PMN Activation Research Articles

Infection and Activation of Human Neutrophils with Fluorescent Leishmania infantum.

Neutrophils (PMNs) are recruited in high numbers to sites of host infection by the protozoan parasites of the genus Leishmania. Although PMNs are capable of phagocytizing Leishmania parasites and are potent producers of anti-microbial compounds including reactive oxygen species (ROS), they are unable to control the establishment of infection. Prior studies document production of ROS in isolated PMNs incubated with Leishmania under conditions allowing phagocytosis, but without a measure of single cells' responses it cannot be discerned whether PMN activation and ROS production is suppressed or ineffective in the cells that internalize the parasite. To address these interactions, we engineered a strain of fluorescent, mCherry-expressing Leishmania infantum (mCherry-Li). By infecting isolated human PMNs in vitro with mCherry-Li, we observed ready association of the parasites with PMNs in a time- and dose-dependent fashion. We also examined production of PMN ROS (using the fluorescent compound DHR123) and PMN activation (as evidence by loss of surface CD62L expression). Whereas many Li-associated (mCherry+) PMNs responded to parasite interactions and uptake with ROS production and/or activation, a proportion exhibited neither response. Furthermore, a large proportion of mCherry - "bystander" PMNs displayed both ROS production and activation. The heterogeneous response of PMNs to Leishmania exposure leads us to hypothesize, first, that some PMNs exhibit decreased activation upon phagocytosis of Leishmania, and could support their maintenance. Second, responses of bystander PMNs may contribute to a local inflammatory environment that is ineffective at parasite clearance.

A Complement-Optimized EGFR Antibody Improves Cytotoxic Functions of Polymorphonuclear Cells against Tumor Cells.

Complement-dependent cytotoxicity (CDC) has been suggested to be an important mechanism of action of tumor-targeting Abs. However, single unmodified epidermal growth factor receptor (EGFR)-targeting IgG1 Abs fail to trigger efficient CDC. For the current study, we generated a CDC-optimized variant of the EGFR Ab matuzumab (H425 wt) by introducing amino acid substitutions K326A/E333A (H425 mt). This Ab was then used to elucidate the impact of complement activation on the capacity of effector cells such as mononuclear cells (MNC) and polymorphonuclear cells (PMN) to exert Ab-dependent cell-mediated cytotoxicity (ADCC). H425 mt, but not H425 wt, significantly induced complement deposition, release of anaphylatoxins, and CDC against distinct tumor cell lines, whereas no differences in ADCC by MNC or PMN were detected. Notably, stronger cytotoxicity was induced by H425 mt than by H425 wt in whole blood assays and in experiments in which MNC or PMN were combined with serum. Although MNC-ADCC was not affected by C5 cleavage, the cytotoxic activity of PMN in the presence of serum strongly depended on C5 cleavage, pointing to a direct interaction between complement and PMN. Strong cell surface expression of C5a receptors was detected on PMN, whereas NK cells completely lacked expression. Stimulation of PMN with C5a led to upregulation of activated complement receptor 3, resulting in enhanced complement receptor 3-dependent PMN-ADCC against tumor cells. In conclusion, complement-optimized EGFR Abs may constitute a promising strategy to improve tumor cell killing by enhancing the interaction between humoral and cellular effector functions in Ab-based tumor therapy.

Role of the Yersinia YopJ protein in suppressing interleukin-8 secretion by human polymorphonuclear leukocytes

