[Corpuscular antigens in the blood neutrophil reaction (Neutrophil Injury Index test) in children with dysentery].
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Adolescent↗
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Neutrophilic dermatosis versus neutrophilic eccrine hidradenitis.
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Dermis↗
Studies on the centrifugal fractionation of leukocyte granules, especially on the isolation of a pure fraction of neutrophilic granules and on the fine structure of neutrophilic granules as observed under the electron microscope.
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Cytoplasmic Granules↗
Neutrophil sequestration in rat lungs.
BACKGROUND: The transit of neutrophils through the pulmonary microvasculature is prolonged compared with red blood cells and is increased further during cigarette smoking and in exacerbations of chronic obstructive pulmonary disease. The increased residence time (sequestration) of neutrophils in the pulmonary capillaries in these conditions may be the first step leading to the accumulation of cells within the lung interstitium and in the bronchoalveolar space, so potentiating lung damage. A rat model has been developed to investigate the factors which may influence neutrophil transit through the lung microvasculature. METHODS: Intratracheal instillation of the heat killed organism Corynebacterium parvum was used to induce an acute neutrophil alveolitis. Neutrophils and red blood cells were isolated from donor rats, labelled with two distinct radioisotopes, and then reinjected into recipient rats to assess their transit through the pulmonary circulation. To ascertain whether peripheral blood neutrophils were minimally altered by the isolation procedure their functional status in vitro was compared with that of inflammatory neutrophils in a number of assays commonly used as descriptors of neutrophil activation. The influence of neutrophil activation on the accumulation of cells in the lungs was assessed by comparing the lung sequestration of control neutrophils, isolated from peripheral blood, with that of inflammatory neutrophils obtained from bronchoalveolar lavage of inflamed rat lungs. Lung sequestration of neutrophils was defined as the fold increase in the ratio of neutrophils labelled with chromium-51 to red blood cells labelled with technetium-99m in lung tissue compared with the same ratio in peripheral blood. RESULTS: Sequestration of peripheral blood neutrophils occurred in control rat lungs as shown by a 17.5 (2.1) fold increase in the ratio of neutrophils to red blood cells in the pulmonary circulation compared with the ratio of these cells in the peripheral circulation. When inflammatory neutrophils, obtained by bronchoalveolar lavage from C parvum-treated animals, were injected into control rats, the increase was 90.6 (11.0) fold. Induction of an inflammatory response in the lung tissue of the recipient rat also caused an increase in the sequestration of control neutrophils compared with the same cells in control rat lungs which was, however, less marked than when inflammatory neutrophils were used (34.7 (4.7) fold). The mean (SE) pressure developed on filtration of inflammatory neutrophils in vitro through a millipore filter (7.53 (0.2) cm H2O) was greater than that of peripheral blood neutrophils (1.18 (0.2) cm H2O). Increased filtration pressure indicates a decrease in cell deformability and suggests that this may be a contributory factor to the increased sequestration of inflammatory neutrophils in the pulmonary vasculature. CONCLUSIONS: This study shows that there is sequestration of neutrophils in the pulmonary vasculature in normal rat lungs which increases in acute lung inflammation and when inflammatory neutrophils are injected into control animals. In this model changes in the neutrophil, such as cell deformability, may have a more important role in inducing increased neutrophil sequestration than the inflammatory response in the lungs.
Animals↗
Interactions of neutrophils with epithelial cells: lessons from the intestine.
Active inflammatory processes in organs lined by columnar epithelial cells are often characterized by abundant neutrophil migration. To reach an epithelial surface, neutrophils must leave the vasculature and cross the interstitium where signals may be generated that influence migration and/or epithelial function. Subsequently neutrophils migrate across epithelial linings by impaling intercellular junctions between epithelial cells and, in doing so, diminish the ability of epithelial linings to serve as a barrier. Decreased barrier function brought about by neutrophil migration across epithelia appears to be a reversible process after the removal of the chemotactic stimulus. The molecular events underlying neutrophil-epithelial interactions are at present poorly understood. With recent information regarding neutrophil-endothelial interactions, it has been possible to better understand issues of neutrophil interactions with epithelial cells. In general, there appear to be different rules governing the interaction of neutrophils with endothelial cells as compared with epithelial cells. Neutrophil migration across endothelial monolayers involves a series of at least three steps, each regulated by glycoproteins expressed on the cellular surface. In considering neutrophil-epithelial interactions, the single common denominator is the requirement for surface expression of neutrophil beta 2 integrins, and specifically CD11b/18, as shown by functional inhibition through the use of antibodies to neutrophil beta 2 integrins and the use of neutrophils from patients lacking the beta-chain of this integrin. An understanding of the regulation of neutrophil-epithelial interactions, although in its infancy, appears to involve inflammatory cytokines. Recent in vivo evidence suggests that interferon-gamma production at the level of the intestine may correlate with the regulation of neutrophil-epithelial interactions in vitro by interferon-gamma. Finally, considerable evidence exists that neutrophils may contribute significantly to fluid transport during inflammatory diseases such as secretory diarrhea. When placed in contact with epithelial monolayers, activated neutrophils bring about the transport of ions, with concomitant water loss across epithelial surfaces. This transport process brought about by the presence of neutrophils is generated by a metabolite produced and secreted from neutrophils. This metabolite was originally termed neutrophil-derived secretagogue and has recently been identified as 5'-AMP. The mechanism of 5'-AMP action on epithelial cell ion transport is discussed.
