Biochemical analysis of the activation of adherent neutrophils in vitro

Cerasoli, Franklin; Gee, Marlys H.; Ishihara, Yoko; Albertine, Kurt H.; Tahamont, Maria V.; Gottlieb, Jonathan E.; Peters, Stephen P.

doi:10.1016/0040-8166(88)90053-5

Cited by 23 publications

(9 citation statements)

References 6 publications

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“…Blood neutrophils were isolated by the technique described by Cerasoli et al (11). Twenty to 30 mL of whole blood was anticoagulated with 0.1 M EDTA, then centrifuged at 300× g for 20 min to obtain platelet-rich plasma, which was removed and centrifuged at 2,500×g for 15 min to obtain platelet-poor plasma (PPP).…”

Section: Methodsmentioning

confidence: 99%

Chemotaxis of Blood Neutrophils from Patients with Primary Ciliary Dyskinesia

et al. 2003

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Primary ciliary dyskinesia is characterized by chronic upper and lower respiratory infections which are caused by the grossly impaired ciliary transport. Since the cilia and neutrophils both utilize microtubular system for their movement, it has been speculated that neutrophil motility such as chemotaxis might be impaired in patients with primary ciliary dyskinesia. Neutrophils were purified from whole blood from 16 patients with primary ciliary dyskinesia and from 15 healthy controls. Chemotactic responses of neutrophils to leukotriene B4 (LTB4), complement 5a (C5a), and formylmethionylleucylphenylalanine (fMLP) were examined using the under agarose method. The chemotactic differentials in response to LTB4, C5a, and fMLP in neutrophils from the patient group were significantly lower than the corresponding values in neutrophils from the control group (p<0.05 for all comparisons). The difference in chemotactic index between the two groups was statistically significant for LTB4 and fMLP (p<0.05 for both comparisons), but not for C5a (p=0.20). Neutrophils from patients with primary ciliary dyskinesia showed a decreased chemotactic response as compared with those from normal subjects. It is concluded that the increased frequency of respiratory tract infection in patients with primary ciliary dyskinesia is possibly due to the defective directional migration of neutrophils, as well as to the defective mucociliary clearance of the airways.

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Section: Methodsmentioning

confidence: 99%

Chemotaxis of Blood Neutrophils from Patients with Primary Ciliary Dyskinesia

et al. 2003

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“…This procedure and our proce dure for the isolation of PMNs typically results in a preparation of PMNs with high purity, low spontane ous release of superoxide anion, and morphologic characteristics similar to those of PMNs found in the vascular space [10]. Therefore, we consider these cells to be well preserved, both structurally and function ally.…”

Section: Resultsmentioning

confidence: 99%

“…In the case o f human cells, gradients of 80, 76,62, and 51% (v/v, plasma/Percoll™) were used [6] following the general procedure of Haslett et al [7]. Purities were routinely > 95% (human) and > 90% (sheep), and viability was > 95% by try pan blue exclusion.…”

Section: Isolation O F Neutrophilsmentioning

confidence: 99%

“…Human and sheep PMNs were isolated using gradients o f auto logous plasma and Percoll™ from EDTA-anticoagulated blood as previously described [6,8). In the case o f human cells, gradients of 80, 76,62, and 51% (v/v, plasma/Percoll™) were used [6] following the general procedure of Haslett et al [7].…”

Section: Isolation O F Neutrophilsmentioning

confidence: 99%

“…Functional characterization of PMNs in the airspaces of individuals at risk for and with established inflam matory lung disorders may provide useful informa tion about the contribution of PMNs to the observed injury. For example, we recently observed that PMNs obtained from the airspace of an individual with ARDS showed diminished superoxide anion (Oj) generation in comparison with his circulating PMNs [5,6]. It has recently been reported that some methods used to isolate neutrophils, for example those which do not exclude lipopolysaccharide (LPS) from buf fers, or those which use gradients prepared in saline solutions, can alter the functional and morphologic characteristics of purified PMNs [5,7], Therefore, it is important to demonstrate that methods used to har vest PMNs from the air space (BAL, usually per formed using saline) do not adversely affect the func tional characteristics of isolated cells.…”

Section: Introductionmentioning

confidence: 99%

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Prolonged Exposure of Neutrophils to Saline or Bronchoalveolar Lavage Fluid Does Not Alter Superoxide Anion Generation

