Mac-1 Expression Research Articles

Pleiotrophin, a multifunctional cytokine and growth factor, induces leukocyte responses through the integrin Mac-1

Pleiotrophin (PTN) is a multifunctional, cationic, glycosaminoglycan-binding cytokine and growth factor involved in numerous physiological and pathological processes, including tissue repair and inflammation-related diseases. PTN has been shown to promote leukocyte responses by inducing their migration and expression of inflammatory cytokines. However, the mechanisms through which PTN mediates these responses remain unclear. Here, we identified the integrin Mac-1 (αMβ2, CD11b/CD18) as the receptor mediating macrophage adhesion and migration to PTN. We also found that expression of Mac-1 on the surface of human embryonic kidney (HEK) 293 cells induced their adhesion and migration to PTN. Accordingly, PTN promoted Mac-1-dependent cell spreading and initiated intracellular signaling manifested in phosphorylation of Erk1/2. While binding to PTN, Mac-1 on Mac-1-expressing HEK293 cells appears to cooperate with cell-surface proteoglycans because both anti-Mac-1 function-blocking mAb and heparin were required to block adhesion. Moreover, biolayer interferometry and NMR indicated a direct interaction between the αMI domain, the major ligand-binding region of Mac-1, and PTN. Using peptide libraries, we found that in PTN the αMI domain bound sequences enriched in basic and hydrophobic residues, indicating that PTN conforms to the general principle of ligand-recognition specificity of the αMI domain toward cationic proteins/peptides. Finally, using recombinant PTN-derived fragments, we show that PTN contains two distinct Mac-1-binding sites in each of its constitutive domains. Collectively, these results identify PTN as a ligand for the integrin Mac-1 on the surface of leukocytes and suggest that this interaction may play a role in inflammatory responses.

Soluble CD40L is associated with increased oxidative burst and neutrophil extracellular trap release in Beh\xe7et\u2019s disease

BackgroundStudies have suggested that soluble factors in plasma from patients with active (aBD) and inactive (iBD) Behçet’s disease (BD) stimulate neutrophil function. Soluble CD40 ligand (sCD40L) is an important mediator of inflammation in BD. Its expression and effect on neutrophil oxidative burst and neutrophil extracellular trap (NET) release have not been characterized. In this study, we sought to investigate the role of plasma and the CD40L pathway on NET release and the oxidative burst profile in patients with aBD and iBD.MethodsNeutrophils and peripheral blood mononuclear cells (PBMCs) were obtained from patients with aBD (n = 30), patients with iBD (n = 31), and healthy control subjects (HCs; n = 30). sCD40L plasma concentration was determined in individual samples. A pool of plasma for each group was created. In some experiments, plasma pools were treated with recombinant CD40 (rhCD40-muIg) for sCD40L blockade. NET release and H2O2/O2− production were determined after stimulation with phorbol 12-myristate 13-acetate, sCD40L, or plasma pool. Flow cytometric analysis was performed to evaluate the expression of (1) CD40, Mac-1, and phosphorylated NF-κB p65 on neutrophils and monocytes and (2) CD40L on activated T cells and platelets. CD40L gene expression in PBMCs was determined by qRT-PCR.ResultssCD40L plasma levels were significantly higher in patients with iBD (median 17,234, range 2346–19,279 pg/ml) and patients with aBD (median 18,289, range 413–19,883 pg/ml) than in HCs (median 47.5, range 33.7–26.7 pg/ml; p < 0.001). NET release was constitutively increased in BD compared with HC. NET release and H2O2/O2− were higher after stimulation with sCD40L or BD plasma and decreased after sCD40L blockade. Mac-1 expression was constitutively increased in neutrophils of patients with aBD (88.7 ± 13.2% of cells) and patients with iBD (89.2 ± 20.1% of cells) compared with HC (27.1 ± 18.8% of cells; p < 0.01). CD40 expression on phagocytes and CD40L expression on platelets were similar in the three groups. PBMCs as well as nonactivated and activated CD4+ T cells from patients with BD showed higher CD40L expression.ConclusionsPlasma from patients with aBD exerts a stimulus on NET release and oxidative burst, probably induced by sCD40L.

The Specific Mitogen- and Stress-Activated Protein Kinase MSK1 Inhibitor SB-747651A Modulates Chemokine-Induced Neutrophil Recruitment.

Mitogen-activated protein kinase (MAPK) signaling is involved in a variety of cellular functions. MAPK-dependent functions rely on phosphorylation of target proteins such as mitogen- and stress-activated protein kinase 1 (MSK1). MSK1 participates in the early gene expression and in the production of pro- and anti-inflammatory cytokines. However, the role of MSK1 in neutrophil recruitment remains elusive. Here, we show that chemokine macrophage inflammatory protein-2 (CXCL2) enhances neutrophil MSK1 expression. Using intravital microscopy and time-lapsed video analysis of cremasteric microvasculature in mice, we studied the effect of pharmacological suppression of MSK1 by SB-747651A on CXCL2-elicited neutrophil recruitment. SB-747651A treatment enhanced CXCL2-induced neutrophil adhesion while temporally attenuating neutrophil emigration. CXCL2-induced intraluminal crawling was reduced following SB-747651A treatment. Fluorescence-activated cell sorting analysis of integrin expression revealed that SB-747651A treatment attenuated neutrophil integrin αMβ2 (Mac-1) expression following CXCL2 stimulation. Both the transmigration time and detachment time of neutrophils from the venule were increased following SB-747651A treatment. It also decreased the velocity of neutrophil migration in cremasteric tissue in CXCL2 chemotactic gradient. SB-747651A treatment enhanced the extravasation of neutrophils in mouse peritoneal cavity not at 1–2 h but at 3–4 h following CXCL2 stimulation. Collectively, our data suggest that inhibition of MSK1 by SB-747651A treatment affects CXCL2-induced neutrophil recruitment by modulating various steps of the recruitment cascade in vivo.

