Percentage Of Peripheral Blood Neutrophils Research Articles

Abnormal Cytokine Production By Mast Cell Cultures from Sickle Cell Anemia Patients in Response to Inflammatory Stimuli and to Co-Culture with Eosinophils

Mast cell function (MC) in pathologic states can be studied through their ability to secrete mediators in vitro depending on MC phenotype and the nature of the stimuli. Reports on MC mediators in sickle cell anemia (SCA) patients are scarce, but clinical signs of MC activation syndrome, such as increased plasma histamine in vaso‐occlusive crisis (VOC), and normal or slightly elevated serum tryptase have been reported. However, assessing the biological relevance of MC as a cytokine source is more challenging because it is unclear under which circumstances they secrete those products in vivo, or if the cytokines measured systemically stem from a different cell type. We aimed to investigate the profile of mediators involved in the inflammatory process produced by MC in SCA. Methods:The supernatant of 5-week old MC cultures (17 SCA, 8 HV) obtained from peripheral blood CD34+ cells from 29 SCA patients and 13 healthy volunteers (HV) was analyzed using a multiplex platform and colorimetric assays for endothelin-1 (ET-1) and substance P (SP) (10 SCA, 6 HV). A correlation matrix (Pearson correlations, R software, v. 3.6.1) was generated using laboratory and clinical data chosen based on their value as inflammatory or prognosis markers (hydroxyurea [HU] treatment, fetal hemoglobin [HbF], hemoglobin [Hb], vaso-occlusive crisis [VOC], percentage of peripheral blood neutrophils, eosinophils (Eos), basophils, erythroblasts, and reticulocytes), MC surface expression of CD117, CD48 and CD63, and the supernatant content of 11 cytokines. To investigate MC cytokine release, we tested the supernatants from Eos-MC co-cultures (3:1 ratio), and after stimulation with ET-1 (20 nM), SP (10 µM) and imatinib (20 µg/ml)(n=3 per treatment). Results: Out of 26 cytokines, we found elevated levels of the following in the supernatants of SCA-MC cultures (data represented as mean in pg/ml±SE): TNFα: SCA=88.7±18.4, HV=32.6±3.8; IFNγ: SCA=55.3±11.2, HV=15.7±1.8; MCP1: SCA=555.0±147.2, HV=145.3±35.2; RANTES: SCA=24.7±3.9, HV=10.7±1.8 (p<0.05). However, SCA-MC from patients treated with HU (n = 11) showed higher values of IL-1b, IL-4, IL-5, IL-9, IL-15, and FGF than HV (n=8) and HU-free patients (n=6) (p<0.05). Supernatants from SCA-MC had higher ET-1 production compared to HV-MC (SCA=16.3±1.2, HV=11.93±1.3, pg/ml, p=0.02) but SP production was similar (SCA=27.9±1.3; HV=31.49±0.7 pg/ml). ET-1 stimulation of MC cultures caused 2-fold increase in IL-1AR production on HV-MC, but failed to produce any effect on SCA-MC. Similarly, imatinib reduced FGF only in HV-MC samples (HV: 15.1±3.5, HV-HU: 4.0±1.6, pg/ml). No effect on cytokine production was observed with SP. Conversely, Eos-MC cocultures showed a 10- and 4-fold increase of IL-5 and IL-9, respectively, regardless of the origin of Eos (HV or SCA). SCA-MC/SCA-Eos co-cultures had elevated proinflammatory (IL-1b, IL-12, TNF-α) and angiogenic (FGF, VEGF) cytokines, RANTES, IL-7, IL-4, and IL1-RA compared with SCA-MC/HV-Eos and HV-MC/HV-Eos (p<0.05). Preliminary multiparametric analysis on data from SCA patients showed a strong negative correlation between HU therapy and VGEF production, and between HbF levels and CD63 expression (MC activation marker).We also found a positive correlation between history of VOC and eotaxin-1 produced by SCA-MC. Conclusions: We found that MC responses depend both on the origin of the cultured cell and the stimuli utilized. Despite differences between in vitro and in vivo MC populations, our data show that cultured SCA-MC have a sustained activated state and produce a repertoire of mediators that could contribute to a perivascular microenvironment in favor of leukocyte and endothelium activation. In terms of cytokine production, cultured SCA-MC were more sensitive to stimulation by SCA-Eos than by HV-Eos, which may be relevant to the pathophysiology of airway inflammation in SCA patients with asthma. Differences in cytokine production between SCA-MC cultures from patients treated or not with HU may reflect the variability in adherence to treatment, individual response to each compound, or epigenetic modifications during the MC differentiation process that affect the phenotype of the mature MC. These results support that mediators produced by MC can contribute to the chronic inflammatory state and may be implicated in exacerbated responses to eosinophil activation in SCA. Disclosures Fertrin: Agios Pharmaceuticals, Inc.: Research Funding.

