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PubMed1997

Neutrophil rheologic changes in septic shock.

Decreases in effective capillary blood flow during septic shock may be related to changes in neutrophil rheology which contribute to microvascular occlusion. The purpose of this study was to examine neutrophil deformability, adherence, and aggregation in patients with severe sepsis and septic shock. Neutrophils were isolated from six patients with septic shock (SS), 12 patients with severe sepsis (S), six noninfected critically ill patients (CINS), and nine normal volunteers (N). Neutrophil deformability was determined by examining filtration through 5-microm filters. Neutrophil aggregation was measured by aggregometry and leukergy. Neutrophil adherence was examined by assessing the binding of latex beads to neutrophils. Patients with S and SS demonstrated decreased neutrophil filterability of 27 +/- 2% and 16 +/- 5%, respectively (p < 0.01), in comparison with N subjects, 55 +/- 4% and CINS patients, 58 +/- 2%. Preincubation of neutrophils from S and SS patients with cytochalasin D significantly increased the percent filtration of neutrophils. Neutrophil aggregation, measured by aggregometry, was increased in SS patients, 16 +/- 4% (p < 0.01) compared with N subjects, 1 +/- 0.2% and CINS patients, 1 +/- 0.2%. Incubation of neutrophils of SS patients with anti-CD11/CD18 significantly increased the filtration of isolated neutrophils to 46 +/- 3% (p < 0.01) and decreased aggregation to 7 +/- 2%. Neutrophil adherence was not increased in S or SS patients. These observations suggest that neutrophil deformability is decreased in patients with S and SS. Increased leukoaggregation may also contribute to decreased filterability of neutrophils in SS patients. These mechanisms may play a role in impaired microvascular flow in septic shock.

Aged↗
PubMed2006

Induction of neutrophil chemotaxis by leptin: crucial role for p38 and Src kinases.

Leptin is involved in energy homeostasis, hematopoiesis, inflammation, and immunity. Although hypoleptinemia characterizing malnutrition has been strictly related to increased susceptibility to infection, other hyperleptinemic conditions, such as end-stage renal disease (ESRD), are highly susceptible to bacterial infections. On the other hand, ESRD is characterized by neutrophil functional defects crucial for infectious morbidity, and several uremic toxins capable of depressing neutrophil functions have been identified. In the present study, we investigated leptin's effects on neutrophil function. Our results show that leptin inhibits neutrophil migration in response to classical chemoattractants. Otherwise, leptin is endowed with chemotactic activity toward neutrophils. The two activities, inhibition of the cell response to chemokines and stimulation of neutrophil migration, could be detected at similar concentrations. On the contrary, neutrophils exposed to leptin did not display detectable [Ca2+]i mobilization, oxidant production, or beta2-integrin upregulation. The results demonstrate that leptin is a pure chemoattractant devoid of secretagogue properties but capable of inhibiting neutrophil chemotaxis to classical neutrophilic chemoattractants. This effect is dependent on the activation of intracellular kinases involved in F-actin polymerization and neutrophil locomotion. Indeed, p38 mitogen-activated protein kinase (MAPK) and Src kinase, but not extracellular-regulated kinase (ERK), were activated by short-term incubation with leptin. Moreover, p38 MAPK inhibitor SB203580 and Src kinase inhibitor PP1, but not MEK inhibitor PD98059, blocked neutrophil chemotaxis toward leptin. Serum from patients with ESRD inhibits migration of normal neutrophils in response to N-formyl-methionine-leucyl-phenylalanine (FMLP) with a strict correlation between serum leptin levels and serum ability to suppress neutrophil locomotion. The serum inhibitory activity can be effectively prevented by immune-depletion of leptin. Taking into account the crucial role of neutrophils in host defense, we show that leptin-mediated ability of ERSD serum to inhibit neutrophil chemotaxis appears to be a mechanism contributing to neutrophil dysfunction in ESRD.

Chemotaxis, Leukocyte↗
PubMed1996

[Flow cytometric measurement of DCFH-oxidation of neutrophils in peripheral whole blood, isolated leukocytes and cerebrospinal fluid cells].

