Polymorphonuclear Function Research Articles

Vitamin C supplementation reduces expression of circulating miR-451a in subjects with poorly controlled type 2 diabetes mellitus and high oxidative stress.

BackgroundVitamin C is an essential element required for normal metabolic function. We investigated the effect of vitamin C supplementation on circulating miRNA (miR) expression in subjects with poorly controlled type 2 diabetes mellitus (T2DM). Changes in miR expression were also correlated with clinical measures of disease.MethodsPre- and post-vitamin C supplementation samples from five participants who had increased vitamin C levels, improved oxidative status and polymorphonuclear (PMN) function after receiving 1,000 mg of vitamin C daily for six weeks were screened for miRNA expression using the NanoString miRNA assay. Differences in miRNA expression identified from the miRNA screen were validated by qRT-PCR.ResultsFour miRNAs showed significantly different expression post-vitamin C supplementation relative to baseline, including the down-regulation of miR-451a (−1.72 fold change (FC), p = 0.036) and up-regulation of miR-1253 (0.62 FC, p = 0.027), miR-1290 (0.53 FC, p = 0.036) and miR-644a (0.5 FC, p = 0.042). The validation study showed only miR-451a expression was significantly different from baseline with vitamin C supplementation. MiR-451a expression was negatively correlated with vitamin C levels (r = − 0.497, p = 0.049) but positively correlated with levels of malondialdehyde (MDA) (r = 0.584, p = 0.017), cholesterol (r = 0.564, p = 0.022) and low-density lipoproteins (LDL) (r = 0.522, p = 0.037). Bioinformatics analysis of the putative target genes of miR-451a indicated gene functions related to signaling pathways involved in cellular processes, such as the mammalian target of rapamycin (mTOR) signaling pathway.ConclusionsVitamin C supplementation altered circulating miR-451a expression. The results from this pilot study suggest that miRNAs could be used as biomarkers to indicate oxidative status in subjects with T2DM and with poor glycemic control and could lead to a novel molecular strategy to reduce oxidative stress in T2DM.

Enhancement of vitamin C on PMN function in healthy individuals

Abstract Vitamin C (ascorbic acid, ascorbate) promotes immune cell activities by supporting various cellular functions especially, in neutrophils or polymorphonuclear (PMN) leukocytes, which play an important role in bacterial infection and acute inflammation. Little data have been reported in impact of vitamin C on PMN function during bacterial phagocytosis in healthy individuals. Here, we have elucidated the effect of vitamin C on PMN phagocytosis and oxidative burst to Staphylococcus aureus by whole blood assay and flow cytometry after pre-incubation without and with vitamin C in vary concentrations. The results showed that vitamin C concentration at 20 mM induced a significant increase phagocytosis (p-value=0.03) and had trend to significant increase oxidative burst (p-value=0.06) while vitamin C concentration of 30 mM showed significant increase both phagocytosis and oxidative burst (p-value=0.04 and 0.02, respectively) at only 15 min of exposure to the bacteria. As a minimal concentration that can increase phagocytosis, we selected vitamin C at 20 mM as optimal concentration to investigate more data by enrollment of healthy volunteers (n=17). The results revealed significant difference both phagocytosis and oxidative burst after 20 mM vitamin C pre-incubation (p<0.0001). In conclusion, we showed that vitamin C at 20 mM can boost phagocytosis and oxidative burst of neutrophils in healthy adults within 15 min after bacterial exposure. This rapid activation may be applied to fight bacterial infection. Nevertheless, further research in clinical trial and underlying mechanisms await further studies.

Cardiac and Vascular Surgery-Associated Acute Kidney Injury: The 20th International Consensus Conference of the ADQI (Acute Disease Quality Initiative) Group.

Acute kidney injury (AKI) occurs in 7% to 18% of hospitalized patients and complicates the course of 50% to 60% of those admitted to the intensive care unit, carrying both significant mortality and morbidity.[1][1] Even though many cases of AKI are reversible within days to weeks of occurrence, data

The Effect of Bifidobacterium animalis ssp. lactis HN019 on Cellular Immune Function in Healthy Elderly Subjects: Systematic Review and Meta-Analysis.

