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PubMed1997

Changes of neutrophil migration without modification of in vitro metabolism and adhesion in Behçet's disease.

OBJECTIVE: Increase of neutrophil chemotaxis in Behçet's disease (BD) has been described, but it is not clear whether there is a correlation with other variables of neutrophil function and whether these modifications correlate with disease activity. METHODS: We studied neutrophil functions in patients with BD in the acute phase in comparison with healthy control subjects and with the same patients during disease remission, with or without therapy. We investigated in vivo neutrophil migration by Senn's skin window technique and measured adhesion assay and superoxide production in circulating and migrating neutrophils after different stimuli. RESULTS: Neutrophil migration in vivo was 101.3 +/- 17.9 x 10(6) polymorphonuclear lymphocytes (PMN)/cm2/24 h in patients with BD in the acute phase and 66.1 +/- 7.8 x 10(6) PMN/cm2/24 h in controls (p < 0.001). No correlation was found between leukocyte counts and neutrophil migration. Neutrophil migration evaluated in the same patients in a phase of disease remission was 58.3 +/- 10.3 x 10(6) PMN/cm2/24 h (p < 0.001 vs acute phase, not significant vs controls). The neutrophils of the exudate were normally primed to response to the chemotactic peptide fMLP. No differences between the 2 groups were found in superoxide production, adhesion under basal conditions, or in response to different stimuli by circulating and migrating neutrophils. CONCLUSION: Abnormally high migration of neutrophils in the active phase of BD is the only consistent neutrophil dysfunction. Since this modification is reversed by therapy, the evaluation of in vivo neutrophil migration may be useful in diagnosing and monitoring disease activity. Blood neutrophils have normal responses to different stimuli, indicating they are not primed by the disease state.

Adult↗
PubMed1998

Complement fragment-induced release of neutrophils from bone marrow and sequestration within pulmonary capillaries in rabbits.

Infusion of complement fragments induces rapid sequestration of neutrophils within the pulmonary capillaries. This study examined the contributions of the bone marrow (BM) and the liver to the accumulation of neutrophils within the lungs. Complement fragments induced the release of neutrophils from the BM within 7 minutes of infusion, and these neutrophils sequestered in the lungs immediately upon reaching the pulmonary capillaries. Neutrophils expressing high levels of L-selectin were preferentially retained within the pulmonary microvasculature. By 30 minutes after the infusion was stopped, the circulating neutrophil counts had increased, primarily because of release from the BM. The number of neutrophils sequestered in the lung had decreased by only 27%, and the number of neutrophils in the liver increased by 223%. These studies indicate that complement fragments induce the release of neutrophils from the BM far more rapidly than previously described. These newly released neutrophils immediately sequester within the lung, increasing the number of neutrophils available to injure the lung many fold beyond the number that were circulating before infusion. The preferential retention of L-selectin-expressing neutrophils likely reflects the requirement for L-selectin-mediated adhesion in maintaining sequestered neutrophils within the pulmonary microvasculature. The number of circulating neutrophils reflects a balance between pulmonary sequestration, rapid release from the BM, and uptake by the liver and other organs.

Animals↗
PubMed1999

Absence of lipopolysaccharide-induced inhibition of neutrophil apoptosis in patients with diabetes.

BACKGROUND: Patients with diabetes mellitus are considered to be at increased risk for infections and often experience more serious and prolonged infections. Defects in neutrophil microbicidal function have been identified in diabetic patients and are believed to contribute to this problem. Neutrophil apoptosis or programmed cell death regulates functional longevity, and is an integral component of inflammation and its resolution. It remains unknown whether neutrophils from diabetic patients demonstrate defects in apoptosis. HYPOTHESIS: Neutrophils from diabetic patients have defects in spontaneous and/or endotoxin (lipopolysaccharide [LPS])-induced apoptosis. DESIGN: Peripheral venous blood samples were collected from healthy volunteers and insulin-dependent diabetic patients to harvest neutrophils for in vitro study. INTERVENTIONS AND MAIN OUTCOME MEASURES: Neutrophils were cultured for 0 and 24 hours in the absence and presence of LPS. Normal neutrophils were also cultured under conditions of normal or high glucose concentration. Percentage of apoptotic cells was determined by flow cytometry. RESULTS: Normal neutrophils undergo significant apoptosis after 24 hours, and the percentage of apoptotic cells is reduced in the presence of LPS. Diabetic neutrophils undergo normal spontaneous apoptosis, but do not demonstrate LPS-induced inhibition of apoptosis. The LPS-induced inhibition of apoptosis in normal neutrophils is prevented under high-glucose conditions. CONCLUSIONS: Neutrophils from diabetic patients demonstrated defects in LPS-induced apoptosis. High-glucose environment may mediate these findings. The inability of diabetic neutrophils to reduce apoptosis following LPS exposure resulted in relatively increased apoptosis. This would cause decreased functional longevity of neutrophils and increased neutrophil clearance from infectious sites, possibly contributing to the increased susceptibility and severity of infections in diabetic patients.

