Cytochalasin B triggers a novel pertussis toxin sensitive pathway in TNF-alpha primed neutrophils - PubMed (original) (raw)
Cytochalasin B triggers a novel pertussis toxin sensitive pathway in TNF-alpha primed neutrophils
Johan Bylund et al. BMC Cell Biol. 2004.
Abstract
Background: Cytochalasin B does not directly activate the oxygen-radical-producing NADPH oxidase activity of neutrophils but transfers desensitized G-protein coupled receptors (GPCR) into an active signaling state by uncoupling GCPR from the cytoskeleton. The receptor uncoupling results in respiratory burst activity when signals generated by reactivated formyl peptide receptors trigger the NADPH-oxidase to produce superoxide anions.
Results: Tumor necrosis factor alpha (TNF-alpha) primes neutrophils for subsequent activation by cytochalasin B. Pretreatment with TNF-alpha induced mobilization of receptor-storing neutrophil organelles, suggesting that receptor up-regulation significantly contributes to the response, but the receptor mobilization was not sufficient for induction of the cytochalasin B sensitive state. The TNF-alpha primed state resembled that of the desensitized non-signaling state of agonist-occupied neutrophil formyl peptide receptors. The fact that the TNF-alpha primed, cytochalasin B-triggered activation process was pertussis toxin sensitive suggests that the activation process involves a GPCR. Based on desensitization experiments the unidentified receptor was found to be distinct from the C5a receptor as well as the formyl peptide receptor family members FPR and FPRL1. Based on the fact the occupied and desensitized receptors for interleukin-8 and platelet activating factor could not be reactivated by cytochalasin B, also these could be excluded as receptor candidates involved in the TNF-alpha primed state.
Conclusions: The TNF-alpha-induced priming signals could possibly trigger a release of an endogenous GPCR-agonist, amplifying the response to the receptor-uncoupling effect of cytochalasin B. However, no such substance could be found, suggesting that TNF-alpha can transfer G-protein coupled receptors to a signaling state independently of agonist binding.
Figures
Figure 1
Cytochalasin B stimulation of TNF-α primed neutrophils. Neutrophils were pre-incubated with TNF-α (25 ng/ml, 20 min, 37°C) after which they were stimulated with cytochalasin B The extracellular release of superoxide anions was measured by isoluminol-amplifled chemiluminescence (CL) given as Mcpm (106 counts per minutes). The figure shows the kinetics of a representative experiment. The indicated value shows the mean peak value ± SD, n = 3. The ability of cytochalasin B to induce NADPH-oxidase activity was dependent on the priming concentration of TNF-α (inset; comparing the peak values of the responses at varying concentrations to the value obtained with 25 ng/ml of TNF-α).
Figure 2
fMLF stimulation of TNF-α primed neutrophils. Neutrophils were pre-incubated in the absence (dashed line) or presence (solid line) of TNF-α (25 ng/ml) for 20 min at 37°C after which they were stimulated with fMLF. The extracellular production of superoxide anion after addition of the peptide (10-7M) was measured by isoluminol-amplified CL. Responses of CL are given as Mcpm (106 counts per minutes). The figure shows the kinetics of representative experiments.
Figure 3
Time dependency of the TNF-α effect Responses to cytochalasin B after incubation with TNF-α for different periods of time. Neutrophils were pre-incubated with TNF-α (25 ng/ml, 37°C) under various time-periods (5–40 min), before they were stimulated with cytochalasin B. The production of superoxide anions was measured by isoluminol-amplified CL as described above.
Figure 4
Pertussis toxin (PtX) sensitivity of cytochalasin B induced superoxide anion production and CR3 upregulation in TNF-α primed neutrophils. Neutrophils were preincubated with TNF-α (25 ng/ml, 20 min, 37°C) before they were incubated with PtX (500 ng/ml) for various time-periods (20–140 min). The cells were either paraformaldehyde-fixed and analyzed by flow cytometry for CR3 upregulation (closed squares), or stimulated with cytochalasin B, and the superoxide anion production were measured by CL (open squares). All analyzed populations of PtX incubated cells were compared to TNF-α primed cells not exposed to PtX.