Using 2',7'-dichlorofluorescein diacetate (DCFH-DA) as an indicator for the intracellular formation of H2O2 and free radicals, we measured DCFH-oxidation (DCF fluorescence) of neutrophils by flow cytometry. The DCF fluorescence intensity of unstimulated neutrophils in whole blood was the same as the autofluorescence of neutrophils. However, that of unstimulated isolated neutrophils was higher than autofluorescence. The DCF fluorescence of isolated neutrophils was higher than that of neutrophils in whole blood with or without stimuli, and was decreased to the intensity of neutrophils in whole blood by the addition of plasma or erythrocytes. The DCF fluorescence of neutrophils in whole blood indicates the oxidative state of neutrophils in vivo. The determination of DCFH-oxidation of neutrophils in isolated leukocytes is also necessary to evaluate precisely the oxidative state of neutrophils. In cerebrospinal fluid (CSF) cells from patients with meningitis, the DCF fluorescence intensity of unstimulated neutrophils was higher than the autofluorescence of CSF neutrophils. The DCF fluorescence of CSF neutrophils stimulated with PMA showed increased intensity over that of unstimulated CSF neutrophils. The DCF fluorescence of CSF neutrophils, both unstimulated and stimulated with PMA, were decreased with the addition of plasma but not with the addition of CSF.

Cerebrospinal Fluid↗
PubMed1999

Impaired innate immunity in the newborn: newborn neutrophils are deficient in bactericidal/permeability-increasing protein.

OBJECTIVE: The mechanisms by which newborns are at increased risk for invasive bacterial infections have been incompletely defined. A central element of innate immunity to bacterial infection is the neutrophil-a cell that contains cytoplasmic granules replete with antibiotic proteins and peptides. The activity of adult neutrophils against gram-negative bacteria is believed to depend to a significant degree on the presence in neutrophil primary (azurophilic) granules of the 55-kDa bactericidal/permeability-increasing protein (BPI), which binds with high affinity to bacterial lipopolysaccharides and kills gram-negative bacteria. In light of the importance of BPI to antibacterial host defense and to investigate possible factors underlying the risk of neonatal bacterial infections, we determined the relative content of BPI in the neutrophils of adults and newborns. DESIGN: The cellular content of BPI was determined by Western blotting of neutrophils derived from full-term newborn cord blood (n = 21; mean gestational age: 38.6 weeks) and from adult peripheral blood (n = 22; mean age: 29 years). Extracellular levels of BPI in adult and newborn plasma were assessed by enzyme-linked immunosorbent assay. Neutrophil content of other azurophil granule markers also was assessed: myeloperoxidase by Western blotting and defensin peptides by acid-urea polyacrylamide gel electrophoresis and Coomassie staining. Acid extracts of newborn and adult neutrophils were analyzed for antibacterial activity against serum-resistant encapsulated isolate Escherichia coli K1/r. RESULTS: The neutrophils of newborns contain at least threefold to fourfold less BPI per cell than adult neutrophils (67 +/- 13 ng per 10(6) cells vs 234 +/- 27 ng per 10(6) cells). The relative BPI-deficiency of newborn neutrophils apparently was not attributable to perinatal stress-related degranulation of intracellular BPI stores because: 1) newborn and adult neutrophils contained nearly identical amounts of 2 microbicidal constituents derived from the same primary (azurophil) granule compartment as BPI (the enzyme myeloperoxidase as well as defensin peptides), and 2) levels of extracellular BPI in newborn plasma, measured by enzyme-linked immunosorbent assay, represent only approximately 2% of cellular BPI content. As predicted by their lower BPI content, newborn neutrophil acid extracts demonstrated significantly lower antibacterial activity against E coli K1/r than did adult neutrophil acid extracts. CONCLUSION: These data suggest that the neutrophils of newborns are selectively deficient in BPI, a central effector of antibacterial activity against gram-negative bacteria. BPI deficiency correlates with decreased antibacterial activity of newborn neutrophil extracts against serum-resistant E coli and could contribute to the increased incidence of gram-negative sepsis among newborns relative to healthy adults.neonatal sepsis, gram-negative bacteria, endotoxin, neutrophil, polymorphonuclear leukocyte, innate immunity, bactericidal/permeability-increasing protein, defensin, myeloperoxidase.

Adult↗
PubMed1983

The neutrophil response to polyvinyl sponge implantation.