Apoptosis↗
PubMedDate not supplied

Investigation of neutrophil--endothelial cell interaction in multiple trauma by chemiluminescence.

The trauma-induced activation of neutrophils and their functional alterations, i.e., increase in adherence and release of oxygen derived metabolites, is considered to play a central role in the initiation and amplification of capillary endothelial cell damage and following organ failure. In the present study neutrophil-endothelial cell interaction was studied using an in vitro model of human umbilical cord vein endothelial cells and human neutrophils. Production of oxygen-derived metabolites was determined by comparing mean peak chemiluminescence of neutrophils from multiply traumatized patients (n = 40) and mean peak chemiluminescence of neutrophils from blood donors (n = 160). Adherence and endothelial injury by neutrophils of multiply traumatized patients were compared with data of healthy blood donors. Chemiluminescence response of 70,000 neutrophils isolated from healthy control individuals was 699 +/- 98 x 10(3) cpm and could be increased significantly by endothelial cells to 1410 +/- 135 x 10(3) cpm (p < 0.05). Chemiluminescence response to neutrophils of polytraumatized patients was 1174 +/- 94 x 10(3) cpm and could not be significantly increased by endothelial cells (1419 +/- 120 x 10(3) cpm). Adherence of neutrophils of blood donors to endothelial cells was 12.31 +/- 0.77%. Adherence of neutrophils of polytraumatized patients was significantly increased to values of 24.83 +/- 2.03%. Injury of endothelial cells was not detectable with neutrophils from blood donors (1.11 +/- 1.09% 111in-release). Significantly increased 111in-release was apparent upon incubation with neutrophils of polytraumatized patients (5.76 +/- 1.28%). The data shows evidence of in vivo preactivation of neutrophils of polytraumatized patients, and supports the hypothesis that endothelial cells play an active role in neutrophil-endothelial cell interactions by modulating production of oxygen-derived metabolites.

Abdominal Injuries↗
PubMed1994

Quantification of neutrophil migration following myocardial ischemia and reperfusion in cats and dogs.

Endothelial cell dysfunction and cardiac myocyte injury resulting from ischemia and reperfusion have been associated with accumulation of neutrophils in the myocardium. To determine whether the accumulation is related primarily to intravascular sequestration or extravascular infiltration of neutrophils during the early period of reperfusion, we morphometrically quantified the tissue distribution of neutrophils in cats and dogs. At the end of the reperfusion period, the base of the heart was cross-clamped to preserve neutrophil location at the moment of death. Point-counting methods were used to determine the distribution of neutrophils inside and outside coronary arterioles and venules (< or = 100 microns in diameter) as well as coronary capillaries 5-10 microns in diameter in 0.5-microns-thick, plastic-embedded sections. Ischemia-reperfusion resulted in a threefold increase in neutrophil number in the lumen of arterioles and venules at 60 min of reperfusion and up to a sevenfold increase at 270 min of reperfusion (P < .05) compared to time-matched control nonischemic hearts. The ratio of intravascular neutrophils in venules to arterioles was 2:1. Intracapillary neutrophils increased, but not significantly, at 60 min of reperfusion. At 270 min of reperfusion, intracapillary neutrophils increased 11-fold (P < .05). The percentage of total neutrophils that accumulated outside arterioles and venules in cat hearts was 8% at 60 min of reperfusion (not significant, NS) and 28% at 270 min of reperfusion (P < .05). In dog hearts, the percentages were 26% (NS) and 44% (P < .05), respectively. The percentage of total neutrophils that accumulated outside capillaries was < 6% in both cat and dog hearts (NS). The combination of rapid intravascular sequestration, delayed extravascular infiltration, and low incidence of neutrophil-cardiac myocyte contact in situ in these two species suggests that neutrophil-mediated cardiac myocyte injury during early reperfusion may initially depend on diffusion of inflammatory mediators and subsequently require direct contact between neutrophils and cardiac myocytes.

