Cytochalasin B, a cytoskeleton disrupting compound, does not induce a neutrophil response by itself 18 but is known to augment the neutrophil response to many stimuli. We investigated whether this was true also for the minimal neutrophil response induced by a direct stimulation with TNF-α. We found that cytochalasin B had no effect on the NADPH-oxidase response when added to neutrophils prior to TNF-α treatment (data not shown). However, when the cells were first treated with TNF-α and subsequently challenged with cytochalasin B, a pronounced respiratory burst activity was noted (Fig 1). The time course of the induced response was similar to that seen with chemoattractants such as the formylated peptide fMLF, an agonist that activates cells through the G-protein coupled formyl peptide receptor, FPR. The peak of activity was reached 1–2 minutes after the addition of cytochalasin B and the response then rapidly declined to reach a base-line level after 3–4 min that remained constant throughout the observation period.

In agreement with the results reported by others 89 we found that TNF-α alone only poorly activated the neutrophil NADPH-oxidase, determined as the release of superoxide anions (3.2 ± 0.3 × 10⁶ cpm; mean peak value ± SD, n = 3; corresponding to a maximum value that was less than 5% of that induced by fMLF). The oxidase activity induced by cytochalasin B in TNF-α-treated cells was of the same magnitude as that triggered by fMLF (Figs 1 and 2). In agreement with previous reports 202122, TNF-α was found to prime neutrophils to a subsequent stimulation with fMLF (Fig 2).

The level of superoxide production induced by cytochalasin B was dependent on the concentration of TNF-α. (Fig 1) as well as on the duration of TNF-preactivation. When the time between the addition of TNF-α and cytochalasin B was less than 5 minutes, no respiratory burst activity was observed upon cytochalasin B challenge. The response to cytochalasin B gradually increased with the time allowed for TNF-α to interact with the neutrophils, reaching a plateau after approximately 20 min (Fig 3). The protein synthesis inhibitor cycloheximide did not inhibit the cytochalasin B-induced NADPH-oxidase activity in TNF-α primed cells (data not shown), suggesting that de novo protein synthesis was not required for the observed neutrophil activation.

All neutrophil chemoattractant receptors including C5aR (for the complement component C5aR), PAFR (for platelet activating factor), CXCR1 and 2 (for IL-8), FPR (for fMLF) and FPRL1 (for WKYMVM) belong to the GPCR family 2. Interaction between these receptors and their respective ligands results in activation of the neutrophil NADPH-oxidase (Table 1). Neutrophils that were allowed to interact with either of these agonists at 15°C and then transferred to 37°C became deactivated, i.e., there was no burst in oxidase activity, and the cells did not respond to further stimulation with the same chemoattractant. Furthermore, cytochalasin B induced a robust burst of oxidase activity in C5a- as well as in fMLF- and WKYMVM-deactivated cells i.e., these cells (or rather the FPR, FPRL1 and C5aR) were reactivated. In contrast, no such reactivation was induced by cytochalasin B in IL-8 or in PAF deactivated cells (Table 1).

The cytochalasin B induced superoxide production in TNF-α-primed neutrophils is similar to that induced by uncoupling and reactivation of occupied and deactivated FPR, FPRL1 or C5aR from the cytoskeleton, induced by the same drug. The most direct interpretation of these results is therefore that the TNF-α primed cells express a GPCR, of hitherto unknown identity, which gradually becomes occupied and then deactivated. When uncoupled from the cytoskeleton by cytochalasin B, the receptor is reactivated and the signals generated induce an activation of the oxidase. To test this hypothesis, we pretreated the cells with pertussis toxin, which inactivates the heterotrimeric G-proteins coupled to the GPCRs. Since no NADPH-oxidase activity could be induced by cytochalasin B in TNF-α primed cells that were first treated with pertussis toxin (Fig 4), our hypothesis is valid, i.e., a GPCR is involved in the TNF-α-primed cytochalasin B response. It should be noted that pertussis toxin had no effect on the ability of TNF-α to mobilize CR3 to the neutrophil surface (Fig 4). On the basis of these results, we hypothesized that a preformed endogenous agonist may be released from neutrophils during interaction with TNF-α, and we therefore attempted to verify the existence of such an agonist.

To test the hypothesis of an endogenous GPCR-agonist, we first determined the basic reactivation characteristics of occupied and desensitized receptors, using a model system in which desensitized neutrophils were pre-incubated with fMLF at 15°C. When pre-warmed neutrophils (37°C for 20 min) were added to a measuring system containing desensitized cells, the oxidase of the non-desensitized cells was activated, suggesting that free ligands are present in the measuring system (data not shown). In order to determine if these free ligands are of importance for the FPR reactivation induced by cytochalasin B, we added the receptor specific antagonist cyclosporine H to fMLF-desensitized cells a few seconds before cytochalasin B. In agreement with the specificity of the antagonist for FPR, a reduced NADPH-oxidase activity was obtained. No such inhibition by cyclosporine H was seen upon reactivation, when using cells that were incubated with the FPRL1 specific agonist WKYMVM or TNF-α (Fig 5).