Neutrophil release and migration in mice were studied over a 24-hr period after the sc implantation of a single polyvinyl sponge. The release of neutrophils from the marrow was evaluated by directly counting the residual neutrophils in the femoral marrow of animals with sponges. Sponge and tissue neutrophil content was determined by extraction and assay of myeloperoxidase (MPO), a marker enzyme for neutrophils. A maximum depletion of 48% of the mature neutrophils in the marrow was observed 5 hr after sponge implantation, in keeping with significant release of neutrophils for migration to the sponge. The released cells were not found in the circulating granulocyte pool, since neutropenia was noted. The accumulation of neutrophils in the sponge increased throughout the 24-hr period, whereas in the tissue adjacent to the sponge maximum accumulation of neutrophils occurred within 7 hr. In fact, neutrophils migrated to at least three sites--the sponge, the skin overlying the sponge, and the skin in which an incision had been made to insert the sponge. The sponge content of neutrophils represented 0.3-33% of the neutrophils migrating to the combined lesion (sponge and skin sites). Therefore, if the neutrophil response to foreign body implantation is to be measured in its entirety, it is necessary to quantify not only the neutrophils within the foreign body but also those in the tissues surrounding it. These studies describe an animal model for neutrophil release and migration to tissues following a standard stimulus. It is proposed that this model may be useful in exploring the factors which influence the release and migration of neutrophils in vivo.

Animals↗
PubMed2005

Comparison of morphology, viability, and function between blood and milk neutrophils from peak lactating goats.

The morphological features of blood and milk neutrophils from peak lactating goats were compared using light microscopy, scanning electron microscopy and flow cytometry in order to investigate the cytological changes of neutrophils after migration into the mammary gland. The kinetics of reactive oxygen intermediates (ROI) generation and gelatinase release of blood and milk neutrophils, with or without stimulation of phorbol 12-myristate, 13-acetate ester (PMA), were used to characterize their responses to inflammatory stimuli. Neutrophils isolated from goat milk were highly segmented and contained multi-lobed nuclei. Ultrastructurally, milk neutrophils were more ruffled on the surface compared to blood neutrophils. Approximately 30% of milk neutrophils were undergoing cell death, either necrosis or apoptosis, in contrast to 8% of blood neutrophils. The ROI production of activated milk neutrophils peaked earlier than blood neutrophils, but the duration and the intensity were much less. Neutrophils from both sources augmented the release of gelatinase in response to PMA (1 ng/mL). However, the amount of gelatinase released from milk neutrophils was lower (P < 0.05) than that of blood neutrophils. In summary, more neutrophils become apoptotic and necrotic in the mammary gland, presumably due to spontaneous aging, the process of diapedesis, and the interaction with milk components. Milk neutrophils have impaired functionalities in comparison with blood neutrophils. The information is relevant when studying mammary gland immunity and related diseases, such as mastitis.

Animals↗
PubMed1990

Priming of neutrophils by lipopolysaccharide for enhanced release of superoxide. Requirement for plasma but not for tumor necrosis factor-alpha.

When human neutrophils are incubated with LPS, they become primed for enhanced release of O2- in response to stimulation by FMLP. We investigated two aspects of LPS priming: 1) whether priming depends on secretion of TNF-alpha by monocytes present in neutrophil preparations, and 2) whether plasma is required for priming. Using plasma-Percoll gradients, we isolated neutrophils that contained only 0.1% monocytes. At 37 degrees C, these neutrophils were significantly primed by LPS (100 ng/ml) within 30 min. In contrast, LPS-treated monocytes required 60 min to secrete significant neutrophil-priming activity, the major component of which was TNF-alpha. Further, antibody against TNF-alpha failed to inhibit priming of neutrophils by LPS at 15, 30, and 45 min, and inhibited only 15% at 60 min. The results suggested that TNF-alpha or other factors from monocytes were not essential for priming of neutrophils by LPS. Neutrophils that had been washed free of plasma by centrifugation through 50% Percoll responded only weakly to LPS with respect to priming for enhanced O2- release and increased expression of alkaline phosphatase activity on the cell surface. Priming of washed neutrophils could be restored by adding back plasma (0.1 to 1.0%). This effect of plasma was not blocked by heating the plasma to 56 degrees C but was blocked at 100 degrees C. LPS priming could be blocked by polymyxin B, even in the presence of plasma. Thus, priming required both LPS and plasma. Neutrophils incubated with LPS in the absence of plasma were not primed by subsequent addition of plasma, but were primed by addition of plasma and LPS. Culture supernatants from neutrophils incubated with 20 ng/ml LPS in the absence of plasma failed to prime fresh neutrophils, but supernatants from neutrophils incubated with LPS in the presence of 1% plasma were able to prime fresh neutrophils. These results implied that neutrophils inactivated LPS and that plasma protected LPS from inactivation. Nevertheless, such inactivated LPS retained the ability to gel Limulus lysate at 10 pg/ml, and the ability to prime monocytes at 100 pg/ml. Thus, plasma prevented a neutrophil-specific inactivation of LPS.