SIRT1 suppresses atherosclerosis induced by TLR2-mediated monocyte adhesion to endothelium through MAC-1 expression

Aim: Sirtuin (SIRT) 1 has been demonstrated to act protectively during the progression of atherosclerosis, but the underlying mechanisms involved are unclear. The authors investigated the role played by SIRT1 in TLR2-mediated monocyte adhesion to vascular endothelium; a key early event during the progression of atherosclerosis.

Abstract 2643: Thymic precursor cells generate acute myeloid leukemia in NP23-NHD13 double transgenic mice

Abstract Oncogenic fusion genes have been identified in a large number of hematologic malignancies. We previously generated mice that expressed either a NUP98-PHF23 (NP23) or NUP98-HOXD13 (NHD13) fusion in the hematopoietic compartment. Both NP23 and NHD13 mice develop a wide variety of leukemias, including myeloid, erythroid, megakaryocytic and lymphoid, at 9-14 months of age. NP23-NHD13 double transgenic mice were generated by interbreeding NP23 and NHD13 mice. Surprisingly, 100% of the NP23-NHD13 double transgenic mice showed rapid onset of acute myeloid leukemia (AML) within 3 months. The leukemias were characterized by extraordinarily high WBC and replacement of the thymus with Mac1+/Gr1+ myeloblasts; the percent of malignant myeloid cells in the thymus was often higher than the bone marrow (BM). It led to the intriguing hypothesis that the AML generated in NP23-NHD13 mice arose in the thymus, as opposed to the BM. We transplanted unfractionated cells from the thymus of a NP23-NHD13 mouse, invaded by Mac1+/Gr1+ AML cells or residual CD4-/CD8- double negative (DN) thymocytes into sub-lethally irradiated recipients. All mice (transplanted with either unfractionated leukemic cells or DN cells from same leukemic mouse) developed Mac1+/Gr1+ AML within 26 days, indicating that the AML was aggressive and transplantable, and could be transmitted by DN thymocytes. The observation that the AML was transmitted by the DN thymocytes or Mac1+/Gr1+ AML suggested that the DN thymocytes may be the cell of origin for this AML. To rule out the possibility that the leukemia in this experiment was transmitted by rare, contaminant Mac1+/Gr1+ cells, we repeated the experiment, twice, using DN thymocytes from 4-5 wk old mice with no signs of leukemia. DN thymocytes again transmitted a Mac1+/Gr1+ AML. We further fractioned DN thymocytes into DN1, DN2, DN3 and DN4 populations; the recipients of DN1 and DN2 populations developed a Mac1+/Gr1+ AML, but DN3 and DN4 recipients were healthy with no signs of engraftment in the peripheral blood. DN thymocytes from non-leukemic NP23-NHD13 mice were cultured on an OP9 stromal layer, which has been shown to support myeloid differentiation in vitro. DN thymocytes from non-leukemic NP23-NHD13 mice cultured on a OP9 stromal layer showed a markedly enhanced ability to differentiate into Mac1+/Gr1+ myeloid lineage cells compared to WT (56% vs 1.4 % at day 10). The NP23-NHD13 cells lost expression of Mac1 and Gr1 after 26 days; with immunophenotype now CD4-, CD8-, CD25-, CD44+, Thy-1.2+ and cKit+, consistent with a self-renewing DN1 thymocyte. These cells were transplanted; all recipients were anemic, and demonstrated engraftment of NP23-NHD13 myeloid cells as well as a less prominent (7.85%-38.5%) population of CD71+/Ter119+ erythroid cells in the BM and spleen. Taken together, these results indicate that NP23-NHD13 thymic progenitors are potently leukemogenic, and retain myeloid and erythroid differentiation potential. Citation Format: Subhadip Kundu, Eun Sil Park, Yang Jo Chung, Trevor Stephen Barlowe, Peter D. Aplan. Thymic precursor cells generate acute myeloid leukemia in NP23-NHD13 double transgenic mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2643. doi:10.1158/1538-7445.AM2017-2643

Effects of intrauterine infusion of Escherichia coli lipopolysaccharide on uterine mRNA gene expression and peripheral polymorphonuclear leukocytes in Jersey cows diagnosed with purulent vaginal discharge

The objectives of the current experiment were to investigate the effects of intrauterine treatment of cows with purulent vaginal discharge (PVD) using lipopolysaccharide (LPS) from Escherichia coli on uterine mRNA expression of genes related to inflammatory responses, peripheral polymorphonuclear leukocyte (PMN) function, hematological parameters, and blood concentrations of cortisol, haptoglobin, and progesterone (P4). Jersey cows (n = 3,084) were examined for PVD at 35 ± 6 d postpartum using the Metricheck device (Simcro, Hamilton, New Zealand). At examination, 310 cows had PVD (10.1%), but to ensure proper collection and processing of samples, 267 cows were used in this experiment. Cows were balanced for lactation number, body condition score, and milk yield, and randomly assigned to the control treatment [intrauterine infusion of 20 mL of phosphate-buffered saline (PBS); n = 87] or to receive intrauterine infusion of 20 mL of PBS containing 150 µg (LPS150; n = 91) or 300 µg (LPS300; n = 89) of E. coli LPS. Uteri were biopsied in a subgroup of cows at 6 h after infusion and in another subgroup of cows at 24 h after infusion. Peripheral PMN expression of adhesion molecules (L-selectin and MAC-1) and phagocytosis and oxidative burst were evaluated at 0, 2, and 6 h after infusion. Blood sampled 0, 2, 6, 24, 48, and 168 h after infusion was used for complete hemogram and to determine concentrations of cortisol, haptoglobin, and P4. Treatment did not affect uterine mRNA expression of adhesion molecules [endothelial leukocyte adhesion molecule (E-selectin), intracellular adhesion molecule-1 (ICAM-1), and vascular cell adhesion molecule-1 (VCAM-1)], cytokines (tumor necrosis factor-α, IL-1β, IL-6, IL-8, and IL-10), and toll-like receptor-4. Treatment did not affect PMN expression of L-selectin, but intensity of expression of MAC-1 was higher for LPS150 cows than PBS cows, and tended to be higher in LPS150 than LPS300 cows. Furthermore, a greater percentage of PMN from LPS300 cows were positive for phagocytosis and oxidative burst compared with PBS and LPS150 cows. No effects were observed of treatment on hematological parameters and concentrations of cortisol, haptoglobin, and P4. These observations suggest that intrauterine infusion of E. coli LPS moderately stimulates peripheral PMN function, but further research is needed to better understand the immunomodulatory effects of LPS in the uterus of cows with PVD.