Animals↗
PubMed1999

Neutrophil apoptosis in the lung after hemorrhage or endotoxemia: apoptosis and migration are independent of IL-1beta.

Hemorrhage and endotoxemia are associated with neutrophil accumulation in the lungs and the development of acute inflammatory lung injury. Because alterations in the rate of apoptosis may affect the number and function of neutrophils in the lungs, we determined the percentage of neutrophils undergoing apoptosis in the lungs of control, hemorrhaged, or endotoxemic mice. In control mice, 18.5 +/- 1.2% of pulmonary neutrophils were apoptotic. The proportion of apoptotic neutrophils in the lungs was significantly decreased 1 h after hemorrhage (6.5 +/- 1.6%, P < 0.01 compared to control) or endotoxemia (7.0 +/- 0.9%, P < 0.01 compared to control). Between 1 and 24 h after endotoxemia or hemorrhage, the proportion of apoptotic neutrophils in the lung remained significantly depressed compared to that in control, unmanipulated mice. By 48 h, the proportion of apoptotic neutrophils returned to baseline levels in the lungs of hemorrhaged (21.4 +/- 1.4%) or endotoxemic (16.4 +/- 1. 6%) mice. Lung neutrophil IL-1beta mRNA was significantly increased from that of control mice [i.e., 0.12 +/- 0.06 relative absorbance units (RAU)] 1 h after hemorrhage (5.19 +/- 0.068 RAU, P < 0.05 compared to control) or endotoxemia (8.90 +/- 1.53 RAU, P < 0.01 compared to control). In IL-1beta-deficient mice, there was no significant difference in lung neutrophil apoptosis or neutrophil entry into the lung after hemorrhage or endotoxemia compared to wild-type mice. Our results show that apoptosis among lung neutrophils is decreased for more than 24 h after hemorrhage or endotoxemia. Although IL-1beta expression is increased in lung neutrophils under these conditions, IL-1beta is not responsible for either the influx of neutrophils into the lung or the reduction of apoptosis in neutrophil populations after hemorrhage or endotoxemia.

Animals↗
PubMed1990

Interactions between the contact system, neutrophils and fibrinogen.

Since plasma kallikrein activates human neutrophils, and in plasma prekallikrein (PK) circulates complexed with high molecular weight kininogen (HK), we determined whether HK could mediate kallikrein's association with neutrophils. Human neutrophils were found to possess surface-membrane binding sites for HK but no internalization was detected at 37 degrees C. 125I-HK binding to neutrophils was dependent upon Zn++, specific, saturable and reversible with a Kd of 9-18 nM and 40,000-70,000 sites per cell. Furthermore, HK found in neutrophils (240 ng/10(7) neutrophils) also served as a cofactor for HNE secretion since neutrophils deficient in HK have reduced HNE secretion when stimulated in plasma deficient in HK or with purified kallikrein. Thus, neutrophil surface HK may serve as a receptor for kallikrein. Fibrinogen inhibited 125I-fibrinogen bound specifically and reversibly to human neutrophils. Zn++ (50 microM) was required for binding of 125I-fibrinogen to neutrophils and the addition of Ca++ (2 mM) increased the binding 2-fold. Excess HK completely inhibited binding of and displaced labeled fibrinogen as well as unlabeled fibrinogen. Binding of 125I-fibrinogen was saturable with an apparent Kd of 170 nM and 140,000 sites/neutrophil. The binding of 125I-fibrinogen to neutrophils was not inhibited by the peptide RGDS derived from the alpha-chain of fibrinogen, nor by the monoclonal antibodies (MAB) 10E5 to the platelet glycoprotein IIb/IIIa heterodimer. Fibrinogen binding was inhibited by a gamma-chain peptide CYGHHLGGAKQAGDV and by MAB OKM1 but was not inhibited by OKM10, a MAB to a different domain of the adhesion glycoprotein Mac-1 (CR3). HK binding to neutrophils was not inhibited by OKM1. These observations were consistent with a further finding that fibrinogen is a noncompetitive inhibitor of 125I-HK binding to neutrophils. These studies indicate that fibrinogen specifically binds to an integrin receptor (Mac-1) on the neutrophil surface through the carboxy terminal of the gamma-chain and that HK inhibits this interaction.

