated that LILRB4 knockdown-promoted fibrosis; inflammation and apoptosis were largely via the NF-κB signaling. Therefore, the findings above identified LILRB4 might be a negative regulator of cardiac remodeling, illustrating that LILRB4 represented as a therapeutic target for the prevention of cardiac hypertrophy and heart failure.

A Novel Anti-LILRB4 CAR-T Cell for the Treatment of Monocytic AML.

To effectively improve treatment for acute myeloid leukemia (AML), new molecular targets and therapeutic approaches need to be identified. Chimeric antigen receptor (CAR)-modified Tcells targeting tumor-associated antigens have shown promise in the treatment of some malignancies. However, CAR-T cell development for AML has been limited by lack of an antigen with high specificity for AML cells that is not present on normal hematopoietic stem cells, and thus will not result in myelotoxicity. Here we demonstrate that leukocyte immunoglobulin-like receptor-B4 (LILRB4) is a tumor-associated antigen highly expressed on monocytic AML cells. We generated a novel anti-LILRB4 CAR-T cell that displays high antigen affinity and specificity. These CAR-T cells display efficient effector function invitro and invivo against LILRB4+ AML cells. Furthermore, we demonstrate anti-LILRB4 CAR-T cells are not toxic to normal CD34+ umbilical cord blood cells in colony-forming unit assays, nor in a humanized hematopoietic-reconstituted mouse model. Our data demonstrate that anti-LILRB4 CAR-T cells specifically target monocytic AML cells with no toxicity to normal hematopoietic progenitors. This work thus offers a new treatment strategy to improve outcomes for monocytic AML, with the potential for elimination of leukemic disease while minimizing the risk for on-target off-tumor toxicity.

Hepatic leukocyte immunoglobulin-like receptor B4 (LILRB4) attenuates nonalcoholic fatty liver disease via SHP1-TRAF6 pathway.

Nonalcoholic fatty liver disease (NAFLD) is an increasingly prevalent liver pathology characterized by hepatic steatosis and commonly accompanied by systematic inflammation and metabolic disorder. Despite an accumulating number of studies, no pharmacological strategy is available to treat this condition in the clinic. In this study, we applied extensive gain‐ and loss‐of‐function approaches to identify the key immune factor leukocyte immunoglobulin‐like receptor B4 (LILRB4) as a negative regulator of NAFLD. The hepatocyte‐specific knockout of LILRB4 (LILRB4‐HKO) exacerbated high‐fat diet–induced insulin resistance, glucose metabolic imbalance, hepatic lipid accumulation, and systematic inflammation in mice, whereas LILRB4 overexpression in hepatocytes showed a completely opposite phenotype relative to that of LILRB4‐HKO mice when compared with their corresponding controls. Further investigations of molecular mechanisms demonstrated that LILRB4 recruits SHP1 to inhibit TRAF6 ubiquitination and subsequent inactivation of nuclear factor kappa B and mitogen‐activated protein kinase cascades. From a therapeutic perspective, the overexpression of LILRB4 in a genetic model of NAFLD, ob/ob mice, largely reversed the inherent hepatic steatosis, inflammation, and metabolic disorder. Conclusion: Targeting hepatic LILRB4 to improve its expression or activation represents a promising strategy for the treatment of NAFLD as well as related liver and metabolic diseases. (Hepatology 2018;67:1303‐1319)

LILRB4 deficiency aggravates the development of atherosclerosis and plaque instability by increasing the macrophage inflammatory response via NF-κB signaling.