Role of neutrophils in treatment of rats with D-galactosamine-induced acute liver failure with bone marrow mesenchymal stem cells

Objective: To investigate the therapeutic effect of bone marrow mesenchymal stromal cell (BMSC) transplantation on D-galactosamine-induced acute liver failure in Sprague-Dawley (SD) rats, as well as the mechanism of neutrophils in this process. Methods: A total of 39 male SD rats were divided into control group (8 rats, intraperitoneal injection of isotonic saline), model group (10 rats, intraperitoneal injection of D-galactosamine), solvent group (9 rats, tail vein injection of isotonic saline at 2 hours after intraperitoneal injection of D-galactosamine), and treatment group (12 rats, tail vein injection of MSCs at 2 hours after intraperitoneal injection of D-galactosamine). The rats were sacrificed at 24 hours after the model of D-galactosamine-induced acute liver failure was established, and the blood and liver tissue were harvested. The serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) and total bilirubin (TBil) were measured, and blood analysis was performed to measure the number and percentage of neutrophils in peripheral blood. Immunofluorescence assay was used to measure the expression of the neutrophil marker Ly6g in the liver, the myeloperoxidase (MPO) kit was used to measure the activity of MPO in liver, and RT-PCR was used to measure the mRNA expression of inflammatory cytokines and chemokines in the liver, i.e., tumor necrosis factor-α(TNF-α), interleukin-1β(IL-1β), interferon-γ(IFN-γ), interleukin-10 (IL-10), CXC chemokine ligand 1 (CXCL1), and CXC chemokine ligand 2 (CXCL2). Another 64 male SD rats were randomly divided into groups, and the survival rates of rats in each group were observed for 7 days. The independent samples t-test was used for comparison between any two groups (Levene homogeneity test of variance, and the corrected t-test was used for a P value of < 0.05), and the log-rank test was used for comparison of survival rates between any two groups. Results: At 24 hours after acute liver failure was induced by D-galactosamine in the SD rats, there were significant increases in the liver function parameters (ALT: 2884.1±541.0 U/L vs 45.4±11.0 U/L,P< 0.001; AST: 3634.9±755.9 U/L vs 143.9±23.7 U/L,P< 0.001; TBil: 44.4±8.4μmmol/L vs 0.9±0.2μmmol/L,P< 0.001) and the number and percentage of peripheral blood neutrophils [number: (4.7±1.1)×109 vs (1.4±0.4)×109,P< 0.001; percentage: 44.9%±8.0% vs 18.3%±4.4%,P< 0.001]. A large number of neutrophils aggregated in the liver tissue, and there were significant increases in the MPO activity (4.72±1.09 U/g vs 1.13±0.24 U/g,P< 0.001), inflammatory cytokines, and chemokines. Compared with the model group, the treatment group showed significant improvements in liver function (ALT: 1 823.9±389.2 U/L vs 2 884.1±541.0 U/L,P< 0.001; AST: 2173.0±567.3 U/L vs 3634.9±755.9 U/L,P< 0.001; TBil: 30.9±6.5μmmol/L vs 44.4±8.4μmmol/L,P< 0.001) and survival rate (50% vs 12.5%,P= 0.023). Meanwhile, the treatment group also showed significant reductions in the number and percentage of peripheral blood neutrophils [number: (3.5±1.0)×109 vs (4.7±1.1)×109,P= 0.012; percentage: 35.9%±8.9% vs 44.9%±8.0%,P= 0.021], number of neutrophils in the liver, and MPO activity (3.52±1.03 U/g vs 4.72±1.09 U/g,P= 0.040), as well as significantly inhibited expression of inflammatory cytokines and chemokines (TNF-α: 2.458±0.762 vs 3.778±1.046, P = 0.005; IL-1β: 2.498±0.547 vs 4.065 ± 0.953,P= 0.002; IFN-γ: 3.977±1.039 vs 5.418±1.255, P = 0.025; IL-10: 6.056±1.542 vs 3.368±0.952,P= 0.001; CXCL1: 7.988±1.911 vs 10.366±1.239,P= 0.010; CXCL2: 3.441±1.005 vs 4.847±1.113,P= 0.019). Conclusion: BMSC transplantation has a therapeutic effect on D-galactosamine-induced acute liver failure in rats, and this process is accompanied by reduced aggregation and activity of neutrophils in peripheral blood and liver. Inflammatory cytokines and chemokines may be involved in the mechanism of regulation of these two aspects.

