Neutrophil CD11b Research Articles

Amniotic fluid induces platelet-neutrophil aggregation and neutrophil activation

Amniotic fluid embolism syndrome is a fatal disease in pregnant women. The exact role of platelets and neutrophils in amniotic fluid embolism syndrome is not clear. We examined whether amniotic fluid could affect platelet-neutrophil aggregation and activation and the possible mechanisms. Blood samples from the pregnant women were pretreated ex vivo with their own amniotic fluid. Flow cytometry was used to measure platelet-neutrophil aggregation and activation. Neutrophil-mediated activity of p38 mitogen-activated protein kinase and extracellular signal-regulated protein kinases 1 and 2 was analyzed by Western blotting. Amniotic fluid significantly induced platelet-neutrophil aggregation, neutrophil CD11b expression, and reactive oxygen species production. Amniotic fluid induced minimal platelet P-selectin expression. The increase of intracellular calcium level of neutrophils and the activity of p38 mitogen-activated protein kinase were enhanced by amniotic fluid stimulation. Amniotic fluid was able to induce neutrophil activation and platelet-neutrophil aggregation with minimal effect on platelet activation. These findings may provide a new insight in the understanding of the pathophysiologic condition of amniotic fluid embolism syndrome.

Simvastatin abrogates inflamed neutrophil adhesive properties, in association with the inhibition of Mac-1 integrin expression and modulation of Rho kinase activity

Leukocytes play a primary role in vascular inflammation, and thus an understanding of the pathways involved in the activation of these cells and means to inhibit their consequent adhesion to the vessel wall is of significant interest. This study aimed to determine whether statins have a direct effect upon neutrophil adhesive properties under inflammatory conditions. Neutrophils from healthy individuals were subjected to adhesion assays (with fibronectin as ligand) and flow cytometry. In the presence of a TNF-α inflammatory stimulus, neutrophils displayed a rapid and substantial enhancement in their adhesive properties that was abrogated by preincubation of cells with simvastatin. Neutrophil surface expression of the Mac-1 integrin subunit, CD11b, was augmented by TNF-α, and this increased expression was also inhibited by simvastatin. TNF-α also induced neutrophil LFA-1 and Mac-1 activation, but this activation was not blocked by simvastatin. Interestingly, while addition of the isoprenoids, geranygerayl pyrophosphate and farnesyl pyrophosphate, to cells did not alter the effect of simvastatin on TNF-α-stimulated adhesion, concurrent incubation of cells with the Rho kinase (ROCK) inhibitor reversed the effects of simvastatin on neutrophil adhesion and CD11b expression. Simvastatin appears to have direct anti-inflammatory effects in neutrophils that may be mediated by modulation of ROCK activity.

Comparative Studies On Branded Enoxaparin and a US Generic Version of Enoxaparin.