Elderly people have increased susceptibility to infections and cancer that are associated with decline in cellular immune function. The objective of this work was to determine the efficacy of Bifidobacterium (B.) animalis ssp. lactis HN019 (HN019) supplementation on cellular immune activity in healthy elderly subjects. We conducted a systematic review of Medline and Embase for controlled trials that reported polymorphonuclear (PMN) cell phagocytic capacity or natural killer (NK) cell tumoricidal activity following B. lactis HN019 consumption in the elderly. A random effects meta-analysis was performed with standardized mean difference (SMD) and 95% confidence interval between probiotic and control groups for each outcome. A total of four clinical trials were included in this analysis. B. lactis HN019 supplementation was highly efficacious in increasing PMN phagocytic capacity with an SMD of 0.74 (95% confidence interval: 0.38 to 1.11, p < 0.001) and moderately efficacious in increasing NK cell tumoricidal activity with an SMD of 0.43 (95% confidence interval: 0.08 to 0.78, p = 0.02). The main limitations of this research were the small number of included studies, short-term follow-up, and assessment of a single probiotic strain. In conclusion, daily consumption of B. lactis HN019 enhances NK cell and PMN function in healthy elderly adults.

Abstract 170: Aldosterone Activates ex vivo Human Polymorphonuclear Leukocytes

An important role for the mineralocorticoid receptor (MR) in vascular inflammation has been proposed. Polymorphonuclear (PMN) leukocytes are critical players following an inflammatory response. We studied the role of aldosterone (ALDO) on ex vivo PMN function by isolation of cells from circulating human blood following density gradient sedimentation with polymorphprep from otherwise healthy subjects. These cells express MR as confirmed by western blot, qRT-PCR and 3H-aldosterone binding analyses. Incubation of ex vivo PMN cells with ALDO (1-10 nM) showed a rise in superoxide production (P&lt;0.01, n=9), an event that was blunted by pre-incubation with 500 nM canrenoic acid (CA), a MR antagonist (P&lt;0.03, n=6). Consistent with these results, we observed that 10 nM ALDO led to rapid increases in cytosolic Ca2+ levels using FURA-2AM fluorescence and was associated with decreases in cellular Mg2+ levels using MagFURA-2AM following 1.5 hr of ALDO. These events were associated with increases in VEGF secretion from PMN as determined by ELISA of supernatants (260.64±44.6 vs. 134.03±1.99 pg/ml, P&lt;0.01 when compared to vehicle, n=8) that was likewise sensitive to CA (P&lt;0.04, n=8); thus suggesting that ALDO-activated PMN cells increase factors involved in migration and endothelial cell activation. Indeed, we showed that 10 nM ALDO increased PMN cell migration in a time-dependent manner by CyQuant fluorescence that lasted up to 60 min and could be blocked by CA (P&lt;0.01, n=3). We then studied the effect of 10 nM ALDO-stimulated PMN cells followed by co-incubation of these cells with human endothelial cells for 4 hr. We observed that incubation of ALDO-stimulated PMNs vs vehicle treated cells led to a 2.1 fold increase in ICAM-1 expression levels using qRT-PCR but not VCAM-1 (P&lt;0.01, n=3) in endothelial cells. We observed similar findings in DMSO-differentiated HL-60 cells, a neutrophil-like human cell line. We detected the presence of MR by western blot and qRT-PCR in these cells. Incubation with 10 nM ALDO led to increases in cellular calcium, superoxide production (P&lt;0.01, n=3) and HL-60 migration (P&lt;0.03, n=4) when compared to vehicle. Thus our results suggest that activation of MR leads to PMN cell activation that may contribute to vascular inflammatory responses in vivo.

Effects of indomethacin and diclofenac on some functions of polymorphonuclear neutrophils

The effects in-vitro of two non-steroidal anti-inflammatory drugs, diclofenac and indomethacin, have been studied on human polymorphonuclear functions. Adhesivity and chemotaxis were decreased by the two drugs, but in a manner that varied with the attractant used. As shown by the nitro blue tetrazolium slide test, all cells remained active but the production of oxygen metabolites by opsonized zymosan was decreased in the presence of diclofenac.

