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PubMed2002

A role for apoptosis in the control of neutrophil homeostasis in the circulation: insights from CD18-deficient mice.

The control of neutrophil turnover in the circulation is a key event in homeostasis and inflammation. Using CD18- deficient (CD18(-/-)) mice that show a 19-fold increase of blood neutrophil counts when compared with wild-type animals (CD18(+/+)), we found that apoptosis of peripheral neutrophils was significantly reduced from 27.4% in the wild-type to 4.8% in CD18(-/-) mice within 4 hours after isolation as measured by analysis of DNA content. This was confirmed by detecting CD16 expression, nuclear morphology, and internucleosomal DNA degradation. In contrast, no difference in apoptosis was observed in neutrophils derived from the bone marrow. Neutrophilia and delayed neutrophil apoptosis were also present in CD18(-/-)/interleukin 6 (IL-6(-/-)) double knockout mice. Moreover, plasma of CD18(-/-) mice was not able to delay apoptosis of CD18(+/+) neutrophils and plasma of CD18(+/+) mice did not augment apoptosis of CD18(-/-) neutrophils. However, CD18(-/-) neutrophils revealed an up-regulation of the antiapoptotic gene bcl-X(l) and a down-regulation of the proapoptotic gene bax-alpha compared with CD18(+/+) neutrophils suggesting that this delayed apoptosis. Accordingly, down-regulation of Bax-alpha using antisense technique delayed apoptosis and prolonged neutrophil survival. The replacement of the hematopoietic system of CD18(+/+) mice by a 1:1 mixture of CD18(+/+) and CD18(-/-) hematopoietic cells abolished the delay of apoptosis in peripheral CD18(-/-) neutrophils and prevented neutrophilia. Altogether, this suggests that a delay of neutrophil apoptosis in CD18(-/-) mice causes an alteration of neutrophil homeostasis, which may induce the massive increase of peripheral neutrophil counts. Thus, apoptosis seems to be critically involved in the control of neutrophil turnover in the circulation.

Animals↗
PubMed2002

Mechanical properties of rat bone marrow and circulating neutrophils and their responses to inflammatory mediators.

Neutrophils are continuously released from the bone marrow (BM), and this release is accelerated during inflammation. This study compared the mechanical properties of mature neutrophils within the BM and the circulating blood, as well as the role of microtubule rearrangement in the release of neutrophils from the BM in rats. Neutrophils isolated from the BM were stiffer than neutrophils in the circulating blood, using magnetic twisting cytometry. BM neutrophils also contained more F-actin within the submembrane region than circulating neutrophils when examined using confocal microscopy, suggesting that mature quiescent neutrophils within the BM are stiffer than circulating neutrophils because of increased formation of F-actin beneath the plasma membrane. Complement protein 5 fragments or formylmethionyl-leucylphenylalanine (fMLP) induced a stiffening response within 2 minutes that was greater in circulating than in BM neutrophils. This stiffening required F-actin formation within the submembrane region but not microtubule rearrangement in both circulating and BM neutrophils. fMLP-induced shape changes were more pronounced in circulating than in BM neutrophils, which showed fewer and smaller pseudopods and fewer membrane irregularities. In vivo, fMLP induced neutropenia, sequestration of neutrophils within the pulmonary capillaries, and release of neutrophils from the BM. Studies using colchicine demonstrated that rearrangement of microtubules was not required for any of these processes but was required for normal trafficking of neutrophils through the pulmonary capillaries.

Actins↗
PubMed1998

Neutrophil serine proteinases and defensins in chronic obstructive pulmonary disease: effects on pulmonary epithelium.