Adult↗
PubMed1992

Age-related changes in neutrophil structure and function.

The elderly suffer higher rates of morbidity and mortality from infectious disease than younger adults. Since neutrophils are the first line of defense against infection the vulnerability to infection of the elderly may be due, at least in part, to age-related changes in neutrophils (PMNs). Previous studies have suggested that there is an age-related increase in plasma membrane microviscosity in lymphocytes and neurons of the elderly. We have explored the hypothesis that there is a similar change in plasma membrane viscosity in the neutrophil and that this alteration in the neutrophil plasma membrane leads to diminished neutrophil function. When we studied plasma membrane viscosity of neutrophils by determining the fluorescence anisotropy of 1-(4-trimethylaminophenyl)-6-diphenyl-1,3,5-hexatriene labeled cells, we were surprised to find that plasma membrane viscosity is decreased in the neutrophils of the elderly as compared to young controls (microviscosity parameter, 1.33 +/- 0.07, in the elderly, n = 21, versus 1.62 +/- 0.06, in young controls, n = 19, P less than 0.004). As expected, stimulated (FMLP 0.1 microM) O2- generation by neutrophils from the elderly was significantly decreased compared with young controls (34 +/- 7% decrease, n = 6, P less than 0.04). Both resting and stimulated neutrophils demonstrated an age-related decline in adherence to a component of the extracellular matrix, denatured collagen (gelatin, P less than 0.04, n = 22 elderly subjects). In contrast, neutrophils from the elderly adhered to fibrinogen at least as well as neutrophils from young controls. Moreover, chemotaxis to activated complement components (in zymosan activated plasma) and FMLP did not change with increased age whether studied in the Boyden chamber (92 +/- 7% control, n = 14) or under agarose (90 +/- 13% control, n = 11). These studies suggest that an age-related decrease in plasma membrane viscosity is associated with a decrease in O2- production and adherence of neutrophils to components of the extracellular matrix. Thus, age-related alterations in neutrophil structure may result in diminished neutrophil function and increased susceptibility to infection with pyogenic bacteria.

Aged↗
PubMed1991

Enhanced adherence activity of OK-432-induced peritoneal neutrophils to tumor cells correlates to their increased expression of CD11b/CD18.

We previously found that activated peritoneal neutrophils adhered to tumor cells and destroyed them in the cancer ascites of patients who had received intraperitoneal (ip) OK-432 injection therapy. Since tight adhesion to the tumor cell is essential for effective neutrophil-mediated tumor cell destruction, we investigated the mechanism of peritoneal neutrophil adhesion to tumor cells, using a microplate adhesion assay. An in vitro study demonstrated that the adherence activity of the peritoneal neutrophils of patients who received OK-432 injection therapy to tumor cells increased greatly compared to that of blood neutrophils. The expression of the adhesion molecules (CD11a,b,c/CD18) of peritoneal neutrophils, which was determined by an immunofluorescence study, was about four times as much in CD11b and twice as much in CD11c and CD18 compared to that in blood neutrophils. In vitro OK-432 stimulation of normal blood neutrophils increased neither the adhesion to PLC nor the CD11b expression. The enhanced adherence activity of peritoneal neutrophils to tumor cells was significantly inhibited by pretreatment of the neutrophils with anti-CD11b and anti-CD18 monoclonal antibodies (mAb), but not by pretreatment with anti CD11a or anti-CD11c mAb. These results indicated that the increased adhesiveness of OK-432-induced peritoneal neutrophils to tumor cells was due to the enhanced expression of CD11b/CD18. We concluded that CD11b/CD18 molecules on OK-432-induced peritoneal neutrophils play a crucial role in the neutrophil adherence activity against tumor cells, and these results are the first demonstration in the field of human neutrophil function.

Antigens, CD↗
PubMed1997

Whole-blood incubation method to study neutrophil cytoskeletal dynamics.