Animals↗
Abnormal regulation in the signal transduction in neutrophils from patients with severe congenital neutropenia: relation of impaired mobilization of cytosolic free calcium to altered chemotaxis, superoxide anion generation and F-actin content.
Severe congenital neutropenia (SCN) can be corrected in vivo by treatment with pharmacological dosages of recombinant human granulocyte colony-stimulating factor (rhG-CSF). In order to analyze the decreased chemotaxis of neutrophils from SCN patients receiving rhG-CSF, neutrophil functions essential for chemotaxis were investigated. The mobilization of cytosolic calcium ([Ca2+]i) and the functional state of cytoskeletal proteins in neutrophils from SCN patients were compared with either neutrophils from healthy donors (or, in selected experiments, from patients with cyclic neutropenia) and neutrophils from patients with chemotherapy-induced neutropenia also receiving rhG-CSF. Using flow cytometric analysis, two neutrophil subpopulations were detected in SCN patients in response to N-formylmethionine leucyl-phenylalanine (FMLP) (10(-9) M to 10(-7) M), one of which was unable to respond to this stimulus with an increase in [Ca2+]i. Whereas a homogeneous increase in [Ca2+]i in normal neutrophils occurred at 10(-9) M FMLP, neutrophils from SCN patients required 10(-6) M FMLP to respond homogeneously with an increase in [Ca2+]i. In contrast, G-CSF induced neutrophils from patients with cyclic neutropenia and from patients with chemotherapy-induced neutropenia showed a normal increase in [Ca2+]i after stimulation. The [Ca2+]i-dependent superoxide anion (O2-) generation in response to FMLP was also significantly diminished in neutrophils from SCN patients compared to normal neutrophils. However, O2- generation elicited by phorbolester (PMA), which directly activates protein kinase C (PKC), was not affected in SCN neutrophils. The total immunoreactive actin content and basal F-actin content in neutrophils from SCN patients were elevated as compared to normal neutrophils and neutrophils from patients with chemotherapy-induced neutropenia. The increase in F-actin content following FMLP activation was much lower in neutrophils from SCN patients as compared with normal neutrophils. These data suggest a defect in the signal transduction pathway in neutrophils from SCN patients between FMLP ligand-receptor interaction and Ca2+ mobilization, whereas upstream of PKC, triggered events seem to be unaffected. Therefore, [Ca2+]i-dependent neutrophil function in response to FMLP, such as actin disassembly, chemotaxis and O2- generation are diminished in SCN neutrophils. The pathomechanism responsible for the defective [Ca2+]i increase might be an initial step in understanding the underlying pathophysiology of SCN.
Actins↗
Thermal injury-induced priming effect of neutrophil is TNF-alpha and P38 dependent.