Abstract 2264 Background:Low molecular weight heparins (LMWHs) are complex biologic drugs whose heterogeneity in saccharide chain length and in the composition (sulfate, acetyl), content, and location of functional groups can impact their multiple biologic activities. Enoxaparin (Lovenox®) is validated for multiple indications and is the most widely used LMWH in the US. Several generic versions of enoxaparin are currently available in the US. We undertook this study to compare the activity profile of branded and a generic enoxaparin. Methods:Five batches each of branded (B; Sanofi-aventis; Bridgewater, NJ) and generic (G; Sandoz US; Princeton, NJ) enoxaparin were studied. Drugs were purchased through hospital pharmacies as pre-filled syringes containing 40 mg drug. The molecular weight profile of each batch of LMWH was determined using HPLC. To analyze in vitro activities, LMWHs were supplemented to normal human plasma and assessed using amidolytic anti-FXa and anti-FIIa, fibrinokinetic, and thrombin generation assays. Human whole blood was supplemented with LMWHs and the dynamic blood coagulation process was analyzed by thrombelastography (TEG). Whole blood flow cytometry was used to assess the ability of the LMWHs to inhibit tissue factor (TF)-induced platelet activation and lipopolysaccharide (LPS)-induced neutrophil activation. To assess the in vivo effect of the LMWHs, primates treated subcutaneously with a dose of 1 mg/kg LMWH had blood samples drawn pre-treatment and at 4, 6, 12 and 24 hours. Ex vivo pharmacodynamic activities of TFPI release, TAFI inhibition, and thrombin generation inhibition were evaluated. Results:Molecular weight parameters and IC50 values for FXa and FIIa inhibition by branded and generic enoxaparin were comparable. In the in vitro thrombin generation and fibrinokinetic assays, branded enoxaparin exhibited a more potent anticoagulant effect demonstrating slower clot formation with a weaker final clot structure (p=0.01) than generic enoxaparin. Although both the branded and generic enoxaparin produced a concentration-dependent anticoagulant effect in the TEG, there was greater degree of variability for the generic product between blood donors and between batches resulting in a less predictable linear response as drug concentration increased. When the increase in TEG R-time was plotted vs. concentration, branded enoxaparin showed a stronger anticoagulant effect (p=0.05). A concentration-dependent reduction in TF-induced platelet P-selectin expression was observed with branded and generic enoxaparin producing a similar effect. Incubation of whole blood with LPS resulted in a dramatic increase in neutrophil CD11b expression (MFI: 13.1±2.8 vs. 249.0±42.1) which was reduced by increasing concentrations of LMWH. This effect appeared to be stronger for generic than branded enoxaparin (MFI: 165.2±31.9 vs. 208.9±25.9). In primates treated with branded and generic enoxaparin, anti-FXa activity assessed by AUC 0–24hrs was similar. Anti-FIIa activity, however, was significantly higher in primates treated with generic enoxaparin (135±28 vs. 91±20 (μg*hr)/ml; p=0.023). AUC for thrombin generation inhibition was (B) 932±59 vs. (G) 775±119 %inhibition*hr; p=0.029. AUC for TFPI release was (B) 1101±98 vs. (G) 822±13 (ng/*hr)/ml; p=0.006. AUC for inhibition of TAFI activation was (B) 780±73 vs. 906±69 % inhibition*hr; p=0.023. Conclusions:This investigation demonstrated a wider variation in anticoagulant response to generic enoxaparin in comparison to branded enoxaparin. This variation was due to the response of the individual subject as well as to the batch of the product. In addition, both in vitro and in vivo/ex vivo activity differences were observed between branded and generic enoxaparin in several parameters relevant to the antithrombotic effect of LMWH. These findings suggest that simple analytical characterization can establish good quality control in manufacturing but may not assure similarity in biological performance between branded and generic enoxaparin. Thus beside the routinely required characterization, inclusion of additional tests for biologic activities and pharmacodynamic profiling of generic products in animal models may provide useful information on the bioequivalence of the generic versions of enoxaparin. Disclosures:Walenga:Sanofi-Aventis, Paris, France: Research Funding. Jeske:Sanofi-Aventis, Paris, France: Research Funding. Hoppensteadt:Sanofi-Aventis, Paris, France: Research Funding. Cunanan:Sanofi-Aventis, Paris, France: Research Funding. Escalante:Sanofi-Aventis, Paris, France: Research Funding. Khan:Sanofi-Aventis, Paris, France: Research Funding. Bailey:Sanofi-Aventis, Paris, France: Research Funding. Fareed:Sanofi-Aventis, Paris, France: Research Funding. Bakhos:Sanofi-Aventis, Paris, France: Research Funding.

Resuscitation of Traumatic Hemorrhagic Shock Patients With Hypertonic Saline—Without Dextran—Inhibits Neutrophil and Endothelial Cell Activation