DioxolaneA3-phosphatidylethanolamines are generated by human platelets and stimulate neutrophil integrin expression

Activated platelets generate an eicosanoid proposed to be 8-hydroxy-9,10-dioxolane A3 (DXA3). Herein, we demonstrate that significant amounts of DXA3 are rapidly attached to phosphatidylethanolamine (PE) forming four esterified eicosanoids, 16:0p, 18:0p, 18:1p and 18:0a/DXA3-PEs that can activate neutrophil integrin expression. These lipids comprise the majority of DXA3 generated by platelets, are formed in ng amounts (24.3±6.1ng/2×108) and remain membrane bound. Pharmacological studies revealed DXA3-PE formation involves cyclooxygenase-1 (COX), protease-activated receptors (PAR) 1 and 4, cytosolic phospholipase A2 (cPLA2), phospholipase C and intracellular calcium. They are generated primarily via esterification of newly formed DXA3, but can also be formed in vitro via co-oxidation of PE during COX-1 co-oxidation of arachidonate. All four DXA3-PEs were detected in human clots. Purified platelet DXA3-PE activated neutrophil Mac-1 expression, independently of its hydrolysis to the free eicosanoid. This study demonstrates the structures and cellular synthetic pathway for a family of leukocyte-activating platelet phospholipids generated on acute activation, adding to the growing evidence that enzymatic PE oxidation is a physiological event in innate immune cells.

Early and Late Leukemic Stem Cells in Murine Leukemia Models

Acute leukemia is uncontrolled proliferation of leukemic stem cells (LSCs). Murine models of leukemia suggest that LSCs arise from lineage-committed progenitor cells. However, whether LSCs also directly arise from long-term (LT) and short-term (ST) hematopoietic stem cells (HSCs) and other hematopoietic progenitor cells (HPCs) and whether differentiation potentials influence the leukemia types are poorly understood.In this study, we used two murine leukemia models (AML with MLL-AF9 fusion protein and T-ALL with Notch-1 intracellular domain, ICN-1). Two HSC and three HPC populations were sorted from B6 (CD45.1) mouse bone marrow (BM) by flow cytometry: HSC1, CD150+CD41-CD34-Lin-Sca-1+c-Kit+ (LSK) cells; HSC2, CD150-CD41-CD34-LSK cells; HPC1, CD150+CD41+CD34-LSK cells; HPC2, CD150+CD41+CD34+LSK cells; HPC3, CD150-CD41-CD34+LSK cells. HSC1, HSC2, HPC1, and HPC3 are enriched in LT-HSCs, ST-HSCs, repopulating common myeloid progenitors, and lymphoid-primed multipotent progenitors, respectively. 400-600 cells were sorted from each population, pre-stimulated for 24 hrs, and transduced with MLL-AF9 or ICN-1 retrovirus for another 24 hrs. Cells were mixed with 3-5 x 105 BM cells (CD45.2), and injected into the lethally irradiated mice (CD45.2). The recipient mice were monitored by detection of GFP+ cells in the peripheral blood (PB). When the recipient mice showed > 50% GFP+ cells in the PB or became ill, mice were killed and analyzed for leukemia.In the MLL-AF9 AML model, leukemia developed in all recipient mice injected with HSC1, 2, HPC1, 2, or 3 around 6 weeks after transplantation. Leukemic cells in PB and BM appeared positive for Mac-1/Gr-1, but negative for CD3 and B220. We detected a new LSC population in the BM: Lin-Mac-1+c-Kit+Sca-1+CD150-CD16/32+ cells. Both CD34-negative and CD34-positive cells were detected in this population. We named this population as "early LSCs" because it was phenotypically similar to ST-HSCs and/or LMPPs in normal mouse BM cells except Mac-1 expression. Early LSCs differed from previously identified LSCs (L-GMP) because they are Sca-1-positive. We now consider L-GMP as one of "late LSC" types. Early LSCs formed leukemic colonies in vitro and initiated the AML in serial transplantation. These results show the similar AML develops regardless of HSC and HPC types transduced. Both early and late LSCs likely play a role in establishment of AML.In the ICN-1 T-ALL model, leukemia developed in all recipient mice injected with HSC1, 2, HPC1, 2, or 3 around 4 weeks after transplantation. Most leukemic cells in PB and BM appeared positive for CD4/CD8, but negative for Mac-1/Gr-1 and B220. We did not detect early LSCs in BM. To identify late LSCs in BM, we injected the CD3+CD8+CD4- and CD3+CD8+CD4+ cells into the sub-lethally irradiated mice. T-ALL developed from both populations (median latency, 23 vs 35 days). These results show the similar T-ALL develops regardless of HSC and HPC types transduced. In this case, late LSCs can be directly generated without early LSCs from HSC or HPC populations.Taken together, we found that the similar types of leukemia develop regardless of different types of initiating cells in both models. We also found new LSC populations, early LSCs in the AML model and late LSCs in the T-ALL model. Both early and late LSCs were able to re-initiate leukemia after secondary transplantation.In conclusion, highly purified murine HSCs and HPCs were used for the first time to initiate leukemia. Both MLL-AF9 AML and ICN-1 T-ALL models suggest that gene mutations at all differentiation stages from HSCs to HPCs potentially induce the similar types of leukemia. Of note is that distinct lineage differentiation potentials of HSCs and HPCs do not affect leukemia types. More importantly, early LSCs may serve as the earliest event in leukemogenesis in AML. In the case of ALL, gene mutations seem carried over until reaching the developmental stage of late LSCs. Our results suggest that both early and late LSCs should be eradicated to achieve complete remission and prevent relapse. DisclosuresNo relevant conflicts of interest to declare.