Priming response of neutrophil in clinical-related conditions and its mechanism has not been clarified. This study is to determine if thermal injury-induced priming effect of neutrophil is TNF-alpha and p38 dependent. In Experiment 1, bone marrow neutrophil of wild-type (WT) mice and TNF receptor superfamily, member 1A (Tnfrsf1a-/-) mice were harvested and treated with TNF-alpha, platelet activating factor (PAF) first, then with or without N-formyl-Met-Leu-Phe (fMLP). Reactive oxygen species (ROS) production and p38 phosphorylation were evaluated. In Experiment 2, ROS of neutrophil from WT and Tnfrsf1a-/- mice at 3 or 15 h after thermal injury with or without fMLP treatment were assayed. In Experiment 3, p38 and p44/42 phosphorylation, CXCR2 and macrophage inflammatory protein-2 expression, apoptotic ratio, and activating protein-1 (AP-1) and nuclear factor-kappa B (NF-kappaB) activation of neutrophil from WT and Tnfrsf1a-/- mice at 3 h after thermal injury were tested. FMLP treatment after TNF-alpha or PAF incubation of neutrophil increased ROS of PAF-treated but not TNF-alpha-treated neutrophil. PAF treatment increased ROS of neutrophil in WT and Tnfrsf1a-/- mice. FMLP increased ROS of neutrophil of WT mice at 3 h after thermal but not that of Tnfrsf1a-/- mice. TNF-alpha and PAF increased p38 phosphorylation of neutrophil in WT but not that in Tnfrsf1a-/- mice. Thermal injury increased p38 phosphorylation, NF-kappaB activation, and decreased apoptosis of neutrophil at 3 h after thermal injury in WT but not in Tnfrsf1a-/- mice. Thermal injury also induced AP-1 activation and ROS production on neutrophil at 3 and 15 h after thermal injury, respectively, in WT and Tnfrsf1a-/- mice. Collectively, fMLP stimulates ROS of neutrophil through TNF-alpha signaling; PAF stimulates that of neutrophil through both TNF-alpha-dependent and TNF-alpha-independent pathway. Thermal injury induces a TNF-alpha-dependent priming effect and a TNF-alpha-independent activation effect on neutrophil at 3 and 15 h after thermal injury, respectively. NF-kappaB signaling pathway plays an important role in neutrophil activation. Thermal injury also induces TNF-alpha-dependent delay apoptosis and TNF-alpha-independent AP-1 activation of neutrophil at 3 h after thermal injury. Taken together with the TNF-alpha-dependent p38 and NF-kappaB activation in primed neutrophil, we conclude that thermal injury-induced priming effect of polymorphonuclear neutrophil is TNF-alpha and p38 dependent.
Animals↗
Ischemia increases neutrophil retention and worsens acute renal failure: role of oxygen metabolites and ICAM 1.
UNLABELLED: The role of neutrophils in acute renal failure (ARF) is controversial. Although ARF occurs in neutropenic subjects, we found that ischemic kidneys activated neutrophils to cause ARF in isolated perfused rat kidneys. To further define the interaction between neutrophils and renal ischemia, we performed quantitative assessment of neutrophil accumulation during renal ischemia. Non-ischemic and ischemic rat kidneys were perfused by the isolated kidney technique with unstimulated, primed, or fully activated, indium-labeled neutrophils. Neutrophil accumulation was quantitated by measuring indium retention after 60 minutes of perfusion. In non-ischemic kidneys, only activated neutrophils were retained while after 20 minutes of renal ischemia, unstimulated as well as primed neutrophils were retained. Following 10 minutes of ischemia, primed neutrophils (but not unstimulated neutrophils) were retained. In the presence of neutrophil retention, there were decreases in GFR and tubular sodium reabsorption. To determine the role of ICAM 1 in ischemic injury, rats were treated with anti-ICAM 1 prior to ischemia and ischemic kidneys were reperfused with unstimulated neutrophils and anti-ICAM 1. After ischemia, the neutrophil component of reperfusion injury in isolated kidneys was prevented with anti-ICAM 1. Oxygen metabolites have been shown to induce EC expression of ICAM 1. To determine the role of ICAM 1 in oxidant-mediated renal injury, ischemic isolated kidneys were reperfused with catalase (CAT) and non-ischemic kidneys were perfused with hydrogen peroxide. Following ischemia, reperfusion with CAT prevented neutrophil retention and injury. In non-ischemic kidneys, hydrogen peroxide caused primed neutrophil retention, activation and renal injury which were completely prevented with anti-ICAM 1. IN CONCLUSION: (1) Ischemic kidneys cause neutrophil retention, activation, and worsening of renal injury in isolated kidneys; and 2) neutrophil retention is dependent on the state of neutrophil activation, duration of renal ischemia and is mediated by oxygen metabolites and ICAM 1. This synergism could account for the high frequency of ARF in conditions such as sepsis where there is both renal hypoperfusion and neutrophil priming.
Acute Kidney Injury↗
Enhanced neutrophil apoptosis in neutropenic patients with hepatosplenic schistosomiasis: evidence of serum Fas ligand.
