Simultaneous assay for oxidative metabolism and adhesion of human neutrophils: evidence for correlations and dissociations of the two responses (original) (raw)
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Activation mechanisms of adherent human neutrophils
Blood, 1990
The mechanism by which unstimulated human neutrophils initiate a respiratory burst on adherence to a surface has been examined. When neutrophils adhere to a plastic surface, they immediately generate a sustained burst of superoxide (O2-). However, this respiratory burst is not initiated by adherence alone, since neutrophils attached to fibronectin fail to mount a response. Adhesion to plastic is calcium (Ca2+) independent, but O2- production requires Ca2(+)-containing buffer in the initiation phase, that is, during adhesion and the early phase of O2- production. The Ca2(+)-dependent step was shown to involve protein kinase C (PK-C) in that the O2- production, but not adherence, was blocked with 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7), and PK-C was found to translocate from the cytosol to the membrane on adhesion. Furthermore, it may be inferred that this translocation results in the generation of a Ca2+ independent form of PK-C, PK-M, since leupeptin, which inhibits the ...
1981
Human neutrophils were incubated with zymosan particles coated with either C3, IgG, or both. The cells formed rosettes with and ingested all 3 kinds of particle. Although C3-coated zymosan bound to about 80% of the neutrophils, the uptake of these particles was slow in comparison with IgGcoated or (C3 + 1gG)coated zymosan. All particles initiated an increase in the respiration of the cells as well as a specific release of lysosomal enzymes from the cells. IgG and C3 on zymosan reacted with different cell structures, as judged by the specific inhibition of F(ab'>anti-lgG on the IgG-mediated reactions and of F(ab')2-anti-C3 on the C3-mediated reactions. Moreover, trypsin treatment of the cells inhibited only the C3-mediated reactions.
The effects of chemotactic factors on the adhesiveness of rabbit neutrophil granulocytes
Experimental Cell Research, 1979
A range of chemotactic factors has been shown to affect the adhesion of rabbit peritoneal neutrophil granulocytes to cultured endothelial cells and to serum-coated glass. At chemotactically optimal concentrations, cYs-casein, p-casein, alkali denatured human serum albumin (HSA) and several synthetic formyl-peptides reduced the number of adherent neutrophils after 30 min to around 50% of control values. These effects were still observed after neutrophils, but not endothelium or serum-coated glass had been exposed to chemotactic factors and washed before use in assays. Two non-chemotactic analogues, native HSA and a non-formyl-peptide were ineffective. The dose responses for adhesion after 30 min in the presence of Lu,-casein and formyl-methionylleucyl-phenylalanine (FMLP) were found to be inversely related to those for migration towards these substances. After incubation for 60 min in high (10-8-10-7 M) concentrations of FMLP, neutrophil adhesion was found to be enhanced. Neutrophil aggregation was also affected by the presence of chemotactic factors in a similar time-and dose-dependent manner to the adhesion to substratum assays. Using FMLP, it was also shown that the timing of the adhesive changes depended on the concentration of chemotactic factor present.
Dual effects of formylpeptides on the adhesion of endotoxin-primed human neutrophils
Cell Biochemistry and Function, 1993
Neutrophils, treated with sequential additions of bacterial products such as endotoxin (E. Coli lipopolysaccharide, LPS) and the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (fMLP), undergo to metabolic activation and express membrane-anchoring proteins that promote adhesion to serum-coated culture wells. By investigating the dose–response relationships of these phenomena, we have found that: (a) resting neutrophils do not produce a significant amount of superoxide (O) and show only minimal adhesion to serum-coated plastic surfaces; (b) fully activatory doeses (> 5 × 10−8M) of fMLP induce the release of O and a significant increase of the cell adhesion; (c) pretreatment of the cells for 1 h with LPS augments cell adhesion to serum-coated culture wells in the absence of further stimulation and primes the neutrophils to enhanced fMLP-dependent O release; (d) addition of low, substimulatory doses of fMLP (from 10−10M to 5 × 10−9M) inhibits and reverses the adhesion of LPS-treated cells, (e) high fMLP doses (> 10−7M) are additive to LPS in promoting adhesion. Phorbol-myristate acetate (> 10−9M) increased adhesion in both normal and LPS-treated neutrophils, but low doses of this stimulant did not inhibit adhesion. Low doses (10−9M) of fMLP increased intracellular cyclic AMP in both normal and LPS-treated neutrophils, suggesting that stimulus-induced rises in cAMP may be the negative signal responsible for down-modulation of adhesion. Low (5 × 10−9M) and high (5 × 10−7M) fMLP doses induced the same increase of expression of CD11/CD18 integrins, indicating that the inhibition of adhesion caused by low doses is not due to quantitative down-regulation of integrins. These findings may provide an in vitro model of the complex biological events involved in the regulation of neutrophil adhesion.