Posttraumatic inflammation and excessive neutrophil activation cause multiple organ dysfunction syndrome (MODS), a major cause of death among hemorrhagic shock patients. Traditional resuscitation strategies may exacerbate inflammation; thus, novel fluid treatments are needed to reduce such posttraumatic complications. Hypertonic resuscitation fluids inhibit inflammation and reduce MODS in animal models. Here we studied the anti-inflammatory efficacy of hypertonic fluids in a controlled clinical trial. Trauma patients in hypovolemic shock were resuscitated in a prehospital setting with 250 mL of either 7.5% hypertonic saline (HS; n = 9), 7.5% hypertonic saline + 6% dextran 70 (HSD; n = 8), or 0.9% normal saline (NS; n = 17). Blood samples were collected on hospital admission and 12 and 24 h after resuscitation. Multicolor flow cytometry was used to quantify neutrophil expression of cell-surface activation/adhesion (CD11b, CD62L, CD64) and degranulation (CD63, CD66b, CD35) markers as well as oxidative burst activity. Circulating concentrations of soluble intercellular adhesion molecule-1 (sICAM-1), vascular cell adhesion molecule-1 (sVACM-1), P- and E-selectins, myeloperoxidase (MPO), and matrix metalloproteinase 9 (MMP-9) were assessed by immunoassay. Multiple organ dysfunction syndrome, leukocytosis, and mortality were lower in the HS and HSD groups than in the NS group. However, these differences were not statistically significant. Hypertonic saline prevented priming and activation and neutrophil oxidative burst and CD11b and CD66b expression. Hypertonic saline also reduced circulating markers of neutrophil degranulation (MPO and MMP-9) and endothelial cell activation (sICAM-1, sVCAM-1, soluble E-selectin, and soluble P-selectin). Hypertonic saline + 6% dextran 70 was less capable than HS of suppressing the upregulation of most of these activation markers. This study demonstrates that initial resuscitation with HS, but neither NS nor HSD, can attenuate posttraumatic neutrophil and endothelial cell activation in hemorrhagic shock patients. These data suggest that hypertonic resuscitation without dextran may inhibit posttraumatic inflammation. However, despite this effect, neither HS nor HSD reduced MODS in trauma patients with hemorrhagic shock.

1190 Activated Protein C Alters Neutrophil Reactive Oxygen Intermediates in Preterm Neonates

BackgroundInflammation and infection are important aetiological factors in development of preterm birth. Inflammation is associated with many disorders of preterm infants including periventricular leukomalacia, chronic lung disease and necrotising eneterocolitis.AimsTo...

Monocytic and neutrophilic CD11b and CD64 in severe sepsis

Monocytic and neutrophilic CD11b and CD64 in severe sepsis

In Vivo Effect of Recombinant Human Granulocyte Colony-Stimulating Factor on Neutrophilic Expression of CD11b in Septic Neonates: A Randomized Controlled Trial

Neonates are susceptible to septicemia secondary to quantitative and qualitative neutrophilic defects. Granulocyte colony-stimulating factor (G-CSF) stimulates myeloid progenitor cell proliferation and induces selective neutrophil functions. The authors aimed to evaluate the effect of G-CSF administration in septic neonates on neutrophil production and CD11b expression. Sixty septic neonates were randomized to receive intravenous G-CSF 10 μg/kg/day for 3 days (G-CSF group, n = 30), or not to receive G-CSF (non–G-CSF group, n = 30). Thirty healthy newborns were included as controls. Laboratory investigations included complete blood count, C-reactive protein, blood culture, renal and liver function tests, and assessment of neutrophilic expression of CD11b. Total leukocytes count (TLC), absolute neutrophil count (ANC), and immature myeloid cell count in G-CSF group showed significant difference between post–and pre–G-CSF levels. TLC, ANC, immature myeloid cell count and immature/total myeloid cells ratio were higher in G-CSF group compared to non–G-CSF group on days 1 and 3. Higher neutrophilic expression of CD11b was reported in both septic groups on day 0 compared to control group. On day 5, CD11b was higher in G-CSF group than non–G-CSF group. G-CSF improved CD11b% in neutropenic and non-neutropenic septic neonates. No significant difference was found between pre- and posttreatment renal and liver function tests. Lower duration of antibiotic intake and hospitalization was observed in G-CSF group compared to non–G-CSF group. G-CSF administration as an adjuvant therapy for neonatal septicemia, whether neutropenic or not, improves neutrophilic count and function and contributed to early healing from sepsis.

EuroSCORE and neutrophil adhesion molecules predict outcome post-cardiac surgery

This study tested the hypothesis that surgical stress and the host response to this trauma trigger an inflammatory cascade in which the neutrophil plays a central role. We hypothesised that pre-operative neutrophil migratory responses will correlate with post-operative clinical outcome in our shock model of open-heart surgery patients. We also tested the hypothesis that surface expression of adhesion molecules involved in the migratory process - CD11b, CD47 and CD99 - could be used to predict outcome. We believe that combining neutrophil migratory response, CD11b, CD47 and CD99 with the logistic European System for Cardiac Operative Risk Evaluation (EuroSCORE) will strengthen the power of the EuroSCORE not only in predicting post-operative mortality but also other clinical endpoints. Neutrophils were isolated pre-operatively from n = 31 patients undergoing open-heart surgery and allowed to migrate across endothelial monolayers in response to N-formyl-methionine-leucine-phenylalanine (fMLP). Isolated neutrophils were also assessed for surface expression of CD11b, CD47 and CD99 in response to fMLP by flow cytometry. Post-operative clinical parameters collected included days 1-5 white cell count and creatinine levels as well as intensive care unit (ICU) and post-operative hospital stay. Pre-operative surface expression of CD99 and CD47 correlates with post-operative creatinine levels (P < 0·05), a measurement of renal injury. We also show that while the logistic EuroSCORE alone can be used as a predictor of ICU stay, when combined with pre-operative CD99 surface expression, it improves its AUC value (0·794). Immunological markers, specifically the ability of the neutrophil to migrate, combined with the logistic EuroSCORE lead to improved sensitivity and specificity to predict patient outcome.