Moreover, when the fMLF specific desensitization procedure was performed in a dense cell population (10⁷ cells/ml desensitized with 10^-7M fMLF) and the cells were diluted to 10⁵ cells/ml in pre-warmed measuring vials containing either a high concentration of peptide (final concentration of fMLF being 10^-7M) or no peptide (final concentration of fMLF being 10^-9M), the addition of cytochalasin B induced a burst in NADPH-oxidase activity only in samples with a high concentration of fMLF (Fig 6). Taken together these results suggest that in order for cytochalasin B to function as an inducer of respiratory burst, the fMLF desensitized neutrophils have to be continuously exposed to a high concentration of free ligand.

Neutrophils contain several preformed agonists/chemoattractants 2324 that could participate in an autocrine amplification loop. As described above, binding of neutrophil chemoattractants to their respective neutrophil receptors induces a rapid desensitization of the receptor, leading to an inability of the cells to respond to a second challenge by the same agonist. Accordingly, if TNF-α would induce secretion of an autocrine activator, this should be expected to induce a desensitized state of the specific receptor population involved. The finding that TNF-α triggered cells were primed rather than desensitized to fMLF, WKYMVM/m and C5a, suggests that the potential endogenous utilizes neither FPR, FPRL1 nor C5aR.

To identify the hypothetical endogenous agonist, we prepared cell free supernatants of TNF-α treated neutrophils and added these to new populations of primed or unprimed cells. No oxidase activation could be induced by compounds secreted from the TNF-α primed cells (data not shown). As stated above, neutrophils added to a population of fMLF desensitized cells were rapidly activated to generate superoxide by the fMLF present in the medium. When repeating this experiment with TNF-primed instead of fMLF desensitized cells, no such activity was seen (Fig 7). Furthermore, the newly added cells (non TNF-α primed) were not triggered by the addition of cytochalasin B (Fig 7), as illustrated by the finding that the NADPH-oxidase activity in mixed population of cells (50% primed and 50% non-primed) was only half of that of a cell population (with the same number of cells) in which all the cells were primed. Taken together these data suggest that no free agonist is present in the TNF-α primed cell suspension that could be responsible for transfer of the cells into a cytochalasin B-sensitive state.

Previous studies have shown that different receptor structures as well as potential activating agonists are stored in secretory granules of peripheral blood neutrophils 172526, suggesting that mobilization of these organelles will prime neutrophils to certain stimuli. We monitored the amount of the complement receptor 3 (CR3) and found that TNF-α-primed cells exposed an increased number of CR3 on their surface (Fig 8). In addition, TNF-α priming was accompanied by an increased specific binding of radiolabeled fMLF (10.2 ± 3.3 moles/10⁶cells bound to TNF-α treated cells compared to 4.0 ± 1.6 fmoles/10⁶ cells for corresponding control cells; mean ± SEM, n = 6), reflecting an increased amount of formyl peptide receptors (FPR) on the neutrophil surface. Hence, FPR and integrin-storing organelles were significantly mobilized upon treatment of neutrophils with TNF-α. We also found it of interest to determine whether induction of the cytochalasin B sensitive state is unique for TNF-α, or if it occurs also with other secretagogues. To investigate the precise role of granule mobilization in induction of the cytochalasin B sensitive state, we monitored the amount of the complement receptor 3 (CR3) on the surface of neutrophils. The finding that neutrophil receptors for IL-8 were not reactivated by cytochalasin B (Table 1) suggested that IL-8 is a suitable control priming agent in these experiments. No respiratory burst was obtained in response to cytochalasin B when TNF-α was replaced by IL-8 (Table 1), despite an induction of storage organelle mobilization also by IL-8 (Fig 9). It should be pointed out that the activation potency in relation to cytochalasin B was retained also when the concentration of TNF-α was reduced to concentrations that gave a level of CR3 mobilization similar to that induced by IL-8.

There is a well described link between TNF-α-induced activation of the p38 mitogen-activated protein kinase (MAPK) and superoxide anion formation in adherent neutrophils (reviewed by Berton 27). We investigated the effect of the specific p38 MAPK inhibitor SB203580 in our model system. As shown in Fig 8, SB203580 significantly attenuated the TNF-α priming, whereas no reduction was observed when neutrophils were first incubated with SB203580 and then stimulated with PMA, a phorbol ester known to activate the NADPH oxidase directly through protein kinase C (Fig 10A). Furthermore, the p38 MAPK inhibitor attenuated the effect of TNF-α induced granule mobilization, as determined by a reduced exposure of CR3 on the surface of cells challenged with TNF-α in the presence of SB 203580 (Fig 10B). Hence, p38MAPK is involved not only in the TNF-α-induced NADPH-oxidase activation in adherent cells, but also in the priming effects (degranulation and cytochalasin B-sensitivity) induced by the cytokine.