Polymorphonuclear leukocytes, in addition to their direct bactericidal activities, produce cytokines involved in the activation and regulation of the innate and adaptive immune response to infection. In this study we evaluated the cytokine response of human PMNs following incubation with the pathogenic Yersinia species. Yersinia pestis strains with the pCD1 virulence plasmid, which encodes cytotoxic Yop proteins that are translocated into host cells, stimulated little or no cytokine production compared to pCD1-negative strains. In particular, PMNs incubated with pCD1-negative Y. pestis secreted 1000-fold higher levels of interleukin-8 (IL-8 or CXCL8), a proinflammatory chemokine important for PMN recruitment and activation. Deletion of yopE, -H, -T, -M or ypkA had no effect on pCD1-dependent inhibition, whereas deletion of yopJ resulted in significantly increased IL-8 production. Like Y. pestis, the enteropathogenic Yersinia species inhibited IL-8 secretion by PMNs, and strains lacking the virulence plasmid induced high levels of IL-8. Our results show that virulence plasmid-encoded effector Yops, particularly YopJ, prevent IL-8 secretion by human PMNs. Suppression of the chemotactic IL-8 response by Y. pestis may contribute to the delayed PMN recruitment to the infected lymph node that typifies bubonic plague.

Neutrophil microvesicles resolve gout by inhibiting C5a-mediated priming of the inflammasome

ObjectivesGout is a highly inflammatory but self-limiting joint disease induced by the precipitation of monosodium urate (MSU) crystals. While it is well established that inflammasome activation by MSU mediates acute...

Neutrophil activation byCandida glabratabut notCandida albicanspromotes fungal uptake by monocytes

Candida albicans and Candida glabrata account for the majority of candidiasis cases worldwide. Although both species are in the same genus, they differ in key virulence attributes. Within this work, live cell imaging was used to examine the dynamics of neutrophil activation after confrontation with either C. albicans or C. glabrata. Analyses revealed higher phagocytosis rates of C. albicans than C. glabrata that resulted in stronger PMN (polymorphonuclear cells) activation by C. albicans. Furthermore, we observed differences in the secretion of chemokines, indicating chemotactic differences in PMN signalling towards recruitment of further immune cells upon confrontation with Candida spp. Supernatants from co-incubations of neutrophils with C. glabrata primarily attracted monocytes and increased the phagocytosis of C. glabrata by monocytes. In contrast, PMN activation by C. albicans resulted in recruitment of more neutrophils. Two complex infection models confirmed distinct targeting of immune cell populations by the two Candida spp.: In a human whole blood infection model, C. glabrata was more effectively taken up by monocytes than C. albicans and histopathological analyses of murine model infections confirmed primarily monocytic infiltrates in C. glabrata kidney infection in contrast to PMN-dominated infiltrates in C. albicans infection. Taken together, our data demonstrate that the human opportunistic fungi C. albicans and C. glabrata are differentially recognized by neutrophils and one outcome of this differential recognition is the preferential uptake of C. glabrata by monocytes.

The impact of cationic solid lipid nanoparticles on human neutrophil activation and formation of neutrophil extracellular traps (NETs)

Cationic solid lipid nanoparticles (cSLNs) are extensively employed as the nanocarriers for drug/gene targeting to tumors and the brain. Investigation into the possible immune response of cSLNs is still lacking. The aim of this study was to evaluate the impact of cSLNs upon the activation of human polymorphonuclear neutrophil cells (PMNs). The cytotoxicity, pro-inflammatory mediators, Ca2+ mobilization, mitogen-activated protein kinases (MAPKs), and neutrophil extracellular traps (NETs) as the indicators of PMN stimulation were examined in this work. The cSLNs presented a diameter of 195nm with a zeta potential of 44mV. The cSLNs could interact with the cell membrane to produce a direct membrane lysis and the subsequent cytotoxicity according to lactate dehydrogenase (LDH) elevation. The interaction of cSLNs with the membrane also triggered a Ca2+ influx, followed by the induction of oxidative stress and degranulation. The cationic nanoparticles elevated the levels of superoxide anion and elastase by 24- and 9-fold, respectively. The PMN activation by cSLNs promoted the phosphorylation of p38 and Jun-N-terminal kinases (JNK) but not extracellular signal-regulated kinases (ERK). The imaging of scanning electron microscopy (SEM) and immunofluorescence demonstrated the production of NETs by cSLNs. This phenomenon was not significant for the neutral SLNs (nSLNs), although histones in NETs also increased after treatment of nSLNs. Our results suggest an important role of cSLNs in governing the activation of human neutrophils.