Nguyen¹,

Shusterman²,

Barnett

et al. 1989

Int Arch Allergy Immunol

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The role played by neutrophils (PMNs) in the genesis of lung injury in diverse clinical situations, such as bronchial asthma, idiopathic pulmonary fibrosis, and the adult respiratory distress syndrome, is an area of intensive investigation. Functional studies of PMNs, particularly those obtained from the alveoli by bronchoalveolar lavage, should shed light on their contribution to lung injury. However, it has not been demonstrated whether procedures used to harvest cells from the lung (bronchoalveolar lavage), particularly the potentially prolonged exposure to saline, commonly used to perform lavage, and other components of lavage fluid, can alter the functional characteristics of PMNs. In this report we demonstrate that a 2- to 3-hour exposure of neutrophils to saline from both humans and sheep in vitro does not alter the functional characteristics of PMNs as determined by superoxide anion generation after activation with phorbol myristate acetate (PMA; 6.96 ± 0.44 vs. 7.60 ± 0.32 nmol O₂/250,000 PMNs for control and saline-treated human cells, respectively, after a 45-min incubation with 10^––⁷M PMA, and 4.73 ± 0.30 vs. 4.50 ± 0.42 nmol O₂/250,000 PMNs for control and saline-treated sheep cells). In a second series of experiments, we studied the effect of exposure of human PMNs to bronchoalveolar lavage fluid supernatants obtained from normal volunteers on superoxide anion generation by neutrophils. Again, spontaneous (1.27 ± 0.49 vs. 1.65 ± 0.71 nmol O₂/200,000 PMNs for control and lavage fluid-treated cells, after a 45-min incubation), PMA-triggered (10^––6M; 5.67 ± 0.27 vs. 5.18 ± 0.55 nmol O₂/200,000 PMNs), and f-met peptide-triggered (10^––6M; 3.34 ± 0.41 vs. 3.04 ± 0.64 nmol O₂/200,000 PMNs) generation of superoxide anion was not affected. Therefore, we conclude that prolonged exposure of neutrophils to either saline or bronchoalveolar lavage fluid supernatants does not alter their capacity to generate superoxide anion and that biochemical studies of neutrophils obtained from the air spaces by bronchoalveolar lavage with saline should provide useful information about the functional characteristics of these cells.

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“Autoregulation” of Human Neutrophil Activation In Vitro: Regulation of Phorbol Myristate Acetate-Induced Neutrophil Activation by Cell Density

Peters

Cerasoli

Albertine

et al. 1990

Journal of Leukocyte Biology

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Phorbol myristate acetate (PMA, 10(-7) M) activation of adherent neutrophils (PMNs) led to a markedly attenuated release of superoxide anion (O2-) per cell when PMNs were activated at high density (2.85 fmol O2-/PMN at 2 million in 0.1 ml) in comparison with cells activated at low cell density (12.0 fmol O2-/PMN at 250,000 in 0.1 ml). This "autoregulatory" phenomenon was not due to a defect in the superoxide anion assay employed, to a differential adherence of neutrophils at high vs. low density, or to substrate (cytochrome c) or cell stimulus (PMA) limitation. It was associated with an inhibition of apparent NADPH oxidase activity and a leftward shift (toward a lower level of activation) in the activation profile of PMNs (as determined by FACS analysis using PMNs preloaded with 2'7'-dichlorofluorescin diacetate in which H2O2 production results in the production of the fluorescent product 2'7'-dichlorofluorescein intracellularly). Other aspects of the neutrophil activation response including arachidonic acid mobilization, phospholipid metabolism, and perhaps phosphatidylinositol turnover were also attenuated when PMNs were activated at high cell density. Studies with cells in solution, cells treated with cycloheximide, and cells treated with 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid suggest that PMN contact with a surface, neutrophil protein synthesis, and an increased surface expression of the heterodimer CD11b/CD18 on PMNs all were not required for autoregulation. Finally, morphometric and morphologic examination of PMNs activated at low vs. high density revealed histologic and structural correlates associated with the attenuated PMN activation response of cells triggered at high cell density. We conclude that multiple structural and functional aspects of the PMN activation response are modulated by cell density and suggest that this property is important both in the physiologic control of neutrophil activation and in the design of in vitro assays of the neutrophil activation response.

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Biochemical analysis of the activation of adherent neutrophils in vitro

Cited by 23 publications

References 6 publications

Chemotaxis of Blood Neutrophils from Patients with Primary Ciliary Dyskinesia

Chemotaxis of Blood Neutrophils from Patients with Primary Ciliary Dyskinesia

Prolonged Exposure of Neutrophils to Saline or Bronchoalveolar Lavage Fluid Does Not Alter Superoxide Anion Generation

“Autoregulation” of Human Neutrophil Activation In Vitro: Regulation of Phorbol Myristate Acetate-Induced Neutrophil Activation by Cell Density

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