Neutrophils have the capacity to accumulate in high numbers in the lung during infection and inflammation. Because they play an important role in host defence against infection, but may also cause tissue injury, these cells are thought to be involved in the pathogenesis of various inflammatory lung disorders, including chronic bronchitis and chronic obstructive pulmonary disease. Neutrophil products that may mediate tissue injury at sites of neutrophil-dominated inflammation include the neutrophil serine proteinases elastase, cathepsin G and proteinase 3, and the nonenzymatic defensins. One of the targets of the neutrophil is the lung epithelium, and in vitro studies have revealed that both the serine proteinases and neutrophil defensins markedly affect the integrity of the epithelial layer, decrease the frequency of ciliary beat, increase the secretion of mucus, and induce the synthesis of epithelium-derived mediators that may influence the amplification and resolution of neutrophil-dominated inflammation. Both neutrophil elastase and defensins induce the release of the neutrophil chemoattractant chemokine interleukin-8 from respiratory epithelial cells. The alpha1-proteinase inhibitor (alpha1-PI) is a well-characterized inhibitor of neutrophil elastase, that also blocks the cytotoxic and stimulatory activity of defensins towards epithelial cells. The elastase inhibitory activity of alpha1-PI is also abrogated by the binding of defensins to this inhibitor. Incubation of epithelial cells with neutrophil defensins in combination with either elastase or cathepsin G resulted in decreased effects on the epithelial cells compared with those observed when the cells were incubated with defensins, elastase or cathepsin G separately. These results suggest that neutrophil defensins and serine proteinases cause injury and stimulate epithelial cells to produce chemokines that attract more neutrophils to the site of inflammation. The effects of neutrophil defensins and serine proteinases on epithelial cells appear to be restricted by proteinase inhibitors and by inhibitory interactions between these sets of neutrophil granule proteins.

Cathepsin G↗
PubMed2004

Effects of glucocorticoids on Fas gene expression in bovine blood neutrophils.

Blood neutrophils are extremely short-lived cells that are programmed for rapid apoptosis after differentiation in bone marrow. Recently, glucocorticoids have been shown to prolong survival of human and rodent neutrophils, but the mechanisms and implications for leukocyte homeostasis and health are unclear. In this study, we investigated the effects of endogenous and exogenous glucocorticoids on Fas expression in bovine neutrophils because Fas is a major death receptor that stimulates apoptosis in circulating cells. Our study subjects were four periparturient dairy cows whose blood concentrations of cortisol peaked at calving, 15 dexamethasone-treated steers and three untreated steers whose neutrophils were exposed to dexamethasone in vitro. Fas mRNA abundance changes in collected neutrophils were monitored numerous times relative to the in vivo glucocorticoid challenges, and the relationships between these data and circulating neutrophil counts were estimated by correlation analyses. Fas mRNA and protein abundance, caspase 8 activity, and survival of neutrophils in vitro were also monitored in the presence and absence of dexamethasone. In the periparturient cows, Fas mRNA abundance in circulating neutrophils showed a sharp decrease between calving and 12 h postpartum. Based on PROC CORR analysis (SAS), this correlated negatively with blood neutrophil count (r=-0.634; P=0.0009) and serum cortisol concentration (r=-0.659; P<0.0001), but showed no relationship with serum progesterone or estradiol concentrations (P > or =0.09). Administration of dexamethasone to steers also caused a pronounced reduction in neutrophil Fas mRNA abundance that persisted for 12 h and correlated negatively with blood neutrophil count (r=-0.748; P=0.0021). In vitro, dexamethasone caused dose-dependent loss of GR proteins from the cytosol of neutrophils concurrently with Fas mRNA downregulation, which was inhibited by the glucocorticoid receptor (GR) antagonist, RU486. Dexamethasone treatment of cultured neutrophils also reduced surface Fas expression, spontaneous and sFasL-induced caspase 8 activity, and rate of apoptosis in the cells. Taken together, these in vivo and in vitro results suggest that glucocorticoids inhibit Fas expression in bovine blood neutrophils via GR activation, possibly contributing to the cells' increased longevity in culture and the pronounced neutrophilia observed in parturient cows and hormone-treated steers. We thus conclude that glucocorticoid-activated GR may change the homeostasis of circulating neutrophils, in part through its negative effects on Fas gene expression and downstream apoptosis signaling pathways.

Animals↗
PubMed2000

Influence of bispecific antibodies on the in vitro bactericidal activity of bovine neutrophils against Staphylococcus aureus.

We conducted the following study to determine if bispecific antibodies enhance the bactericidal activity of bovine neutrophils. Bispecific antibodies were synthesized by chemically crosslinking bovine neutrophil monoclonal antibodies to Staphylococcus aureus 305 capsule polysaccharide monoclonal antibodies. The efficiency of chemically coupling monoclonal antibody monomers was approximately 50% for each bispecific antibody produced. Monoclonal antibodies against neutrophils enhanced the respiratory burst activity of neutrophils by 2.3- to 2.5-fold. To determine the influence of bispecific antibodies on neutrophil function, S. aureus 305 was preincubated with various concentrations of bispecific antibodies and neutrophils were then added to the opsonized bacteria at different bacteria to neutrophil ratios. The bactericidal activity of neutrophils was expressed as a percentage reduction in colony-forming units in test cultures compared with the number of colony-forming units in control test cultures that did not contain bispecific antibodies or neutrophils. The addition of bispecific antibodies to test cultures increased the bactericidal activity of neutrophils. A reduction in colony-forming units as a function of increasing the S. aureus 305 to neutrophils ratio was observed in both the absence and presence of bispecific antibodies. However, a greater reduction was observed in the presence of bispecific antibodies. Increasing concentrations of bispecific antibodies enhanced the bactericidal activity of neutrophils at a constant S. aureus 305 to neutrophil ratio of 1:500. The results indicate that bispecific antibodies that recognize both S. aureus 305 capsular polysaccharide and neutrophil antigens potentiate the bactericidal activity of neutrophils.