Adherence of human neutrophils changes Ca2+ signaling during activation with opsonized particles
FEBS Letters, 1990
Changes in the cytosolic free Ca*+ concentration ([Ca*']i) upon activation of human neutrophils by opsonized particles (serum-treated zymosan; STZ) were evaluated by three different methods: (i) measurement of total fluorescence changes in indo-l loaded neutrophils activated in suspension; (ii) measurement of fluorescence changes in individual indo-l loaded neutrophils in a flow cytometer and (iii) measurement of fluorescence changes in individual fura-loaded neutrophils adherent to serum-coated coverslips. Our study shows that the opsonized particle-induced change in [Ca*+li in neutrophils is altered during adherence of the cells to a serum-coated surface. These observations might be of importance for neutrophil function in vivo, since adherence is a prerequisite for diapedesis and chemotazis.
Neutrophil activation by adhesion: mechanisms and pathophysiological implications
International journal of clinical & laboratory research, 1996
Neutrophil adhesion plays an essential role in the formation of an inflammatory exudate. Moreover, adhesion activates selective neutrophil functions and regulates the cell response to additional stimuli. In this review we summarize the information available on adhesion molecules involved in neutrophil adhesion to endothelial cells and extracellular matrix proteins and the experimental approaches which have been developed to block neutrophil adhesion and neutrophil mediated tissue damage. We also address the mechanisms of activation of selective neutrophil functions by adhesion molecules and, in particular the mechanisms of signal transduction by neutrophil integrins. On the basis of recent results obtained in our and other laboratories we propose a model hypothesizing mechanisms of signaling by neutrophil integrins involved in regulation of selective functions.
Effects of ex vivo manipulation on the expression of cell adhesion molecules on neutrophils
Journal of Immunological Methods, 1995
In vitro studies of ncutrophil adhesion generally utilise purified populations and are often pcrformcd at 37°C. This study determines the effects of temperature changes and neutrophil separation procedures on the expression of ccl1 adhesion molecules on neutrophils. We found that neutrophil separation procedures involving erythrocyte sedimentation and hypotonic lysis are associated with a significant increase in the expression of both a structural and functional epitope of the & integrin CDllb, an increase in the expression of sialyl Lewi? (CDlSs) and the hyaluronate receptor (CD44) as well as a significant decrease in L-selectin (CD62L) expression. Separated neutrophils are also more resistant than unseparated neutrophils to PMA induced upregulation of a functional epitope of CDllb. Incubating neutrophils at 37°C is associated with increases in the expression of structural and functional epitopes of CDllb. Neutrophil separation is also associated with increases in the expression of both structural and functional epitopes of CDllb which is greater when neutrophil separation is performed at room temperature compared with neutrophil separation at 0-4°C. However, this difference is lost when the latter are incubated at 37°C. Furthermore, neutrophil separation at both 0-4°C and room temperature is associated with a significant increase in CD& expression. This increase is less when separation is performed at room temperature. These findings suggest that neutrophil separation should be performed at room temperature unless the cells are going to be used at 0-4°C. Researchers using purified neutrophil populations need to be aware of these significant structural and functional changes when extrapolating in vitro results to in vivo situations. . Tel.: (081-204-4722; Fax: (08)-204-5450. 0022-1759/95/$09.50 0 1995 Elsevier Science B.V. All rights reserved SSDI 0022-I759(95)00 146-8
The effect of enkephalins and prostaglandins on O2− release by neutrophils
Journal of Surgical Research, 1986
Derivatives of superoxide (O;), produced by phagocytic cells, are thought to play a role in the adult respiratory distress syndrome (ARDS) and other disease states. Control of the release of 0; may prove beneficial. Using human neutrophils as a source of OF, and an assay for OF based upon the reduction of cytochrome C, we found that prostaglandin D2 (PGD,), leucine enkephalin (LE), and methionine enkephalin (ME) inhibited 02 release. The Escherichia coli product, N-formyl methionyl leucyl phenylalanine (FMLP), was employed to stimulate 0: release. PGDz was most potent while there was no significant difference between LE and ME. Another peptide, thyrotropin releasing hormone (TRH), had no effect on 0; release There was no correlation between the potency of the inhibitory effect on OF release and the effect of these agents on the binding of [3H] FMLP to human neutrophils. Comparison of different but structurally related prostaglandins (PGDr , PGEz, and PGF*,) revealed that PGDz was more potent than PGE2 in inhibiting 0; and that PGFr, had no effect. This result suggested that the presence and position of the carbonyl group was an important determinant of the magnitude of inhibition.