Increase In Peripheral Blood Neutrophils Research Articles

2379Endothelial cell derived extracellular vesicles mediate immune cell deployment from the spleen and transcriptional programming following acute myocardial infarction

Abstract Background Acute myocardial infarction (AMI) induces transcriptional activation of monocyte en route to the injured myocardium, possibly through interactions involving plasma liberated extracellular vesicles (EVs), which are enriched for proteins and microRNAs (miRNAs) post-AMI. Neutrophils are the first immune cells to arrive at sites of injury and mediate further damage to the ischaemic myocardium. Here, we describe neutrophil-deployment from the spleen in AMI and by endothelial cell (EC) derived-EVs. Methods Patients presenting AMI provided informed consent as part of the Oxford Acute Myocardial Infarction Study (OxAMI). Plasma EV were isolated by differential ultra-centrifugation (120,000g, 2 hours) followed by washing and characterised for: morphology using transmission electron microscopy (TEM), size and concentration profiling by Nanoparticle Tracking Analysis, EV markers (TSG101, ALIX, CD9, HSP70) by western blot, and miRNAs by RT-qPCR. Mouse and human EC were used in vitro to derive EC-EV under control conditions or after inflammatory stimulation with tumour necrosis factor-alpha (TNF-α) (10ng/mL) and from CRISPR-edited miRNA-126 knock-out ECs. EC-EVs were tail vein injected into wild-type mice or exposed to primary human peripheral blood neutrophils in vitro. Results Patients presenting with AMI (N=15) have significantly more plasma EV at time of injury vs a 6-month follow-up measurement (2.2-fold more, P=0.008). Plasma EVs at the time of AMI presentation correlate significantly with the extent of ischaemic injury (R=0.046, P=0.006) and plasma neutrophils (R=0.37, P=0.017). Experimental AMI in wild-type mice induced a significant increase in peripheral blood neutrophils and a simultaneous reduction in splenic-neutrophils, suggesting splenic-neutrophil deployment (P=0.004). Human plasma EV are enriched for vascular cell adhesion molecule-1 (VCAM-1) and EC-associated miR-126 post-AMI (Akbar et al 2017). miRNA-126-mRNA targets are significantly over represented when compared to neutrophil Gene Ontology terms for: degranulation (P<0.001), activation (P<0.001), chemotaxis (P=0.008) and migration (P=0.008). Human and mouse EC release more EV after inflammatory stimulation and show enrichment for miRNA-126. CRISPR-edited miRNA-126 deficient human EC express more VCAM-1 (P<0.001) and release more EC-EVs (P<0.001). EC-EV exposure to primary human neutrophils alters inflammatory gene expression (IL-6 (P<0.05), CCL7 (P<0.001) and CCL18 (P<0.001)). EC-EV tail vein injected into wild-type mice mobilise splenic-neutrophils to peripheral blood (P<0.001). Conclusions Neutrophil deployment from the spleen is a novel finding in acute injury and interactions with EC-EV may mediate their splenic liberation and transcriptional programming following AMI, en route to the injured myocardium. The splenic neutrophil reserve may be a novel therapeutic target in AMI to modulate the inflammatory response before recruitment of cells to sites of injury. Acknowledgement/Funding British Heart Foundation Project Grant and Centre for Research Excellence Awards (RE/13/1/30181), Nuffield Benefaction for Medicine and ISSF

CD2-iCre mice reveal a role for ABCB7 in B cell development and bone marrow homeostasis

Abstract ABCB7 is a mitochondrial transporter that exports iron-sulfur (Fe-S) cluster intermediates from the mitochondria to the cytoplasm. Using CD2-iCre ABCB7 cKO mice, we found ABCB7 is required for early B cell development. These mice have a severe block in pro-B cell development at the Fr. C to Fr. C′ transition, with reduced expression of intracellular μ and pre-BCR. CD19-cre and CD23-cre ABCB7 cKO mice have normal B cell development and peripheral B cell populations, indicating ABCB7 is required only during early B cell development. We observed reduced expression of Runx1, Ikaros, Foxo1, E47 (E2A), and Pax5 in CD2-iCre ABCB7 cKO pro-B cells. Interestingly, we also observed decreased BACH2 expression, which indicates elevated heme synthesis is occurring in the absence of ABCB7. We hypothesize that in the absence of ABCB7, developing B cells enhance heme synthesis to dispose of accumulating mitochondrial iron. Using a hen egg lysozyme BCR transgene, we were able to rescue B cell development in CD2-iCre ABCB7 cKO mice. Intriguingly, at the age of 5–8 weeks old, CD2-iCre ABCB7 cKO mice become lethargic and hunched. CBCs reveal an increase in peripheral blood neutrophils. Both the spleen and bone marrow become highly infiltrated by CD11b+Ly6Glow/+Ly6C− cells that are immature and mature neutrophils. As other knockout mice with pro-B cell blocks do not have neutrophilia, we considered if increased iron or heme is responsible for our observation. As heme can stimulate TLR4, we propose that excess heme from developing ABCB7-deficent B cells activates macrophages. In support of this, hemin induced GM-CSF production in BMDM cultures. We are currently exploring the mechanism by which excess heme production can stimulate the production of neutrophils.