Alkaline Phosphatase↗
PubMed1989

Generation of biologically active C-reactive protein peptides by a neutral protease on the membrane of phorbol myristate acetate-stimulated neutrophils.

The association of human C-reactive protein (CRP) with nonstimulated and PMA-stimulated human neutrophils and the concomitant degradation of CRP (monitored by TCA-soluble peptides and SDS-PAGE analysis) has been studied. Maximum association of 125I-labeled CRP with neutrophils and 125I-labeled CRP degradation during association with these cells was achieved by stimulating the neutrophils with PMA at 10 ng/ml; a concentration in which azurophil granule release was not significant. For PMA-stimulated neutrophils, the association of 125I-labeled CRP was 1.8 times higher and PMA-stimulated neutrophil-mediated degradation of the ligand was three times faster than that for nonstimulated cells. The neutrophil-associated 125I-labeled CRP in the absence and presence of PMA proved on SDS-PAGE analysis to be approximately 50% degraded. There was a positive correlation between the extent of CRP degradation and the association of 125I-labeled CRP with neutrophils. In addition to generation of neutrophil associated CRP intermediates, small soluble CRP peptides were generated during association of CRP with neutrophils. These peptides inhibited superoxide production from opsonized zymosan-activated neutrophils by approximately 40% at 10 micrograms/ml. 125I-labeled CRP degradation mediated by nonstimulated neutrophils, and neutrophil-conditioned medium (from both non-stimulated and PMA-stimulated cells) was inhibitable by alpha 1-antitrypsin and approximately seven times less at 1 h than that occurring during 125I-labeled CRP-association with PMA-stimulated neutrophils. The degradation of 125I-labeled CRP mediated by PMA-stimulated neutrophils was not fully inhibitable by alpha 1-antitrypsin. The data point to the involvement of a membrane-associated serine protease, which is maximally activated by PMA, in the degradation of 125I-labeled CRP during association with neutrophils. Our results indicate that at an inflammatory site CRP-derived peptides can be produced that inhibit the action of activated neutrophils.

C-Reactive Protein↗
PubMed1987

Prevention of pulmonary injury in isolated perfused rat lungs by activated human neutrophils preincubated with anti-Mo1 monoclonal antibody.

Neutrophil activation results in neutrophil adherence and may subsequently cause lung injury through the generation of oxidants, release of granule proteases, and generation of a variety of mediator substances. We hypothesized that inhibition of neutrophil adherence and subsequent lung sequestration would attenuate the lung injury caused by activated neutrophils. Using isolated perfused rat lungs, we determined if anti-Mo1 monoclonal antibody (binds to the alpha subunit of a neutrophil glycoprotein [gp 155.94] that facilitates adherence) would attenuate lung neutrophil sequestration and lung injury caused by human neutrophils stimulated by phorbol myristate acetate (PMA). PMA-stimulated neutrophils but not PMA or neutrophils alone caused lung injury as assessed by accumulation of 125I-bovine serum albumin into lung parenchyma and alveolar lavage fluid. Incubation of neutrophils with anti-Mo1 antibody prior to stimulation with PMA attenuated lung injury and neutrophil sequestration. Furthermore, a histological survey revealed that anti-Mo1 antibody inhibited neutrophils present in the lung from spreading following exposure to PMA. Anti-Mo1 antibody did not inhibit PMA-stimulated neutrophil release of granule constituents or toxic O2 metabolites as evidenced by lysozyme and lactoferrin release or the reduction of ferricytochrome c in the lung perfusate. The inhibition of lung injury caused by the anti-Mo1 antibody was not likely due to a nonspecific effect of the antibody, since another murine monoclonal antibody of the same class (anti-Mo5) did not inhibit lung neutrophil sequestration or lung injury. Thus, in this experimental model, interference with the close approximation of the neutrophil to its target site inhibited the ability of the activated human neutrophil to cause injury.

Animals↗
PubMed1986

Effects of human colony-stimulating factor on the uptake and destruction of a pathogenic parasite (Trypanosoma cruzi) by human neutrophils.