Figure 5
Cyclosporin H sensitivity of cytochalasin B induced superoxide anion production in fMLF desensitized, WKYMVM desensitized or TNF-α primed neutrophils. Neutrophils were preincubated with fMLF (10-7M; 10 min A), WKYMVM (10-7M; 10 min B) or TNF-α (25 ng/ml; 20 min C). The cells were challenged with cytochalasin B in samples without (solid lines) and with cyclosporine H (final concentration in A, 10-7M and in B and C, 10-6M; dashed lines) that was added 1 min before cytochalasin B. The production of superoxide anions was measured by isoluminol-amplified CL as described above. The figure shows the kinetics of representative experiments and the ratios between the peak response in the absence and presence of cyclosporine H are given.
Figure 6
Dilution effects on cytochalasin B induced activation Neutrophils were incubated in the presence of fMLF (10-7M; A) or TNF-α (25 ng/ml; B) for 20 min at 37°C and then diluted in measuring vials containing the same concentration (high; solid lines) or no agonist (low; dashed lines), respectively. The cells were then immediately challenged with cytochalasin B. The production of superoxide anion was measured by isoluminol-amplified CL as described above. The figure shows the kinetics of representative experiments and the ratios, determined from the peak response in the presence of high and low concentrations of agonist.
Figure 7
Addition of non-primed cells to TNF-α primed cells and activation by cytochalasin B. Neutrophils incubated for 20 min at 37°C for 20 min were added to a population of TNF-α primed cells. The amount of superoxide release by the newly added cells was determined (A; dashed line). For comparision the NADPH-oxidase activity induced by cytochalasin B when added to the TNF-primed cells (A; solid line) is also shown. The NADPH-oxidase activity induced by cytochalasin B in a cell population where all the cells were primed with TNF-α for 20 min (B; solid line) was compared to that of a cell population where 50% of the cells were primed with TNF-α for 20 min and the other 50% for 1 min (B; dashed line). The production of superoxide anions was measured by isoluminol-amplified CL as described above. The figure shows the kinetics of representative experiments and the ratios between the peak responses in the populations are also given.
Figure 8
CR3-upregulation in TNF-α primed neutrophils. Neutrophils were incubated with TNF-α (25 ng/ml, 20 min, 37°C, solid line) and were paraformaldehyde-fixed, incubated with phycoerythrin-conjugated anti-CR3 antibodies (CD18/CD11b) and analysed by flow cytometry and compared with control cells (dashed line). Representative histograms of binding are shown, and the inset shows the exposure of CR3 on fMLF-primed neutrophils. The numbers indicate the mean fluorescence intensity as percent of control ± SD, n = 3.
Figure 9
CR3-upregulation in IL-8 and TNF-α primed neutrophils. Neutrophils were incubated with or without IL-8 (100 ng/ml) or TNF-α (25 ng/ml) for 20 min at 37°C. The cells were then paraformaldehyde-fixed, incubated with phycoeryterin-conjugated anti-CR3 antibodies (CD18/CD11b), analysed by flow cytometry and compared with control cells. The data is calculated from the mean fluorescence intensity of each cell population and expressed as percentage of the value obtained in control cells, n = 6.
Figure 10
Effects of SB203580 on TNF-α priming. Neutrophils were primed by TNF-α (25 ng/ml, 20 min, 37°C) in the absence or presence of the p38 MAPK inhibitor SB203580 (1 μM). (A) The cells were stimulated with cytochalasin B (5 μg/ml) and the extracellular production of superoxide anion was measured by CL. The effect of SB203580 on PMA-induced superoxide anion production was used as control. (B) Neutrophils were incubated with TNF-α (25 ng/ml) in the absence or presence of SB203580 (1 μM)), or with SB203580 alone (20 min, 37°C) and were paraformaldehyde-fixed, incubated with phycoerythrin-conjugated anti-CR3 antibodies (CD18/CD11b), analysed by flow cytometry and compared with control cells. Calculation were made from the mean fluorescence intensity of each cell population and expressed as percentage of the value obtained in control cells. The results are given as mean ± SE of three independent experiments.
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