Effects of repeated anaesthesia on gill and general health of Atlantic salmon, Salmo salar.

Fish are the second most widely utilized vertebrate group used for scientific procedures in the United Kingdom, but the development and application of 3Rs (the principles of replacement, reduction, and refinement) in aquaculture disease research lags behind methodologies in place for mammalian studies. With a need for individual monitoring and non-lethal sampling, the effect of repeat anaesthesia on experimental fish needs to be better understood. This study analyses the effect of repeat anaesthesia with MS-222, metomidate and AQUI-S upon the gill and general health of post-smolt Atlantic salmon Salmo salar. A single, lethal dose of anaesthetic was compared with seven anaesthetizing time points over 28 days, terminating in a lethal dose. No anaesthetic showed significant differences in accumulation in the muscle tissue, or changes in plasma glucose after repeated or single dosing. Fish repeatedly anaesthetized with MS-222 or AQUI-S exhibited upregulation of osmoregulatory genes in the gill and AQUI-S-treated individuals showed, histologically, epithelial lifting from the lamellae capillary irrespective of whether they had a single or repeated dose history. No significant changes were seen in inflammatory or stress genes in the head kidney of fish repeatedly anaesthetized with AQUI-S or metomidate, however MS-222 treatment resulted in upregulation of tnfα3. Repeated anaesthesia with MS-222 and metomidate gave a significant decrease and increase in peripheral blood neutrophils, respectively. This study concludes that no increase in cumulative stress or inflammation is induced by the repeated anaesthetization of S. salar with any of the tested anaesthetics, however gill osmotic regulation and blood parameters may be affected.

Novel Small-Molecule Human G-CSF Receptor Agonist Stimulates Neutrophil Counts In Monkeys and Displays Anti-Proliferative Effects On Tumor Cells Mediated By a Reduction Of Intracellular Iron

Previously we reported on the discovery of a series of novel, non-peptidyl, small molecules that selectively activate the human granulocyte colony stimulating factor receptor (hG-CSFR) in a manner distinct from G-CSF and similar to the mechanism of small-molecule human thrombopoietin receptor (hTPOR) agonists [Marschke KB, et al. ASH 53rd Annual Meeting Abstracts, Blood 2011 118(21):1453, Abstract 3391]. The lead compound, LG7455, activates STAT5 and STAT3 signal transduction and induces G-CSF-dependent hematopoietic cell growth, but not TPO-dependent or erythropoietin-dependent cell growth. LG7455 promotes the differentiation of human bone marrow cells into granulocytes, significantly increasing expression of the granulocyte-specific marker CD15 (FUT4). The G-CSF-like effect of LG7455 requires the expression of human or monkey G-CSFR, and a histidine in the transmembrane domain is needed for activity, similar to what has been found for small-molecule hTPOR agonists, such as eltrombopag. LG7455 increases the binding of GCSF in a manner consistent with allosteric receptor modulation. To test the ability of LG7455 to stimulate neutrophil counts in vivo, cynomolgus monkeys were dosed intravenously (IV) 3 times per day (TID) to increase LG7455 exposure. Increases in peripheral blood neutrophils were observed in male cynomolgus monkeys with LG7455 (2 mg/kg/dose, TID). Because of the potential for LG7455 to interact with metal ions, their role in the activity of LG7455 was investigated. The ability of LG7455 to activate luciferase expression in cells transiently transfected with an hG-CSFR expression vector and a STAT5-responsive luciferase reporter was dependent on the addition of zinc (II) ion (up to 30 µM) to the cell culture media. The addition of Zn(II) also significantly enhanced the efficacy (63% vs. 23% relative to rhG-CSF) and potency ( EC50 of 28 nM vs. 300 nM) by which LG7455 stimulated the growth of UT-7 cells made dependent on G-CSF by stable transfection of hGCSFR (UTP-hGCSFR). LG7455 did not activate luciferase expression or stimulate growth of cells incubated in the presence of additional iron (III), copper (II), magnesium (II) or calcium (II) ions. Similar to the antitumor effect of the well-described iron chelator salicylaldehyde isonicotinyl hydrazine (SIH), LG7455 inhibited the growth of a selection of human tumor cell lines, including the myeloid leukemia cell lines HL-60 and Kasumi-1, the lung carcinoma cell line A549, the embryonic kidney cell line HEK293, and the hepatocellular carcinoma cell line HepG2 (IC50's of 5.0, 12.1, 6.1, 1.4 and 1.0 µM, respectively). SIH did not stimulate but instead inhibited the growth of UTP-hGCSFR cells. The anti-proliferative effect of LG7455 on HL-60 cells was accompanied by an increase in apoptosis as determined by Annexin V/propidium iodide staining and FACS analysis. The addition of excess Fe(III) to the tumor cells was able to overcome the anti-proliferative effect of LG7455 in a concentration-dependent manner, indicating that a reduction in intracellular iron plays a role in the effect. The addition of Zn(II) and Cu(II) were less efficient at blocking the anti-proliferative effect of LG7455 than Fe(III). Addition of Mg(II) or Ca(II) had no effect. LG7455 increased the fluorescence of HL-60 cells loaded with calcein-AM, a fluorescent dye quenched when bound to iron, further supporting that a reduction of free intracellular iron occurs in cells treated with LG7455. Lastly, as has been shown with other iron chelators with anti-proliferative activity, LG7455 increased intracellular reactive oxygen species (ROS) formation in HL-60 cells loaded with the cell-permeant ROS indicator, CM-H2-DCFDA, and evaluated by flow cytometry. Further optimization of the LG7455 series may provide a novel, orally-available adjuvant anticancer therapy that is also a supportive care agent to treat neutropenia in patients receiving bone-marrow suppressive treatments. Disclosures:Marschke:Ligand Pharmaceuticals Incorporated: Employment. Vajda:Ligand Pharmaceuticals Incorporated: Employment. Zhi:Ligand Pharmaceuticals Incorporated: Employment.