To reduce artifactual effects in the study of filamentous (F)-actin dynamics in neutrophils, we have developed a whole-blood incubation method. Neutrophils in whole blood contained significantly less basal F-actin than did separated neutrophils. Although the peak relative F-actin content of neutrophils in whole blood after formyl-methionyl-leucyl-phenylalanine (fMLP) stimulation was significantly higher than that of separated neutrophils at 10(-9) to 10(-6) M fMLP concentrations (p < 0.05), there was no significant difference in increase in mean fluorescence intensity and the EC50 (concentration of stimulant giving a half-maximum response). On the other hand, the EC50 of platelet-activating factor (PAF) between separated neutrophils and whole-blood-incubated neutrophils differed significantly (1.6 +/- 1.1 x 10(-9) M in separated neutrophils and 2.0 +/- 0.7 x 10(-8) M in whole-blood-incubated neutrophils, p < 0.05). The whole-blood incubation method described presently reduces the sample volume, cost and time needed to separate neutrophils, prevents neutrophil activation during separation, and reserves all blood components that may affect neutrophil function. For these reasons, the conditions adopted in the present method are thought to simulate well neutrophils circulating in vivo and the method would be preferable to other neutrophil function tests performed to study actin dynamics.

Actins↗
PubMed1997

Comparison of in vitro function of neutrophils from cattle deficient in plasma factor XI activity and from normal animals.

Cattle, homozygous for the genetic disorder of factor XI (FXI) deficiency, exhibit less than 2% of normal plasma FXI activity, display an increased bleeding tendency and are more prone to infectious diseases. FXI is one of the protein components of the contact activation system of coagulation that assembles on the surface of circulating neutrophils. Because of the central role of neutrophils in inflammation, the in vitro responses of neutrophils from normal and FXI deficient cattle were compared. Neutrophil degranulation was evaluated by measuring the release of myeloperoxidase and alkaline phosphatase, and the respiratory burst was evaluated by determining superoxide anion production. Neutrophils from FXI deficient animals exhibited a significant increase (P < 0.05) in the spontaneous release of granule contents compared to the cells from normal cattle. Following stimulation with C5a complement derived from normal serum, the neutrophils from the FXI deficient animals exhibited a greater increase (P < 0.05) in both alkaline phosphatase release and superoxide production. In these neutrophils, following stimulation with C3b complement from normal serum, the relative increase in myeloperoxidase release compared to the unstimulated neutrophils was lower than that observed in the neutrophils from normal animals. There was minimal superoxide production in unactivated neutrophils from either normal or FXI deficient cattle and the response to phorbol ester stimulation was similar in both groups of animals. The C5a complement from FXI deficient serum was more effective (P < 0.05) in stimulating alkaline phosphatase release and superoxide production in normal neutrophils than the equivalent fraction from FXI deficient serum while the C3b complement from the FXI deficient serum was less effective than the normal serum fraction at inducing myeloperoxidase release from normal neutrophils. The results indicate that the differences in the in vitro neutrophil function are likely related to a variation in the function of the contact activation system on the neutrophil surface between normal and FXI deficient animals.

Animals↗
PubMed1997

Impaired actin polymerization and depolymerization in neutrophils from patients with thermal injury.

Acquired neutrophil dysfunction is considered an important cause of increased susceptibility to infection in patients with burns. In the early postinjury phase, large amounts of circulating chemo-attractants, cytokines and endotoxins induce strong systemic activation of neutrophils which may impair their motile functions. Actin is the most prevalent component of the microfilament lattice that generates force for the neutrophil motile responses, and in the present study we examined the dynamics of actin polymerization and depolymerization in neutrophils from 11 patients with large burns. At admission, the amount of polymerized actin in unstimulated neutrophils was 39.9 per cent higher than that of parallel controls. In addition, there was a positive correlation between the amount of polymerized actin and the total body surface area (TBSA) burn. The time course of patient neutrophil actin polymerization in response to FMLP, C5a, (Ser-IL-8)72, (Ala-IL-8)77 and crosslinking of surface Fc gamma RII was similar to that of controls, and the maximal amount of neutrophil F-actin was demonstrated after 30 s stimulation. At the peak of actin polymerization, however, patient neutrophils contained 27.3, 24.0, 24.7 and 25.6 per cent more polymerized actin than control cells stimulated with FMLP, (Ser-IL,-8)77, (Ala-8)77 and Fc gamma RII crosslinking, respectively. However, the relative increase of neutrophil F-actin following stimulation was significantly lower in patients than in controls. Moreover, the rate of patient neutrophil actin depolymerization was 39.0, 23.5, 63.3 and 51.7 per cent lower than that of controls after stimulation with FMLP, C5a (Ser-IL-8)72 and Fc gamma RII crosslinking, respectively. At discharge, the dynamics of neutrophil actin polymerization and depolymerization were similar to that of controls. The results demonstrate that in neutrophils during the early postburn phase, there are increased basal levels of polymerized actin, a lower responsiveness to stimulation and a reduced rate of actin depolymerization. As periodic polymerization and depolymerization of actin is essential for all neutrophil motile responses, it is probable that the alterations observed may contribute significantly to the overall neutrophil dysfunction following thermal injury.