Atherosclerosis is a chronic inflammatory disease. LILRB4 is associated with the pathological processes of various inflammatory diseases. However, the potential function and underlying mechanisms of LILRB4 in atherogenesis remain to be investigated. In this study, LILRB4 expression was examined in both human and mouse atherosclerotic plaques. The effects and possible mechanisms of LILRB4 in atherogenesis and plaque instability were evaluated in LILRB4-/-ApoE-/- and ApoE-/- mice fed a high-fat diet. We found that LILRB4 was located primarily in macrophages, and its expression was up-regulated in atherosclerotic lesions from human coronary arteries and mouse aortic roots. LILRB4 deficiency significantly accelerated the development of atherosclerotic lesions and increased the instability of plaques, as evidenced by the increased infiltration of lipids, decreased amount of collagen components and smooth muscle cells. Moreover, LILRB4 deficiency in bone marrow-derived cells promoted the development of atherosclerosis. In vivo and in vitro analyses revealed that the pro-inflammatory effects of LILRB4 deficiency were mediated by the increased activation of NF-κB signaling due to decreased Shp1 phosphorylation. In conclusion, the present study indicates that LILRB4 deficiency promotes atherogenesis, at least partly, through reduced Shp1 phosphorylation, which subsequently enhances the NF-κB-mediated inflammatory response. Thus, targeting the "LILRB4-Shp1" axis may be a novel therapeutic approach for atherosclerosis.

Tolerogenic immunoreceptor ILT3/LILRB4 paradoxically marks pathogenic auto-antibody-producing plasmablasts and plasma cells in non-treated SLE.

Plasmablasts and plasma cells (PBs and PCs) producing pathogenic auto-antibodies in patients with systemic autoimmune diseases could be a better target for specific therapies for the disease than general immunosuppression or pan- or activated B-cell targeting. Our previous study indicated that leukocyte immunoglobulin-like receptor (LILR) B4 (B4, also known as ILT3/LIR-5/CD85k), a tolerogenic receptor in antigen-presenting cells, is ectopically expressed on the PB/PC surface in healthy individuals. Here, we show that the enlarged population size of PBs/PCs with augmented B4 expression is characteristic in non-treated systemic lupus erythematosus (SLE). Paradoxically, the transcription frequency of the anti-double-strand DNA immunoglobulin-coding VH sequence in the B4+ population of non-treated SLE was significantly higher than that in B4- cells. B4+ and B4- PBs/PCs were suggested to be developmentally equivalent based on the simultaneous generation of these populations upon activation of memory B cells in vitro B4 expression was found to be induced efficiently by IL-2, while IFN-α effectively induced B4+ PBs/PCs in vitro Utilizing the elevated B4 will support opening a new avenue for identifying the mechanism for generation of, and additional molecular markers for, pathogenic cells.

Leukocyte immunoglobulin-like receptor B4 regulates key signalling molecules involved in FcγRI-mediated clathrin-dependent endocytosis and phagocytosis.

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 middle tyrosine residue of tyrosine-based inhibitory signalling motifs is a key molecule in inhibitory functions of the leukocyte immunoglobulin-like receptor B4 in FcγRI mediated cytokine production and bacterial phagocytosis. (CCR3P.211)

Abstract The leukocyte immunoglobulin-like receptor B4 (LILRB4) is an inhibitory cell surface receptor, primarily expressed on mono-myeloid cells. LILRB4 is a potent inhibitor of FcγRI-mediated pro-inflammatory cytokine production by monocytes and induces tolerogenic dendritic cells in vitro. It is believed that LILRB4 regulates monocyte/macrophage activation through its three intracellular immunoreceptor tyrosine-based inhibitory motifs (ITIMs) by dephosphorylation of non-receptor tyrosine kinases via recruitment of Src homology phosphatase-1 (SHP-1). However, the relative contribution of these ITIMs to LILRB4-mediated inhibition of monocyte/macrophage functions is poorly characterised. To address this, the three functional tyrosine residues of LILRB4 ITIMs at positions 337, 389 and 419 were single, double or triple mutated to non-functional phenylalanine and stably transfected into THP-1 cells. The effects of these mutants on FcγRI-mediated monocyte functions were then assessed. We show that the middle tyrosine residue (Y389) alone maximally inhibited but the proximal tyrosine residue (Y337) enhanced FcγRI-mediated TNF-α production and Fc-dependent bacterial phagocytosis. These results for the first time show that LILRB4 may have variable functions in vitro dependent on the position of the tyrosine residues in its ITIMs. The underlying mechanisms and in vivo implications of these adaptable functions require further investigations.

