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(Journal of Leukocyte Biology. 2002;72:401-409.)
© 2002 by Society for Leukocyte Biology

Physiological levels of interleukin-18 stimulate multiple neutrophil functions through p38 MAP kinase activation

Travis H. Wyman*,{dagger}, Charles A. Dinarello{ddagger}, Anirban Banerjee§, Fabia Gamboni-Robertson§, Andrew A. Hiester*, Kelly M. England{dagger}, Marguerite Kelher§ and Christopher C. Silliman*,{dagger},§

* Bonfils Blood Center, and the Departments of
{ddagger} Medicine,
§ Surgery, and § Pediatrics, University of Colorado School of Medicine, Denver

Correspondence: Christopher C. Silliman, M.D., Ph.D., Research Department, Bonfils Blood Center, 717 Yosemite Circle, Denver, CO 80230. E-mail: chistopher.silliman{at}uchsc.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Patients with sepsis and acute lung injury have increased interleukin (IL)-18 levels systemically. We hypothesize that IL-18 stimulates neutrophils (PMNs) at physiologic concentrations. IL-18 primed the oxidase at 15 min (10–100 ng/ml), 30 min (0.1–100 ng/ml), and 60 min (100 ng/ml; P<0.05) and caused translocation of p47phox to the membrane similar to lipopolysaccharides. CD11b surface expression was increased by IL-18 in a time- and concentration-dependent manner. IL-18 caused up-regulation of the formyl-Met-Leu-Phe receptor, changes in PMN size, and elastase release. Investigation of signaling demonstrated IL-18-mediated activation of p38 mitogen-activated protein (MAP) kinase in a concentration (0.1–100 ng/ml)-, time (5–15 min)-, and Ca2+-dependent manner. IL-18 directly increased cytosolic Ca2+ concentration. IL-18 activation of PMNs was blocked by inhibition of p38 MAP kinase activity (SB203580) or by inhibition of p38 MAP kinase activation by chelation of cytosolic Ca2+. We conclude that IL-18, at physiologic concentrations, is an effective PMN priming agent that requires p38 MAP kinase activity.

Key Words: inflammation • cytokines • translocation of oxidase components


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Neutrophils (PMNs) are a vital part of host defense, particularly against bacterial and fungal infections [1 2 3 ]. Normal PMN function involves a progression of events that result in migration to the extravascular site of infection [4 5 6 ]. Priming of PMNs, which is distinct from oxidase activation, is part of normal PMN physiology and begins with the attraction of PMNs to activated vascular endothelium and continues through emigration to the tissues [1 , 4 5 6 ]. Primed PMNs have enhanced microbicidal capacity as compared with quiescent PMNs, and although priming may be beneficial to effectively eradicate pathogens, it is associated with syndromes of inappropriate PMN activation including acute lung injury (ALI) [1 , 4 , 5 ]. Sepsis is one of the leading predisposing conditions to ALI, and recent clinical studies have documented increases (0.1–2.0 ng/ml) of interleukin-18 (IL-18) systemically [2 , 7 8 9 10 11 12 ].