Thymic Precursor Cells Generate Acute Myeloid Leukemia in NUP98-PHF23/NUP98-HOXD13 Double Transgenic Mice

Introduction: Oncogenic fusion genes have been identified in a large number of hematologic malignancies. Using Vav regulatory elements, we previously developed transgenic mice that expressed either a NUP98-PHF23 (NP23) or NUP98-HOXD13 (NHD13) fusion in the hematopoietic compartment. Both NP23 and NHD13 mice develop a wide variety of leukemias, including myeloid, erythroid, megakaryocytic and lymphoid, with an age of onset typically between 9 and 14 months, but rarely before 6 months of age.Results: NP23-NHD13 double transgenic mice were generated by interbreeding NP23 and NHD13 single transgenic mice. Surprisingly, 100% of the NP23-NHD13 double transgenic mice showed rapid onset of acute myeloid leukemia (AML) within three months. In contrast, none of the single transgenic mice had developed leukemia by 3 months of age. The leukemias were characterized by extraordinarily high WBC (>500,000/uL) and almost complete replacement of the thymus with Mac1+/Gr1+ myeloblasts, with the percent of malignant myeloid cells in the thymus often higher than the percent in the bone marrow or spleen. These findings led to the intriguing hypothesis that the AML generated in NP23-NHD13 mice arose in the thymus, as opposed to the bone marrow.We harvested cells from the thymus of a NP23-NHD13 mouse that had been invaded by Mac1+/Gr1+ AML cells, and transplanted unfractionated cells (10E06 per recipient) as well as residual CD4-/CD8- double negative (DN) thymocytes (10E05 per recipient; these thymocytes were negative for Mac1 and Gr1 as well as CD4 and CD8) into sub-lethally(600 rds) irradiated recipients. All mice (transplanted with unfractionated thymocytes or DN cells from same leukemic mouse) developed Mac1+/Gr1+ AML within 26 days, indicating that the AML was aggressive and transplantable, and could be transmitted by DN thymocytes. The observation that there was no difference in onset of AML transmitted by the DN thymocytes or Mac1+/Gr1+ AML suggested that the DN thymocytes may be the cell of origin for this leukemia. To rule out the possibility that the leukemia in this experiment was transmitted by rare, contaminant Mac1+/Gr1+ cells, we repeated the experiment, twice, using DN thymocytes from 4-5 wk old mice with no signs of leukemia. In these experiments, the DN thymocytes again transmitted a Mac1+/Gr1+ AML. Finally, we further fractioned DN thymocytes into DN1, DN2, DN3 and DN4 populations; the recipients of DN1 and DN2 populations developed a Mac1+/Gr1+ AML, whereas the DN3 and DN4 recipients were healthy with no signs of engraftment in the peripheral blood.To gain further insight into this phenomenon, DN thymocytes from non-leukemic NP23-NHD13 mice were co-cultured on an OP9 stromal layer, which has been shown to support myeloid, B, and T lymphocyte (when supported with Notch1 ligands) differentiation in vitro. DN thymocytes from non-leukemic NP23-NHD13 mice co-cultured on a OP9 stromal layer showed a markedly enhanced ability to differentiate into Mac1+/Gr1+ myeloid lineage cells compared to WT (56% vs 1.4 % at day 10). However, this "wave" of myeloid differentiation was extinguished by 26 days. The NP23-NHD13 cells lost expression of Mac1 and Gr1; an expanded immunophenotype was CD4-, CD8-, CD25-, CD44+, Thy-1.2+ and cKit+, consistent with a self-renewing DN1 thymocyte. These cells were transplanted; all recipients were anemic, and demonstrated engraftment of NP23-NHD13 myeloid cells as well as a less prominent (7.85%-38.5%) population of CD71+/Ter119+ erythroid cells in the BM and spleen. There was no evidence of T-cell differentiation of the transplanted NP23-NHD13 cells. Further thymocyte fractionation experiments using the OP9 co-culture system demonstrated that myeloid differentiation potential resided in the DN1 population.Conclusions: Taken together, these results indicate that NP23-NHD13 thymic progenitors are potently leukemogenic, and retain myeloid and erythroid differentiation potential. DisclosuresNo relevant conflicts of interest to declare.

Flavonoid-enriched fraction from Echinodorus macrophyllus aqueous extract exhibits high in-vitro and in-vivo anti-inflammatory activity.

Echinodorus macrophyllus (Kunth) Micheli (Alismataceae) is popularly used as an infusion to treat inflammatory diseases. This work fractionated the aqueous extract of E. macrophyllus (AEEm) to improve its anti-inflammatory effects. Aqueous extract of E. macrophyllus was fractionated by Sephadex LH-20 and analysed by HPLC-DAD. Anti-inflammatory action was evaluated, in vivo, by air pouch model (total leucocyte, protein and leukotriene B4 (LTB4 )), and, in vitro, by neutrophil migration (transwell assay) and its Mac1 expression (flow cytometry), and RAW 264.7 nitric oxide (NO) production (Griess reaction). Fr20 reduced total leucocyte at 2.5 mg/kg (29.7%) while ethanolic extract of E. macrophyllus (EAEm) increased it (94.0%). Fr20 showed higher (P < 0.05) inhibition (89.8%) of LTB4 in exudate than EAEm (75.0%). Fr20 and EAEm decreased exudate protein and inflammatory infiltrate in pouch tissues, in-vitro neutrophil migration, and NO production. Otherwise, Fr40 did not reduce leucocytes and exudate protein (until 50 mg/kg) nor tissue inflammation, and increased in-vitro NO production. The inhibition of neutrophil migration by EAEm, but not Fr20, was dependent on reduced Mac-1 expression. The fractionation of AEEm provided a more potent anti-inflammatory fraction containing flavonoids (Fr20) that reduces the migration of neutrophils and LTB4 release, probably contributing to its mechanism of action.

B7H3 ameliorates LPS-induced acute lung injury via attenuation of neutrophil migration and infiltration.

Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are characterized by an excessive inflammatory response within the lungs and severely impaired gas exchange resulting from alveolar-capillary barrier disruption and pulmonary edema. The costimulatory protein B7H3 functions as both a costimulator and coinhibitor to regulate the adaptive and innate immune response, thus participating in the development of microbial sepsis and pneumococcal meningitis. However, it is unclear whether B7H3 exerts a beneficial or detrimental role during ALI. In the present study we examined the impact of B7H3 on pulmonary inflammatory response, polymorphonuclear neutrophil (PMN) influx, and lung tissue damage in a murine model of lipopolysaccharide (LPS)-induced direct ALI. Treatment with B7H3 protected mice against LPS-induced ALI, with significantly attenuated pulmonary PMN infiltration, decreased lung myeloperoxidase (MPO) activity, reduced bronchoalveolar lavage fluid (BALF) protein content, and ameliorated lung pathological changes. In addition, B7H3 significantly diminished LPS-stimulated PMN chemoattractant CXCL2 production by inhibiting NF-κB p65 phosphorylation, and substantially attenuated LPS-induced PMN chemotaxis and transendothelial migration by down-regulating CXCR2 and Mac-1 expression. These results demonstrate that B7H3 substantially ameliorates LPS-induced ALI and this protection afforded by B7H3 is predominantly associated with its inhibitory effect on pulmonary PMN migration and infiltration.

Inhaled corticosteroids increase blood neutrophil count by decreasing the expression of neutrophil adhesion molecules Mac-1 and L-selectin

ObjectiveThe objective was to investigate the effect of commonly used inhaled corticosteroids on white blood cell count (WBC) and to examine the mechanisms involved. MethodsThis randomized comparative study comprised 60 healthy adults. We measured the effects of budesonide (by face mask inhalation or aerosol inhaler), fluticasone (by inhaler), and saline inhalation (control) on WBC and the differential leukocyte count, especially the absolute neutrophil count (ANC). To elucidate the mechanisms involved, we measured the expression of the adhesion neutrophil ligands Mac-1 (CD11b) and L-selectin (CD62L), and granulocyte colony-stimulating factor serum levels. ResultsSix hours after a single-dose inhalation of budesonide, mean increases of 23.4% in WBC (95% confidence interval [CI], 11.3-35.4) and 30.1% in ANC (95% CI, 7.2-53.0) were noted. The percentage of neutrophils increased from 54.6% to 58.1% (P< .001). Inhaled fluticasone increased WBC and ANC by 12.6% (95% CI, 1.5-23.7) and 22.7% (95% CI, 6.2-39.2), respectively (P< .01 for both). The absolute lymphocyte and eosinophil counts did not change significantly from baseline.The expression of Mac-1 and L-selectin decreased by 51.0% (P< .01) and 30.9% (P= .02), respectively, following face mask inhalation of budesonide and by 39.8% (P= .01) and 17.4% (P= .17), respectively, following inhalation of fluticasone. No significant changes in granulocyte colony-stimulating factor levels were noted. ConclusionsGlucocorticoid inhalation increases WBC by increasing ANC. Reduced neutrophil adhesion to the endothelial surface, mediated by decreased adhesion molecule expression on neutrophils, is a plausible mechanism. Physicians should be aware of the effect of inhaled corticosteroids on WBC, as it may influence clinical decisions, especially in the emergency department.

Interference with purinergic signalling: an explanation for the cardiovascular effect of abacavir?

The association of abacavir (ABC), a guanosine analogue, with cardiovascular toxicity is a long-lasting matter of controversy engendered by the lack of a mechanism of action. Clinical data point to an acute mechanism of vascular inflammation. Previous studies have shown that ABC induces leukocyte-endothelial cell interactions, an indicator of vascular inflammation. These effects are reproduced by another purine analogue, didanosine, but not by pyrimidine or acyclic nucleotide analogues, hinting at an interference with the purinergic system. The aim of the present study was to assess the role of ATP-receptors in leukocyte accumulation induced by ABC. Clinical concentrations of ABC were analysed in an animal model in vivo (intravital microscopy using male C57BL/6 wild-type or P2rx7 knockout mice), in human endothelial cells and leukocytes in vitro (flow chamber), or in leukocyte Mac-1 expression (flow cytometry). ABC reduced leukocyte rolling velocity and increased rolling flux and adhesion both in vivo and in vitro. These effects were absent in P2rx7 knockout mice and following the specific blockade of ATP-P2X7 receptors in wild-type animals. Further pharmacological characterization in flow chamber experiments confirmed the role of ATP-P2X7 receptors and suggested that those located on leukocytes were particularly implicated. Activation of ATP-P2X7 receptors is needed for expression of leukocytic Mac-1. Similar effects were obtained with didanosine. ABC induces leukocyte-endothelial cell interactions through a mechanism involving interference with purine-signalling pathways via ATP-P2X7 receptors located mainly on leukocytes. Our data are compatible with existing clinical data revealing an increased cardiovascular risk in ABC-treated patients.

Human Platelets Utilize Cycloxygenase-1 to Generate Dioxolane A3, a Neutrophil-activating Eicosanoid

Eicosanoids are important mediators of fever, pain, and inflammation that modulate cell signaling during acute and chronic disease. We show by using lipidomics that thrombin-activated human platelets generate a new type of eicosanoid that both stimulates and primes human neutrophil integrin (Mac-1) expression, in response to formylmethionylleucylphenylalanine. Detailed characterization proposes a dioxolane structure, 8-hydroxy-9,11-dioxolane eicosatetraenoic acid (dioxolane A3, DXA3). The lipid is generated in nanogram amounts by platelets from endogenous arachidonate during physiological activation, with inhibition by aspirin in vitro or in vivo, implicating cyclooxygenase-1 (COX). Pharmacological and genetic studies on human/murine platelets revealed that DXA3 formation requires protease-activated receptors 1 and 4, cytosolic phospholipase A2 (cPLA2), Src tyrosine kinases, p38 MAPK, phospholipase C, and intracellular calcium. From data generated by purified COX isoforms and chemical oxidation, we propose that DXA3 is generated by release of an intermediate from the active site followed by oxygenation at C8. In summary, a new neutrophil-activating platelet-derived lipid generated by COX-1 is presented that can activate or prime human neutrophils, suggesting a role in innate immunity and acute inflammation.