Ineffective Erythropoiesis With Increased Neutrophils In Old Mice Overexpressing Hmga2 cDNA

HMGA2 is frequently overexpressed in hematopoietic cells of patients with paroxysmal nocturnal hemoglobinuria (PNH) and primary myelofibrosis (PMF), and occasionally in myelodysplastic syndromes/myeloproliferative neoplasms (MDS/MPN). We recently reported that transgenic mice overexpressing truncated Hmga2 cDNA (ΔHmga2 mice) showed increased numbers in all lineages of peripheral blood (PB) cells, hypercellular bone marrow (BM), and splenomegaly in young adults of 3 months old (Ikeda et al, Blood, 2011). ΔHmga2 mice also showed growth advantage of hematopoietic stem cells (HSCs) in serial BM transplants and of committed progenitors in colony-replating assays. Thus, overexpression of HMGA2 may contribute to clonal expansion in PNH or myeloproliferation in PMF and MDS/MPN. Although these disorders show disease progression and development of leukemia in the long term, consequences of long-term overexpression of HMGA2 in hematopoiesis is unknown. Therefore, in this study, we investigated hematopoiesis in old ΔHmga2 mice at 15 months old. We found an increase in PB neutrophils in 16 old ΔHmga2 mice compared with 8 old wild-type (WT) mice (mean ± SD; 4.3 ± 1.7 vs. 2.4 ± 0.6 x109/L, p<0.01) as well as young mice. Strikingly, in contrast to young mice, we found a decrease in PB red blood cells of older ΔHmga2 mice compared with WT mice (8.5 ± 0.5 vs. 9.3 ± 0.4 x1012/L, p<0.01), which was associated with an increase in the mean corpuscular volume (MCV, 48.5 ± 3.7 vs. 42.1 ± 1.8 fL, p<0.01). PB total leukocyte and platelet counts were similar between old ΔHmga2 mice and old WT mice. Total BM cell counts were higher in 11 old ΔHmga2 mice compared with 12 WT mice (2.1 ± 0.4 vs. 1.4 ± 0.5 x107/femur, p<0.01). Spleens were 2-fold heavier in old ΔHmga2 mice (n=11) compared with old WT mice (n=12, p<0.01). Histology showed hypercellular BM with erythroid dysplasia, but neither fibrosis nor leukemia, in 5 old ΔHmga2 mice. Absolute numbers of BM lineage-Sca-1+c-kit+ HSCs were higher in 5 old ΔHmga2 mice compared with 3 old WT mice (73 ± 31 vs. 23 ± 10 x103/femur, p=0.036). In addition, proportions of CD71+Ter119+ erythroblasts were higher in 3 old ΔHmga2 mice compared with 3 WT mice (11.8 ± 1.2 vs. 5.1 ± 1.1%, p<0.01), although absolute number of megakaryocyte-erythrocyte progenitors did not show a statistical difference (137 ± 36 vs. 96 ± 18 x103/femur, p=0.1). Progenitor assay also showed increased numbers of BFU-E colonies in 3 old ΔHmga2 mice compared with 3 WT mice (25 ± 2 vs. 13 ± 2, p<0.01), but new BFU-E colonies did not grow from replating of these colonies of old ΔHmga2 mice, in contrast to young ΔHmga2 mice. Annexin V staining showed increased spontaneous apoptosis after 24-hour incubation in Ter119+CD71+ cells of 3 old ΔHmga2 mice compared with 3 old WT mice (26.6 ± 2.7 vs. 14.3 ± 7.0%, p=0.033). In MACS-sorted Ter119+ erythroid cells, expression of anti-apoptotic Bcl-xl mRNA in old ΔHmga2 mice was lower compared with old WT mice (3.3 ± 1.2 vs. 5.3 ± 0.3, p=0.016), while that in young ΔHmga2 mice were higher than young WT mice (6.9 ± 0.4 vs. 3.5 ± 0.3, p<0.01), suggesting the possibility that some changes, including expression of Bcl-xl affected sensitivity of erythroid cells to apoptosis during aging, resulted in ineffective erythropoiesis. In conclusion, long-term expression of HMGA2 may lead to neutrophilia with ineffective erythropoiesis, which may mimic MDS/MPN rather than myelofibrosis, possibly through impairment of Bcl-xl in erythroid cells during a long course. Ineffective erythropoiesis may also in part explain BM failure in PNH. Disclosures:No relevant conflicts of interest to declare.