Enhanced neutrophil apoptosis has been reported in neutropenic hepatosplenic schistosomiasis. The shortening of neutrophil survival via apoptosis may explain the neutropenia that occur in these patients. However, the regulation of neutrophil apoptosis in hepatosplenic schistosomiasis has not been clearly defined. Neutrophils harvested from neutropenic patients with hepatosplenic (HS) schistosomiasis, (n=25), non-neutropenic patients with hepatointestinal (HI) schistosomiasis (n=10), and age-/gender-matched healthy control subjects (n=10) were incubated with autologous serum. Neutrophils apoptosis was quantified by flow cytometry through determination of propidium iodide nuclear staining and confirmed by DNA gel electrophoresis at 0 (i.e. fresh neutrophils), 4 and 24 h culture. Neutrophils from healthy subjects were also incubated with either 10% heterologous normal or neutropenic serum, with and without anti-Fas ligand antibody. Fas expression was assessed in fresh neutrophils using flow cytometry. Compared with normal healthy neutrophils, and HI neutrophils, neutropenic neutrophils demonstrated greater apoptosis in the presence of autologous serum (P<0.01, 0.05, respectively). Furthermore, compared with normal neutrophils exposed to heterologous normal serum, those exposed to heterologous neutropenic serum exhibited higher apoptosis rates ( P<0.01). Moreover, anti-Fas L antibody attenuated the neutropenic serum-induced neutrophil apoptosis in normal neutrophils. Fas expression was significantly higher in the neutropenic group when compared to both HI and normal healthy controls (P<0.05). In addition, Fas expression by neutrophils was paralleled by high neutrophil apoptosis. On the other hand, neutrophil apoptosis was not correlated to the size of spleen in neutropenic group. In conclusion, the rate of neutrophil apoptosis is accelerated in patients with neutropenic hepatosplenic schistosomiasisis. These findings suggest that the enhanced neutrophil apoptosis that occurs in neutropenic HS patients is triggered by a serum factor, which is mostly a Fas ligand.
Adult↗
Beta 2-integrin (CD11b/CD18) is the primary adhesive glycoprotein complex involved in neutrophil-mediated immune injury to human sperm.
The pathogenesis of antisperm antibody (ASA)-mediated infertility is postulated to be related in part to complement (C)-dependent neutrophil-mediated injury to sperm in the female genital tract. We have reported that sperm-bound IgG activated human C and deposited C3 fragments on motile sperm. We also demonstrated that IgG and C3-bound motile sperm adhered to human neutrophils in vitro, and that this adhesion potentiated the localized release of oxygen radicals at the site of neutrophil/sperm membrane contact. The goal of the present study was to identify the neutrophil surface receptor(s) involved in neutrophil/sperm adhesion and to evaluate their relevance to the pathogenesis of neutrophil-mediated immune injury to sperm. Neutrophils were coincubated with motile sperm in the presence of C-fixing ASA+ sera or control sera. After defined incubation periods, the following neutrophil variables were evaluated: 1) surface expression of Fc (Fc gamma RII and Fc gamma RIII) and C receptors (CR1[CD35], CR3 [CD11b/CD18], and CR4 [CD11c/CD18]) by flow cytometry, 2) neutrophil aggregation by flow cytometry, 3) tyrosine phosphorylation of neutrophil proteins by flow cytometry, and 4) the immune adherence and ingestion of sperm by light and scanning electron microscopy (SEM). The functionality of adhesion receptors was studied by use of a panel of anti-leukocyte monoclonal antibodies (mAbs) for their ability to block neutrophil/sperm adhesion and neutrophil aggregation. Only the incubation of neutrophils and sperm in the presence of C-fixing ASA+ sera resulted in marked (> 70%) sperm binding to neutrophils. Consistent with this pattern, a significant (p < 0.0001) increase in surface expression of neutrophil CD11b and neutrophil aggregation was evident. According to SEM, most of the sperm were linked to the neutrophil by the acrosomal region of sperm head. Maximum expression of CD11b antigen was obtained when neutrophils were coincubated with sperm in the presence of C-fixing ASA+ sera. CD11b up-regulation correlated with a significant (p < 0.05) increase in tyrosine phosphorylation of neutrophil proteins during sperm phagocytosis. Only the combination of mAbs directed to the beta 2-integrin, CD11b (D12/SHCL-3), or CD18 (MHM23) subunits maximally inhibited ASA- and C-mediated sperm adhesion to neutrophils (by 70%) or sperm phagocytosis (by 75%), as well as neutrophil aggregation (by 96%). These findings strongly implicate the CD11b/CD18 glycoprotein complex (CR3) in the adhesive events involved in ASA- and C-mediated immune destruction of motile sperm by neutrophils.
Adult↗
Superoxide anion release from blood and bone marrow neutrophils is altered by endotoxemia.