Development of a fluorescence-based in vivo phagocytosis assay to measure mononuclear phagocyte system function in the rat

The mononuclear phagocyte system (MPS) which provides protection against infection is made up of phagocytic cells that engulf and digest bacteria or other foreign substances. Suppression of the MPS may lead to decreased clearance of pathogenic microbes. Drug delivery systems and immunomodulatory therapeutics that target phagocytes have a potential to inhibit MPS function. Available methods to measure inhibition of MPS function use uptake of radioactively-labeled cells or labor-intensive semi-quantitative histologic techniques. The objective of this work was to develop a non-radioactive quantitative method to measure MPS function in vivo by administering heat-killed E. coli conjugated to a pH-sensitive fluorescent dye (Bioparticles®). Fluorescence of the Bioparticles® is increased at low pH when they are in phagocytic lysosomes. The amount of Bioparticles® phagocytosed by MPS organs in rats was determined by measuring fluorescence intensity in livers and spleens ex vivo using an IVIS® Spectrum Pre-clinical In Vivo Imaging System. Phagocytosis of the particles by peripheral blood neutrophils was measured by flow cytometry. To assess method sensitivity, compounds likely to suppress the MPS [clodronate-containing liposomes, carboxylate-modified latex particles, maleic vinyl ether (MVE) polymer] were administered to rats prior to injection of the Bioparticles®. The E. coli particles consistently co-localized with macrophage markers in the liver but not in the spleen. All of the compounds tested decreased phagocytosis in the liver, but had no consistent effects on phagocytic activity in the spleen. In addition, administration of clodronate liposomes and MVE polymer increased the percentage of peripheral blood neutrophils that phagocytosed the Bioparticles®. In conclusion, an in vivo rat model was developed that measures phagocytosis of E. coli particles in the liver and may be used to assess the impact of test compounds on MPS function. Still, the detection of inhibition of splenic macrophage function will require further assay development.

The maternal autoimmune environment affects the social behavior of offspring

Autism spectrum disorders (ASD) are neurodevelopmental disorders with unknown etiology. BTBR-T+tf/J (BTBR) mice, a mouse strain with behaviors that resemble autism and with elevated levels of anti-brain antibodies (Abs), have enhanced activation of peripheral B cells and CD4+ T cells and an expanded percentage of CD4+ T cells expressing Vβ6 chains. The CD4+CD25+Vβ6+ and Vβ6-splenic cells of BTBR mice have elevated levels of IL-4, IFN-γ and IL-17, but there appears to be no preferential CD4+ T subset skewing/polarization. The high level of IgG production by BTBR B cells was dependent on T cells from BTBR mice. The CD4+ T cells of BTBR mice, especially those expressing Vβ6 become spontaneously activated and expanded in an autoimmune-like manner, which occurred in both BTBR and B6 hosts that received an equal number of BTBR and B6 bone marrow cells. BTBR mice also have an elevated percentage of peripheral blood neutrophils, which may represent their elevated inflammatory state. B6 offspring derived from B6 dams that were gestationally injected with purified IgG from sera of BTBR mice, but not IgG of B6 mice, developed significantly impaired social behavior. Additionally, B6 offspring that developed in BTBR dams had impaired social behavior, while BTBR offspring that developed in B6 dams had improved social behavior. All of the immunological and behavioral parameters of BTBR mice were compared with those of B6 mice, which have relatively normal behaviors. The results indicate maternal Abs and possibly other maternal influences affect the social behavior of offspring.

Low-dose carbon monoxide treatment attenuates early pulmonary neutrophil recruitment after acid aspiration

Exogenous carbon monoxide (CO) has anti-inflammatory and cytoprotective properties that show promise in the treatment of numerous pulmonary diseases. However, the effectiveness of CO in acute pulmonary injury associated with direct lung insult has not been shown conclusively. The purpose of this study was to determine if exogenous CO would modulate the pulmonary inflammation and lung injury that develops after acid aspiration. Groups of mice were given intratracheal (IT) injections of either saline or an acidic solution. After the IT injection, some of the mice in each group were allowed to spontaneously inhale CO (500 ppm). Mice exposed to CO for 6 h after IT acid had a significant decrease in bronchoalveolar lavage (BAL) fluid neutrophil counts and in histological evidence of lung injury. These results could not be explained by changes in BAL fluid chemokine levels or altered CXCR2 expression. The reduced neutrophil recruitment was associated with a decrease in the percentage of peripheral blood neutrophils expressing CD11b protein. However, within 24 h, the BAL neutrophil counts increased and were not different from animals without CO exposure. In addition, indices of vascular integrity were not different between animals with acid aspiration regardless of CO exposure at the later time point. These results showed that CO can modulate the early development of acute lung inflammation in this model of acid aspiration. Although these effects were eventually overwhelmed, the results suggest that CO may have efficacy during the initial treatment of aspiration lung injury.