Effect of Aminoglutethimide on Neutrophils in Rats: Implications for Idiosyncratic Drug-Induced Blood Dyscrasias

Aminoglutethimide (AMG) is an aromatic amine aromatase inhibitor associated with a high incidence of idiosyncratic blood dyscrasias, especially agranulocytosis. Animal models of idiosyncratic drug reactions (IDRs) represent essential tools to study these reactions; however, there is currently no valid model of idiosyncratic drug-induced agranulocytosis. Although AMG does not cause agranulocytosis in most animals or humans, drugs associated with serious IDRs generally cause a higher incidence of mild reactions that resolve despite continued treatment. Therefore, the effects of AMG on neutrophils and bone marrow in rats were studied to understand the mechanisms of more serious IDRs. An increase in peripheral blood neutrophils occurred as early as 24 h after AMG treatment with minimal changes to the total leukocyte count. Further investigation using 5-bromo-2'-deoxyuridine (BrdU) found an increased release of neutrophils from the bone marrow. Histologically, this corresponded to an increase in myeloid cells in the bone marrow, which was confirmed by differential staining with CD45 and CD71. AMG treatment stimulated an increase in colony forming unit granulocyte-macrophage and colony forming unit granulocyte ex vivo. There was also a marked increase in the number of activated neutrophils in the circulation expressing the extravasation marker CD62L. These findings indicate that AMG affects neutrophil production, release, and function. Similar effects on neutrophil kinetics in clozapine-treated rats have previously been found, and transient neutrophilia has been observed in patients taking other drugs associated with idiosyncratic agranulocytosis; therefore, the changes observed with AMG may be biomarkers to predict the risk that a drug will cause agranulocytosis.

P111 Bronchoalveolar Lavage does not affect the Acute Inflammatory Response Following Bronchoscopy and Mediastinoscopy

ObjectiveBronchoalveolar lavage (BAL) may be used to investigate acute inflammation following thoracic surgery. However, BAL has previously been found to induce an acute phase response in healthy and critically-ill patients...

Phase 2 Trial of the Farnesyltransferase Inhibitor Tipifarnib in Previously Untreated Older Adults with AML and Baseline Presence of a Specific 2-Gene Expression Signature Ratio

AbstractAbstract 1508 Background:Tipifarnib is an orally bioavailable farnesyltransferase inhibitor with well-established antileukemic activity in a minority of unselected patients with AML. Seeking to identify patients most likely to have tipifarnib-responsive AML, we applied a PCR-based quantitative 2 gene expression signature ratio (RASGRP1:APTX), which had been tested and validated retrospectively as a predictor of response and survival in previous large studies of tipifarnib, as a molecular screening tool for selection of patients to this pilot trial. Objectives:1) primary: to determine the complete response (CR +CRi) rate in AML patients prospectively selected for tipifarnib treatment on the basis of a 2-gene signature (RASGRP1:APTX ratio) in bone marrow aspirates. 2) secondary: to assess safety, overall survival, and immunophenotypic expression of RASGRP1 on baseline bone marrow blasts for correlation with PCR-based detection. Methods:This was a multicenter, open-label, phase 2 study. Key eligibility criteria included 1) patients with previously untreated AML, age ≥65 years and deemed unsuitable for or refusing traditional induction chemotherapy, and 2) RASGRP1: APTX ratio of ≥5.0 (measured in bone marrow aspirate) based upon qRT-PCR. Other inclusion criteria included: ECOG PS 0–2, adequate end-organ function, and WBC < 30,000/microliter. Treatment consisted of Tipifarnib 300 mg BID × 21 days, with 7 days off. Dose escalation to 600 mg BID following cycle 1 was permitted for patients who had stable disease (SD) after cycle 1. Patients who achieved CR/CRi or PR after 2 cycles were eligible to continue treatment for up to 6 cycles maximum. Results:A total of 62 patients were consented to the trial. Of these, 21 (34%) were eligible based upon the required 2-gene ratio. Assay results were reported within 72 hours for all patients. Median age was 75 years. Fourteen (67%) patients had adverse karyotype and 14 (67%) had secondary AML (including 7 patients who received prior therapy for MDS). Three patients were replaced during cycle 1, due to withdrawal of consent or early unrelated death, such that 18 patients were considered evaluable for response. Median RASGRP1:APTX was 14.8. Two patients (11%) achieved CR and 6 (33%) additional patients had both SD and achievement of ≥50% reduction in bone marrow blasts by completion of cycle 2. Two of these 6 patients also had ≥50% increase in peripheral blood neutrophils or platelets. All 8 patients with CR or SD received at least 2 cycles of therapy. There was no correlation between baseline RASGRP1:APTX ratio and response. Overall, tipifarnib was well tolerated. Early death (≤ 30 days) was observed in only 1 patient (5%). The most common therapy-related non-hematologic toxicities (mainly grade 1–2) included: anorexia (33%), nausea (33%), fatigue (28%), febrile neutropenia (23%) and diarrhea (23%). Due to the trial not meeting its primary endpoint of at least 3 CR/CRi after 2 cycles, accrual to the trial was suspended. Conclusion:Monotherapy with tipifarnib in preselected elderly patients with AML and a RASGRP1:APTX ratio of ≥5 was associated with significant antileukemic activity and good tolerance. Although the primary endpoints during the first stage of the trial were not met, this study demonstrated the ability to rapidly and efficiently execute a biomarker-driven trial in an aggressive malignancy. Given the observed antileukemic activity in this selected population, along with a favorable toxicity and bioavailability profile for chronic dosing, further study of tipifarnib with less rigid endpoints than CR/CRi is warranted. Updated survival and RASGRP1 protein expression data will also be presented. Disclosures:Off Label Use: Tipifarnib in AML. Knoblauch:Johnson & Johnson: Employment. Karkera:Johnson & Johnson: Employment.