In vivo endotoxin infusion produces neutrophil-mediated acute lung injury and increases superoxide anion release from phorbol myristate acetate (PMA)-stimulated blood neutrophils collected 18-24 hours after the infusion. Because the turnover time of circulating blood neutrophils is only 6-8 hours, it was hypothesized that the prolonged increase in superoxide anion release from peripheral blood neutrophils is associated with increased superoxide anion release from bone marrow neutrophils. To test this hypothesis, two doses of Escherichia coli endotoxin (5.0 and 0.5 micrograms/kg) were infused into chronically instrumented awake sheep. Blood and bone marrow neutrophils were collected 24 hours after the infusion, and superoxide anion release from unstimulated and PMA-stimulated neutrophils was measured in vitro. Endotoxin infusion produced an increase in pulmonary microvascular permeability, in intravascular activation (degranulation) of blood neutrophils, and in circulating blood neutrophils 24 hours after the infusion. High-dose endotoxin (5.0 micrograms/kg; n = 4) increased superoxide anion release from unstimulated peripheral blood neutrophils (2.25 +/- 0.38 times baseline [p less than or equal to 0.05]) and from peripheral blood neutrophils stimulated with 10(-9) M PMA in vitro (1.46 +/- 0.55 times baseline). Low-dose endotoxin (0.5 micrograms/kg; n = 5), on the other hand, did not alter superoxide anion release from peripheral blood neutrophils. Bone marrow neutrophils could not be isolated reproducibly after high-dose endotoxin because of leukoaggregation. Bone marrow neutrophils were isolated after low-dose endotoxin infusion. Stimulation of these cells with 10(-9) M PMA in vitro resulted in a two- to fourfold increase above control release (p less than or equal to 0.05). Increased superoxide anion release from both peripheral blood and bone marrow neutrophils occurred in the absence of circulating endotoxin, as measured by a Limulus assay. These results show that the prolonged increase in superoxide anion release from peripheral blood neutrophils is associated with an increase in the superoxide anion release from bone marrow neutrophils. Furthermore, the recruitment of affected bone marrow neutrophils into peripheral blood may explain the increased superoxide anion release from blood neutrophils 24 hours after endotoxin infusion.
Alkaline Phosphatase↗
Biochemical analysis of the activation of adherent neutrophils in vitro.
The role played by neutrophil oxidative responses in host defense and injury is an area of active investigation. In order to study neutrophil responses in vitro, methods are required for cell purification, enumeration, and quantification of activation responses, which mimic the in vivo situation as closely as possible. In this communication (and its companion paper, Albertine et al., 1988) improved methods for all of these tasks are described and applied to investigate neutrophil structure-function relationships in vitro and in vivo. Human neutrophils were purified by using a series of platelet-poor plasma-Percoll gradients (51, 62, 76 and 80% in Percoll). This modification of previously published procedures results in consistently successful neutrophil purification and has allowed us to purify neutrophils from bronchoalveolar lavage fluid as well as blood. Activation of human and sheep neutrophils (superoxide anion production) was quantitated by the reduction of ferricytochrome c using a microtiter plate reader to measure the increase in absorbance at 550 nm from adherent neutrophils. Adherence of neutrophils was quantitated by measurement of LDH in cells lysed with Triton X-100 using a new method which uses readily available commercial reagents and can quantitate the LDH content of as few as 5000 neutrophils (or the LDH released from 5% of 100,000 neutrophils). Assay conditions for superoxide anion were optimized, limitations both in assay design and instruments used to measure OD were explored and enumerated, and these methods were used to quantitate sheep and human neutrophil activation responses. Using methods described in Albertine et al. (1988) for fixing neutrophils in microtiter wells after assay of their functional capacity, we have studied the same cells functionally and morphologically. We have used these techniques to study blood and alveolar neutrophils from a patient with acute respiratory failure. His alveolar neutrophils displayed 67% of the activation response as peripheral neutrophils (4.31 +/- 0.12 nmol superoxide released per 250,000 neutrophils at 60 min vs. 6.38 +/- 0.18 in blood, P less than 0.01) and structural changes which suggested previous activation in vivo. These studies demonstrate that similar morphological changes are observed in neutrophils activated with phorbol myristate acetate in vitro, as are observed in cells which have been activated by pathophysiologic processes in vivo.
Animals↗
In vivo activation of equine eosinophils and neutrophils by experimental Strongylus vulgaris infections.