IL‐8 from Local Subcutaneous Wounds Regulates CD11b Activation

The cellular and soluble mediators of a dermal inflammation can be studied by the skin chamber technique. The aim of this study was to address the physiological effect of soluble mediators, released into the skin chamber, with special focus on neutrophil CD11b activation. Mediators released at the inflammatory site were studied by Milliplex and enzyme-linked immunosorbent assay (ELISA) and correlated with transmigration and CD11b activation in vivo and in vitro. Transmigration was studied by the skin chamber technique and by the transwell method, and expression of the CBRM1/5 epitope on activated CD11b was analysed by flow cytometry following in vivo and in vitro incubation with chamber fluid or recombinant interleukin-8 (IL-8). Leucocyte in vivo and in vitro transmigration both correlated with the concentrations of IL-1β, tumour necrosis factor alpha (TNFα) and IL-8 at P < 0.05 (R > 0.7). Furthermore, CD11b was activated, in terms of exposure of the activation epitope, on neutrophils after 30 min of in vitro incubation with chamber fluid and correlated solely with the concentration of IL-8, P < 0.05 (R = 0.72). In vitro incubation with recombinant IL-8 confirmed a concentration-dependent expression of the activation epitope; however, induction of CBRM1/5 by recombinant IL-8 required a concentration that was significantly higher compared with that in chamber fluid. In addition, the CBRM1/5 epitope was analysed on in vivo extravasated neutrophils that displayed a significantly higher expression compared with circulating neutrophils, P = 0.04. We conclude that IL-8 is the major factor regulating the expression of CD11b activation epitope in neutrophils.

Microparticles from Stored Red Blood Cells Activate Neutrophils and Cause Lung Injury after Hemorrhage and Resuscitation

Transfusion of stored blood is associated with increased complications. Microparticles (MPs) are small vesicles released from RBCs that can induce cellular dysfunction, but the role of RBC-derived MPs in resuscitation from hemorrhagic shock is unknown. In the current study, we examined the effects of RBC-derived MPs on the host response to hemorrhage and resuscitation. MPs were isolated from murine packed RBC units, quantified using flow cytometry, and injected into healthy mice. Separate groups of mice underwent hemorrhage and resuscitation with and without packed RBC-derived MPs. Lungs were harvested for histology and neutrophil accumulation and assessed by myeloperoxidase content. Human neutrophils were treated with human RBC-derived MPs and CD11b expression, superoxide production, and phagocytic activity were determined. Stored murine packed RBC units contained increased numbers of RBC-derived MPs compared with fresh units. Hemorrhaged mice resuscitated with MPs demonstrated substantially increased pulmonary neutrophil accumulation and altered lung histology compared with mice resuscitated without MPs. Intravenous injection of MPs into normal mice resulted in neutrophil priming, evidenced by increased neutrophil CD11b expression. Human neutrophils treated with RBC-derived MPs demonstrated increased CD11b expression, increased superoxide production, and enhanced phagocytic ability compared with untreated neutrophils. Stored packed RBC units contain increased numbers of RBC-derived MPs. These MPs appear to contribute to neutrophil priming and activation. The presence of MPs in stored units can be associated with adverse effects, including lung injury, after transfusion.