The ability of granulocyte-macrophage colony-stimulating factor (CSF-H) to modulate human neutrophil functions was studied by using an in vitro system in which this cell type interacted with intracellular (amastigote [AMA]) forms of Trypanosoma cruzi. The presence of CSF-H during the 30-min period of neutrophil incubation with the AMA markedly enhanced parasite internalization. This effect was evidenced by significant increases in both the percentage of neutrophils incorporating AMA and the average number of AMA per 100 neutrophils with respect to mock-treated neutrophils. Pretreatment of the neutrophils with CSF-H reproduced the enhancement effect, whereas pretreatment of the AMA had no detectable consequence. The minimal neutrophil CSF-H pretreatment period required to significantly increase the number of AMA per 100 neutrophils was 20 min--suggesting that CSF-H induced time-dependent events ultimately leading to the manifestation of the noted effect--but neutrophil treatment with CSF-H for longer periods of time (up to 60 min) caused a much greater enhancement. Consistent with the notion of a regulatory action of CSF-H on neutrophils was the fact that the enhancing effect subsided gradually after removal of the factor and was no longer detectable after 16 hr. When 3H-labeled AMA were used, CSF-H-treated neutrophils released greater amounts of radiolabeled substances than mock-treated cells, indicating a stimulatory effect of CSF-H on the killing capacity of neutrophils. This was confirmed by the fact that untreated neutrophils that had internalized 3H-AMA killed the parasites at a faster rate when subsequently incubated with CSF-H. Catalase, but not superoxide dismutase, mannitol, benzoate, or histidine, inhibited neutrophil killing of the 3H-AMA whether the granulocytes had been exposed to CSF-H or not. This indicated that the cytotoxic mechanism involved the production of hydrogen peroxide in both cases, but possibly at a higher rate in the CSF-H-treated neutrophils. These results point to a regulatory effect of CSF-H on neutrophils that promotes cellular activities that might be relevant to the mechanisms of clearance of T. cruzi in vivo.

Animals↗
PubMed1996

The greater omentum is the primary site of neutrophil exudation in peritonitis.

BACKGROUND: Peritonitis remains a major infectious problem. Neutrophil influx into the peritoneal cavity is one of the most important host defense mechanisms. However, no studies have focused on the site of neutrophil exudation. This study examined the primary anatomic site of neutrophil exudation in bacterial peritonitis. STUDY DESIGN: Fifty-five rats were injected intraperitoneally with saline solution (control group) or 10(7) Escherichia coli (peritonitis group). In experiment 1, 1 x 10(6) fluorescein-labeled neutrophils were infused 3 hours after the challenge. Then, peritoneal-lavaged fluids and peritoneal tissues (the greater omentum, mesentery, parietal peritoneum, colon, and ileum) were obtained. Subpopulations of peritoneal exudative cells and numbers of labeled neutrophils in tissues were counted. In experiment 2, labeled neutrophils were infused at 10 minutes and at 1 and 5 hours after the challenge. Peritoneal tissues were also harvested. The number of labeled neutrophils in each tissue was determined. RESULTS: In experiment 1, numbers of labeled peritoneal neutrophils and exudative neutrophils were higher in the peritonitis group than in the control group. Numbers of exudative neutrophils showed a positive correlation with numbers of labeled peritoneal neutrophil. In experiment 2, at 1 and 5 hours after the challenge, the number of labeled neutrophils was higher in the peritonitis group than in the control group. The number of neutrophils in the omentum was higher than the number in other peritoneal tissues. CONCLUSIONS: Our fluorescence microscopic method is useful for detecting neutrophil adhesion. Neutrophil exudation into the peritoneal cavity was most marked in the omentum. The greater omentum may play an important role in host defense as a source of exudative neutrophils.

Animals↗
PubMed1998

The role of neutrophils in acute renal failure.

The role of neutrophils in acute renal failure is controversial. Acute renal failure can clearly occur in the absence of neutrophils. However, recent studies using specific neutrophil markers indicate that neutrophils accumulate in postischemic kidneys. Moreover, reperfusion of ischemic kidneys with neutrophils worsens ischemic injury and causes kidney neutrophil retention. Neutrophil retention is dependent on the state of neutrophil activation and the duration of renal ischemia. This interaction could account for the high frequency of acute renal failure in conditions associated with prolonged prerenal asotemia and neutrophil priming such as the adult respiratory distress syndrome, or sepsis. Neutrophil retention is mediated by interaction of neutrophil integrins and endothelial cell ICAM-1 because maneuvers reducing the expression and/or function of these adhesion molecules is protective in experimental models of ischemia. Nitric oxide is a key modulator of neutrophil worsening of ischemic injury because maneuvers that decrease nitric oxide production worsen and those which increase nitric oxide protect ischemic kidneys from neutrophil effects. The clinical significance of neutrophils may relate to the observation that bioincompatible membranes activate complement, and retard recovery from acute renal failure. In conclusion, neutrophils are an important contributor to ischemic acute renal failure. It remains to be determined whether decreasing neutrophil function accelerates recovery in acute renal failure.