Sirt1 decreases TLR2-mediated Mac-1 expression in atherosclerosis

Abstract Sirtuin (Sirt) 1 has been demonstrated to exerts protective effects in the progression of atherosclerosis, however, the underlying mechanisms are unclear. Thus, this study investigated the the role of Sirt1 in TLR2-mediated monocyte adhesion to vascular endothelium, which is a key early event in macrophage foam cell formation. The atherosclerotic lesion formation in aortic sinus was significantly increased in Pam3CSK4-treated wild type (WT) mice, which was significantly inhibited in Sirt1 Tg mice group. An en face immunohistochemistry of endothelial surfaces revealed a marked increase in monocyte adhesion to aortic endothelium in WT mice treated with Pam3CSK4, which was significantly attenuated in Sirt1 Tg mice. Likewise, the adhesion capacity of primary monocytes isolated from Pam3CSK4-treated WT mice was higher than those of monocytes from Sirt1 Tg mice. In in vitro study, Pam3CSK4 increased monocyte adhesion to endothelial cells with an enhanced Mac-1 expression. These were attenuated by a recombinant Sirt1, as well as by molecular overexpression of Sirt1 in monocytes. In addition, we found that Pam3CSK4-reduced Sirt1 expression in monocytes was regulated by the transcription factors, NF-kB and CREB. The role of NF-kB and CREB in Pam3CSK4-reduced Sirt expression was confirmed by siRNA knockdown of Sp1 and NF-kB. Sirt1 supresses atherosclerosis progression by TLR2-mediated monocyte adhesion to endothelium through Mac-1 expression, which is mediated by NF-kB and CREB pathways in monocytes, consequently leading to the development of therapeutic interventions for regulating Sirt1 expression in atherosclerosis.

Platelet-derived CXCL4 regulates neutrophil infiltration and tissue damage in severe acute pancreatitis

Platelets are known to play an important role in acute pancreatitis (AP) via promotion of neutrophil accumulation, although mechanisms behind platelet-dependent accumulation of neutrophils in the pancreas remain elusive. Platelets contain a wide spectrum of different pro-inflammatory compounds, such as chemokines. CXCL4 (platelet factor 4) is one of the most abundant chemokine in platelets, and we hypothesized that CXCL4 might be involved in platelet-dependent accumulation of neutrophils in the inflamed pancreas. The aim of this study was to examine the role of CXCL4 in severe AP. Pancreatitis was provoked by infusion of taurocholate into the pancreatic duct or by intraperitoneal administration of L-arginine in C57BL/6 mice. Animals were treated with an antibody against platelets or CXCL4 before induction of pancreatitis. Plasma and lung levels of CXCL2, CXCL4, and interleukin (IL)-6 were determined by use of enzyme-linked immunosorbent assay. Flow cytometry was used to examine surface expression of macrophage-1 (Mac-1) on neutrophils. Plasma was obtained from healthy individuals (controls) and patients with AP. Challenge with taurocholate increased plasma levels of CXCL4, and depletion of platelets markedly reduced plasma levels of CXCL4 indicating that circulating levels of CXCL4 are mainly derived from platelets in AP. Inhibition of CXCL4 reduced taurocholate-induced neutrophil recruitment, IL-6 secretion, edema formation, amylase release, and tissue damage in the pancreas. However, immunoneutralization of CXCL4 had no effect on CXCL2-evoked neutrophil expression of Mac-1 or chemotaxis invitro, suggesting an indirect effect of CXCL4 on neutrophil recruitment in AP. Targeting CXCL4 significantly attenuated plasma and lung levels of CXCL2, which is a potent neutrophil chemoattractant, and inhibition of the CXCL2 receptor attenuated neutrophil infiltration and tissue damage in the inflamed pancreas. A significant role of CXCL4 was confirmed in an alternate model of AP induced by L-arginine challenge. Moreover, patients with AP had significantly increased plasma levels of CXCL4 compared with healthy controls. These findings' results suggest that platelet-derived CXCL4 is a potent stimulator of neutrophil accumulation in AP and that this is mediated via generation of CXCL2 in the inflamedpancreas. We conclude that CXCL4 plays an important role in pancreatic inflammation and that targeting CXCL4 might be a useful way to ameliorate tissue damage in AP.

043 Regulatory natural killer cells in alopecia areata

Alopecia areata (AA) is a T-cell mediated autoimmune disease of hair follicle. Although the polymorphisms in the ULBP3/6 ligands of activating receptor NKG2D has been shown to associate with AA, the role of natural killer (NK) cells in AA is not fully defined. NK cells are major components of innate immunity and play critical roles in early defense against microbial pathogens and tumors. Recent reports indicate that NK cells play opposite roles with both pathogenic and regulatory activity in some autoimmune disorders. The protective role of NK cells was previously reported in C3H/HeJ mouse model of AA, however, little is known about the mechanisms of this protective function. Here we also found that NK cell depletion accelerates the development of AA in C3H/HeJ mice. Murine NK cells can be divided into distinct subsets based on the expression of Mac-1 and CD27. Mac-1high CD27high NK cells exhibit higher cytotoxicity against target cells than other NK cell subsets. We detected a higher percentage of Mac-1high CD27high NK cells in skin draining lymph nodes from new-onset AA mice (after grafting) than from control C3H/HeJ mice without AA. The expression of CD69, LAG-3, KLRG1 and TIGIT was also significantly increased on this population of NK cells in AA mice, indicative of activation of these NK cells in AA. It has been suggested that NK cells may homeostatically control inflammation by targeting activated T cells via engagement of NK activating receptors by T cell expression of NKG2DLs, Qa-1 and/or through LFA-1/ICAM-1 interactions. We detected higher levels of ICAM-1 on activated CD4 and CD8 T cells than on their naïve counterparts, which could render activated T cells vulnerable to NK-mediated cytolysis. Our data suggests that Mac-1high CD27high NK cells may inhibit autoimmune T cell responses by regulating activated autoantigen-specific T cells.