Mesenchymal stem cell attenuates neutrophil-predominant inflammation and acute lung injury in an in vivo rat model of ventilator-induced lung injury.

Background:Subsequent neutrophil (polymorphonuclear neutrophil [PMN])-predominant inflammatory response is a predominant feature of ventilator-induced lung injury (VILI), and mesenchymal stem cell (MSC) can improve mice survival model of endotoxin-induced acute lung injury, reduce lung impairs, and enhance the repair of VILI. However, whether MSC could attenuate PMN-predominant inflammatory in the VILI is still unknown. This study aimed to test whether MSC intervention could attenuate the PMN-predominate inflammatory in the mechanical VILI.Methods:Sprague-Dawley rats were ventilated for 2 hours with large tidal volume (20 mL/kg). MSCs were given before or after ventilation. The inflammatory chemokines and gas exchange were observed and compared dynamically until 4 hours after ventilation, and pulmonary pathological change and activation of PMN were observed and compared 4 hours after ventilation.Results:Mechanical ventilation (MV) caused significant lung injury reflected by increasing in PMN pulmonary sequestration, inflammatory chemokines (tumor necrosis factor-alpha, interleukin-6 and macrophage inflammatory protein 2) in the bronchoalveolar lavage fluid, and injury score of the lung tissue. These changes were accompanied with excessive PMN activation which reflected by increases in PMN elastase activity, production of radical oxygen series. MSC intervention especially pretreatment attenuated subsequent lung injury, systemic inflammation response and PMN pulmonary sequestration and excessive PMN activation initiated by injurious ventilation.Conclusions:MV causes profound lung injury and PMN-predominate inflammatory responses. The protection effect of MSC in the VILI rat model is related to the suppression of the PMN activation.

A second stimulus required for enhanced antifungal activity of human neutrophils in blood is provided by anaphylatoxin C5a.

Polymorphonuclear neutrophilic granulocytes (PMN) as cellular components of innate immunity play a crucial role in the defense against systemic Candida albicans infection. To analyze stimuli that are required for PMN activity during C. albicans infection in a situation similar to in vivo, we used a human whole-blood infection model. In this model, PMN activation 10 min after C. albicans infection was largely dependent on the anaphylatoxin C5a. Most importantly, C5a enabled blood PMN to overcome filament-restricted recognition of C. albicans and allowed efficient elimination of nonfilamentous C. albicans cph1Δ/efg1Δ from blood. Major PMN effector mechanisms, including oxidative burst, release of secondary granule contents and initial fungal phagocytosis could be prevented by blocking C5a receptor signaling. Identical effects were achieved using a humanized Ab specifically targeting human C5a. Phagocytosis of C. albicans 10 min postinfection was mediated by C5a-dependent enhancement of CD11b surface expression on PMN, thus establishing the C5a-C5aR-CD11b axis as a major modulator of early anti-Candida immune responses in human blood. In contrast, phagocytosis of C. albicans by PMN 60 min postinfection occurred almost independently of C5a and mainly contributed to activation of phagocytically active PMN at later time points. Our results show that C5a is a critical mediator in human blood during C. albicans infection.

Mitochondrial damage-associated molecular patterns from fractures suppress pulmonary immune responses via formyl peptide receptors 1 and 2.

