Poster group 3. Although human airway epithelial cells are the main target of respiratory syncytial virus RSV , it also infects immune cells, such as macrophages and B cells. Whether T cells are permissive to RSV infection is unknown. Infection, phenotype, and cytokine production by T cells were analyzed by flow cytometry or enzyme-linked immunosorbent assay.
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Neutrophils are crucial in the response to a broad range of clinically relevant pathogens, a fact underscored by the occurrence of severe, and often fatal, infections in patients with congenital neutrophil deficiencies 2. These cells engulf and kill bacteria by producing reactive oxygen species into a phagocytic vacuole 3. Neutrophils have been traditionally considered within the innate immune response setting for its antimicrobial capacity.
However, recent studies performed with highly purified neutrophils have shown that these cells also produce and release different cytokines that may potentially influence the course of an immune response 5. However, the definition of these pathways still remains controversial Autophagy macroautophagy has been often defined as a degradative process and a tributary of the lysosomal pathway, which contributes to remove defunct or disused organelles, particulate targets and invading microbes Rabbit polyclonal antibody anti-LC3B cat.
LS-C and cat. All other chemicals employed were purchased from Sigma Aldrich St. Neutrophils were isolated from heparinized human blood from healthy donors who gave written informed consent, by centrifugation on Ficoll-Paque, dextran sedimentation, and hypotonic lysis 7.
Cells were used immediately after isolation. Then, they were stimulated or not with 2. In some experiments, where indicated, the time points of addition of inhibitors and evaluation of the results were modified according to the aims of the assay. In a set of experiments, at 3. PLB cells kindly provided by Dr.
Then, neutrophils were incubated with the primary antibodies in blocking buffer for 1 h at room temperature, washed, and then incubated with the corresponding secondary antibodies for 1 h at room temperature.
Images were analyzed using Fiji software and macros for automatized image quantification were designed. A low threshold that included the fluorescence of the whole cell area was applied for the generation of a mask image. Fields of low cellular density were selected for imaging; the tool watershed was applied for separation of attached cells in the mask image and to generate individual ROIs corresponding to each cell.
For each image, a background value was subtracted. This value was obtained by averaging the intensity inside the nuclear region. Two masks were created: one for the vesicular and another for the cellular compartment. Both masks were applied to the background subtracted-images, and the integrated density of the intensity in both compartments was measured in each cell. The colocalizing areas were measured and expressed as percentage of the total area of the cell.
In some experiments, after immunostaining, cell fluorescence was determined by flow cytometry using a FACscalibur BD Pharmingen flow cytometer or a Partec Cyflow cytometer.
Data were analyzed by using the software FlowJo. The statistical calculations were performed using GraphPad Prism version 6. However, we also detected donor specific variability as some samples exhibited earlier responses, probably due to differences in donor responsiveness to ATP, as was previously reported We then evaluated the effect of Baf A1, a compound which disrupts autophagic flux and as a consequence impairs LC3B-II autolysosomal degradation We first evaluated the impact of 3-MA and Wortmannin, two autophagy inhibitors that block an early stage of the process by inhibiting the PtdIns 3-kinase.
To rule out that these compounds were exerting an overall inhibitory effect on protein secretion, we also determined their impact on the secretion of IL-8, a cytokine that is released following the canonical ER-Golgi pathway. The values of these coefficients range from 0 to 1, for no colocalization to perfect colocalization, respectively.
In fact, IL-8 was hardly found under our experimental conditions, and monensin had to be added to neutrophil cultures in order to detect it Figure S7 in Supplementary Material. Images were acquired with a confocal microscope A and quantifications B—D were performed by using specific macro with Fiji software.
A Representative images of 3—5 experiments. B—D Values were derived from five independent experiments with at least 35 cells analyzed at each time point for each experiment. We then performed additional assays with Baf A1, a compound which not only inhibits V-ATPase-dependent autolysosome acidification but also disrupts the autophagic flux Images were acquired with a confocal microscope.
Arrows indicate compartments with overlapping fluorescent signals near the cell membrane A and in a cell pole B. Images are representative of experiments with 10 different donors.
C Viability of neutrophils treated as indicated. Images are representative of four experiments with different donors. F,G Quantifications of images of experiments depicted in D , performed by using a specific macro with Fiji software. Previous studies determined that autophagosomes can fuse to endosomes forming amphisomes Other studies showed that granules occasionally fuse with each other in the cytosol prior to their subsequent fusion with the plasma membrane Values were derived from five independent experiments with at least 60 cells analyzed per experiment.
Results were evaluated by Mann—Whitney test analysis and differences were not statistically significant. Neutrophils are terminally differentiated short-lived cells that are refractory to transfection; thus, they are not amenable for gene silencing and fluorescence protein expression. Probably for these reasons information about human neutrophil vesicular trafficking and autophagy molecular machinery in these cells is scarce.
These findings are in agreement with studies by Zhang et al. However, disparate observations have been reported probably arising from different experimental systems. Our studies partially agree with those reported by Dupont et al.
In contrast to Dupont et al. Our findings also contrast with those reported by Nakahira et al. These authors determined that disruption of LC3B generates a defect in mitochondrial homeostasis, which results in a greater basal amount of mitochondrial ROS production and renders mitochondria more susceptible to damage by treatment with LPS and ATP.
However, in human blood neutrophils, mitochondria not only are scarce but also are different from those of other myeloid cells, because they preserve mainly death-mediating abilities.
Neutrophils mainly use glycolysis rather than mitochondrial oxidative phosphorylation for their energy supply, and mitochondria hardly participate in ATP synthesis. Moreover, neutrophils have 10—15 times less copies of the mitochondrial genome in comparison to peripheral blood mononuclear cells In fact, we previously reported that neutrophil NLRP3 expression is not modulated by LPS priming in contrast to the observation we made in human monocytes 7 and to what was reported for mouse macrophages In contrast to these findings, recent studies in differentiated THP-1 cells have identified distinctive molecular components involved in secretory autophagy that distinguishes it from degradative autophagy.
All authors reviewed the manuscript. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
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Autophagy Mediates Interleukin-1β Secretion in Human Neutrophils.
Background: Although human airway epithelial cells are the main target of respiratory syncytial virus RSV , it also infects immune cells, such as macrophages and B cells. Whether T cells are permissive to RSV infection is unknown. Infection, phenotype, and cytokine production by T cells were analyzed by flow cytometry or enzyme-linked immunosorbent assay. All rights reserved. For permissions, e-mail: journals.