Animals↗
PubMed1998

In vitro effects of GM-CSF on mature peripheral blood neutrophils.

GM-CSF can play a crucial role in regulating the neutrophil-mediated inflammatory response. This growth factor is a proliferative stimulus for bone marrow neutrophil stem cell precursors and has at least 3 important roles in regulating neutrophil-mediated immunity: a) a direct effect on the proliferation and development of neutrophil progenitors; b) synergistic activity with other haemopoietic growth factors; c) stimulation of the functional activity of mature neutrophils. The production of GM-CSF may be triggered directly by exogenous factors such as antigens and endotoxins, or indirectly through the release of cytokines by a variety of cells including lymphocytes, activated macrophages and endothelial cells exposed to products of mononuclear phagocytes. Such production of GM-CSF may serve to quickly release mature neutrophils from the bone marrow in response to infections. Moreover, enhancement of the function of mature neutrophils may also augment their ability to migrate to infective sites and then phagocytose and kill pathogens. Increased expression of CD11b/CD18 may play a fundamental part in this mechanism because this receptor is essential for the adhesion of neutrophils to the endothelium. Both phagocytosis and oxidative burst activity increase as a result of the action of GM-CSF and the increased expression of complement- and Fc-receptors can augment opsono-phagocytosis. A further level of neutrophil up-regulation occurs by increasing the functional life span of neutrophils by GM-CSF. Thus, by delaying neutrophil apoptosis, GM-CSF greatly extends the time over which neutrophils may function at inflammatory sites. GM-CSF can thus exert a variety of important regulatory controls of neutrophil function during bacterial infections. Both the number and the functional status of neutrophils is highly regulated by GM-CSF. It is also possible that GM-CSF produced within localised sites of acute inflammation or infection may attract, trap and then activate neutrophils within this site.

Annexin A5↗
PubMed1990

Reversible lung neutrophil accumulation can cause lung injury by elastase-mediated mechanisms.

Neutrophils have been implicated in multiple models of end-organ injury. The purposes of this study were to determine whether (1) a sublethal septic insult promotes lung neutrophil accumulation, (2) this pulmonary neutrophil accumulation is reversible, (3) these accumulated neutrophils can be activated to injure lung, and (4) this pulmonary neutrophil accumulation obligates lung injury. Rats were administered low-dose endotoxin, 500 micrograms/kg, intraperitoneally, and at 6 or 12 hours, lungs were harvested and assayed for myeloperoxidase, a marker of neutrophil accumulation, and iodine 125-labeled albumin uptake, a marker of lung injury. A second set of rats were administered low-dose endotoxin and at 6 or 12 hours were given a neutrophil activator formyl-norleucyl-leucyl-phenylalanine (FNLP) 250 micrograms/kg, intravenously. At 8 or 14 hours, lungs were harvested and assayed for 125I-labeled albumin uptake. A third set of rats were administered low-dose endotoxin, and at 5 1/2 hours 30 minutes before FNLP administration, they were given a neutrophil elastase inhibitor, methyoxysuccinyl-L-alanine-L-alanine-L-proline-L-valine-chlorometh yl ketone, 2.5 mg/kg, intraperitoneally. At 6 hours rats were given FNLP, and at 8 hours lungs were harvested and assayed for 125I-labeled albumin uptake. The following results were obtained: (1) low-dose endotoxin caused a transient increase (p less than 0.05) in lung neutrophil accumulation at 6 hours, which was resolved by 12 hours; (2) lung 125I-labeled albumin uptake was unchanged both 6 and 12 hours after isolated low-dose endotoxin administration; (3) neutrophil activation increased (p less than 0.05) lung 125I-labeled albumin uptake when imposed 6 but not 12 hours after low-dose endotoxin administration; and (4) elastase inhibition decreased (p less than 0.05) the lung 125I-labeled albumin uptake promoted by endotoxin and FNLP. We conclude that sublethal endotoxemia causes a reversible lung neutrophil accumulation and that this lung neutrophil accumulation does not obligate lung injury; but activation of these accumulated neutrophils can promote lung injury, and this neutrophil-associated lung injury is mediated in part by neutrophil elastase.