Eosinophils and neutrophils from ponies with Strongylus vulgaris-induced eosinophilia (eosinophilic ponies; activated eosinophils and neutrophils) were assayed in vitro for chemotactic and chemokinetic responses to zymosan-activated serum (ZAS) using the filter system in Boyden chambers, for Fc and complement (C) receptors using the EA and EAC-rosette assays, respectively, and for phagocytic and bactericidal activities using opsonized Escherichia coli and the acridine orange method. The responses of activated eosinophils and neutrophils in the above assays were compared with those of eosinophils and neutrophils from S. vulgaris-naive ponies without eosinophilia (noneosinophilic ponies; nonactivated eosinophils and neutrophils). Differences in cell density following centrifugation in a continuous Percoll gradient were used to further characterize the heterogeneity of activated eosinophils and neutrophils. Activated and nonactivated eosinophils demonstrated similar chemotactic responses to ZAS while activated and nonactivated neutrophils demonstrated similar chemokinetic responses to ZAS. A higher percentage of activated eosinophils and neutrophils expressed Fc and C receptors compared with nonactivated cells (P less than 0.05). Generally, higher percentages of eosinophils and neutrophils expressed C than Fc receptors. However, the percentage of neutrophils with both receptors was higher than that of eosinophils. Phagocytosis and killing of E. coli by either type of eosinophil were not consistently observed. Both activated and nonactivated neutrophils phagocytized E. coli and significant differences between the two cell types were not observed. The bacterial activity, however, of activated neutrophils was significantly greater than that obtained using nonactivated neutrophils (P less than 0.05). Activated eosinophils and neutrophils were both separated into two distinct fractions based on differences in cell densities. A higher percentage of band 2 eosinophils (density of 1.106) expressed C receptors than did band 1 eosinophils (density of 1.049) (P less than 0.05). A higher percentage of band 1 neutrophils (density of 1.072) expressed both Fc and C receptors and these neutrophils were more phagocytic and bactericidal than were band 2 neutrophils (density of 1.082) (P less than 0.05). These data suggest that equine eosinophils and neutrophils are activated by chronic S. vulgaris infections.
Animals↗
Comparison of the response of bovine and human neutrophils to various stimuli.
Elastase release, oxidant production and cytoplasmic Ca2+ fluxes by bovine and human neutrophils were compared using sensitive kinetic assays on a photon-counting spectrofluorometer. The stimulants used were phorbol myristate acetate (PMA), cytochalasin B, zymosan opsonized with bovine complement (bOZ) or human complement (hOZ), calcium ionophore, formyl-methionyl-leucyl-phenylalanine (FMLP) and concanavalin A (Con A). The respiratory burst of bovine and human neutrophils was stimulated by PMA and OZ but not by cytochalasin B, or calcium ionophore. Con A weakly stimulated this response in human neutrophils but not bovine. FMLP stimulated the respiratory burst of human but not bovine neutrophils. For evaluation of elastase release, human neutrophils were pretreated with cytochalasin B for 5 min and then stimulated. Cytochalasin B alone did not stimulate elastase release from human neutrophils. Phorbol myristate acetate, calcium ionophore, hOZ, FMLP and Con A did stimulate human neutrophils pretreated with cytochalasin B to release elastase. Human serum OZ was also able to stimulate elastase release from human neutrophils not pretreated with cytochalasin B. Some bovine neutrophils released elastase in response to cytochalasin B alone. Those bovine neutrophils that did not release elastase in response to cytochalasin B alone released elastase when stimulated with Con A or calcium ionophore after cytochalasin B pretreatment. Bovine neutrophils did not release elastase in response to FMLP or PMA with or without cytochalasin B pretreatment, but did release elastase in response to bOZ alone. Total elastase activity of bovine neutrophils was determined to be about 50 times less than that of human neutrophils. Intracellular calcium fluxes were stimulated in human neutrophils by calcium ionophore, FMLP, hOZ and Con A but not by PMA or cytochalasin B. Bovine neutrophil calcium fluxes were stimulated by calcium ionophore, Con A and bOZ; cytochalasin B also stimulated bovine neutrophils to increase cytoplasmic calcium concentration. Cytoplasmic calcium fluxes were not stimulated in bovine neutrophils by PMA or FMLP. In summary, human and bovine neutrophils respond similarly to calcium ionophore and OZ, but differently to PMA, cytochalasin B, Con A and FMLP.
Animals↗
Interference of neutrophil-platelet interaction by YC-1: a cGMP-dependent manner on heterotypic cell-cell interaction.