Cancers predispose neutrophils to release extracellular DNA traps that contribute to cancer-associated thrombosis

Cancer-associated thrombosis often lacks a clear etiology. However, it is linked to a poor prognosis and represents the second-leading cause of death in cancer patients. Recent studies have shown that chromatin released into blood, through the generation of neutrophil extracellular traps (NETs), is procoagulant and prothrombotic. Using a murine model of chronic myelogenous leukemia, we show that malignant and nonmalignant neutrophils are more prone to NET formation. This increased sensitivity toward NET generation is also observed in mammary and lung carcinoma models, suggesting that cancers, through a systemic effect on the host, can induce an increase in peripheral blood neutrophils, which are predisposed to NET formation. In addition, in the late stages of the breast carcinoma model, NETosis occurs concomitant with the appearance of venous thrombi in the lung. Moreover, simulation of a minor systemic infection in tumor-bearing, but not control, mice results in the release of large quantities of chromatin and a prothrombotic state. The increase in neutrophil count and their priming is mediated by granulocyte colony-stimulating factor (G-CSF), which accumulates in the blood of tumor-bearing mice. The prothrombotic state in cancer can be reproduced by treating mice with G-CSF combined with low-dose LPS and leads to thrombocytopenia and microthrombosis. Taken together, our results identify extracellular chromatin released through NET formation as a cause for cancer-associated thrombosis and unveil a target in the effort to decrease the incidence of thrombosis in cancer patients.

Cyclin-Dependent Kinases Phosphorylate AML1 (RUNX1) and Regulate AML1 Transactivation.

The transcription factor AML1 (RUNX1) plays a critical role in normal hematopoiesis and in the development of leukemia. The phenotype generated by knockout of the AML1 gene demonstrates that AML1 is necessary for normal development of fetal hematopoietic cells. Conditional knockout of AML1 expression in adult bone marrow leads to lineage-specific effects on B- and T-cell maturation and pronounced inhibition of common lymphocyte progenitor production. In addition, the AML1-deficient adult mice exhibited inefficient platelet production and a mild myeloproliferative phenotype characterized by an increase in peripheral blood neutrophils, an increase in myeloid progenitor populations, and extramedullary hematopoiesis composed of maturing myeloid and erythroid elements. Previous studies have shown that phosphorylation of AML1, particularly at serines 276 and 303, can affect transcriptional activation. We have now shown that phosphorylation of AML1 serines 276 and 303 can be blocked in vivo by inhibitors of the cyclin-dependent kinases (Cdks) Cdk1 and Cdk2. In addition, these two sites can be phosphorylated in vitro by purified, active Cdk1/cyclin B and Cdk2/cyclin A, but not by Cdk4/cyclin D. Mutation of AML1 serines 276 and 303 reduces AML1 activity during most phases of the cell cycle. In addition, mutation of serines 276 and 303 prevents phosphorylation from occurring at two nearby sites (serine 293 and threonine 300). Mutation of serine 293 and threonine 300 causes an increase of AML1 activity, especially in cells arrested in G2/M by treatment with nocodazole. Since Cdk phosphorylation can be stimulatory (serines 276 and 303) or inhibitory (serine 293, threonine 300), the interplay of Cdk phosphorylation at different sites might confer a very fine level of regulation of AML1 activity.

Loss of Runx1 perturbs adult hematopoiesis and is associated with a myeloproliferative phenotype

AbstractHomozygous loss of function of Runx1 (Runt-related transcription factor 1 gene) during murine development results in an embryonic lethal phenotype characterized by a complete lack of definitive hematopoiesis. In light of recent reports of disparate requirements for hematopoietic transcription factors during development as opposed to adult hematopoiesis, we used a conditional gene-targeting strategy to effect the loss of Runx1 function in adult mice. In contrast with the critical role of Runx1 during development, Runx1 was not essential for hematopoiesis in the adult hematopoietic compartment, though a number of significant hematopoietic abnormalities were observed. Runx1 excision had lineage-specific effects on B- and T-cell maturation and pronounced inhibition of common lymphocyte progenitor production. Runx1 excision also resulted in inefficient platelet production. Of note, Runx1-deficient mice developed a mild myeloproliferative phenotype characterized by an increase in peripheral blood neutrophils, an increase in myeloid progenitor populations, and extramedullary hematopoiesis composed of maturing myeloid and erythroid elements. These findings indicate that Runx1 deficiency has markedly different consequences during development compared with adult hematopoiesis, and they provide insight into the phenotypic manifestations of Runx1 deficiency in hematopoietic malignancies.

