Ultraviolet irradiation accelerates apoptosis in human polymorphonuclear leukocytes: protection by LPS and GM‐CSF

Polymorphonuclear leukocytes (PMN) play a central role in host response to injury and infection. Understanding factors that regulate PMN survival may therefore have a major influence on the development of novel treatment strategies for controlling life‐threatening infections, as well as local and systemic inflammatory responses. Unfortunately, the presently utilized in vitro culture model of PMN apoptosis makes the examination of early biochemical events surrounding PMN apoptosis very difficult. This study demonstrates that a short course of UV irradiation (15 min) can be used to induce rapid progression of PMN through the apoptotic process with 70–90% of PMN displaying features of apoptosis by 4 h after UV exposure. Bacterial lipopolysaccharide and granulocyte‐macrophage colony‐stimulating factor, which are known to prolong PMN survival during in vitro culture, also protected PMN from UV‐accelerated apoptosis. The UV‐accelerated model of PMN apoptosis provides another valuable tool for the investigation of early signaling pathways associated with inducing or delaying PMN apoptosis. J. Leukoc. Biol. 62: 517–523; 1997.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/142305/1/jlb0517.pd

Membrane-Proximal Calcium Transients in Stimulated Neutrophils Detected by Total Internal Reflection Fluorescence

ABSTRACT A novel fluorescence microscope/laser optical system was developed to measure fast transients ofmembraneproximal versus bulk cytoplasmic intracellular calcium levels in cells labeled with a fluorescent calcium indicator. The method is based on the rapid chopping of illumination of the cells between optical configurations for epifluorescence, which excites predominantly the bulk intracellular region, and total internal reflection fluorescence, which excites only the region within-100 nm of the cell-substrate contact. This method was applied to Fluo-3-loaded neutrophils that were activated by the chemoattractant N-formyl-met-leu-phe. Chemoattractant-activated cells showed 1) transient increases in both membraneproximal and bulk cytosolic Ca2+ that peaked simultaneously; 2) a larger fractional change (20-60%) in membrane-proximal Ca2+ relative to bulk cytosolic Ca2+ that peaked at a time when the main Ca2+ transient was decreasing in both regions and that persisted well after the main transient was over. This method should be applicable to a wide variety of cell types and fluorescent ion indicators in which membrane-proximal ionic transients may be differentfrom those deeper within the cytosol

Desensitization of the actin polymerization response in human neutrophils at low cell density

Many chemoattractant‐activated responses in neutrophils show transient kinetics, suggesting that rapid desensitization occurs during the time course of the response. We found that desensitization of the actin polymerization response to N‐formyl peptides is, in a large part, due to inhibition by adenosine released from cells to the medium and depletion or a chemical inactivation of the agonist. To reduce the influence of these factors, we stimulated neutrophils in a very diluted suspension, sometimes with continuous replacement of the medium. The actin polymerization response to a high agonist concentration was greatly enhanced and prolonged under these conditions, often without any tendency to subside within 10 min at 25°C. It has previously been shown that the N‐formyl peptide receptor converts from a rapidly dissociating to a slowly dissociating and presumably inactive form during activation. Under the conditions of low cell concentration, the conversion to a slowly dissociating receptor still occurred. Thus the prolonged response was not due to prolonged presence of rapidly dissociating receptors. We conclude either that a low number of rapidly dissociating receptors, which we failed to see, is sufficient to maintain actin polymerization or that slowly dissociating receptors can support the actin response. In contrast to responses stimulated by high agonist concentrations, the responses to low concentrations of the agonists were transient. The results of other authors indicate that low concentrations of N‐formyl peptides do not desensitize the receptors. Other mechanisms, which are specific for the actin polymerization response, must be involved in response termination to low concentrations of N‐formyl peptides. Activation at low cell density will be a useful approach for studying other processes (Ca2+ elevation, oxidant production, etc.) and chemoattractants (leukotriene B4, interleukin 8, etc.) for which an understanding of the kinetics due to desensitization of the components of the receptor‐mediated activation pathway is desired.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/141841/1/jlb0331.pd

Ultraviolet irradiation accelerates apoptosis in human polymorphonuclear leukocytes: protection by LPS and GM-CSF

Polymorphonuclear leukocytes (PMN) play a central role in host response to injury and infection. Understanding factors that regulate PMN survival may therefore have a major influence on the development of novel treatment strategies for controlling life‐threatening infections, as well as local and systemic inflammatory responses. Unfortunately, the presently utilized in vitro culture model of PMN apoptosis makes the examination of early biochemical events surrounding PMN apoptosis very difficult. This study demonstrates that a short course of UV irradiation (15 min) can be used to induce rapid progression of PMN through the apoptotic process with 70–90% of PMN displaying features of apoptosis by 4 h after UV exposure. Bacterial lipopolysaccharide and granulocyte‐macrophage colony‐stimulating factor, which are known to prolong PMN survival during in vitro culture, also protected PMN from UV‐accelerated apoptosis. The UV‐accelerated model of PMN apoptosis provides another valuable tool for the investigation of early signaling pathways associated with inducing or delaying PMN apoptosis. J. Leukoc. Biol. 62: 517–523; 1997.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/142305/1/jlb0517.pd