Association Between Bronchiectasis, Systemic Inflammation, and Tumor Necrosis Factor α

The relationship between systemic inflammation and different measures of bronchiectasis severity has not been described. The objective of this study was to analyze the relationship between plasma concentrations of tumor necrosis factor alpha (TNF-alpha), as a marker of systemic inflammation, and some commonly used criteria for quantifying bronchiectasis severity in clinically stable patients whose disease was not caused by cystic fibrosis. Sixty-eight clinically stable patients with bronchiectasis and 19 age- and sex-matched healthy control subjects were included in the study. Data on disease history, symptoms, severity, functional variables, sputum volume, and microbiological cultures, laboratory findings, and other indicators of disease course were collected. Plasma concentrations of TNF-alpha were measured using high-resolution enzyme-linked immunoabsorbent assay. Plasma concentrations of TNF-alpha were higher in patients than controls (8.28 vs 5.67 pg/mL; P=.001). This observation correlated with other markers of systemic inflammation such as erythrocyte sedimentation rate (r=0.42; P=.001), C-reactive protein (rho=0.45; P=.001), and percentage of peripheral blood neutrophils (rho=0.45; P=.001). Patients with high plasma concentrations of TNF-alpha (>8.1 pg/dL) had more severe disease (5.19 vs 3.21; P=.001), were more likely to have respiratory failure (37.5% vs 8.3%; P=.003), and a higher rate of Pseudomonas aeruginosa colonization (34.3% vs 8.3%; P=.008). High plasma concentrations of TNF-alpha were associated with several criteria usually used to assess severity of bronchiectasis in clinically stable patients with disease not caused by cystic fibrosis.

Immunological, hematological, and biochemical responses in immature white-footed mice following maternal Aroclor 1254 exposure: A possible bioindicator.

A number of hematological, immunological, and biochemical parameters were measured in Peromyscus leucopus pups born from dams exposed to a single dose (300 mg/kg body weight) of Aroclor 1254. To increase the chances of uncovering even modest consequences of the exposure, in one protocol the pups were weaned at 3 weeks and examined at 6 weeks of age, while in a second protocol the pups were kept with their mother for 4 weeks, at which time they were examined. The older pups showed significant decreases in body weight, ratio of spleen weight to body weight, numbers of peripheral white blood cells and lymphocytes, and number and percentage of monocytes. They also showed significant increases in the stimulation index in response to the mitogen phytohemagglutinin (PHA), percentage of peripheral blood neutrophils and liver EROD induction. Pups sacrificed at 4 weeks of age showed even more significant differences. Their body and liver weights, percentage and number of peripheral blood lymphocytes, and serum antibody titers were significantly lower than those of their controls, while spleen to body weight ratios, percent of neutrophils in their peripheral blood, and liver EROD, PROD, and BROD levels were significantly higher than those of the controls. The primary implication of this work is that white-footed mouse pups could be used as biomonitors of contaminated sites. Females could be captured at the sites and bred in captivity with normal males. The vulnerable parameters identified in this study could then be measured in the resulting offspring and compared with a database collected from normal pups.

Unique human neutrophil populations are defined by monoclonal antibody ED12F8C10

A mouse IgG1 monoclonal antibody ED12F8C10 (C10) binds a constant percentage of peripheral blood neutrophils in the same individual when studied over time, defining a distinct subset of neutrophils in all normal individuals studied to date. Bone marrow studies confirm that the heterogeneity is present to the same degree at all stages of neutrophil development from the myelocyte to the mature neutrophil. Neither in vivo nor in vitro activation of neutrophils explains or significantly alters the relative percentages of C10-positive and -negative neutrophils in the same individual. With both activation and exudation, however, expression of the C10-defined epitope increases in intensity in the C10 binding subpopulation. Studies of NBT reduction, phagocytosis, adherence, light scattering characteristics, and monoclonal antibody surface binding have failed to demonstrate physical or functional differences between the C10-defined populations. We examined C10 binding in patients with different defects of phagocyte function. In two patients with neutrophil-specific granule deficiency, less than 1% of the neutrophils were found to be C10 positive, while neutrophils from a patient with idiopathic leukemoid reaction and recurrent infections demonstrated greater than 99% C10 binding. Although the present study does not delineate the physiologic significance of C10 binding heterogeneity, it firmly supports the concept of neutrophil heterogeneity at the level of surface antigen expression.

The nitroblue tetrazolium test in Crohn's disease and ulcerative colitis.

In subjects with Crohn's disease and ulcerative colitis, an increased percentage of peripheral blood neutrophils reduces the dye nitroblue tetrazolium. This is probably a non-specific index of inflammatory activity. There is no evidence that defective neutrophil function, as measured by this test, contributes to granuloma formation in Crohn's disease.