Amino Acid Chloromethyl Ketones↗
PubMed1988

Perturbation of beta-glucan receptors on human neutrophils initiates phagocytosis and leukotriene B4 production.

Normal human neutrophils were stimulated with the yeast cell wall product, zymosan, and examined for two biologic responses, ingestion of particles and production of leukotriene B4 (LTB4), under conditions that were comparable and optimal for the quantitation of each response. Monolayers of adherent neutrophils ingested unopsonized zymosan particles, at particle-to-cell ratios of 12.5:1 to 125:1, in a dose- and time-related manner. At a ratio of 125:1, the percentages of neutrophils ingesting greater than or equal to 1 and greater than or equal to 3 zymosan particles reached plateau levels of 55 +/- 6 and 32 +/- 9% (mean +/- SD, n = 8), respectively, within 30 min. At this same ratio, neutrophils during gravity sedimentation with zymosan particles synthesized LTB4 in a time-dependent manner for at least 45 min. The maximum amount of immunoreactive LTB4 released into supernatants was 3.8 +/- 1.2 ng per 10(6) neutrophils (mean +/- SD, n = 5) and the corresponding total immunoreactive LTB4 was 6.2 +/- 1.9 ng per 10(6) neutrophils. Treatment of 2 x 10(7) suspended neutrophils with 250 micrograms of trypsin for 20 min before concurrent assessment of neutrophil phagocytosis and LTB4 production reduced both of these responses by about 50%. Pretreatment of neutrophils with 800 micrograms/ml of soluble yeast beta-glucan inhibited their ingestion of zymosan by 84% (mean +/- SD, n = 3), with 50% inhibition occurring with 100 micrograms/ml of soluble beta-glucan; 800 micrograms/ml of soluble yeast alpha-mannan had no inhibitory effect. Pretreatment of neutrophils with 400 micrograms/ml of soluble yeast beta-glucan inhibited neutrophil synthesis of LTB4 by 90%, with 50% occurring with 200 micrograms/ml; 400 micrograms/ml of soluble yeast alpha-mannan had no inhibitory effect. The presence of 1.25 micrograms/ml of cytochalasin B during incubation with zymosan particles reduced neutrophil phagocytosis from 65 to 6%, and neutrophil synthesis of LTB4 from total levels of 6.0 +/- 0.3 ng/10(6) cells to zero (mean +/- SD, n = 3). Pretreatment with either cytochalasin B or vinblastine did not alter neutrophil generation of LTB4 induced by calcium ionophore. Neutrophils pretreated with vinblastine, at 4 x 10(-6) to 4 x 10(-4) M, and then maintained at one-half these concentrations during incubation with unopsonized zymosan particles exhibited no diminution in particle ingestion, but were markedly reduced in zymosan-induced synthesis of LTB4.(ABSTRACT TRUNCATED AT 400 WORDS)

Cytochalasin B↗
PubMed1994

Association between neutrophil functions and periparturient disorders in cows.

Neutrophil functions were examined in healthy periparturient dairy cows (n = 46) and in cows with retained placenta and metritis complex (n = 20); metritis (n = 18); or mastitis (n = 13). Blood samples (50 ml) were collected from each cow via jugular vein twice weekly from 1.5 weeks before to 4 weeks after parturition. Neutrophil function was evaluated, using 6 tests: random migration, chemotaxis, ingestion, myeloperoxidase activity (iodination), superoxide production (cytochrome C reduction), and antibody-dependent cell-mediated cytotoxicity. Ability to ingest bacteria and random migration activity of neutrophils from clinically normal cows were high around parturition and increased immediately after parturition, whereas myeloperoxidase activity and antibody-dependent cell-mediated cytotoxicity ability of neutrophils from these cows decreased after parturition. Measurement of neutrophil function in 4 ovariectomized cows revealed significant (P < 0.0005) seasonal changes in results of all 6 functional assays. We observed various defects of neutrophil function in all cows with abnormal conditions after parturition. Before parturition, superoxide production activity by neutrophils from cows with metritis and chemotaxis by neutrophils from cows with mastitis were significantly (P < 0.001 and P < 0.05, respectively) lower, indicating that a defect of neutrophil function may be a predisposing factor in the development of these disorders. In conclusion, the host defense role of neutrophils in periparturient cows was impaired, principally because of a defect in killing capacity, which may increase susceptibility to infections. We also investigated the in vitro effects of arachidonic acid metabolites and recombinant human colony-stimulating factors (rhCSF) on functions of neutrophils from clinically normal and postparturient cows with abnormalities, including retained placenta, metritis, or mastitis (n = 5/group). Each abnormal cow was matched for postpartum period with a clinically normal cow. Neutrophils from individual cows were preincubated with arachidonic acid metabolites (prostaglandin F2 alpha, 10(-7) M; prostaglandin E2, 10(-6) M; leukotriene B4, 10(-8) M; and lipoxin B, 10(-8) M) and rhCSF (rh-granulocyte-CSF, 1,000 or 6,000 U/ml; rh-granulocyte-macrophage-CSF, 5 or 15 ng/ml) in a 37 C water bath for 30 minutes before submitting them to function assays. There was no response by neutrophils from either clinically normal or abnormal postparturient cows to treatment with either arachidonic acid metabolites or rhCSF in any of the 6 functional assays. However, preincubation of neutrophils alone in a 37 C water bath for 30 minutes resulted in some alteration of neutrophil function.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1995