Automated assessment of the neutrophil and platelet activation status in patients with essential thrombocythemia

Neutrophil and platelet activation are consistently found in essential thrombocythemia (ET), but the techniques employed to demonstrate such abnormalities are complex. To ascertain whether the ADVIA 120 analyzer can be employed to assess neutrophil and platelet activation status in ET, 55 such patients and the same number of matched healthy individuals were studied and the results correlated with neutrophil CD11b and platelet P-selectin expressions measured by flow cytometry. Compared with controls, ET patients had significantly higher values of neutrophil myeloperoxidase index (MPXI), mean platelet volume (MPV), platelet distribution width (PDW), and platelet component distribution width, and significantly lower values of neutrophil lobularity index and mean platelet component (MPC). Patients with the JAK2 mutation had significantly lower values of MPC and higher values of MPV and PDW than those with wild-type allele. A positive correlation was observed between MPXI and neutrophil CD11b expression and a negative correlation between MPC and platelet P-selectin expression. The intensity of the agreement between the variables obtained by the two methods was moderate. These results support the possible value of MPC as surrogate parameter of platelet activation in ET.

Evaluation of adhesion molecules CD64, CD11b and CD62L in neutrophils and monocytes of peripheral blood for early diagnosis of neonatal infection

This study was undertaken to assess the value of neutrophils CD11b, CD64, and CD62L for the early diagnosis of neonatal infection. Eighty-four neonates who were followed up for a suspected neonatal infection were included in this study. They were assigned into an infection group (n=49) and a non-infection group (n=35). Healthy neonates served as controls (n=35). A full sepsis screening was performed and neutrophil and monocyte expressions of CD11b, CD64 and CD62L were determined by flow cytometry. The expressions of CD64 and CD11b were significantly enhanced in the infection group compared to the non-infective group and the controls. CD64 expression on neutrophils and monocytes is a useful diagnostic marker for the early diagnosis of neonatal infection. Combination of CD64, CD11b and C reactive protein further enhances the sensitivity of the expression and its negative predictive value.

Expression of inflammatory and apoptosis factors following coronary stent implantation in coronary heart disease patients

We investigated the changes in characteristics of neutrophil CD11b, monocyte CD11b, platelet CD62P, endothelin (ET), and neutrophil CD178 in patients with coronary heart disease (CHD) before and after primary coronary stenting. A total of 41 patients with CHD who underwent coronary stenting and 40 control subjects were enrolled in the study. In CHD patients, peripheral blood samples were taken 24h before and 30min, 24h, and 72h after successful coronary stenting. All markers were significantly elevated in patients with CHD compared with controls (P<0.05). Time-course studies revealed that the expressions of neutrophil CD11b, monocyte CD11b, platelet CD62P, and ET were lower at 30min post-operation (PO) compared with that at 24h before operation (BO)(P<0.05). All levels significantly increased from 30min PO to 24h PO (P<0.05) and decreased thereafter until 72h PO (P>0.05). Time course changes in neutrophil CD11b levels after coronary stenting were significantly higher in patients with unstable angina pectoris than in patients with stable angina pectoris (P<0.05). CD11b levels were related to CD62P in patients with CHD (P<0.05). Neutrophil CD11b and monocyte CD11b levels were significantly increased in patients with CHD who underwent coronary stenting compared with controls (P<0.05). Results show that CD11b levels increased, meanwhile, the levels of CD62P and ET increased in CHD patients after coronary stenting. In addition, neutrophil CD178 levels of apoptosis factor in patients, which is important for regression of inflammation, remained high for a period of time after coronary stenting.

Effect of Prolonged In Vivo Administration of Progesterone in Pregnancy on Myometrial Gene Expression, Peripheral Blood Leukocyte Activation, and Circulating Steroid Hormone Levels

We aimed to investigate the effects of progesterone on gene expression and function of both myometrium and circulating leukocytes. We recruited women participating in a randomized clinical trial of progesterone to prevent preterm delivery. These participants had a twin pregnancy and were managed in 1 of 2 tertiary referral centers. Participants were treated with progesterone (90 mg vaginally) or placebo from 24 to 34 weeks of pregnancy. The outcome measures were myometrial and leukocyte gene expression and expression of cell surface markers in circulating leukocytes, all quantified ex vivo. Prolonged in vivo administration of progesterone inhibited myometrial expression of connexins 26 and 43, endothelial nitric acid synthase (eNOS), and the prostaglandin receptor EP2 ex vivo. Administration of progesterone also increased numbers of circulating neutrophils while decreasing lymphocyte proportions and decreasing neutrophil CD11b expression. The observed effects of prolonged in vivo administration of progesterone will minimize the ability of the uterus to contract as a synctium and the ability of peripheral blood leukocytes to migrate into the myometrium during parturition. We suggest that these are putative mechanisms by which progesterone might prevent preterm birth in women at high risk.