IKZF2, a Novel Target of MSI2 RNA-Binding Protein Plays an Oncogenic Role in Myeloid Leukemia

Deregulated epigenetic program is found in many cancers, and genetic aberrations of histone methyltransferases contribute to transformation in myeloid leukemias. Post-transcriptional regulation in leukemia has recently been highlighted as a novel way for maintaining the leukemia stem cell (LSC) program. We have recently demonstrated that Msi2 is required for LSC function in a murine MLL-AF9 leukemia model. We determined that MSI2 maintains the mixed-lineage leukemia (MLL) self-renewal program by interacting and retaining efficient translation of critical MLL regulated transcription factors including Hoxa9, Myc and Ikzf2. Despite extensive studies implicating Myc and Hoxa9 in leukemia, the role for Ikzf2 in myeloid leukemia is not known.Ikzf2 is a member of the Ikaros transcription factor family and regulates lymphocyte development by controlling regulatory T-cell function. Ikzf2 is highly expressed in Hematopoietic Stem Cells (HSC) and to investigate if Ikzf2 is involved in HSC function, we utilized mice that have a specific deletion of Ikzf2 in the hematopoietic system through the Vav-cre system. We found similar frequencies for different populations in the stem, progenitor and mature cells in the bone marrow of Ikzf2f/f and Ikzf2D/D mice. Colony assays of isolated Lin- Sca1+ c-Kit+ (LSK) cells from Ikzf2f/f and Ikzf2D/D mice resulted in a comparable number of myeloid progenitor colonies. Furthermore, noncompetitive transplant of Ikzf2f/f and Ikzf2D/D bone marrow cells showed similar chimerism after 34 months indicating that I kzf2D/D mice have normal HSC function and hematopoiesis.To interrogate the role of Ikzf2 in acute myeloid leukemia we utilized the MLL-AF9 retroviral transduction model. Intracellular flow cytometry showed that IKZF2 is highly expressed in the LSC population compared to the non-LSCs. We then transduced Ikzf2f/f and Ikzf2D/D LSK cells with MLL-AF9 and found that Ikzf2 deletion results in a ten-fold reduction in colony formation compared to Ikzf2f/f cells. Transplantation of transduced cells results in delayed leukemia progression with reduced disease burden. Secondary transplantation of the initiation experiment exhibited a significant delay in leukemogenesis in the Ikzf2D/D compared to the Ikzf2f/f mice (median survival of 32 and 19.5 days, respectively). The role for Ikzf2 in maintenance was assessed with an inducible puro-creER system, which resulted in 80% decrease in viable cell number within 24hrs of 4-hydroxytamoxifen (4-OHT) treatment. Flow cytometric analysis showed that the Ikzf2-deficient cells had increased apoptosis and differentiation, shown by AnnexinV/7-AAD and Mac1 expression respectively. Furthermore, inducible deletion of Ikzf2 using puro-creER system in vivo revealed that Ikzf2 deletion leads to a delay in leukemia after tamoxifen administration in mice. These results indicate that Ikzf2 is required for both leukemia initiation and maintenance.To determine a role for IKZF2 in human leukemia cells, we performed Ikzf2 knockdown experiments with shRNAs in Kasumi-1, KG1, KCL22 and MOLM13 cells. Ikzf2 depletion resulted in decreased cell growth and increased apoptosis compared to cells infected with scramble shRNA.To determine the mechanism for how IKZF2 controls leukemia cell survival and self-renewal, we performed gene expression profiling of the Ikzf2-deficient Vav-cre LSCs and demonstrated enrichment in signatures for self-renewal loss, increased differentiation, loss of Myc-regulated genes and loss for targets of Hoxa9 and Meis1. Further analysis overlapping our MSI2 HITS-CLIP data and our differentially regulated genes revealed a strong enrichment suggesting that the MSI2 bound targets are transcriptionally regulated by IKZF2. Lastly, ATAC-sequencing of Ikzf2f/f and Ikzf2D/D LSCs revealed alterations in chromatin accessibility that correlated closely with differentially expressed genes. Utilizing the ATAC-seq data we predicted that HOXA9 and MYC sites were significantly altered. We validated that MYC RNA and protein levels were reduced in both murine and human AML cell lines.In contrast to its known tumor suppressor role in hypodiploid B-ALL and T-ALL, these results suggest that Ikzf2 contributes to MLL leukemia cell initiation and maintenance. Thus, we provide evidence that Ikzf2 can regulate c-MYC expression helping in maintaining the stem cell self-renewal program in LSCs. DisclosuresNo relevant conflicts of interest to declare.

A Tumor Suppressor microRNA Defines the Leukemic Hierarchy in Acute Myeloid Leukemia