Endothelial cell associated platelet-activating factor (PAF), a costimulatory intermediate in TNF-alpha-induced H2O2 release by adherent neutrophil leukocytes.

TNF is a strong secretagogue for surface-contacting neutrophils. During inflammation, endothelium offers the first substrate for neutrophil adherence and for modulation of the toxic response of neutrophils to soluble agonists such as TNF. In this in vitro study, evidence is presented that endothelium participates actively in TNF-induced neutrophil respiratory burst activity by expressing platelet-activating factor (PAF) in response to initial neutrophil H2O2 release. Three findings are shown that favor such a mechanism. First, PAF receptor antagonists reduced H2O2 release by TNF-activated neutrophils placed on endothelium approximately by 50%, whereas H2O2 responses by neutrophils placed on serum-coated polystyrene remained intact. Second, preincubation of HUVEC with known PAF-inducing agents PMA, H2O2, and thrombin, followed by fixation, enhanced neutrophil H2O2 release in response to TNF. H2O2 release by these neutrophils was sensitive to the presence of PAF receptor antagonists, whereas H2O2-release from neutrophils placed on fixed nonactivated endothelial cells was not. Finally, replacing endothelium by monolayers of human renal cortical epithelial cells and human fibroblasts, cells that are known to produce less PAF than endothelial cells, reduced the effect of PAF receptor antagonists. P-selectin expression and IL-8 release, two other ways by which endothelial cells might influence H2O2-release by TNF preincubated neutrophils, were examined in parallel, and were found not to influence TNF-induced neutrophil H2O2-release. We conclude that during neutrophil-endothelial interaction in inflammation, endothelium modulates the toxic response of neutrophils to TNF. Endothelial cell-associated PAF, but not endothelial cell IL-8 release and P-selectin expression, is likely to participate in TNF-induced neutrophil respiratory burst activity.

Cell Adhesion↗
PubMed1994

Neutrophil accumulation in the large colon of horses during low-flow ischemia and reperfusion.

Histomorphologic/morphometric evaluation, leukocyte scintigraphy, and myeloperoxidase activity were used to determine whether neutrophils accumulate in the large colon of horses during low-flow ischemia and reperfusion. Twenty-four adult horses were assigned to 1 of 3 groups: group 1, sham-operated (n = 6); group 2, 6 hours of ischemia (n = 9); and group 3, 3 hours of ischemia and 3 hours of reperfusion (n = 9). Low-flow ischemia of the large colon was induced in horses of groups 2 and 3 by reducing colonic arterial blood flow to 20% of baseline. Radiolabeled (99mTc) autogenous neutrophils were injected at 175 minutes, which corresponded to 5 minutes prior to reperfusion in group-3 horses. Full-thickness biopsy specimens of the left ventral colon were collected at baseline and at 30-minute intervals for 6 hours; a portion of the biopsy specimen was placed in formalin for histologic examination, and the remainder was used to measure mucosal radioactivity and myeloperoxidase activity. There were no differences in baseline mucosal neutrophil index, mucosal neutrophil numbers, submucosal venular neutrophil numbers, mucosal radioactivity, or mucosal myeloperoxidase activity among groups, or over time in group-1 horses. Neutrophils accumulated in the colonic mucosa during ischemia and further increased at reperfusion, as indicated by neutrophil index (morphology) and mucosal neutrophil numbers (morphometry); mucosal neutrophil index was significantly (P < 0.05) greater in group-3 horses during reperfusion than at the corresponding periods of ischemia in group-2 horses. Neutrophil numbers were significantly (P < 0.05) increased in submucosal venules at 10 minutes of reperfusion in group-3 horses and were significantly (P < 0.05) greater in group-3 than in group-2 horses during the interval from 3 to 6 hours. Mucosal radioactivity significantly (P < 0.05) increased at reperfusion in group-3 horses; there was a trend (P = 0.076) toward greater mucosal radioactivity in group-3, compared with group-2 horses, throughout the 3- to 6-hour interval. There were no differences in mucosal myeloperoxidase activity among or within any of the 3 groups over time. Neutrophils accumulated in the large colon of horses during low-flow ischemia and reperfusion. Neutrophil infiltration was detected by histologic examination and leukocyte scintigraphy, but not by measurement of myeloperoxidase activity. The accumulation of neutrophils during ischemia and the further neutrophil infiltration during reperfusion indicate that neutrophils may contribute to reperfusion injury of the large colon.