Loss of Runx1 Perturbs Adult Hematopoiesis and Is Associated with a Myeloproliferative Phenotype.

Homozygous loss of function of Runx1 during murine development results in an embryonic lethal phenotype characterized by a complete lack of definitive hematopoiesis. In light of recent reports of disparate requirements for hematopoietic transcription factors during development as opposed to adult hematopoiesis, we employed a conditional gene targeting strategy to effect loss of Runx1 function in adult mice. In contrast with the critical role of Runx1 during development, Runx1 was not essential for hematopoiesis in the adult hematopoietic compartment, although there were a number of significant hematopoietic abnormalities observed. Runx1 excision had significant lineage specific effects on B- and T-cell maturation, as well as pronounced inhibition of common lymphocyte progenitor production. Runx1 excision also resulted in inefficient platelet production. Of note, Runx1 deficient mice developed a mild myeloproliferative phenotype characterized by an increase in peripheral blood neutrophils, an increase in myeloid progenitor populations, and extramedullary hematopoiesis comprised of maturing myeloid and erythroid elements. These findings indicate that Runx1 deficiency has markedly different consequences during development compared with adult hematopoiesis, and provides insights into the phenotypic manifestations of Runx1 deficiency in hematopoietic malignancies.

Cholecystokinin blockade alters the systemic immune response in rats with acute pancreatitis.

Acute pancreatitis (AP) is characterized by initial pancreatic injury resulting from the activation of digestive enzymes and, later, widespread inflammation to distant organs. The aim of this study was to study whether the time-course of inflammatory events during AP induced by bile-pancreatic duct obstruction (BPDO) varies after lowering the acinar enzyme content by L364,718 (0.1 mg/kg/day) administration over 7 days before inducing AP. Flow cytometric immunophenotyping was used to analyse the following at different AP stages: distribution of major circulating leucocyte subsets, activation state of circulating neutrophils and monocytes as reflected by CD11b expression and tumour necrosis factor-alpha (TNF-alpha) production and the contribution of T-cell-derived pro-(TNF-alpha) and anti-(IL-10) inflammatory mediators. TNF-alpha plasma levels and neutrophil infiltration in pancreas and lung were also measured. At early BPDO times, L364,718 treatment partially inhibited leukocytosis and increase in peripheral blood neutrophils and monocytes as well as TNF-alpha expression by monocytes. However, from 6 h onwards after BPDO, L364,718 treatment was unable to prevent either pancreatic and lung neutrophil infiltration or the release of TNF-alpha from activated monocytes. By its action on circulating lymphocytes, L364,718 treatment enhanced the severity of the inflammatory response induced by BPDO. Peripheral blood lymphocytes were recruited from earlier BPDO times, and 12 h after BPDO, T cells displayed a significantly higher reserve of TNF-alpha able to be released under stimulation but lower functional reserve of interleukin-10 (IL-10) than observed in untreated rats. It is concluded that lowering the acinar enzyme content through L364,718 treatment prevents earlier systemic immune events in BPDO-induced AP. However, at the point of maximal injury, the inflammatory response became pronounced, largely due to the role played by activated T lymphocytes.

Blockade of S100A8 and S100A9 suppresses neutrophil migration in response to lipopolysaccharide.

Recently, proinflammatory activities had been described for S100A8 and S100A9, two proteins found at inflammatory sites and within the neutrophil cytoplasm. In this study, we investigated the role of these proteins in neutrophil migration in vivo in response to LPS. LPS was injected into the murine air pouch, which led to the release of S100A8, S100A9, and S100A8/A9 in the pouch exudates that preceded accumulation of neutrophils. Passive immunization against S100A8 and S100A9 led to a 52% inhibition of neutrophil migration in response to LPS at 3 h postinjection. Injection of LPS was also associated with an increase in peripheral blood neutrophils and the presence in serum of S100A9 and S100A8/A9. Intravenous injection of S100A8, S100A9, or S100A8/A9 augmented the number of circulating neutrophils and diminished the number of neutrophils in the bone marrow, demonstrating that S100A8 and S100A9 induced the mobilization of neutrophils from the bone marrow to the blood. Finally, passive immunization with anti-S100A9 inhibited the neutrophilia associated with LPS injection in the air pouch. These results suggest that S100A8 and S100A9 play a role in the inflammatory response to LPS by inducing the release of neutrophils from the bone marrow and directing their migration to the inflammatory site.

Contribution of circulating leukocytes to cytokine production in pancreatic duct obstruction-induced acute pancreatitis in rats.