Acute Kidney Injury↗
PubMed2002

Increased neutrophil migratory activity after major trauma: a factor in the etiology of acute respiratory distress syndrome?

OBJECTIVE: Neutrophil infiltration of the lung is characteristic of early posttraumatic acute respiratory distress syndrome (ARDS). This study examines the ability of neutrophils isolated (over the first 24 hrs) from the peripheral blood of patients admitted after major trauma to migrate in response to interleukin-8. Interleukin-8 is elevated in the lung within 2 hrs of major trauma in patients who later develop ARDS, and thus it plays a central role in the recruitment of neutrophils to the lung and their subsequent activation. We hypothesized that enhanced interleukin-8-mediated neutrophil migratory activity in the early postinjury phase, before the development of ARDS, may be a crucial factor in the etiology of ARDS. DESIGN: Prospective observational study. SETTING: University Hospital Wales, the Royal Gwent Hospital, and East Glamorgan General Hospital. Laboratory work was conducted at the Institute of Nephrology. PATIENTS: Adult blunt trauma victims with Injury Severity Score > or = 18. MEASUREMENTS AND MAIN RESULTS: Neutrophils were isolated from citrated blood from 17 adult blunt major trauma patients at admission (0 hrs) and 8 and 24 hrs later. Identical samples were obtained from normal laboratory volunteers (n = 9). The neutrophil count in each specimen was measured, and the number of neutrophils migrating across porous tissue culture inserts in response to defined concentrations of interleukin-8 (0, 10, 30, and 100 ng/mL) was quantitated by peroxidase assay. Neutrophil counts in the whole blood specimens obtained from those later developing ARDS were elevated significantly at admission and declined rapidly throughout the next 24 hrs. Significantly greater numbers of trauma patients' neutrophils migrated to concentrations of interleukin-8 (30 and 100 ng/mL) at each time point when compared with normal volunteers (Mann-Whitney U test, p <.05). Neutrophils isolated from major trauma patients exhibited an enhanced migratory response to high concentrations of interleukin-8 throughout the first 24 hrs of admission, in contrast to the normal physiologic attenuation of migration seen in neutrophils isolated from normal laboratory volunteers. CONCLUSIONS: These data indicate that major blunt trauma enhances the migratory capacity of circulating neutrophils. This is manifest within 2 hrs of admission and may be attributable to alteration in interleukin-8 receptor expression, affinity, or downstream signaling. In patients who later develop ARDS, initially elevated circulating neutrophil counts decrease rapidly, over the same time course. Early enhanced neutrophil migratory activity coupled with elevated pulmonary concentrations of interleukin-8 may be central to the establishment of the neutrophil infiltration that is characteristic of ARDS.

Acute Disease↗
PubMed1995

Differences in oxidative response of subpopulations of neutrophils from healthy subjects and patients with rheumatoid arthritis.

OBJECTIVES: To determine whether blood neutrophils from healthy individuals and blood and synovial fluid neutrophils from patients with rheumatoid arthritis (RA) responded differently to priming agonists and stimuli of the oxidative burst and, if so, whether this was a property of a subpopulation of neutrophils. METHODS: Continuous flow electrophoresis was used to separate neutrophils into subpopulations based upon quantitative differences in net negative surface charge. The generation of superoxide anion (O2-) was used as a measure of oxidative activity using 10(-7) mol/l N-formyl-methionylleucyl-phenylalanine (FMLP) as the stimulating agonist and 10(-8) mol/l platelet activating factor (PAF) as the priming agent. RESULTS: The production of O2- by blood and synovial fluid neutrophils from RA patients in response to FMLP was greater than that observed with control blood neutrophils (p < 0.001). Priming of normal blood neutrophils with PAF increased their FMLP induced oxidative burst (p < 0.001), but PAF treatment had no effect on rheumatoid neutrophils. Neutrophils from synovial fluid of RA patients were less electronegative than paired blood samples and exposure of blood neutrophils to FMLP but not PAF reduced their surface charge. Continuous flow electrophoresis isolated three neutrophil subpopulations: cells of least surface electronegativity were ascribed to pool P1 and cells of greatest surface electro-negativity to P3. Normal blood neutrophils from P3, but not P1, showed increased oxidative activity after PAF priming (twofold increase; p < 0.01), whereas the responsiveness of rheumatoid blood and synovial fluid neutrophils from P1 and P3 was not modified by PAF treatment under the same conditions. CONCLUSION: It is suggested that most of the circulating neutrophils in RA are already in a state of readiness to generate O2- upon activation by an inflammatory stimulus. This is in contrast to normal blood neutrophils, which have both responsive and non-responsive subpopulations with respect to priming agonists.