Animals↗
PubMed1996

Role of neutrophils in the pathogenesis of experimental vasculitis.

In the Brown-Norway rat, mercuric chloride (HgCl2) induces an autoimmune syndrome characterized by high IgE levels. There is widespread necrotizing leukocytoclastic vasculitis involving lung, skin, mucous membranes, pancreas, liver, and gut, with tissue injury being most marked in the cecum. As in systemic vasculitis in man, there are neutrophils at the site of tissue injury and the animals develop anti-neutrophil cytoplasmic antibodies, which in the Brown-Norway rat are directed against myeloperoxidase. To determine whether neutrophils are involved in the pathogenesis of the vasculitis, we have used a monoclonal antibody that was reported to deplete neutrophils in other rat strains. Rats treated with HgCl2 received antibody by intravenous injection at various time points. Serial blood samples were taken for neutrophil counts and to assay for anti-myeloperoxidase and IgE antibodies. The guts of animals killed after antibody therapy were scored for vasculitic changes and neutrophils infiltrate. RP3 (but not the control antibody MAC6) was shown to bind to Brown-Norway rat neutrophils and to block glycogen-induced influx of neutrophils into the peritoneum. When given at peak disease, RP3 caused a dose-dependent reduction in tissue injury with a marked reduction in circulating blood neutrophil numbers and in tissue neutrophil infiltrate. RP3 treatment did not affect the rise in titer of IgE and anti-myeloperoxidase antibodies. The data presented demonstrate that in this model neutrophils are necessary for the induction of vasculitis and that the degree of vasculitis correlates with neutrophil number. To our knowledge, this study is the first to provide direct evidence for a role for neutrophils in vasculitis. We suggest that antibodies directed against neutrophils, especially if they deplete neutrophils, may be useful in the therapy of vasculitis in man.

Animals↗
PubMed2006

In vitro and in vivo effects of salbutamol on neutrophil function in acute lung injury.

BACKGROUND: Intravenous salbutamol (albuterol) reduces lung water in patients with the acute respiratory distress syndrome (ARDS). Experimental data show that it also reduces pulmonary neutrophil accumulation or activation and inflammation in ARDS. AIM: To investigate the effects of salbutamol on neutrophil function. METHODS: The in vitro effects of salbutamol on neutrophil function were determined. Blood and bronchoalveolar lavage (BAL) fluid were collected from 35 patients with acute lung injury (ALI)/ARDS, 14 patients at risk from ARDS and 7 ventilated controls at baseline and after 4 days' treatment with placebo or salbutamol (ALI/ARDS group). Alveolar-capillary permeability was measured in vivo by thermodilution (PiCCO). Neutrophil activation, adhesion molecule expression and inflammatory cytokines were measured. RESULTS: In vitro, physiological concentrations of salbutamol had no effect on neutrophil chemotaxis, viability or apoptosis. Patients with ALI/ARDS showed increased neutrophil activation and adhesion molecule expression compared with at risk-patients and ventilated controls. There were associations between alveolar-capillary permeability and BAL myeloperoxidase (r = 0.4, p = 0.038) and BAL interleukin 8 (r = 0.38, p = 0.033). In patients with ALI/ARDS, salbutamol increased numbers of circulating neutrophils but had no effect on alveolar neutrophils. CONCLUSION: At the onset of ALI/ARDS, there is increased neutrophil recruitment and activation. Physiological concentrations of salbutamol did not alter neutrophil chemotaxis, viability or apoptosis in vitro. In vivo, salbutamol increased circulating neutrophils, but had no effect on alveolar neutrophils or on neutrophil activation. These data suggest that the beneficial effects of salbutamol in reducing lung water are unrelated to modulation of neutrophil-dependent inflammatory pathways.