Actins↗
PubMed1999

Role of neutrophil elastase in hypersecretion in asthma.

Goblet cell (GC) hyperplasia and mucous plugging are common in patients with acute asthma. These patients also show neutrophil recruitment into the airways. Neutrophils contain elastase, a potent secretagogue in airways. Therefore, it was reasoned that neutrophil recruitment, by releasing elastase, could result in GC hypersecretion. When neutrophil chemoattractants were instilled in the airways of guinea-pigs, time-dependent neutrophil recruitment and GC degranulation occurred. An inhibitor of leukocyte infiltration (NPC15669) prevented both responses, implicating neutrophils. An inhibitor of neutrophil elastase (ICI 200,355) abolished GC degranulation, implicating elastase. Further studies implicate movement of elastase from cytoplasmic granules to the neutrophil surface, and they suggest a role for adhesion molecules on neutrophils and on GCs in neutrophil-dependent GC degranulation. Similarly, instillation of ovalbumin (OVA) into airways of OVA-sensitized guinea-pigs caused early recruitment of neutrophils and GC degranulation. GC degranulation was prevented by pretreatment with NPC15669 or ICI 200,355. These results implicate neutrophil release of elastase in allergen-induced hypersecretion. The results suggest a mechanism for the mucous plugging that occurs in acute asthma; prevention of neutrophil recruitment, prevention of neutrophil-GC adhesion, or inhibition of elastase activity could provide effective therapy for this serious pathophysiological abnormality.

Animals↗
PubMed2003

Donor dependent, interferon-gamma induced HLA-DR expression on human neutrophils in vivo.

Neutrophils are effector cells of innate immune responses. Stimulated by interferon-gamma (IFN-gamma) to express HLA-DR, neutrophils acquire accessory cell functions for superantigen-mediated T cell activation. In vitro HLA-DR induction on neutrophils varies in a functionally relevant way as levels of MHC class II expression and magnitude of neutrophil induced T cell responses are correlated functions. The aim of this study was to assess whether IFN-gamma induces HLA-DR on human neutrophils in a donor dependent fashion in vivo and to define regulatory events operative in MHC class II expression of neutrophils. In vivo administration of rhIFN-gamma in 55 patients with renal cell carcinoma resulted in a varying increase of HLA-DR on neutrophils. By setting a cut-off for response at>10% HLA-DR positive neutrophils, HLA-DR responders (51%) were as frequent as nonresponders (49%). In vivo kinetic studies revealed a peak expression of HLA-DR on neutrophils 48 h after rhIFN-gamma application, while nonresponders remained HLA-DR negative over a 72-h period. In vitro IFN-gamma stimulated neutrophils recapitulated the response profiles observed in vivo. No differences in IFN-gamma dependent CD64 and invariant chain expression, and IFN-gamma serum levels were observed among the response subgroups. HLA-DR mRNA was detected in neutrophils from rhIFN-gamma treated responders and nonresponders, HLA-DR protein solely in lysates of responder neutrophils. IFN-gamma stimulated HLA-DR expression on neutrophils is subject to donor dependent variations in vivo, which result from rather post-transcriptional than transcriptional regulation. Due to their abundance in inflammatory reactions heterogeneous HLA-DR expression by neutrophils could determine the outcome of superantigen-driven diseases.

Adult↗
PubMed1995

The chemical and immunoglobulin structural features necessary for reactions of quinine-dependent antibodies to neutrophils.