No known biologic mechanisms link tissue injury with pneumonia (PNA). Neutrophils (PMNs) are innate immune cells that clear bacteria from the lung by migration toward chemoattractants and killing bacteria in neutrophil extracellular traps (NETs). We predicted that tissue injury would suppress PMN antimicrobial function in the lung. We have also shown that mitochondria-derived damage-associated molecular pattern molecules from the bone can alter PMN phenotype and so hypothesized that formyl peptides (FPs) from fractures predispose to PNA by suppressing PMN activity in the lung. Animal studies involved the following. (1) Rats were divided into three groups (10 per condition) as follows: (a) saline injection in the thigh (b) Staphylococcus aureus (SA, 3 × 10) injected intratracheally, or (c) pseudofracture (PsFx; bone supernatant injected in the thigh) plus intratracheally injected SA. (2) Rats were divided into four groups as follows: (a) control, (b) pulmonary contusion (PC), (c) PsFx, and (d) PC + PsFx. Bronchoalveolar lavage was performed 16 hours later. Clinical studies involved the following. (3) Human bone supernatant was assayed for its FP-receptor (FPR) stimulation. (4) Trauma patients' PMN (n = 32; mean ± SE Injury Severity Score [ISS], 27 ± 10) were assayed for chemotaxis (CTX) or treated with Phorbol 12-myristate 13-acetate (PMA, Phorbol ester) and analyzed for NET formation. In the animal studies, (1) SA was rapidly cleared by the uninjured mice and PsFx markedly suppressed lung bacterial clearance (p < 0.01). (2a) PC induces PMN traffic to the lung, but PsFx decreases PC-induced PMN traffic (p < 0.01). (2b) SA increased bronchoalveolar lavage PMN, and PsFx decreased that influx (p < 0.01). In the clinical studies, (3) bone supernatant activates PMN both via FPR-1 and FPR-2. (4) Trauma decreases PMN CTX to multiple chemokines. Circulating PMNs show NETs spontaneously after trauma, but maximal NET formation is markedly attenuated. Fractures may decrease lung bacterial clearance because FP suppresses PMN CTX to other chemoattractants via FPR-1/2. Trauma activates NETosis but suppresses maximal NETosis. Fractures decrease lung bacterial clearance by multiple mechanisms. PNA after fractures may reflect damage-associated molecular pattern-mediated suppression of PMN antimicrobial function in the lung.

Coronary neutrophil extracellular trap burden and deoxyribonuclease activity in ST-elevation acute coronary syndrome are predictors of ST-segment resolution and infarct size.

Mechanisms of coronary occlusion in ST-elevation acute coronary syndrome are poorly understood. We have previously reported that neutrophil (polymorphonuclear cells [PMNs]) accumulation in culprit lesion site (CLS) thrombus is a predictor of cardiovascular outcomes. The goal of this study was to characterize PMN activation at the CLS. We examined the relationships between CLS neutrophil extracellular traps (NETs), bacterial components as triggers of NETosis, activity of endogenous deoxyribonuclease, ST-segment resolution, and infarct size. We analyzed coronary thrombectomies from 111 patients with ST-elevation acute coronary syndrome undergoing primary percutaneous coronary intervention. Thrombi were characterized by immunostaining, flow cytometry, bacterial profiling, and immunometric and enzymatic assays. Compared with femoral PMNs, CLS PMNs were highly activated and formed aggregates with platelets. Nucleosomes, double-stranded DNA, neutrophil elastase, myeloperoxidase, and myeloid-related protein 8/14 were increased in CLS plasma, and NETs contributed to the scaffolds of particulate coronary thrombi. Copy numbers of Streptococcus species correlated positively with dsDNA. Thrombus NET burden correlated positively with infarct size and negatively with ST-segment resolution, whereas CLS deoxyribonuclease activity correlated negatively with infarct size and positively with ST-segment resolution. Recombinant deoxyribonuclease accelerated the lysis of coronary thrombi ex vivo. PMNs are highly activated in ST-elevation acute coronary syndrome and undergo NETosis at the CLS. Coronary NET burden and deoxyribonuclease activity are predictors of ST-segment resolution and myocardial infarct size.