Albuterol↗
PubMed1999

Plaunotol prevents indomethacin-induced gastric mucosal injury in rats by inhibiting neutrophil activation.

BACKGROUND: Activated neutrophils play a critical role in indomethacin-induced gastric mucosal injury. AIM: To investigate the effect of plaunotol, an anti-ulcer agent, on neutrophil activation in vitro and its effect on gastric mucosal injury and gastric accumulation of neutrophils in rats given indomethacin. METHODS: Human monocytes and neutrophils were isolated from the peripheral blood of healthy volunteers. We examined the effect of plaunotol on neutrophil elastase release, production of O2-, intracellular calcium concentration and expression of adhesion molecules CD11b and CD18 in activated neutrophils in vitro. The effect of plaunotol on TNF-alpha production by monocytes stimulated with endotoxin also was investigated in vitro. The effect of plaunotol (100 mg/kg, p.o.) on gastric mucosal injury and neutrophil accumulation was investigated in male Wistar rats given indomethacin (30 mg/kg, p.o.). RESULTS: Plaunotol inhibited the fMLP-induced release of neutrophil elastase from activated neutrophils, as well as the opsonized zymosan-induced production of O2- by neutrophils. Plaunotol significantly inhibited increased levels of intracellular calcium, a second messenger of neutrophil activation, in vitro. The fMLP-induced increases in CD11b and CD18 expression were also inhibited by plaunotol in vitro. Plaunotol inhibited monocytic production of TNF-alpha, a potent activator of neutrophils. Both gastric mucosal injury and gastric neutrophil infiltration in rats given indomethacin were significantly inhibited by the oral administration of plaunotol. CONCLUSIONS: Plaunotol inhibits indomethacin-induced gastric mucosal injury, at least in part by inhibiting neutrophil activation.

Animals↗
PubMed2000

Analysis of the functional characteristics of L-selectin and its expression on normal and CD18-deficient bovine neutrophils.

In vivo responsiveness to epinephrine, expression of L-selectin on neutrophils, changes in intracellular calcium ([Ca2+]i), sulfatide-induced superoxide production and tyrosine phosphorylation in neutrophils were evaluated to elucidate the role of L-selectin-associated functions of normal and CD18-deficient bovine neutrophils. The number of neutrophils in peripheral blood was significantly increased (P < 0.05) in four normal calves at 5-20 min after in vivo administration of epinephrine; however, no significant increase of neutrophils was found in three calves with bovine leucocyte adhesion deficiency (BLAD). Expression of L-selectin on neutrophils from three calves with BLAD was 61-77% of that of normal calves. Pretreatment of neutrophils with phorbol myristate acetate caused a marked decrease in the expression of L-selectin on neutrophils from both normal and BLAD calves. The sulfatide-induced sustained phase of [Ca2+]i concentration in neutrophils from calves with BLAD was significantly (P < 0.05) decreased. Following stimulation with aggregated IgG, the transient phase of [Ca2+]i in neutrophils from normal and BLAD calves was increased; however, the sustained phase of [Ca2+]i in BLAD neutrophils was significantly lower (P < 0.05) than that of controls. Sulfatide-induced O2- production and chemiluminescent response in neutrophils from calves with BLAD were 48-51% of those of normal calves and were inhibited by genistein and wortmannin, respectively, in a dose-dependent manner. The amount of tyrosine phosphorylated 100 kDa protein in neutrophils from BLAD calves stimulated with sulfatides was 57% of that of controls. The degree of L-selectin expression on neutrophils was correlated with the intracellular signalling events and the related superoxide production.

Animals↗
PubMed2001

Neutrophil activation induced by placental factors in normal and pre-eclamptic pregnancies in vitro.