BACKGROUND: Previously described were three patients with quinine-dependent antibodies to neutrophils, platelets, and red cells who had episodic pancytopenia and renal failure. The nature of the antibody-drug-neutrophil interactions was investigated with sera from these patients. STUDY DESIGN AND METHODS: Sera from all three patients were tested against neutrophils in flow cytometry in the presence of several compounds related to quinine. IgG and Fab and F(ab')2 fragments were prepared from the serum of one patient and tested against neutrophils in flow cytometry and immunoprecipitation in the presence of quinine and related compounds. RESULTS: In flow cytometry, sera from all three patients plus quinidine reacted with neutrophils. Sera from Patients 1 and 3 reacted with neutrophils in the presence of cinchonidine (desmethoxy-quinine) and serum from Patient 3 also reacted with neutrophils in the presence of cinchonine (desmethoxy-quinidine). None of the sera reacted with neutrophils in the presence of chloroquine or primaquine. Serum from Patient 3 plus quinolinic acid, a tryptophan metabolite, reacted with neutrophils, but sera from the other two patients did not. Patient 3 serum plus tryptophan or another tryptophan metabolite, quinalidic acid, did not react with neutrophils. IgG from Patient 3 serum reacted with neutrophils in flow cytometry in the presence of quinine, quinidine, cinchonidine, cinchonine, and quinolinic acid. F(ab')2 fragments plus quinine or cinchonidine also reacted with neutrophils, but Fab fragments plus quinine did not. In the presence of quinine, Patient 3 IgG immunoprecipitated the 85- and 60-kDa molecules and F(ab')2 fragments immunoprecipitated the 85-kDa molecule. Patient 3 serum plus quinidine, cinchonidine, cinchonine, and quinolinic acid immunoprecipitated the 130- and 85-kDa molecules, but not the 60-kDa molecule. CONCLUSION: Quinine-dependent neutrophil antibodies often react with neutrophils in the presence of quinidine and related compounds. These reactions were mediated by the F(ab')2 domain of IgG.

Autoantibodies↗
PubMed2005

Role of adenosine receptors in volatile anesthetic preconditioning against neutrophil-induced contractile dysfunction in isolated rat hearts.

BACKGROUND: The authors tested the hypothesis that adenosine receptors in polymorphonuclear neutrophils and the heart mediate the preconditioning effects of volatile anesthetics against neutrophil-induced contractile dysfunction. METHODS: Studies were conducted in buffer-perfused and paced isolated rat hearts. Left ventricular developed pressure served as index of contractility. Neutrophils and platelet-activating factor were added to perfusate for 10 min followed by 30 min of recovery. The effect of selective pretreatment of the neutrophils and the hearts with 1.0 minimum alveolar concentration isoflurane or sevoflurane on the neutrophil-induced contractile dysfunction was assessed. Studies were performed in the absence and presence of the nonselective adenosine receptor antagonist 8-phenyltheophylline (10 microM). Neutrophil retention was determined from difference between those administered and collected in coronary effluent and from myeloperoxidase concentration in myocardial samples. Superoxide production of neutrophils was measured by spectrophotometry. RESULTS: Under control conditions (no anesthetic pretreatment), activated neutrophils caused marked and persistent reductions in left ventricular developed pressure, associated with increases in neutrophil retention and myeloperoxidase activity. Pretreatment of the neutrophils or the heart with either isoflurane or sevoflurane abolished these effects. Pretreatment of the neutrophils also reduced the platelet-activating factor-induced increase in superoxide production by 29 and 33%, respectively. 8-Phenyltheophylline blunted the effects of anesthetic pretreatment of the neutrophils, whereas it did not alter the effects of anesthetic pretreatment of the heart. CONCLUSION: An activation of adenosine receptors in neutrophils, but not in the heart, plays a role in the preconditioning effects of volatile anesthetics against neutrophil-induced contractile dysfunction.

Anesthetics, Inhalation↗
PubMed1990

Release of a neutrophil-derived vasoconstrictor agent which augments platelet-induced contractions of blood vessels in vitro.