N-Formyl-Met-Leu-Phe (fMLP) activated neutrophils and then induced neutrophil-platelet complex formation in co-incubation condition. In addition, fMLP induce intracellular calcium mobilization in platelets, only when it is incubated along with neutrophils. This data established that fMLP-stimulated neutrophils activate platelets. 9E1, a monoclonal antibody of P-selectin, significantly blocks the formation of neutrophil-platelet complex induced by fMLP, indicating the involvement of P-selectin in the neutrophil-platelet complex formation. 3-(5'-hydroxymethyl-2'-furyl-1-benzylindazole (YC-1), an unique nitric oxide-independent activator of soluble guanylate cyclase, was evaluated for its effect on neutrophil-platelet complex. YC-1 inhibits fMLP-induced neutrophil-platelet complex formation in a concentration-dependent manner with an IC50 value of 15.3+/-3.5 microM. However, this effect of YC-1 is partially reversed by pre-treatment of 1H-(1,2,4)oxadiazolo[4,3-a]quinozalin-1-one (ODQ; 10 microM), which is a soluble guanylate cyclase inhibitor. Pre-treatment of either neutrophils or platelets with YC-1 (50 microM) prevent the fMLP-induced neutrophil-platelet complex formation, indicating that YC-1 could potentially exert its effects individually on either neutrophils or platelets alone. Cathepsin G released from fMLP-stimulated neutrophil activates the nearby platelets. YC-1 was also shown to inhibit this release of cathepsin G in a concentration-dependent manner. The IC50 value was 6.2+/-0.2 microM. This inhibitory effect of YC-1 on cathepsin G release is reversed by ODQ (10 microM) and a protein kinase G inhibitor [1-oxo-9.12-epoxy-1H-diindolo[1,2,3-fg:3',2',1'-kl]pyrrolo[3,4-l][1,6]benzodiazocine-10-carbooxylic acid methyl ester (KT5835); 1 microM]. YC-1 inhibits cathepsin G-induced P-selectin expression on human platelet at the IC50 value of 32.5+/-2.6 microM. A further study showed that YC-1 inhibits fMLP-induced neutrophil-platelet complex formation in whole blood at the IC50 value of 35.8+/-8.1 microM in a concentration-dependent manner. According to these data, it was hypothesized that fMLP stimulates neutrophils to release cathepsin G, which subsequently activates the nearby platelets, creating neutrophil-platelet complexes. YC-1 inhibits fMLP-induced neutrophil from releasing cathepsin G via a cGMP-dependent pathway. This inhibitory effect of YC-1 on cathepsin G release is a major mechanism for affecting fMLP-induced neutrophil-platelet complex. YC-1's inhibition P-selectin expression on platelet may potentiate its effects. These inhibitory effects may contribute to the inhibition of neutrophil-platelet complex formation in whole blood.
Adult↗
Neutrophil cytoskeletal rearrangements during capillary sequestration in bacterial pneumonia in rats.
RATIONALE: Neutrophils accumulate in pulmonary capillaries during acute inflammation. Initial events in injury recognition and sequestration do not occur through selectin-mediated rolling. Cytoskeletal rearrangements, as assessed by submembrane F-actin rims, result in poorly deformable neutrophils that may not pass through capillaries. OBJECTIVE: To test the hypothesis that neutrophils sequestering during pneumonia contain F-actin rims and to determine the roles of CD11/CD18, L-selectin expression, and neutrophil-platelet adhesion in neutrophil sequestration. METHODS: Neutrophils were compared in blood obtained simultaneously from venous and arterial sites before and 4 h after instillation of Streptococcus pneumoniae or Escherichia coli in rats. MEASUREMENTS AND MAIN RESULTS: At 4 h of pneumonia, the number of neutrophils was greater in the venous blood entering the lungs than in the arterial blood leaving the lungs, indicating that neutrophil sequestration was occurring. More neutrophils entering the lungs contained F-actin rims than did neutrophils exiting, and the venous-arterial difference in F-actin-rimmed neutrophil counts completely accounted for sequestration. In E. coli pneumonia, in which neutrophil adhesion is mediated by CD11/CD18, CD18 blockade 15 min before blood samples were obtained did not prevent this sequestration of F-actin-rimmed neutrophils. Neutrophils expressing high or low levels of L-selectin or of neutrophils that bound platelets while circulating did not preferentially sequester. CONCLUSIONS: Neutrophils with cytoskeletal rearrangements preferentially sequester within the lungs during pneumonia, and this sequestration is not due to CD11/CD18-mediated adhesion, L-selectin expression, or platelet adhesion to neutrophils, suggesting that cytoskeletal rearrangements result in sequestration of neutrophils.
Actins↗
Effects of exogenous nitric oxide on neutrophil oxidative function and viability.