182: The leukocyte immunoglobulin-like receptor B4 differentially regulates FcγRI-dependent TNF production via its three intracellular immunoreceptor tyrosine-based inhibitory signalling motifs (ITIMs)

The leukocyte immunoglobulin-like receptor (LILR) B4 belongs to a family of activating and inhibitory cell surface receptors that have been increasingly recognised as key regulators of the threshold and amplitude of leukocyte activation. LILRB4 which is highly expressed on monocytes and macrophages transduces inhibitory signals through its three intracellular immunoreceptor tyrosine-based inhibitory motifs (ITIMs). Clinically, increased expression is associated with better clinical outcome in renal transplant recipients contrasted with impaired immune effector cell functions in HIV infection. However, mechanisms of how LILRB4 regulates monocyte activation and the exact contribution of the three ITIMs for its immune-regulatory functions are poorly characterised. In this study we show that co-ligation of LILRB4 with FcγRI on THP-1 cells and primary monocytes markedly inhibited the production of TNF, a key pro-inflammatory cytokine. Cross-linking of FcγRI on THP-1 cells increased phosphorylation key protein tyrosine kinase signaling molecules and an adaptor protein that targets protein tyrosine kinases for degradation (c-Cbl). Co-ligation of LILRB4 and FcγRI considerably reduced FcγRI-mediated phosphorylation of these signaling molecules and terminated cellular activation by recruiting Src homology region 2 domain-containing phosphatase-1 (SHP-1)-like molecule. Importantly, using site-targeted mutagenesis we found for the first time that the middle (Y412) and distal (Y442) tyrosine residues in its ITIM motifs were critical for the inhibitory functions of LILRB4 and Y412 alone was sufficient for maximal inhibition. By contrast, the proximal tyrosine residue at Y360 showed unexpectedly enhanced Fc γ RI-mediated TNF production. Collectively, our results suggest that LILRB4 is a potent immune regulatory receptor in which combined contribution of its multiple ITIMs may fine tune innate immune responses.

Downregulation of Key Early Events in the Mobilization of Antigen-bearing Dendritic Cells by Leukocyte Immunoglobulin-like Receptor B4 in a Mouse Model of Allergic Pulmonary Inflammation

Leukocyte Immunoglobulin-like Receptor B4 (LILRB4) null mice have an exacerbated T helper cell type 2 (Th2) immune response and pulmonary inflammation compared with Lilrb4+/+ animals when sensitized intranasally with ovalbumin (OVA) and low-dose lipopolysaccharide (LPS) followed by challenge with OVA. Moreover, OVA-challenged Lilrb4 −/− mice exhibit greater migration of antigen (Ag)-bearing dendritic cells (DCs) to lymph nodes and accumulation of interleukin 4- and interleukin 5-producing lymph node lymphocytes. The main objective of this study was to determine how the absence of LILRB4 leads to a greater number of DCs in the lymph nodes of Ag-challenged mice and increased lung Th2 inflammation. Mice were sensitized intranasally with PBS alone or containing OVA and LPS; additional cohorts were subsequently challenged with OVA. Expression of chemokine (C-C motif) ligand 21 (CCL21) in the lung was assessed immunohistologically. OVA ingestion and expression of LILRB4 and chemokine (C-C motif) receptor 7 (CCR7) were quantified by flow cytometry. Inhalation of OVA and LPS induced upregulation of LILRB4 selectively on lung Ag-bearing DCs. After sensitization and challenge, the lung lymphatic vessels of Lilrb4 −/− mice expressed more CCL21, a chemokine that directs the migration of DCs from peripheral tissue to draining lymph nodes, compared with Lilrb4+/+ mice. In addition, lung DCs of challenged Lilrb4 −/− mice expressed more CCR7, the CCL21 receptor. The lungs of challenged Lilrb4 −/− mice also contained significantly greater numbers of CD4+ cells expressing interleukin-4 or interleukin-5, consistent with the greater number of Ag-bearing DCs and Th2 cells in lymph nodes and the attendant exacerbated Th2 lung pathology. Our data establish a new mechanism by which LILRB4 can downregulate the development of pathologic allergic inflammation: reduced upregulation of key molecules needed for DC migration leading to decreases in Th2 cells in lymph nodes and their target tissue.

Leukocyte Ig-like Receptor B4 (LILRB4) Is a Potent Inhibitor of FcγRI-mediated Monocyte Activation via Dephosphorylation of Multiple Kinases

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.