Remote Ischemic Preconditioning

Circulatory arrest and stroke are among the leading causes of death and disability worldwide, and they are difficult to treat. Only a limited number of therapeutic approaches have reached the bedside (eg, hypothermia and recombinant tissue plasminogen activator)1 and, unfortunately, relatively few patients are eligible. Virtually all experimental compounds tested so far have failed at some stage of translational development. Preconditioning is an attractive strategy that makes the brain and other visceral organs relatively resistant to tissue injury. It develops when a noxious stimulus, below the damage threshold, leads to reduced tissue injury on subsequent challenge with a stimulus given above injury threshold. It is an appealing strategy to identify novel endogenous protective mechanisms and, as demonstrated by Jensen, Loukogeorgakis, and their colleagues in this issue of Circulation ,2 it may prove useful to prevent brain damage in the clinical context when there is a high probability of cerebral ischemia (eg, before high-risk surgery or in patients with subarachnoid hemorrhage). Article see p 714 Various types of preconditioning stimuli have been used experimentally to protect brain, heart, retina, liver, kidney, and other organs.3 Some use ischemic preconditioning, in which the blood supply to a target organ is temporarily interrupted before introducing a longer infarct-generating insult that would ordinarily produce infarction; other studies use hypoxic preconditioning, in which animals are exposed to oxygen levels around 8% to 9% for a few hours. But ischemia and hypoxia are just 2 examples in a larger list of strategies that induce tolerance to brain injury: hypoglycemia, hypoxia, kainate-induced seizures, and exposure to volatile anesthetics, to name a few.4 Preconditioning can protect the brain either rapidly after stimulation (known as early, first window, or classical preconditioning) or after a 24-hour delay to induce protein synthesis-dependent protection lasting up to 96 hours …

Analysis of the temperature affects on leukocyte surface antigen expression

Flow cytometric analysis of leukocyte surface antigens has been used to characterize infectious and septic processes in patients. We wanted to investigate how the sampling and processing temperature, the anticoagulant used, and the storage of the sample influence leukocyte immunophenotyping. Four blood samples, two using acid citrate dextrose and two using heparin as an anticoagulant, were taken from five intensive-care unit patients with severe sepsis and five healthy volunteers. The samples were collected, stored, and processed either at +4°C or at room temperature (RT). The samples were processed for flow cytometric analysis within 1 hr of collection or after 6 or 24 hr storage. The surface antigens of interest were neutrophilic CD11b and CD64, monocytic CD11b, CD14, CD40, CD64, CD80 and HLA-DR, and lymphocytic CD69 (separately in CD4+ and CD8+ T cells, B cells, and natural killer cells). The fluorescence intensities were higher at RT than at +4°C. During storage the intensities increased at RT, but at +4°C there were only minor changes. The effects were similar with both anticoagulants studied. According to our results, flow cytometric analysis of leukocyte surface antigen expressions should be performed using +4°C temperature throughout the process and within 6 hr.

Tissue factor contributes to neutrophil CD11b expression in alpha-naphthylisothiocyanate-treated mice

Tissue factor contributes to neutrophil CD11b expression in alpha-naphthylisothiocyanate-treated mice

Tissue factor contributes to neutrophil CD11b expression in alpha-naphthylisothiocyanate-treated mice

Cholestatic liver injury induced by alpha-naphthylisothiocyanate (ANIT) is provoked by injury to intrahepatic bile ducts and the progression of hepatic necrosis requires the procoagulant protein tissue factor (TF) and extrahepatic cells including neutrophils. Recent studies have shown that myeloid cell TF contributes to neutrophil activation. We tested the hypothesis that myeloid cell TF contributes to neutrophil activation in ANIT-treated mice. TF activity in liver homogenates increased significantly in TFflox/flox mice treated with ANIT, but not in TFflox/flox/LysMCre mice (TFΔMyeloid mice), which have reduced TF expression in monocytes/macrophages and neutrophils. Myeloid cell-specific TF deficiency did not alter expression of the chemokines KC or MIP-2 but reduced hepatic neutrophil accumulation in ANIT-treated mice at 48h as indicated by tissue myeloperoxidase (MPO) activity. Myeloid cell TF deficiency significantly reduced CD11b expression by blood neutrophils in ANIT-treated mice, and this was associated with reduced plasma MPO protein levels, an index of neutrophil degranulation. However, myeloid cell-specific TF deficiency had no effect on ANIT-induced coagulation cascade activation. The increase in serum ALT and ALP activities in ANIT-treated mice was reduced by myeloid cell TF deficiency (p<0.05), but the myeloid cell TF deficiency did not reduce hepatic necrosis at 48h, as determined by histopathology and morphometry. The results suggest that myeloid cell TF contributes to neutrophil CD11b expression during cholestasis by a coagulation-independent pathway. However, the resultant reduction in neutrophil accumulation/activation is insufficient to substantially reduce ANIT hepatotoxicity, suggesting that myeloid cell TF is only one of many factors modulating hepatic necrosis during cholestasis.