Increased neutrophil activation has been demonstrated in women with pre-eclampsia. Activated neutrophils may play a significant role in the vascular endothelial pathophysiology in this disorder of pregnancy. How neutrophils become activated in pre-eclampsia is unknown. It has been proposed that activating factors could be produced and released by the placenta. To test if placental factors could stimulate neutrophil activation and what mechanism might be involved, neutrophils isolated from healthy female volunteers were exposed to the conditioned medium (CM) derived from either normal (Nor) or pre-eclamptic (PE) placental villous culture. Neutrophil-endothelial adhesion, neutrophil superoxide generation, elastase activity and integrin expression were measured. The data were analysed by ANOVA. A P value less than 0.05 was considered statistically significant. All values are expressed as a mean+/-s.e. We found: (1) neutrophil-endothelial adhesion was significantly increased in neutrophils exposed PE-CM than those exposed to Nor-CM and non-CM, P< 0.01; (2) both Nor-CM and PE-CM could stimulate neutrophils to generate more superoxide radicals; (3) there was no difference in elastase activity after neutrophil exposure to Nor-CM compared to PE-CM, P> 0.1; (4) significant changes in CD62L and CD11b expression were found in neutrophils exposed to PE-CM. We conclude that factors produced by the placenta can activate neutrophils by an increase in superoxide generation and modulation of adhesion molecule expression. Upregulation of surface adhesion molecule CD11 expression may be responsible for the increased neutrophil-endothelial adhesion induced by factors derived from pre-eclamptic placentae.

CD11 Antigens↗
PubMedDate not supplied

Neutrophil activation by murine retroviral infection during chronic ethanol consumption.

AIMS: Neutrophil adhesion molecule CD11b and reactive oxygen species (ROS) are neutrophil activation markers for evaluating the functional activity of neutrophils. The aim of this study was to determine if neutrophils are activated in murine AIDS and/or chronic ethanol consumption and if neutrophil CD11b expression and ROS production vary when progressive retrovirus infection occurs. METHODS: Four groups were studied: control, murine AIDS, ethanol and ethanol plus murine AIDS. Neutrophil activation was assessed by CD11b expression and ROS production using flow cytometry. RESULTS: We found that neutrophils lost their responsiveness to fMLP due to retrovirus or ethanol exposure. In the murine AIDS group, neutrophil CD11b expression was up-regulated along with a significant increase in ROS after 1 month of retroviral infection. After 2 months, neutrophil CD11b and ROS decreased. However, neutrophil CD11b expression further increased after 3 months. In the ethanol consumption group, neutrophil CD11b expression was down-regulated after 2 months, whereas ROS production increased in the first and third months. In the murine AIDS plus ethanol group, there were significant increases in both ROS and CD11b expression during the 3-month observation period. CONCLUSIONS: These findings suggest that neutrophil function is impaired by LP-BM5 retrovirus infection and/or chronic ethanol consumption. The pattern of neutrophil CD11b expression and ROS production might help to predict the stage of murine AIDS. Ethanol may further compromise neutrophil function in AIDS.

Administration, Oral↗
PubMed2005

Effect of cytokines and chemokines on sickle neutrophil adhesion to fibronectin.

A role for leukocytes in sickle cell vaso-occlusive crisis is becoming increasingly recognized. Neutrophil counts are higher in sickle cell patients and neutrophils from these patients demonstrate increased adhesion to endothelial monolayers under certain circumstances. The effects of selected cytokines on the adhesion mechanisms of normal neutrophils and neutrophils from sickle cell anaemia patients (SCA neutrophils) were investigated. Neutrophils were separated from the blood of homozygous (HbSS) SCA patients and healthy controls. Following pre-incubation (25 min, 37 degrees C) of the cells with cytokines, the adhesion of the cells to fibronectin (FN)-coated plates (20 micro) was determined (60 min, 37 degrees C, 5% CO2). Basal adhesion of normal and SCA neutrophils to FN was not statistically different. Pretreatment of normal neutrophils with either IL-6 (10-100 pg/ml), GCSF (1- 10 ng/ml) or IL-8 (1-100 ng/ml) had no significant effect upon their adhesion to FN. In contrast, SCA neutrophil adhesion to FN was increased significantly following pre-incubation with IL-6, G-CSF and IL-8 (p < 0.01). RANTES (1-100 ng/ml) had no significant effect on either normal or SCA neutrophil adhesion to FN. Flow-cytometric analyses demonstrated that IL-8 (10 ng/ml) significantly augments CD11b (Mac-1 integrin subunit) expression on SCA neutrophils, but not normal neutrophils. IL-6 and G-CSF (10 pg/ml and 10 ng/ml, respectively), however, had no effect on SCA neutrophil adhesion molecule expression. In conclusion, SCA neutrophil adhesion mechanisms may increase in the presence of certain cytokines, in vivo, and this activation may contribute to the physiopathology of sickle cell disease.

Adolescent↗