Polymorphonuclear function in naturally occurring renal failure in dogs

Chronic renal failure causes immunosuppression in people and is thought to be one of the causes of non-infectious secondary immunosuppression in dogs. The purpose of this study was to evaluate changes in counts and activity of polymorphonuclears in dogs with chronic renal failure in various stages. Haematological, biochemical examinations and examination of non-specific immune response cells (total and differential leukocyte counts, phagocytosis of methacrylate particles, chemiluminescence test, and level of lysozyme) were performed in blood samples obtained from these dogs. Neutrophilia, lymphopoenia and a decreased number of eosinophils in comparison with healthy control were the main findings in groups with clinical signs. We found the statistically highly significant elevation of lysozyme level; it was in a strong positive correlation with the level of urea, creatinine and phosphorus. We did not find any statistically significant changes in phagocytosis process and other serological factors. In conclusion, despite the reports from human medicine, chronic renal failure in dogs does not alter phagocytosis. From this aspect, the elevation of lysozyme level is the main effect of uraemia.

Immunologic Analysis of HIV-Uninfected Taiwanese Children with BCG-Induced Disease

The aim of this study was to evaluate immunity in HIV-uninfected children with bacille Calmette-Guerin-induced disease (BCG-ID) over an 8-year period, with particular emphasis on underlying diseases. Patient afflicted with BCG-ID proven by clinical courses, dermatologic features, pathology, specific polymerase chain reaction, and/or spoligotyping were enrolled between 2000 and 2007. Lymphocyte proliferation, polymorphonuclear function, interleukin (IL)-12/23-interferons (IFN)-gamma circuit, and Toll-like receptor 2-associated signaling were investigated. Of the 271,618 total live births who received the BCG vaccine, eight patients (seven males) with BCG-ID were enrolled during an 8-year period and presented as three disseminated, two distant, and three regional BCG-ID. Their age at onset ranged from 1 to 28 months. All had a vaccine-injection scar except for one with lower CD3 and natural killer cells, compatible with severe combined immunodeficiency (SCID) identified by IL-2 receptor common gamma chain (IL2RG) mutation (Arg226Lys). The other SCID patient with de novo IL2RG mutation (Trp74Gly) had more recurrent infections. The third patient with primary autoimmune neutropenia had disseminated BCG-ID extending to abdominal wall. The fourth patient with chronic mucocutaneous candidiasis had regional BCG-ID and impaired lymphocyte proliferation to Candida and BCG antigens. No defective evidence of polymorphonuclear functions, IL-12/23-IFN-gamma circuit, and Toll-like receptor 2-associated signaling was detected in the remaining four patients. Immunologic analysis in HIV-uninfected patients with BCG-ID reveals primary immunodeficiency diseases, especially in those with deficiencies in T-cell and neutrophil functions observed in our cohort, including primary autoimmune neutropenia and chronic mucocutaneous candidiasis.

Rabbit polymorphonuclear granulocyte function during ethanol administration--migration and oxidative responses in a joint with immune complex synovitis.

Functional impairments of polymorphonuclear granulocytes (PMN) are believed to contribute to hampered inflammation and host defence in alcoholics. We studied effects of i.v. ethanol administration on PMN responses in rabbits during induction of a knee-joint synovitis. The synovitis conferred systemic effects, since chemiluminescent responses of peripheral blood PMN to opsonized zymosan and phorpbol myristate acetate (PMA) increased 6.4- and 17.9-fold, respectively. Chemiluminescent responses of synovial PMN were further amplified. This up-regulation was reduced to 33% in rabbits treated with ethanol when opsonized zymosan was used as the PMN stimulus; in contrast, PMA responses were unaffected. The appearance and migration of PMN to the synovitis joint were normal despite a blood ethanol concentration of 0.5%. Thus, ethanol impaired release of oxygen metabolites from PMN, but not the delivery of cells at an inflammatory site.