Arthritis, Rheumatoid↗
PubMed2003

Evaluation of activated neutrophils in the blood of horses with colic.

OBJECTIVE: To evaluate the activation status of neutrophils in blood samples obtained from horses with naturally occurring colic associated with strangulating obstruction, nonstrangulating obstruction, or inflammatory bowel disease. ANIMALS: 30 horses with naturally occurring colic and 30 healthy control horses. PROCEDURE: Activation status of neutrophils was determined by assessing the number of neutrophils that could pass through filters with 5-microm pores, cell-surface CD11-CD18 expression, and alterations in size and granularity of neutrophils. RESULTS: Horses with impaction or gas colic did not have evidence of activated neutrophils. Horses with inflammatory bowel disease consistently had evidence of activated neutrophils, including decreased leukocyte deformability, increased CD11-CD18 expression, increased neutrophil size, and decreased neutrophil granularity. Horses with strangulating colic had variable results. Of horses with strangulating colic, 7 of 14 had marked changes in filtration pressures, 5 of 14 had increased CD11-CD18 expression, 6 of 14 had changes in neutrophil size, and 5 of 14 had changes in neutrophil granularity. Among horses with strangulating colic, changes in deformability, size, and granularity of neutrophils correlated with an adverse outcome. CONCLUSIONS AND CLINICAL RELEVANCE: Activated neutrophils were detected in all horses with inflammatory bowel disease and a few horses with strangulating colic. Correlation of activated neutrophils with horses that had strangulating colic that died or were euthanatized indicates that activated neutrophils are a negative prognostic indicator. Additional studies are needed to determine whether activated neutrophils contribute directly to the adverse outcome in horses with strangulating colic.

Animals↗
PubMed2005

Neutrophils promote experimental choroidal neovascularization.

PURPOSE: To investigate the role of neutrophils in the development of laser induced experimental choroidal neovascularization (CNV). METHODS: CNV was induced by laser photocoagulation in adult male C57BL/6J mice. Neutrophil infiltration was evaluated by histology and confocal immunohistology. The expression of neutrophil chemotactic chemokines in the regions of laser injury was determined by quantitative real-time PCR. Animals were treated with NIMP-R14, an anti-murine neutrophil monoclonal antibody (mAb), intraperitoneally to deplete neutrophils. The specific neutrophil depletion was confirmed by flow cytometry. The CNV responses were compared between neutropenic and untreated control mice on the basis of fluorescein angiography (FA), CNV lesion volume and lesion histology, and vascular endothelial growth factor (VEGF) expression by ELISA. Expression of VEGF and Angiopoietin-1 and Angiopoietin-2 protein by murine neutrophils was evaluated by confocal immunohistochemistry. RESULTS: Neutrophils infiltrated the sites of laser injury as early as day 1 after laser treatment and peaked at day 3. The neutrophil infiltration correlated with enhanced mRNA expression of neutrophil chemotactic chemokines MIP-2 and KC in the lesions. Administration of NIMP-R14 mAb specifically depleted neutrophils. Analysis of FA, CNV volume, and lesion histology, all demonstrated a moderate decrease in the CNV response in neutropenic mice compared to control mice (p<0.01). The reduction in the CNV response in neutropenic mice was associated with decreased VEGF protein levels in the ocular posterior segment. Murine neutrophils contained VEGF and Angiopoietin-1 and Angiopoietin-2 proteins. CONCLUSIONS: Neutrophil invasion was part of early inflammatory responses during laser induced CNV. Neutrophil depletion correlated with reduced CNV responses and decreased VEGF protein expression. These data suggest that neutrophils promoted the early development of CNV possibly via secretion of angiogenic growth factors.

Angiopoietin-1↗
PubMed1996

Neutrophil trafficking into inflamed joints in patients with rheumatoid arthritis, and the effects of methylprednisolone.