Little information is available regarding the role of circulating leukocytes in the pathogenesis of acute pancreatitis (AP). Our aim was to explore the time-course of the potential role of inflammatory peripheral blood (PB) cells during AP induced in rats by pancreatic duct obstruction (PDO). Flow cytometry immunophenotyping was used to analyse the distribution of the major circulating leukocyte subsets, the activation state of circulating monocytes as reflected by both CD11b expression and TNF-alpha production and the relative contribution of T-cell derived pro- (TNF-alpha) and anti- (IL-10) inflammatory mediators at different stages of PDO-induced AP. A progressive increase in PB neutrophils and monocytes was observed up to 6h after PDO whereas lymphocytes, as well as CD4(+) and CD8(+) T-cell subsets, rose as early as 1.5 h after PDO and decreased thereafter. Monocytes were activated in PB from 6 h after inducing AP as reflected by increases in both CD11b expression and spontaneous TNF-alpha production; nevertheless, they showed the capability of producing TNF-alpha at earlier AP stages by lipopolysaccharide (LPS) stimulation. In contrast, T-cells were unable to produce TNF-alpha during AP neither spontaneously nor after stimulation with PMA/Ionomycin. Therefore, only PB monocytes contribute to increase TNF-alpha levels in plasma as observed from 12 h onwards after inducing AP. Interleukin-10 was produced by T-cells 6 h after PDO only after PMA/Ionomycin stimulation. We conclude that systemic inflammatory events are triggered off at early stages of PDO-induced AP, with the activation of circulating monocytes, though not T-cells, playing a central role.

BAL Induces an Increase in Peripheral Blood Neutrophils and Cytokine Levels in Healthy Volunteers and Patients With Pneumonia

To examine the peripheral effects of BAL on the neutrophil counts and cytokine levels in the circulation. WBC counts and plasma cytokines were measured before and 4 h after fiberoptic bronchoscopy (FOB) without further interventions (n = 6), or combined with BAL in normal volunteer subjects (n = 6), and in patients with bacterial pneumonia (n = 4). The bronchus of the right middle lobe was wedged, and three 50-mL aliquots of sterile saline solution was instilled. There was no endotoxin contamination in the saline solution or the fluid obtained through the working channel of bronchoscope. In volunteers, peripheral WBC counts and the number of nonsegmented and segmented neutrophils increased after the BAL procedure (p < 0.05) associated with the increase in plasma concentration (mean +/- SEM) of interleukin (IL)-6 (0.99 +/- 0.32 pg/mL before BAL and 20.38 +/- 13.42 pg/mL after BAL; p < 0.05) and granulocyte colony-stimulating factor (G-CSF; 14.1 +/- 1.7 pg/mL before BAL and 38.5 +/- 9.7 pg/mL after BAL; p < 0.05). The increase in WBC counts and neutrophil counts was positively correlated to the increase in IL-6 (p < 0.05) and the increase in G-CSF (p < 0.05). In patients with pneumonia, IL-6 and G-CSF levels were higher after BAL than in normal volunteer subjects (p < 0.05). There was no increase in plasma concentration of IL-1beta, tumor necrosis factor-alpha, or IL-8 after BAL in normal volunteer subjects or in patients with pneumonia. FOB without BAL did not increase the WBC count, neutrophil count, or plasma cytokine levels. The BAL procedure increases the number of WBCs, and segmented and nonsegmented neutrophils in the peripheral circulation as well as circulating IL-6 and G-CSF levels.

The effect of an experimental rhinovirus 16 infection on bronchial lavage neutrophils

Background: Viral respiratory tract infections are the most frequent cause of asthma exacerbations. Of the respiratory viruses associated with these exacerbations, rhinovirus (RV) is the most common. It is proposed that these RV infections may enhance airway inflammation and thus provoke asthma. Objective: It is our hypothesis that RV infections generate nasal proinflammatory mediators that are associated with an initial increase in circulating leukocytes and may contribute to later development of neutrophilic airway inflammation. Methods: To evaluate this hypothesis, subjects with a history of allergic asthma were experimentally inoculated with strain 16 RV (RV16). The effect of this experimental infection was evaluated on circulating leukocytes, nasal-derived mediators, and markers of bronchial inflammation that were obtained by bronchoscopy and lavage. Results: RV16 inoculation was associated with an initial increase in circulating neutrophils. Paralleling these acute changes in circulating neutrophils was an increase in nasal concentrations of IL-8 and granulocyte–colony-stimulating factor (G-CSF). The RV16-associated changes in circulating and nasal G-CSF correlated with increases in peripheral blood neutrophils ( r s = 0.874, P < .001 and r s = 0.898, P < .001, respectively). Bronchial lavage samples showed no increase in neutrophils 48 hours after RV16 inoculation; however, 96 hours after RV inoculation there was a significant increase in bronchial neutrophils compared with preinoculation values. Conclusions: These results suggest that the production of nasal mediators associated with the RV infection, particularly G-CSF, may be important to the eventual development of neutrophilic bronchial inflammation and thus contribute to asthma exacerbations. (J Allergy Clin Immunol 2000;105:1169-77.)