A Flow Cytometric Opsonophagocytic Assay for Measurement of Functional Antibodies Elicited after Immunization with Outer Membrane Vesicle of Neisseria meningitidis serogroup B

Opsonophagocytosis mediated by antibody and complement is the major defense mechanism for clearing Neisseria meningitidis from the host. Therefore, a newly developed phagocytosis assay based on flow cytometry (flow assay) was using sera obtained from rabbit postvaccination with outer membrane vesicle of N. meningitidis serogroup B, was done in order to evaluation of the potential efficacy of (experimental) meningococcal vaccines. The Outer Membrane Vesicles (OMVs) and control were injected intramuscularly into groups of five rabbit with boosters on 14, 28 and 42 days after the primary immunization. The serum on 0, 14, 28, 42 and 56 days were collected and stored at -20 degrees C for next analysis. Phagocytic function of and intracellular oxidative burst generation by rabbit polymorphonuclear (PMN), against N. meningitidis serogroup B, was measured with flow cytometer, using dihydrorhodamine-123 as probes, respectively. We use a Coulter Epics XL-profile (USA) with an argon laser operating at 488 nm. The results of quantitative flow cytometric analysis of rabbit PMN function in hyperimmun sera with OMVs revealed a highly significant increase in opsonophagocytic responses against serogroup B meningococci after 56 day in comparison with the control group (p < 0.05). Present results indicated that OMVs could be as a candidate for vaccine toward serogroup B meningococci and a new standard flow cytometric method to measure the opsonophagocytosis activity by rabbit PMNs was shown by this study.

Effect of acute hyperglycemia and/or hyperinsulinemia on polymorphonuclear functions in healthy subjects

Abnormal immune functions of polymorphonuclear (PMN) cells occur in a variety of pathophysiological conditions. There exists a close link between glucose metabolism and PMN functions. The aim of this study was to assess the effect of short-term hyperglycemia and/or hyperinsulinemia on phagocytosis and respiratory burst of PMN cells in healthy subjects in vivo. The study was performed on 12 healthy subjects (mean age, 26.9 ± 1.6 years; body mass index, 24.4 ± 0.84 kg/m 2). Acute hyperglycemia and/or hyperinsulinemia was induced by three 4-hour-long clamp studies—hyperglycemic hyperinsulinemic clamp (HHC), hyperinsulinemic euglycemic clamp (HEC), and isolated hyperglycemic clamp with insulin secretion blockade (HGC). Polymorphonuclear cell phagocytosis and PMN cell respiratory burst (mean percentage and mean fluorescent intensity of phagocyting/activated PMN cells, phagocytic, and respiratory burst indexes) were evaluated by flow cytometry under basal and stimulated conditions. Results detected during clamp studies were compared with those found during a control study with saline infusion. Significant reductions in the mean percentage of phagocyting cells measured under basal conditions after the HHC (6.7% ± 1.3% vs 12.1% ± 4.3%; P < .05) and HGC (4.5% ± 1.8% vs 9.9% ± 2.1%; P < .05) were found in comparison with the preclamp study period; however, these results did not differ significantly from those detected during the control clamp (CC) study. Significantly higher phagocytic (115.1 ± 65 vs 35.8 ± 18.6; P < .05) and respiratory burst indexes (16.5 ± 3 vs 10.1 ± 1.4; P < .05) measured under basal conditions were found after HEC in comparison with the preclamp data. However, these data did not differ significantly from those found after the CC study. No significant differences in other parameters of detected PMN cell immune functions were found after HHC, HEC, and HGC. In conclusion, immune functions of PMN cells were not significantly influenced by short-lasting hyperglycemia and/or hyperinsulinemia induced in vivo by clamp techniques in healthy subjects compared to changes induced by the CC study. Further studies on the short-term effect of glucose metabolism on PMN functions in diabetic patients should be considered necessary.