In order to identify functionally relevant microRNAs (miRNA) in AML, we profiled global miRNA expression in a murine AML progression model based on Hoxa9 and Meis1 overexpression. We found miR-155 and miR-708 as the most significantly upregulated miRNA species in leukemic Hoxa9/Meis1 cells compared to the preleukemic Hoxa9/ctrl cells (both p<0.01). Subsequent analysis of various AML subtypes (CN-AML, t(11q23), t(8;21), t(15;17), n=38) showed significantly elevated levels of miR-155 and miR-708 in all tested samples (n=5-10) compared to total bone marrow from healthy donors, indicating potential oncogenic roles for these miRNAs.We further investigated, in vivo, the role of miR-155 and miR-708 in AML by retroviral overexpression with Hoxa9 in murine bone marrow (mbm) cells followed by syngeneic transplantation. Overexpression of miR-155 in conjunction with Hoxa9 (Hoxa9/miR-155) caused a significantly accelerated onset of a myelomonocytic leukemia within 93 days (survival 93±23 days, p<0.0001), but still a less aggressive course of disease compared to mice transplanted with Hoxa9/Meis1 (survival 36±4 days, p<0.0001).We then hypothesized that the combination of miR-155 and miR-708 could further replace the oncogenic potential of Meis1. Therefore, mbm cells were retrovirally transduced with Hoxa9, miR-155 and miR-708 (Hoxa9/155/708) or Hoxa9 and miR-708 (Hoxa9/miR-708) and functionally analyzed in vitro and in vivo. To our surprise, miR-708 abrogated the leukemogenic effect of Hoxa9, alone or in combination with Hoxa9/miR-155 in vivo (p=0.0117, p<0.0001, the mice were sacrificed after 197 days), with little or no engraftment.To understand why miR-708, a potent tumor suppressor miRNA, is upregulated in the highly aggressive Hoxa9/Meis1 AML cells, we first showed in vivo that miR-708 does not impair the development of AML by Hoxa9/Meis1 cells. In order to explore the role of miR-708 in leukemia initiating cells (LIC), we FACS-sorted subpopulations enriched for LIC based on c-kit, Mac-1 and Gr-1 expression within the Hoxa9/Meis1 cells. Of the three subpopulations (c-kit+ Gr-1- Mac-1-, c-kit+ Gr-1+ Mac-1+, c-kit- Gr-1+ Mac-1+) the transplanted c-kit+ Gr-1- Mac-1- cells caused a significantly shorter survival (survival 36 days, range: 32-37d) compared to the other sorted subpopulations (p=0.0072 and p=0.0021, respectively), whereas the c-kit- Gr-1+ Mac-1+ subpopulation exhibited the longest survival (survival 44 days, range: 42-51d). Based on these findings, we measured miR-708 and miR-155 levels in the sorted Hoxa9/Meis1 subpopulations by qRT-PCR. Strikingly, no difference compared to bulk cells was detected for miR-155, highlighting its role as an oncogenic driver in all subpopulations. In contrast, miR-708 showed a distinct expression pattern with lower expression in the LIC-enriched c-kit+ Gr-1- Mac-1- subpopulation compared to bulk (p=0.032), allowing discernment of the LIC enriched population and highlighting its role as an orchestrator of the leukemic hierarchy. Adapted from these findings, we developed an AML 4-step model (Hoxa9/miR-708, Hoxa9/ctrl, Hoxa9/miR-155, Hoxa9/Meis1) based on four immortalized Hoxa9 based cell lines with completely different in vivo properties ranging from no engraftment to induction of highly aggressive AML.Considering the central role of Hoxa9/Meis1 in AML, we demonstrate, for the first time, the concept of a tumor suppressor miRNA that stratifies the leukemic hierarchy driven by an oncogenic miRNA. Moreover, we present a new AML 4-step model as a novel tool that will allow detailed investigation of the impact of new oncogenes at every step of leukemogenesis. DisclosuresBuske:CELLTRION, Inc.: Consultancy, Honoraria.

Ibrutinib Targets Nurse-like Cells Supporting an Immunosuppressive Phenotype in Chronic Lymphocytic Leukemia

Introduction: In lymphoid organs, nurse-like cells (NLCs) show properties of tumor-associated macrophages with a M2-like phenotype, associated to prominent immunosuppressive functions, and play a crucial role in CLL survival. Ibrutinib is a potent inhibitor of BTK and ITK kinases and is able to counteract the microenvironmental-derived pro-survival effects in CLL cells. One of the main effects of ibrutinib treatment is attenuated retention and homing of CLL cells to tissue compartments, which ultimately leads to mobilization of CLL cells from microenvironmental niches into the peripheral blood.Aim: Here, we investigated whether ibrutinib treatment may have an effect on the immunosuppressive properties of NLCs.Methods: NLCs were generated in complete medium after 12 days of in vitro culture of CLL-PBMCs. Cell activation status, cell surface markers, real time PCR and functional studies were performed after 1h or 24h of ibrutinib treatment, to test whether ibrutinib influenced M1 or M2-polarization of NLCs.Results: We first observed that, after ibrutinib treatment, the activation status and viability of NLCs were not affected, the morphology of NLCs in vitro analyzed by flow-cytometric FCS and SSC plots was not modified and the number of NLCs generated after 12 days of culture was preserved. Furthermore, treatment with ibrutinib stimulated the expression of the M2-associated markers hemoglobin scavenger receptor CD163 and the mannose receptor C type 1 CD206, and induced expression of the monocyte differentiation antigen CD14. At the gene expression level, ibrutinib induced strong expression of genes involved in M2 polarization such as CD163 (293%, p<0.01), IL10 (279%, p<0.05), MRC1/CD206 (180%, p<0.05) and CCL18 (271%, p<0.05), and concomitantly decreased the expression of M1 polarization genes such as IL1 (53%, p<0.01), TNFα (57%, p<0.01) and IL2 (11%, p<0.01), as compared to the untreated control (100%). Of interest, treatment with ibrutinib stimulated also the expression of the enzyme nicotinamide phosphoribosyltransferase (NAMPT) to 141% (p<0.05) and the programmed death-ligand 1 (PD-L1) to 133% (p<0.01) in NLCs, both important in the induction of an immunosuppressive and protective microenvironment in CLL. Overall these data suggest that ibrutinib could stimulate features related to suppression of the immune system. Next, we tested the ability of ibrutinib to regulate actin cytoskeletal reorganization, which is an essential process during macrophages phagocytosis. Upon ibrutinib stimulation, NLCs showed low actin polymerization levels, since F-actin formation decreased to 66% (p<0.05) as compared to unstimulated control (100%). In addition, ibrutinib impaired the phagocytic activity of NLCs and functionally affected the expression of MAC-1 (CD11b/CD18), which is required for optimal phagocytosis, from 100% to 86% (p<0.01).Conclusions: Collectively, these preliminary data provide new insights into the effects of ibrutinib treatment on the modulation of immune elements in CLL tissue microenvironment. Noteworthy, our data demonstrate that ibrutinib further promotes M2-polarization features of NLCs, suggesting that this drug not only has an effect on the CLL clone but also extensively influences the cellular components of the CLL microenvironment, whether these effects may be related to ibrutinib-resistance warrants further investigations. DisclosuresBurger:Boehringer Ingelheim Pharma: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Noxxon: Membership on an entity's Board of Directors or advisory committees; Portola Pharmaceuticals: Research Funding; Gilead: Research Funding; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees, Research Funding. Marasca:Janssen: Honoraria, Other: support for travel to congresses.