1. The effects of neutrophil-derived products on vascular tone in vitro were examined by adding purified rabbit neutrophils to siliconized organ baths containing rings of rabbit thoracic aorta. 2. Neutrophil-derived products induced a concentration-dependent contraction of the blood vessels generating 3.9 +/- 0.2 g of tension at a cell concentration of 2 X 10(6) ml-1. This contractile response was not dependent on an intact endothelium and was not ameliorated by treatment of the neutrophils with inhibitors of lipoxygenase or cyclo-oxygenase enzymes, by free radical scavengers or by the use of end organ antagonists to angiotensin II, desArg9-bradykinin, histamine, catecholamines or acetylcholine. 3. Neutrophils stimulated with phorbol myristate acetate, formyl methionyl-leucyl-phenylalanine or calcium ionophore A23187 released a contractile factor into the supernatant which produced qualitatively similar contractions compared to those elicited by incubation with intact unstimulated neutrophils. 4. Ten to twenty times more platelets were required to evoke equivalent contractions to those observed with neutrophils. However, neutrophil supernates significantly augmented platelet-mediated contractions (P less than 0.001). 5. Contractions elicited by neutrophils and supernates derived from activated neutrophils were partially antagonized by 5-hydroxytryptamine (5-HT) receptor antagonists, methysergide and ketanserin. However, the inhibition of exogenous 5-HT-induced contractions on rabbit aorta and rat stomach strips by both antagonists was greater than the inhibition of contractions produced by neutrophils and neutrophil-derived supernates. 6. Extraction of the biologically active material from supernatants of activated neutrophils into acetone, but not chloroform-methanol or ethyl acetate, suggests the contractile factor may be a protein/peptide. Partial purification on a Sephadex G100 column yields a contractile factor with a molecular weight of less than 4,000 daltons. 7. This factor may augment vascular tone, either directly or by interactions with platelets, in pathophysiological states associated with neutrophil activation and accumulation.

Animals↗
PubMed1994

Studies on the mechanisms involved in the inflammatory response in a reversed passive Arthus reaction in guinea-pig skin: contribution of neutrophils and endogenous mediators.

1. Mediators of inflammation can increase vascular permeability in at least two different ways: by acting directly on endothelial cells or, indirectly, through an incompletely understood mechanism, dependent on circulating neutrophils. Neutrophil-dependent oedema formation has been described in the skin of rabbits, rats, hamsters, mice and man. In contrast, we presented evidence in a previous study that local oedema formation induced by i.d. injection of chemoattractants in guinea-pig skin was neutrophil-independent. In the present study, we sought evidence of neutrophil-dependent oedema formation in immune-complex-mediated vasculitis, the reversed passive Arthus (RPA) reaction, in guinea-pig skin. We also investigated whether haemorrhage in the RPA reaction was neutrophil-dependent (as it is in other species) and the role of endogenous mediators of inflammation (prostaglandins, nitric oxide, histamine, PAF and leukotrienes) in contributing to the local inflammatory response. 2. In the RPA reaction, most oedema formation occurred over the first 60 min whereas 111In-neutrophil accumulation was still increasing from 60 to 240 min. The different kinetics of these two events suggested that they may be dissociated. 3. Oedema formation was partially inhibited by a long-acting PAF antagonist (UK-74,505) and an H1 histamine receptor antagonist (mepyramine) but not by a 5-lipoxygenase inhibitor (ZM 230487). A nitric oxide synthesis inhibitor (NG-nitro-L-arginine methyl ester, L-NAME) suppressed oedema formation by 68% whereas a cyclo-oxygenase inhibitor suppressed oedema by 27%. 4. 111In-neutrophil accumulation in the RPA reaction was partially suppressed by UK-74,505. In contrast, ZM 230487 was without effect at doses which abrogated arachidonic acid-induced 111In-neutrophil accumulation. 5. The anti-CD18 monoclonal antibody, (mAb) 6.5E F(ab')2, effectively inhibited 111In-neutrophil accumulation induced by PAF, zymosan-activated plasma (ZAP) and in the RPA reaction. However, oedema formation measured in the same sites was not altered. In contrast, oedema formation in the RPA reaction was partially suppressed by 6.5E whole mAb which was 2.5 times more potent than 6.5EF(ab')2 at inhibiting guinea-pig neutrophil adhesion to protein-coated plastic. Haemorrhage induced by PAF and in the RPA reaction was significantly inhibited by 6.5E F(ab')2 pretreatment.6. We conclude that in the RPA reaction in guinea-pig skin, oedema formation is partially neutrophil dependent as assessed by using an anti-CD18 mAb, whereas ZAP-induced oedema formation is neutrophil-independent. Haemorrhage was also dependent on neutrophil accumulation. In addition, our studies support a role for PAF in mediating both oedema formation and "'In-neutrophil accumulation in the RPA reaction. Endogenous release of histamine also appears to be important in mediating oedema formation suggesting that mast cells play a critical role in increases of vascular permeability in inflammatory reactions in guinea-pig skin. Moreover, our results confirm previous findings which suggest a dominant role for nitric oxide in maintaining cutaneous blood flow in the guinea-pig.

Animals↗