IL-18 is a proinflammatory cytokine with multiple immunomodulating properties [13 14 15 16 17 18 19 20 21 ]. A recent report documented that IL-18 (100 ng/ml) primed formyl-Met-Leu-Phe (fMLP) activation of the reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase of PMNs at 60 min and also caused increased expression of CD11b and release of specific granule components, and was not proapoptotic [22 ]. We hypothesize that IL-18 primes the PMN oxidase at physiologic concentrations and stimulates multiple PMN functions through cellular reorganization of oxidase components and activation of p38 mitogen-activated protein (MAP) kinase, respectively. In the present study, we examined the effects of IL-18 on multiple PMN functions, IL-18-mediated translocation of cytosolic oxidase components (p47phox and p67phox), and IL-18 activation of p38 MAP kinase.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
All reagents were lipopolysaccharide (LPS)-free and, unless specified, were purchased from Sigma Chemical Co. (St. Louis, MO). IL-18 was purchased from R&D Systems (Minneapolis MN). All solutions were made from sterile water, United States Pharmacopoeia (USP), purchased along with sterile 0.9% saline, USP, from Baxter Healthcare Corp. (Deerfield, NY). All buffers were made from the following stock USP solutions: 10% CaCl2 (Fujisawa USA, Inc., Deerfield IL), 23.4% NaCl, 20 Meq/ml KCl; 50% MgSO4 (American Regent Laboratories, Inc., Shirley, NY); sodium phosphates (278 mg/ml monobasic and 142 mg/ml dibasic); and 50% dextrose (Abbott Laboratories, North Chicago, IL). All solutions were sterile filtered with NalgeneTM MF75 series disposable sterilization filter units purchased from Fischer Scientific Corp. (Pittsburgh, PA). Phycoerythrin (PE)-labeled monoclonal antibodies (mAb) to CD11b and the fMLP receptor (fMLPr) plus a polyclonal antibody to dual phosphorylated p38 MAP kinase were purchased from Becton Dickinson/Pharmingen (San Diego, CA) and Cell Signaling Technology (Beverly, MA). An LS50B spectrofluorimeter, indo-1-acetoxymethylester (AM), and 1,2-bis(O-aminophenyl-ethane-ethane)-N,N,N',N'-tetraacetic acid (BAPTA)-AM were obtained from Perkin-Elmer Corp. (Norwalk, CT) and Molecular Probes (Eugene, OR). A Leica DRM mechanized fluorescence microscope, equipped with a custom Zeiss 63X water-immersion lens, was purchased from Leica Microsystems, Inc. (Exton, PA). Epifluorescence cubes [fluorescein isothiocyanate (FITC), Cy-3, Cy-5, and AMCA] were obtained from Bioptechs (Butler, PA) and Chroma Technology Corp. (Brattleboro, VT). A cooled, charged coupled device (CCD) camera and SlidebookTM software were purchased from The Cooke Corporation, Ltd. (Tonawanda, NY) and Intelligent Imaging Innovations (Lakewood, CO), respectively. SB203580 was obtained from Calbiochem (La Jolla, CA).

PMN isolation and IL-18 priming of the fMLP-activated respiratory burst
PMNs were isolated from healthy human donors by standard techniques [23 ]. IL-18 was suspended in saline to working concentrations. The priming activity of IL-18 was measured by incubating PMNs with saline or IL-18 (0.001–100 ng/ml) for 5–60 min at 37°C, followed by activation of the oxidase with saline, 1 µM fMLP, or 200 ng/ml phorbol myristate-13-acetate (PMA). The maximal rate of superoxide anion production was measured by the superoxide dismutase (SOD)-inhibitable reduction of cytochrome c [23 24 25 ]. Priming activity was determined as the augmentation, in response to IL-18, of the maximal rate of O2- by fMLP or PMA. To assess synergistic priming with IL-12, PMNs were incubated for 30 min with IL-12 (1 ng/ml) and IL-18 (1 ng/ml), followed by activation with fMLP and measurement of the maximal rate O2-.

Translocation of p47phox and p67phox by digital microscopy
Isolated PMNs were incubated with IL-18 (10–100 ng/ml) or saline controls for 15–60 min, fixed with 4% paraformaldehyde, and smeared onto slides. All manipulations with these slides, unless otherwise indicated, occurred at room temperature. Slides were washed three times with phosphate-buffered saline (PBS), pH 7.4, for 10 min, and the PMNs were porated with 30% acetone, 70% methanol for 3 min. After washing with PBS for 10 min, the slides were blocked with goat serum (the animal vector for the secondary antibody) for 60 min. The serum was replaced with primary antibody to p47phox or p67phox and incubated overnight at 4°C. The slides were washed three times with PBS and then incubated with bis-benzimide, wheat germ agglutinin (WGA), and Cy3-conjugated goat anti-rabbit secondary antibodies for 60 min. The PMNs were washed and mounted in antiquenching media.