Phagocytic Polymorphonuclear Function in Patients with Progressive Uremia and the Effect of Acute Hemodialysis

Susceptibility to infections is a major cause of high morbidity and mortality in patients with chronic kidney disease (CKD). Polymorphonuclear leukocytes (PMNs) play a major role in the host defense against various infections. Although the phagocytic function of PMNs in uremic patients has been subjected to repeated evaluation, results have been inconsistent. Also, the effects of progressive uremia and acute hemodialysis (HD) on polymorphonuclear function have been scarcely evaluated. In this study, we assessed the phagocytic index (PI) of PMNs following their uptake of IgG-coated sheep erythrocytes in patients with varying severity of CKD and in healthy controls. In patients with advanced renal failure, the effects of acute HD on PI pre- and post-HD were also studied. A total of 30 patients with CKD and 15 healthy controls were studied. Patients with CKD were categorized into two groups: mild to moderate renal failure (S. Creatinine 2-6 mg/dL) and advanced renal failure (S. Creatinine >6 mg/dL) (n=15 for each). All three groups were age and sex matched. Patients with advanced renal failure had significantly impaired PI (4.03 +/- 1.15) as compared to patients with mild to moderate renal failure (7.17 +/- 2.62) and normal controls (8.13 +/- 3.31) (p < 0.001 for both). There was a statistically insignificant decline in the PI in patients with mild to moderate RF when compared to controls (7.17 +/- 2.62 versus 8.13 +/- 3.31). In patients with advanced renal failure, acute HD led to a significant improvement in PI (5.52 +/- 1.55 versus 4.03 +/- 1.15, p < 0.01). In patients with CKD, there is a progressive decline in the phagocytic index of the PMNs with increasing severity of uremia. Significant improvement in the phagocytic index following acute hemodialysis suggests the role of a circulating uremic toxin in this PMN dysfunction.

Storage of neutrophil granulocytes (PMNs) in additive solution or in autologous plasma for 72 h

Neutropenia may necessitate polymorphonuclear (PMN) transfusion, but among other reasons, PMN short shelf-life complicates realization of innovative transfusion strategies. In 18 donors, PMNs were mobilized using rHuG-CSF + dexamethasone. (8.3 +/- 1.6) x 10(10) PMNs were harvested in 203 +/- 8.7 mL. PMNs were stored undiluted (1, n = 18) and diluted 1-in-2, 1-in-4, 1-in-8 using T-Sol (2, n = 6), T-Sol + 1% HSA (3, n = 6), or autologous plasma (4, n = 6) for 72 h. Haemograms, pH values, phagocytosis, oxidative burst, and interleukin (IL)-1beta, IL-8 and tumour necrosis factor (TNF)-alpha levels were assessed every 24 h. PMN count decreased from (4.3 +/- 0.8) x 10(10) to (2.2 +/- 1.0) x 10(10), and pH value dropped from 6.4 +/- 0.3 to 5.4 +/- 0.2 within 72 h (1), whereas 1-in-4 and 1-in-8 dilutions exhibited consistent haemograms and pH values above 6.0. 1-in-8 dilution (4) stabilized pH at 7.1 +/- 0.4 after 72 h. Function deteriorated to about 50% within 24 h (1), but 1-in-8 (3), 1-in-4 and 1-in-8 diluted PMNs (4) kept it >90% for 72 h. In all collectives, cytokine levels increased during storage. After all, IL-1beta ranged between 31.0 +/- 16.3 (1-in-4, 4) and 100.0 +/- 21.4 (1-in-4, 2), IL-8 from 513 +/- 454 (1) to 3180 +/- 760 (1-in-8, 2), and TNF-alpha between 3.8 +/- 1.7 (1-in-2, 2) and 23.2 +/- 11.8 (1-in-8, 4) (pg mL(-1)). PMN function may be preserved for 72 h in vitro by dilution of PMN apheresates with, preferably, autologous plasma.