OBJECTIVE: To investigate the trafficking of circulating blood neutrophils and synovial fluid neutrophils in rheumatoid arthritis (RA) patients and the influence of a 1,000-mg intravenous pulse of methylprednisolone succinate (MP). METHODS: Neutrophils were isolated from the circulation and from the knee synovial compartments of subjects with RA. Circulating neutrophils were labeled with technetium-99 hexametazime (99mTc-HMPAO) and reinjected intravenously. Synovial fluid neutrophils were labeled from indium-111 oxine and reinjected into the knee from which they were isolated. Gamma camera images were obtained at intervals up to 24 hours post MP. Each patient had a baseline study (no MP) and a study in which MP was administered either 4 hours before (2 patients), 10 minutes before (1 patient), or 30 minutes to 1.5 hours after (6 patients) injection of the radiolabeled neutrophils. Subsequent analysis allowed quantitation of the neutrophil uptake into and clearance from the knee as a function of time. RESULTS: Nine patients who had not received glucocorticoids in the previous 3 months were studied. MP significantly decreased neutrophil ingress in 13 of the 16 knees studied (almost total inhibition in 5 knees), and this occurred within 1.5 hours of MP administration in all except 1 knee. At 24 hours after MP administration, there was a significant increase in visual analog scale (VAS) scores for well-being and significant decreases in scores on the modified Health Assessment Questionnaire (P<0.05), tender joints (P<0.005), VAS for pain (p<0.005), and generalized stiffness (P<0.005), as well as a decrease in the C-reactive protein level (P<0.05). MP had no effect on neutrophil egress (2 patients). Two additional patients who were receiving oral glucocorticoids were studied. One of them was clinically unresponsive to oral prednisolone, and MP had no effect on neutrophil ingress. The other patient showed no neutrophil ingress during the baseline study. This was confirmed by the presence of a noninflammatory synovial fluid at arthrocentesis. CONCLUSION: Neutrophil ingress into and egress from inflamed joints can be accurately monitored using radiolabeled neutrophils and quantitative gamma camera imaging. MP rapidly and substantially decreases neutrophil ingress into inflamed joints. In contrast, MP has no effect on neutrophil egress from the joint.

Aged↗
PubMed1999

Impaired neutrophil exocytosis in patients with severe pneumonia.

OBJECTIVE: Polymorphonuclear neutrophils (PMN) are one of the major effector cells of pulmonary defence against bacterial infection. To determine whether neutrophil function is impaired in patients with severe pneumonia, we assessed the two main partial functions exocytosis and oxidative response (ROS production) in isolated neutrophils from the peripheral venous blood of pneumonia patients and healthy volunteers. In addition, pulmonary neutrophils and peripheral neutrophils were compared in pneumonia patients. PATIENTS AND METHODS: Twenty-one patients with severe pneumonia were enrolled in the study. Eleven patients were mechanically ventilated, ten patients breathed spontaneously. For comparison, ten healthy adults were studied. The release of two markers of neutrophil exocytosis, lactoferrin and myeloperoxidase (MPO), with and without stimulation by phorbol-myristate-acetate (PMA), was determined using immunoluminometric assays. ROS production was quantified using luminol-enhanced chemiluminescence. In addition, the clinical severity of pneumonia was correlated to neutrophil exocytosis. RESULTS: With regard to blood neutrophils, both basal and PMA-stimulated exocytosis were significantly impaired in pneumonia patients compared to healthy volunteers (basal lactoferrin secretion in pneumonia patients: 0.25+/-0.36 pg/PMN versus controls: 1.17+/-0.78 pg/PMN, p<0.01). In contrast, both basal and PMA-stimulated ROS production were increased in patients compared to controls (spontaneous chemiluminescence in pneumonia patients: 13.6x10(5) cpm versus controls: 5.5x10(5) cpm). In pneumonia patients, the pulmonary neutrophils released significantly more lactoferrin, MPO and ROS compared to blood neutrophils (basal lactoferrin secretion of pulmonary neutrophils: 1.19+/-1.55 pg/PMN; p<0,01). However, after stimulation with PMA the exocytosis of pulmonary and blood neutrophils was similar. The severity of pneumonia and prognostic indices like albumin were inversely correlated to the release of lactoferrin in blood neutrophils (p<0,05). CONCLUSION: In patients with severe pneumonia, the exocytosis of blood neutrophils was significantly impaired. In contrast to this, the oxidative response was increased. Impaired bone marrow maturation of neutrophils during severe infection, perhaps due to shortened maturation time, could explain these findings.

Adult↗