Neutrophils in antiretroviral therapy–controlled HIV demonstrate hyperactivation associated with a specific IL-17/IL-22 environment

Despite control of HIV infection under antiretroviral therapy (ART), immune T-cell activation persists in patients with controlled HIV infection, who are at higher risk of inflammatory diseases than the general population. PMNs play a key role in host defenses against invading microorganisms but also potentiate inflammatory reactions in cases of excessive or misdirected responses. The aim of our study was to analyze PMN functions in 60 ART-treated and controlled HIV-infected patients (viral load, <20 RNA copies/mL; CD4 count, ≥ 350 cells/mm(3)) with (HIV[I] group) and without (HIV[NI] group) diseases related to an inflammatory process and to compare them with 22 healthy control subjects. Flow cytometry was used to evaluate PMN functions in whole-blood conditions. We studied in parallel the activation markers of T lymphocytes and monocytes and the proinflammatory cytokine environment. Blood samples from HIV-infected patients revealed basal PMN hyperactivation associated with deregulation of the apoptosis/necrosis equilibrium. Interestingly, this hyperactivation was greater in HIV(I) than HIV(NI) patients and contrasted with a lack of monocyte activation in both groups. The percentage of circulating cells producing IL-17 was also significantly higher in HIV-infected patients than in control subjects and was positively correlated with markers of basal PMN activation. In addition, the detection of IL-22 overproduction in HIV(NI) patients suggests that it might contribute to counteracting chronic inflammatory processes during HIV infection. This study thus demonstrates the presence of highly activated PMNs in HIV-infected patients receiving effective ART and the association of these cells with a specific IL-17/IL-22 environment.

Effects of Naproxen on Immune Responses in a Colchicine-Induced Rat Model of Alzheimer's Disease

Background: The components of the immune system have been indicated to be linked with the neurotoxicity in Alzheimer's disease (AD). The participation of the immune system in the neurodegeneration in a rat model of colchicine-induced AD has not been explored. Methods: In the present study, hippocampal neurodegeneration along with reactive oxygen species (ROS), nitrite and TNF-α in the hippocampus and some systemic immune responses were measured after 15 and 21 days of intracerebroventricular colchicine injection in rats and again after oral administration of different doses of the anti-inflammatory drug naproxen in AD rats. Results: Chromatolysis and amyloid plaques were found along with higher ROS, nitrite and TNF-α levels in the hippocampus of colchicine-induced AD rats, and these changes were prevented by naproxen in a dose-dependent manner. Alterations in immunological parameters [increased phagocytic activity of white blood cells and splenic polymorphonuclear cells (PMN), increased cytotoxicity and decreased leucocyte adhesive inhibition index (LAI) of splenic mononuclear cells (MNC)] were also observed in colchicine-injected rats, which showed a dose-dependent recovery after oral administration of naproxen in AD rats. The number of plaques, chromatolysis of Nissl granules, TNF-α, nitrite and ROS levels in the hippocampus, phagocytic activity of splenic PMN and LAI of splenic MNC in AD rats showed greater changes in the 21- than in the 15-day study, and the recovery of these parameters after administration of naproxen differed between the two study durations. Conclusion: The present study shows that colchicine-induced neurodegeneration is time dependent and mediated by cyclooxygenase-induced neuroinflammation, which is reflected in the systemic immunological responses.

Reduced bioenergetics and toll-like receptor 1 function in human polymorphonuclear leukocytes in aging