Leukocytes were isolated from freshly prepared leukopacks (The Blood Center, Sahlgrenska University Hospital, Göteborg) obtained from healthy blood donors. After removal of erythrocytes through dextran sedimentation, the leukocyte-rich supernatant was carefully layered onto Ficoll-Paque (Lymphoprep, Nyegaard, Norway). After centrifugation at 380 × g for 30 minutes, the pellet was resuspended in Krebs-Ringer phosphate buffer (KRG, pH 7.3; 120 mM NaCl, 5 mM KC1, 1.7 mM KH₂PO₄, 8.3 mM NaHPO₄ and 10 mM glucose) supplemented with Ca²⁺ (1 mM) and Mg²⁺ (1.5 mM), and kept on melting ice until used in experiments 42.

The hexapeptide Trp-Lys-Tyr-Met-Val-Met-NH₂ (WKYMVM) was synthesized and HPLC-purified by Alta Bioscience (University of Birmingham, Birmingham, United Kingdom). The formylated peptide N-formylmethionyl-leucyl-phenylalanine (fMLF), the chemotactic fragment of complement factor 5 (C5a), platelet activating factor (PAF), and TNF-α were from Sigma Chemical Co., St. Louis. Human recombinant IL-8 was provided by R&D Systems, Oxon, UK. The peptide agonists were dissolved in dimethyl sulfoxide to 10^-2M and stored at -70°C until use. Further dilutions were made in KRG.

Neutrophils (1–2 × l0⁶cells/ml) were incubated at 37°C for 5–40 minutes in the presence or absence (control) of a priming agent. The NADPH-oxidase activity of these cells was then recorded using luminol/isoluminol-enhanced chemiluminescence (CL) systems 43. The CL activity was measured in a six-channel Biolumat LB 9505 (Berthold Co. Wildbad, Germany), using disposable 4-ml polypropylene tubes with a 0.90-ml reaction mixture containing 1–2 × 10⁶ neutrophils. In order to differentiate between intracellularly and extracellularly generated reactive oxygen species, two different reaction mixtures were used. Tubes used for the measurement of extracellular release of superoxide anion contained neutrophils, horseradish peroxidase (HRP; a cell impermeable peroxidase; 4 U) and isoluminol (a cell impermeable CL substrate; 2 × 10^-5M). Tubes used for measurement of intracellular generation of reactive oxygen species contained neutrophils, SOD (a cell impermeable scavenger for O₂^-; 50 U), catalase (a cell impermeable scavenger for H₂O₂; 2000 U), and luminol (a cell permeable CL substrate; 2 × 10^-5M). The tubes were equilibrated at 37°C, after which the stimulus (0.1 ml) was added. The light emission was recorded continuously.

The amount of fMLF-receptors expressed on the cell surface in the different neutrophil populations was determined by incubating the cells with radiolabeled fMLF in the presence or absence of unlabeled fMLF. Unbound peptide was removed by centrifugation of the cells through an oil layer. The oil layer which was composed of a mixture of dibutylphtalate and dinonylphtalate (10:3, v/v; 100 μl) was layered on top of 10 μl of urea (6 M) in Eppendorf tubes. The radiolabeled peptide (50 μl [³H]fMLF; 8 × 10^-8M) was then layered on top of the oil followed by 50 μl of unlabeled fMLF (4 × 10^-5M) in KRG or vehicle alone. Neutrophils (2 × 10⁶, 100 μl) were added to the fMLF-solution and the tubes were incubated on melting ice for 1 h. After centrifugation at 9000 × g for 15 seconds in a Beckman microfuge (Beckman Instruments, Fullerton, CA), the bottom of the centrifuge tubes (containing the pelleted cells) was excised and collected for determination of radioactivity.

To measure surface exposure of CR3, the cells were labeled with phycoerythrin-conjugated monoclonal antibodies specific for CD11b (DAKO M741; 10 μl to a cell pellet of 10⁶). The cells were examined by use of flow cytometry (FACScan; Becton Dickinson, Mountain View, CA).

In order to deactivate neutrophils to GPCR agonists, the cells were first equilibrated for 10 min at 15°C. The agonist, e.g., the chemoattractant fMLF (final concentration 10^-7M) was added and the incubation was continued at 15°C for an additional 5 min 44. The cells were then transferred to 37°C for 5 minutes, and reactivation was subsequently achieved by adding cytochalasin B (5 μg/ml).

Human recombinant TNF-α, fMLF, luminol, isoluminol, cycloheximide, Pertussis toxin, SB 203580 and cytochalasin B were obtained from Sigma Chemical Co., St. Louis. Superoxide dismutase (SOD), catalase and horse radish peroxidase (HRP) were from Boehringer-Mannheim, Germany. Radiolabeled fMLF was from Du Pont NEN (Boston, Mass).

The Student's t-test (two-tailed) was performed to determine statistical significance.