Phagocytic Polymorphonuclear Function in Patients with Progressive Uremia and the Effect of Acute Hemodialysis

Background. Susceptibility to infections is a major cause of high morbidity and mortality in patients with chronic kidney disease (CKD). Polymorphonuclear leukocytes (PMNs) play a major role in the host defense against various infections. Although the phagocytic function of PMNs in uremic patients has been subjected to repeated evaluation, results have been inconsistent. Also, the effects of progressive uremia and acute hemodialysis (HD) on polymorphonuclear function have been scarcely evaluated. Methods. In this study, we assessed the phagocytic index (PI) of PMNs following their uptake of IgG-coated sheep erythrocytes in patients with varying severity of CKD and in healthy controls. In patients with advanced renal failure, the effects of acute HD on PI pre- and post-HD were also studied. Results. A total of 30 patients with CKD and 15 healthy controls were studied. Patients with CKD were categorized into two groups: mild to moderate renal failure (S. Creatinine 2–6 mg/dL) and advanced renal failure (S. Creatinine > 6 mg/dL) (n = 15 for each). All three groups were age and sex matched. Patients with advanced renal failure had significantly impaired PI (4.03 ± 1.15) as compared to patients with mild to moderate renal failure (7.17 ± 2.62) and normal controls (8.13 ± 3.31) (p< 0.001 for both). There was a statistically insignificant decline in the PI in patients with mild to moderate RF when compared to controls (7.17 ± 2.62 versus 8.13 ± 3.31). In patients with advanced renal failure, acute HD led to a significant improvement in PI (5.52 ± 1.55 versus 4.03 ± 1.15, p< 0.01). Conclusion. In patients with CKD, there is a progressive decline in the phagocytic index of the PMNs with increasing severity of uremia. Significant improvement in the phagocytic index following acute hemodialysis suggests the role of a circulating uremic toxin in this PMN dysfunction.

Effects of customized bicarbonate buffered solutions for continuous renal replacement therapies on polymorphonuclear function and viability

We have previously shown that the mixture of bicarbonate and calcium in the solutions for continuous renal replacement therapies (CRRT) leads to the formation of crystals and a decrease in the delivered dose of calcium. The aim of this study was to investigate the impact of bicarbonate/calcium and lactate/calcium solutions for CRRT on polymorphonuclear cell (PMN) viability and function. We tested four customized bicarbonate buffered solutions: the single bag (bicarbonate and calcium mixed 24 hours before testing), the double bag (mixed immediately before), and the filtered single bag and double bag solutions. We also tested one commercial lactate buffered solution. Blood from six volunteers was incubated with the test solution for 30 min followed by PMN isolation. Isolated PMNs were resuspended in 50% RPMI + 50% autologous serum and kept overnight. Viability, necrosis and apoptosis were analyzed by annexin V and PI staining. Phagocytosis was determined by the analysis of Staphylococcus aureus-PI stained PMN and ROS production by the mean fluorescence intensity of DCFH in these cells. There was no difference between the test solutions with respect to PMN viability and function. Lactate and bicarbonate had similar effects on PMN viability and function. More studies are mandatory to elucidate the impact of CRRT solutions on other inflammatory cells and pathways, particularly on the cytokines associated with systemic inflammatory response syndrome and sepsis.

Proteome analysis of rat polymorphonuclear leukocytes: a two-dimensional electrophoresis/mass spectrometry approach.

The development of a two-dimensional (2-D) map of rat polymorphonuclear (PMN) leukocytes is here reported for the first time. The map is built up by utilizing a wide immobilized pH gradient (IPG), pH 3-10, in the first dimension and also a narrower IPG pH 4.5-8.5 gradient. In addition, the map is constructed by adopting the most recent protocols in 2-D mapping, which call for reduction and alkylation of the sample prior to the start of any electrophoretic step, including the IPG dimension. Fifty-two major protein spots have been so far identified by utilizing both matrix assisted laser desorption/ionization-time of flight (MALDI-TOF) and electrospray quadrupole (Q)-TOF mass spectrometry. A large number of house-keeping and cytoskeleton proteins were detected, together with proteins which are specific to PMN organelles or related to PMN functions such as phagocytosis and chemotaxis. The results obtained demonstrate the possibility of obtaining a single 2-D gel based proteomic map of PMN with representative proteins from different cellular compartments, also including membrane components, allowing the study of PMN protein expression on a proteome-wide scale. The aim of this project is to build an extensive database of such proteins, to be utilized for future studies where the expression of PMN proteins is used as a disease- or drug treatment marker.