Aging is associated with a progressive decline in immune function (immunosenescence) resulting in an increased susceptibility to viral and bacterial infections. Here we show reduced expression of Toll-like receptor 1 (TLR1) in polymorphonuclear leukocytes (PMN) and an underlying age-dependent deficiency in PMN bioenergetics. In older (>65 years) adults, stimulation through TLR1 led to lower activation of integrins (CD11b and CD18), lower production of the chemokine IL-8, and lower levels of the phosphorylated signaling intermediate p38 MAP kinase than in PMN from younger donors (21-30 years). In addition, loss of CD62L, a marker of PMN activation, was reduced in PMN of older adults stimulated through multiple pathways. Rescue of PMN from apoptosis by stimulation with TLR1 was reduced in PMN from older adults. In seeking an explanation for effects of aging across multiple pathways, we examined PMN energy utilization and found that glucose uptake after stimulation through TLR1 was dramatically lower in PMN of older adults. Our results demonstrate a reduction in TLR1 expression and TLR1-mediated responses in PMN with aging, and reduced efficiency of bioenergetics in PMN. These changes likely contribute to reduced PMN efficiency in aging through multiple aspects of PMN function and suggest potential therapeutic opportunities.

Streptococcus suis Adenosine Synthase Functions as an Effector in Evasion of PMN-mediated Innate Immunity

Streptococcus suis serotype 2 (S. suis 2) is a highly invasive pathogen in pigs and humans that can cause severe systemic infection. Sepsis and meningitis are the most common clinical manifestations of S. suis 2 infection. However, the mechanisms of S. suis 2 surviving in human blood remains unclear, so to identify novel virulence factors in evasion of polymorphonuclear leukocyte (PMN)-mediated innate immunity play important roles in developing therapies against S. suis 2 infection. Here, we found that S. suis 2 can escape phagocytic clearance by adenosine synthesis in blood. Through bioinformatics-based analyses we identified a cell wall-anchored protein harbors a 5′-nucleotidase signature sequence and evidence strongly indicated that it can convert adenosine monophosphate (AMP) to adenosine. It was designated as Ssads (the adenosine synthase of S. suis 2). Furthermore, we found that Ssads could impair PMN's defense against S. suis 2 with decreasing of oxidative activity and degranulation of PMNs in human blood via A₂a receptors. Additionally, this enzyme-deficient mutant was found to have diminished virulence in the piglet infection model. Taken together, these results indicate that Ssads play an important role in S. suis 2 escaping human innate immunity in the context of inhibiting PMN's activity by synthesis of adenosine.

Low production of reactive oxygen species in granulocytes is associated with organ damage in systemic lupus erythematosus.

IntroductionPolymorphonuclear leukocytes (PMN) are main effector cells in the acute immune response. While the specific role of PMN in systemic lupus erythematosus (SLE) and autoimmunity is still unclear, their importance in chronic inflammation is gaining more attention. Here we investigate aspects of function, bone marrow release and activation of PMN in patients with SLE.MethodsThe following PMN functions and subsets were evaluated using flow cytometry; (a) production of reactive oxygen species (ROS) after ex vivo stimulation with phorbol 12-myristate 13-acetate (PMA) or Escherichia coli (E. coli); (b) capacity to phagocytose antibody-coated necrotic cell material; (c) PMN recently released from bone marrow, defined as percentage of CD10−D16low in peripheral blood, and (d) PMN activation markers; CD11b, CD62L and C5aR.ResultsSLE patients (n = 92) showed lower ROS production compared with healthy controls (n = 38) after activation ex vivo. The ROS production was not associated with corticosteroid dose or other immunotherapies. PMA induced ROS production was significantly reduced in patients with severe disease. In contrast, neither ROS levels after E. coli activation, nor the capacity to phagocytose were associated with disease severity. This suggests that decreased ROS production after PMA activation is a sign of changed PMN behaviour rather than generally impaired functions. The CD10−CD16low phenotype constitute 2% of PMN in peripheral blood of SLE patients compared with 6.4% in controls, indicating a decreased release of PMN from the bone marrow in SLE. A decreased expression of C5aR on PMN was observed in SLE patients, pointing towards in vivo activation.ConclusionsOur results indicate that PMN from SLE patients have altered function, are partly activated and are released abnormally from bone marrow. The association between low ROS formation in PMN and disease severity is consistent with findings in other autoimmune diseases and might be considered as a risk factor.