Previous studies have suggested that nitric oxide (NO) can modulate neutrophil function. Exposure to inhaled NO for pulmonary vasodilation could thus potentially affect neutrophil involvement in lung inflammation and infection. We evaluated the effect of exogenous NO gas exposure at clinically relevant concentrations in vitro on the oxidative function of human neutrophils. Isolated neutrophils were exposed for 2 h to either room air (RA), 80% oxygen (O2), or NO at 20 or 5 ppm blended with room air (NO20/RA, NO5/RA) or blended with 80% oxygen (NO20/O2) (NO5/O2). Neutrophils were then evaluated for superoxide anion generation with the cytochrome c reduction assay, for oxygen consumption with the Clark oxygen electrode technique, and for myeloperoxidase (MPO) release by enzyme-linked immunosorbent assay (ELISA). Neutrophil viability was determined by both trypan blue dye exclusion and fluorescence viability/cytotoxicity assay. Neutrophils exposed to NO at 20 ppm demonstrated a significant decrease in superoxide anion generation in both NO20/RA (97 +/- 46 nmol/10(6) neutrophils) and NO20/O2 (102 +/- 54 nmol/10(6) neutrophils) groups as compared with RA (190 +/- 41 nmol/10(6) neutrophils) (mean +/- SEM, P < 0.005 by analysis of variance [ANOVA] and the Student-Newman-Keuls test). No significant difference was seen at 5 ppm NO exposure. Neutrophil oxygen consumption was decreased with NO20/O2 (6.5 +/- 1.2 nmol O2/ml/min/10(7) neutrophils) as compared with RA (13.7 +/- 3.9 nmol O2/ml/min/10(6) neutrophils) or O2 alone (11.6 +/- 3.1 nmol O2/ml/min/10(7) neutrophils) (P < 0.002). MPO levels were significantly decreased with NO20/O2 (2.3 +/- 0.4 microg/ml) as compared with RA (4.0 +/- 0.4 microg/ml, P < 0.005), and also with NO5/O2. Cell viability as reflected by trypan blue dye exclusion was decreased with O2 (70 +/- 2.3%), NO20/RA (61 +/- 4%), and NO20/O2 (58 +/- 2.5%) exposure as compared with RA control (84.4 +/- 0.9%) (P < 0.0001). Decreased neutrophil viability was confirmed by live/dead assay for O2 (80.8 +/- 2.8%), NO20/RA (62.8 +/- 6.1%), and NO20/O2 (31.7 +/- 5.6%) groups as compared with RA control (95.8 +/- 1.4%, P < 0.0001). Adjusting neutrophil superoxide anion generation, oxygen consumption, and MPO values for cell viability abolished differences between exposure groups. We conclude that exogenous NO exposure at clinically relevant concentrations decreases neutrophil oxidative function, primarily as a result of reduced cell viability. Further studies are necessary to determine if these effects serve an in vivo immunoregulatory or immunosuppressive role in neutrophil response to lung injury and infection.
Adult↗
Role of neutrophils in mucus hypersecretion in COPD and implications for therapy.
Airway mucus hypersecretion is a serious and presently untreatable symptom of COPD. Over the past several years, emerging evidence has implicated epidermal growth factor receptor (EGFR) expression and activation in mucin production by airway epithelial (goblet) cells. Activated neutrophils recruited to the airways (and their secreted products) play several key roles in EGFR-dependent mucus hypersecretion: (i) activated neutrophils secrete tumor necrosis factor (TNF)-alpha, which induces EGFR expression in airway epithelial cells; (ii) activated neutrophils release reactive oxygen species, which activate EGFR; (iii) neutrophil elastase cleaves the EGFR proligand, pro-transforming growth factor (TGF)-alpha, releasing mature TGF alpha which activates EGFR in a ligand-dependent fashion; and (iv) neutrophil elastase causes potent goblet cell degranulation. The secretion of active products by neutrophils appears carefully regulated. The local release of neutrophil elastase requires close contact between the neutrophil and another cell, mediated by surface adhesion molecules, thus limiting proteolysis to the immediate pericellular environment. In the airway lumen, neutrophils undergo apoptosis and are cleared by macrophages without releasing their intracellular contents. In contrast, neutrophils that die by necrosis disgorge proteases and reactive oxygen species into the lumen. In COPD, conditions within the airway lumen promote neutrophil necrosis. It is concluded that neutrophil death via necrosis leads to the high concentrations of free neutrophil elastase and reactive oxygen species in the sputum of patients with airway neutrophilia and mucus hypersecretion. Inflammatory cells (neutrophils), molecules (neutrophil elastase and reactive oxygen species), signaling pathways (EGFR), and cellular processes (neutrophil necrosis) contribute to mucus hypersecretion in COPD, and are potential targets for therapy. Interventions that target EGFR, neutrophil elastase, and reactive oxygen species exist and can be evaluated as treatments for neutrophil-dependent mucus hypersecretion.
Antioxidants↗