Neutrophilic Respiratory Tract Inflammation and Peripheral Blood Neutrophilia After Grain Sorghum Dust Extract Challenge

To determine if inhalation of grain sorghum dust in the laboratory would cause neutrophilic upper and lower respiratory tract inflammation in human volunteers, as well as systemic signs of illness. Prospective. University of Nebraska Medical Center. Thirty normal volunteers. Inhalation challenge with 20 mL of a nebulized solution of filter-sterilized grain sorghum dust extract (GSDE). One group received prednisone, 20 mg for 2 days, prior to the challenge. Bronchoscopy with bronchoalveolar lavage (BAL) was performed 24 h after challenge, with samples collected as bronchial and alveolar fractions. Findings included visible signs of airways inflammation, quantified as the bronchitis index. The percentage of bronchial neutrophils was significantly increased in those challenged with GSDE vs the control solution, Hanks' balanced salt solution (40.3 +/- 4.5% vs 14.3 +/- 5.1%, p < or = .01). Similar findings were seen in the alveolar fraction. Pretreatment with corticosteroids did not prevent the rise in neutrophils recovered by BAL. Peripheral blood neutrophils were also increased in volunteers challenged with the grain dust extract. To explain the increase in peripheral blood neutrophil counts, the capacity of the peripheral blood neutrophils to migrate in chemotaxis experiments was examined. The results demonstrate an increase in peripheral blood neutrophils and an increase in chemotactic responsiveness. Inhalation challenge with a grain dust extract causes respiratory tract inflammation and a peripheral blood neutrophilia. One reason for this may be an increase in activated peripheral blood neutrophils.

Thrombocytopenia in Dogs Induced by Granulocyte-Macrophage Colony-Stimulating Factor: Increased Destruction of Circulating Platelets

Administration of recombinant canine granulocyte-macrophage colony-stimulating factor (rcGM-CSF) to normal dogs in previous studies induced an increase in peripheral blood neutrophils and a dose-dependent decrease in platelet counts. In six dogs that received the highest tested dose of rcGM-CSF (50 micrograms/kg/d) for a minimum of 12 days, the mean nadir of the platelet count was 46,000/microL (range, 4,000 to 91,000/microL) on day 9 +/- 1.1 after starting therapy, compared with a mean baseline platelet count of 398,000/microL (range, 240,000 to 555,000/microL). In three dogs, survival of autologous 111In-labeled platelets was reduced from a mean of 4.9 days to 1.3 days during the administration of rcGM-CSF. Biodistribution studies with gamma camera imaging indicated that there was an increase in mean hepatic uptake during the administration of rcGM-CSF, from 15% to 44% of the total injected 111In-labeled platelets at 2 hours, whereas splenic uptake was not significantly changed. In contrast, in two evaluable dogs who were recipients of 111In-labeled platelets from matched allogeneic donors receiving rcGM-CSF, platelet survival was not reduced and no increased hepatic uptake was noted. A third dog became alloimmunized to the matched donor platelets and was not evaluable. Immunohistologic studies of liver and spleen were performed with monoclonal antibodies specific for canine gpIIb/IIIa and P-selectin in dogs treated with rcGM-CSF and compared with untreated controls. On treatment, a marked reduction of platelets in the red pulp of the spleen was evident, and in general, the presence of platelet antigen in the liver was unchanged. Therefore, platelets were not being sequestered, but destroyed in the liver and spleen. The platelet antigens, P-selectin and gpIIb/IIIa, were identified in association with Kupffer cells in the liver, but no difference in the number of distribution of these Kupffer cells was found between controls and rcGM-CSF-treated dogs. In the spleen during rcGM-CSF treatment, most platelet antigens were associated with large mononuclear cells in the marginal zone. During administration of rcGM-CSF, CD1c and CD11c expression was increased on Kupffer cells. Platelet P-selectin expression and binding of leukocytes to circulating platelets were unchanged from baseline studies with rcGM-CSF treatment. In conclusion, during the administration of rcGM-CSF to dogs, a local process in the liver and spleen is induced resulting in thrombocytopenia.(ABSTRACT TRUNCATED AT 400 WORDS)

Clonal Heterogeneity of a Human Functioning Tumor which Produces Granulocyte Colony-stimulating Factor

Two different clonal strains have been isolated from a human squamous cell carcinoma which produces granulocyte colony-stimulating factor (G-CSF). The clones differed in morphology, growth characteristics, karyotype, production of G-CSF, histology of the tumors produced by inoculating the cultured cells, and in the development of granulocytosis in host mice transplanted with the cultured cells. A well-differentiated squamous cell carcinoma was developed by inoculation with clone 1 cells into athymic nude mice. No CSF activity was detected in the cultured medium of the cells. The host mice with the transplanted cells showed no increase in peripheral blood neutrophils. Clone 2, which formed a poorly-differentiated squamous cell carcinoma in nude mice, produced a large amount of CSF in vitro and developed a marked neutrophilia in host nude mice. Clone 1 cells were more sensitive to bleomycin than were clone 2 cells in vitro. The results suggested the G-CSF producing tumor to be heterologous and the cells with different functional properties, including G-CSF production, sensitivity to bleomycin, and keratinization, to pre-exist in the parental cell population.