Estimation of lactoferrin levels in gingival crevicular fluid before and after periodontal therapy in patients with chronic periodontitis.

Background:The lactoferrin (LF) is an iron binding protein present specifically and in abundance in the secondary granules of polymorphonuclear leukocytes (PMN's). It has been suggested that LF in crevicular fluid is a useful marker of PMN activity. Hence, this study aimed to estimate the levels of LF in gingival crevicular fluid (GCF) before and after surgical therapy in chronic periodontitis patients to assess the validity of LF in monitoring of treatment results.Materials and Methods:A total of 30 patients with chronic periodontitis having probing depth of ≥5 mm who were scheduled for periodontal surgery were included in the study. The clinical parameters were recorded and GCF samples were obtained 2 weeks after scaling and root planing and 2 weeks after conventional flap technique. The samples collected were then assayed for LF using Enzyme-linked immunosorbent assay (ELISA).Results:The results showed that LF levels decreased significantly from 266.53 ± 75.86 to 195.47 ± 74.53 after scaling and root planing. There was further significant reduction in LF levels to 90.42 ± 32.89 following 2 weeks of periodontal surgery, indicating decrease in inflammation.Conclusion:There is a significant reduction in GCF LF levels following periodontal surgery. Hence, LF levels in GCF could serve as a useful marker for monitoring of periodontal treatment results.

CD11b expression on polymorphonuclear leukocytes from patients with ankylosing spondylitis in a lipopolysaccharide-stimulated whole blood ex vivo model

Ankylosing spondylitis (AS) is a chronic inflammatory disease that compromises the axial skeleton, causing pain and disability. The involved mechanisms are not completely understood, but evidence suggests a role of the gut microbiota in eliciting innate immune responses. We conducted an experimental study to determine if AS patients exhibit an enhanced inflammatory response to microbial compounds. We incubated whole peripheral blood of AS patients and healthy controls with phosphate-buffered saline (PBS) or lipopolysaccharide (LPS) for 48 h with saturating levels of labeled antibodies (CD66b, CD11b and human leukocyte antigen type DR [HLA-DR]) for flow cytometry. CD11b and HLA-DR expression levels were assessed in polymorphonuclear leukocytes (PMN) (CD66b high - HLA-DR low). We also measured basal CD11b and HLA-DR levels on circulating PMN (without incubation). We found that CD11b and HLA-DR levels were similarly elevated in response to LPS on PMNs from healthy controls (HC) and AS patients. Basal levels of CD11b and HLA-DR on circulating PMN from AS patients and HC were similar. However, significantly lower levels of CD11b and HLA-DR were observed in PMNs from AS patients than HC in response to incubation with PBS for 48h. We concluded that the response of AS innate immune cells to LPS is similar to that observed in immune cells from HCs, and suggested that the lower PMN activation of AS patients after 48h saline incubation is mainly due to anti-inflammatory medication.

Organ failure in the obese adipocytes prime polymorphonuclear cell inflammation under stress conditions

Obesity is associated with a higher risk of remote organ failure after shock and trauma. The mechanism(s) is poorly understood. Polymorphonuclear cell (PMN) inflammatory responses are important in the pathogenesis of organ injury following shock. Morbid obesity is a low-grade inflammatory state associated with proinflammatory mediator production from adipose tissue. We hypothesized that adipose tissue may modulate PMN inflammatory potential and is dependent on the magnitude of the injury-related stress response. This was studied in an in vitro model. Adipose-derived stem cells (ADSCs) conditioned to behave as mature adipocytes were incubated with physiologic and stress concentrations of adrenaline for 12 hours, and cell culture supernatants were obtained. PMNs from normal human volunteers were cocultured with the ADSC supernatants (priming) followed by addition of 1-μM fMLP (activation). PMNs alone served as control. PMN activation was indexed by superoxide anion (O2) production, elastase release (%) and CD11b expression (mean fluorescent intensity). Physiologic and stress levels of adrenaline resulted in significantly increased PMN activation in the presence or absence of adipocytes. However, the largest increase was noted in PMNs exposed to ADSC culture supernatants that had been cocultured with stress levels of adrenaline for 12 hours, twofold increase in CD11b expression and fourfold increase in superoxide anion and percent elastase release. Adipocyte-derived mediators prime PMNs in vitro. There was a graded PMN response to adrenaline concentration with or without adipocytes in these experiments. The most profound increase in PMN inflammatory potential was noted with the adipocyte supernatant + stress adrenaline group. The clinical impact of obesity on remote organ injury is likely dependent on patient body mass index and the injury-related sympathetic responses. These data suggest a potential role for β blockade in this patient population.