Systemic Neutrophil Activation in a Mouse Model of Ischemic Stroke and Reperfusion

As a natural response to injury and disease, neutrophils activate, adhere to the microvasculature, migrate into brain tissue, and release toxic substances such as reactive oxygen species and proteases. This neutrophil response occurs when blood flow is returned to brain tissue (reperfusion) after ischemic stroke. Thus, the presence of activated systemic neutrophils increases the potential for tissue injury during reperfusion after ischemic stroke. Although experiments in rat models suggest that activated neutrophils play a pivotal role in cerebral ischemia reperfusion injury, little is known about systemic neutrophil activation during reperfusion following ischemic stroke in a mouse model. The purpose of this study was to characterize systemic leukocyte responses and neutrophil CD11b expression 15-min and 24-hr post-reperfusion in a mouse model of ischemic stroke. The intraluminal filament method of transient middle cerebral artery occlusion (tMCAO) with reperfusion or a sham procedure was performed in male C57Bl/6 mice. Automated leukocyte counts and manual white blood cell (WBC) differential counts were measured. Flow cytometry was used to assess systemic neutrophil surface CD11b expression. The data suggest that the damaging potential of systemic neutrophil activation begins as early as 15 min and remains evident at 24 hr after the initiation of reperfusion. In addition, because transgenic mouse models, bred on a C57Bl/6 background, are increasingly used to elucidate single mechanisms of reperfusion injury after ischemic stroke, findings from this study are foundational for future investigations examining the damaging potential of neutrophil responses post-reperfusion after ischemic stroke in genetically altered mouse models within this background strain.

Systemic Evaluation of Platelet and Leukocyte Activation and Interaction in a Rat Model of Pulmonary Arterial Hypertension

Objectives: Thrombosis and inflammation are associated with the pathogenesis of pulmonary arterial hypertension (PAH). However, there are no solid data supporting the involvement of platelet and leukocyte activation and interaction in PAH. The present study thus investigated the activation and interaction of circulating platelets and leukocytes in a rat model of monocrotaline (MCT)-induced pulmonary hypertension. Methods: Mean pulmonary arterial pressure (mPAP) was monitored in rats (n = 24) before and 2, 3 and 7 weeks after MCT (60 mg/kg)injection. In parallel, activation of circulating platelets and leukocytes and platelet-leukocyte aggregates were measured by whole-blood flow cytometry. Results: Two weeks after MCT injection, mPAP had increased significantly, i.e. from 11.25 ± 0.92 mm Hg at baseline to 15.71 ± 1.66 mm Hg (p < 0.05), and it had increased even further at week 7 (26.83 ± 3.29 mm Hg; p < 0.01). Fibrinogen binding of circulating platelets had increased from the basal level of 1.45 ± 0.61 to 4.08 ± 1.59% 3 weeks after MCT injection (p < 0.01). Platelet responsiveness to ADP was also significantly enhanced. CD11b expression of circulating neutrophils was elevated; i.e. mean fluorescence intensity increased from 1.67 ± 0.38 before MCT injection to 2.37 ± 0.31 3 weeks after MCT injection (p < 0.01), and N-formyl-methionyl-leucyl-phenylalanine (1 × 10^–7M) stimulation induced more marked elevation of neutrophil CD11b expression in MCT-treated animals. Circulating platelet-neutrophil aggregates were already increased 2 weeks after MCT treatment (14.93 ± 4.22%; p < 0.01) compared to baseline (6.01 ± 2.91%) and remained elevated at 3 weeks (15.19 ± 4.78%; p < 0.01). Conclusions: MCT-induced PAH in rats is associated with increased platelet and leukocyte activation and platelet-leukocyte interaction in vivo, which may play an important role in the pathogenesis of PAH.