Nicotine effects on polymorphonuclear cell apoptosis and lipopolysaccharide-induced monocyte functions. A possible role in periodontal disease?

Apoptosis provides a mechanism for clearance of unwanted cells in a variety of situations in which programmed or physiological cell death occurs; but the premature death of defensive cells could promote infection, inflammation and concomitant disease. We detected high values of apoptosis in polymorphonuclear cells (PMN) elicited from crevicular sulci of smokers affected by adult periodontitis. To learn more about the effects of nicotine on the periodontal environment, we studied its ability to modulate the apoptosis of two phagocytic lines, PMN and mononuclear cells, which are continuously recruited from gingival vessels to prevent or control plaque extension. Brief exposure of PMN to nicotine concentrations ranging from 0.01 to 0.3% shortened, in a dose-dependent relationship, the lag culture time required to observe at fluorescent microscopy the morphological traces of apoptosis. These observations were confirmed by specific tools of apoptosis: DNA fragmentation on gel electrophoresis and expression of the apoptosis-signaling receptor Fas/Apo-1. The apoptotic effect excited by nicotine on these first line defensive cells may be an important feature of the pathogenesis of periodontal disease. As for mononuclear leukocytes, nicotine was unable to induce apoptotic modifications on cells observed up to 72 h culture time, but the drug inhibited IL-1beta release and procoagulant activity (PCA) expression. The conflicting role played by these lipopolysaccharide (LPS)-induced monocyte functions in the inflammatory process is a further intrigue in the mechanism by which nicotine compromises the oral health.

Activation-dependent proteolytic degradation of polymorphonuclear CD11b

CD11b/CD18 is the principal integrin of polymorphonuclear (PMN) leucocytes and is involved in their adhesion, migration and phagocytosis. In quiescent cells, the receptor is stored in intracellular granules from where it is translocated to the cell surface in response to a variety of stimuli. In this study, we demonstrated that strong stimulation of PMNs not only leads to the upregulation of CD11b surface expression, but also to the subsequent time-dependent apparent loss of this receptor, as detected by fluorescence-activated cell sorting (FACS) using a monoclonal antibody (mAb) against an N-terminal CD11b epitope. This epitope loss was observed following either direct stimulation of protein kinase C (PKC) with phorbol 12-myristate 13-acetate (PMA) or after multiple receptor stimulation using a combination of the agonist N-formylmethionyl-leucyl-phenylalanine (FMLP) and the priming agents granulocyte macrophage-colony stimulating factor (GM-CSF) and platelet factor (PF) 4. However, upregulation following weak stimulation with FMLP alone was not followed by subsequent epitope loss of the receptor. The increases and subsequent decreases in CD11b expression induced by PMA were paralleled by an increase and a decrease in PMN adhesion to CD11b-specific ligands, fibrinogen and intercellular adhesion molecule (ICAM)-1. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis showed that this epitope loss of PMN CD11b was the result of proteolytic degradation of the N-terminal region of the molecule. The use of a range of proteinase inhibitors indicated that this CD11b degradation involves a cell-associated serine proteinase. This is the first demonstration of the proteolytic alteration of CD11b in response to strong PMN stimulation. Given the central role of CD11b/CD18 in all aspects of PMN function, this alteration of the CD11b molecule and its effect on PMN adhesion are probably of considerable pathophysiological importance.

Integrin-mediated signaling in human neutrophil functioning.

Integrins are important signal transducers for virtually all neutrophil functions. Although a variety of signals ultimately result in integrin activation, the intracellular targets of integrin-initiated signals are poorly delineated to date. Polymorphonuclear (PMN) leukocyte responses to inflammation are dependent on both the stimulants and the extracellular environment encountered. Integrin ligation, by cell-cell or cell-matrix interactions, activates a variety of signaling cascades. These events dictate the nature of PMN responses to the encountered stimulus. The complex system of effector molecule recruitment and permissive signaling by integrins serves to strictly regulate PMN functions such as cell adhesion, motility, oxidant production, and protein synthesis. Moreover, there is evidence that cross-talk between integrins exists to prime integrin populations for subsequent functioning. This review summarizes the current understanding of signaling mechanisms for integrin priming and activation. In this connection, the role of specific signaling molecules in key PMN functions are examined.