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Introduction: Adenosine triphosphate (ATP), which is well known as energy currency of cells, is involved in both intracellular and extracellular signaling. Neutrophils (PMN) have a pivotal role in inflammation response. We have shown that ATP release and feedback via ATP receptors are essential for neutrophil activation. Meanwhile, two new fluorescent chemosensors (2-2Zn(II), 3-2Zn(II)), which detect ATP and its delivertives in specific region of living cells, were developed. Although the utility of these chemosensors has been demonstrated in cultured cells, imaging studies using human PMN have not been reported. Our objective is to establish the methods of staining human PMN with 2-2Zn(II), 3-2Zn(II) and quantify the fluorescence intensity by flow cytometry. Methods: PMN were isolated from the peripheral blood of healthy volunteers (n=12). Live PMN with these staining were observed by a confocal microscopy. Fluorescence intensity on plasma membrane (2-2Zn(II)) and in mitochondria (3-2Zn(II)) of PMN with and without fMLP stimulation were evaluated using flow cytometry. Results: These chemosensors could localize on plasma membrane surface and mitochondria in human neutrophils. Mean fluorescence intensity (MFI) of 2-2Zn(II) on plasma membrane of PMN with fMLP stimulation was significantly higher than without stimulation (274.3 ± 100.6 vs 208.4 ± 81.8 ; p< 0.01). On the other hand, MFI of 3-2Zn(II) in mitochondria with stimulation tends to be lower than without stimulation . Conclusions: Fluorescence imaging of ATP in human PMN with chemosensors (2-2Zn(II), 3-2Zn(II)) was established. The higher MFI on plasma membrane with fMLP stimulation suggests the activation of PMN. This method would contribute to understanding the dynamics of ATP in neutrophils and elucidating their diverse physiological functions in inflammation.

Effects of Saffloryellow on survival rate of skin flaps with ischemia-reperfusion injury in rats

Objective To investigate the effects of Saffloryellow (SY) on the flaps with ischemiareperfusion injury (IRI) in rats and the mechanism.Methods Thirty-two SD rats were used to establish IRI skin flaps along superficial epgastric artery.All rats were randomly divided into the control and experimental groups (n =16 each).SY injection [40 mg/(kg· d)] and normal saline with the same volume were injected into the abdominal cavity of rats respectively at 1 h prior to the reperfusion and the next 6 days after operation.The survival rate,pathologic changes,levels of superoxide dismutase (SOD),malondialdehyde (MDA) and polymerphonuclear leucocytes (PMN) of the flaps were analyzed.Results The contents of MDA and PMN were decreased (P ＜0.01),while the activity of SOD increased in the experimental group (P ＜ 0.01).The mean survival rate of the flaps at the 7th day after operation was (38.17 ±8.29) ％ in the control group and (66.53 ± 7.86) ％ in the experimental group (P ＜ 0.01).Conclusion SY can reduce the IRI and increase the survival rate of the flaps significantly in rats probably by inhibiting the activation of PAF and PMN,declining the blood viscosity,improving the activity of SOD,reducing the level of oxygenderived free radical and lipid peroxidation. Key words: Saffloryellow; Flap; Reperfusion injury