Effect of two-chambered bicarbonate lactate-buffered peritoneal dialysis fluids on peripheral blood mononuclear cell and polymorphonuclear cell function in vitro

Low pH, high osmolality, increasing glucose concentration, and glucose degradation products (GDP) formed during heat sterilization of conventional peritoneal dialysis (PD) fluids have been shown to have a detrimental effect on cells involved in peritoneal host defense. The two-chambered PD fluid bag in which glucose at pH ∼ 3 is separated from a bicarbonate (25 mmol/L)-lactate (15 mmol/L) buffer during heat sterilization permits PD fluids with lower GDP to be delivered to the patient at neutral pH. To establish the possible benefit of two-chambered bag PD fluids on peripheral blood mononuclear cell (PBMC) and polymorphonuclear (PMN) cell function, we compared conventional 1.5% Dianeal (1.5%D) with 1.5% two-chambered bag bicarbonate-lactate (1.5%D-B), and conventional 4.25% Dianeal (4.25%D) with 4.25% two-chambered bag bicarbonate-lactate (4.25%D-B). Furthermore, to study the effect of the sterilization process on PBMC and PMN function, we compared filter-sterilized 4.25%D (4.25%D-F) with 4.25%D and 4.25%D-B. PBMC were harvested by Ficoll-Hypaque separation, and 2.5 × 10 6 cells in RPMI were incubated with an equal volume of the test fluids for 4 hours, palleted, and resuspended in RPMI containing 10 ng endotoxin for a further 20 hours. Tumor necrosis factor alpha (TNFα) production by endotoxin-stimulated PBMC was not significantly different ( P = 0.10) between 1.5%D-B and 1.5%D, but was significantly higher ( P = 0.01) with 4.25%D-B compared with 4.25%D. PBMC exposed to filter-sterilized fluid (4.25%D-F) showed significantly higher endotoxin-stimulated TNFα production compared with 4.25%D ( P = 0.02), but was not significantly different from 4.25%D-B ( P = 0.40). PMN were harvested by Ficoll-Hypaque separation and 10 × 10 6 cells incubated with test fluids for 30 minutes. After incubation, phagocytosis (phagocytosis index) was determined by the uptake of 14C-labeled Staphylococcus aureus, oxidative burst by reduction of ferricytochrome C to ferrocytochrome C on stimulation with PMA, and enzyme release by measurement of endotoxin-stimulated bactericidal/permeability increasing protein (BPI). Bicarbonate-lactate two-chambered fluids of similar osmolality and glucose concentration conferred a significant improvement in phagocytosis ( P = 0.02 for 1.5%D-B and P < 0.001 for 4.25%D-B). Oxidative burst and BPI release were significantly higher in 4.25%D-B compared with 4.25%D ( P < 0.001). Filter-sterilized 4.25%D-F conferred a significant improvement in phagocytosis and oxidative burst compared with 4.25%D ( P < 0.001) or 4.25%D-B ( P < 0.001). Furthermore, conventional 4.25%D was associated with significantly lower BPI release compared with 4.25%D-F ( P = 0.01). GDP's acetaldehyde and 5-HMF were analyzed in 4.25%D-B, 4.25%D, and 4.25%D-F. Acetaldehyde was below the lower limit (0.79 ppm) of the standard curve in 4.25%D-B and 4.25%D-F fluids but was detected (3.76 to 5.12 ppm) in all of the 4.25%D fluids. Relative levels of 5-HMF in the 4.25%D-B (0.032 to 0.041 Abs @ 284 nm) and 4.25%D (0.031 to 0.036 Abs @ 284 nm) were similar. The lowest levels (0.001 Abs @ 284 nm) were observed in the filter-sterilized 4.25%D-F. The beneficial effects of two-chambered bicarbonate lactate-buffered PD fluids on PBMC and PMN function are probably related to reduction of GDP from heat sterilization of glucose in a separate chamber at a lower pH. This improvement in biocompatibility could have a beneficial effect on peritoneal defenses.