The PMNs were imaged at 100x (Zeiss NA1.4, working depth, 2.2 µm), and the data from three epifluorescence channels were digitized on a cooled CCD camera at 1280 x 1024 pixels with 12-bit fidelity. The images were examined to ensure that intensity data were not saturated. Regions of interest, cell body, and cell membrane, were selected by threshold masking on the WGA intensity and by subselection of the highest intensity edge, respectively. The individual cells were separated and counted (submasking), each by total area and membrane area. In each region, area (cellular footprint), mean intensities, and total intensities were calculated for each channel. The data for the two regions of interest (7x2) in each cell (30 PMNs per treatment group) were loaded onto Statview software (4.5; Cupertino, CA) and analyzed by ANOVA for the effect of treatments (control, IL-18, and LPS). Translocation was derived from the membrane enrichment of p47phox or p67phox (total protein staining intensity in the membrane region) relative to the total intensity over the entire cell [26 27 28 ]. Selection of the regions of interest with the intensity of WGA staining for sialo-membranes (FITC channel) removed bias in assessing the integrated intensity of p47phox and p67phox (Cy-3 channel) contained in those regions. PMNs from four healthy donors were treated and processed identically and analyzed as a replicate.

Measurement of CD11b and fMLPr surface expression and changes in PMN size and complexity
PMNs (1x106) were warmed to 37°C and incubated for 30 min with saline or IL-18 (0.01–100 ng/ml), and positive controls consisted of identical PMNs incubated with 200 ng/ml PMA or 1 µM fMLP for 5 min. The PMNs were rapidly cooled to 4°C, and saturating concentrations (1 µg/106 PMNs) of PE-labeled mAb to CD11b or to the fMLPr were added. Following 30 min incubation at 4°C, the PMNs were fixed with paraformaldehyde. CD11b and fMLPr surface expression, cell size, and cell complexity were measured by flow cytometry [23 , 24 , 29 , 30 ]. Smears of fixed PMNs incubated with an antibody to the fMLPr were made and examined by digital microscopy.

Determination of elastase release
Elastase release was measured as previously described using 1.5 x 106 PMNs per treatment and an IL-18 incubation of 30 min, followed by activation with buffer or 1 µM fMLP [31 ]. Elastase release was reported as the percentage of total cellular elastase as determined by 0.1% Triton X-paired treatment of an identical number of PMNs.

Measurement of activated p38 MAP kinase
PMNs were treated with IL-18 (0.01–100 ng/ml) or saline for 15 s–15 min and immediately placed in Laemmli digestion buffer with freshly prepared protease inhibitors [32 ]. The proteins from these lysates were separated by sodium dodecyl sulfate (SDS) gel electrophoresis, transferred to nitrocellulose, and immunoblotted with antibodies to dual-phosphorylated p38 MAP kinase [32 ]. The bands of immunoreactivity were visualized using a horseradish peroxidase-labeled secondary antibody and enhanced chemiluminescence. Identical numbers of cell equivalents were used for each treatment as determined by cell counting (Sysmex K-1000, Roche Diagnostics, Indianapolis, IN). As controls, identical lysates were immunoblotted with an antibody to p38 MAP kinase. No differences were observed in the resulting blots, irrespective of treatment group (results not shown).

IL-18-mediated changes in cytosolic Ca2+ concentration
PMN cytosolic Ca2+ levels were determined by indo-1-AM loading of PMNs, washing the PMNs with fresh buffer, and direct measurement over real time as previously reported [24 , 32 , 33 ].

Inhibition of IL-18 priming by BAPTA and SB203580
PMNs were pretreated for 15 min at 37°C with dimethyl sulfoxide (DMSO), 50 µM BAPTA-AM, or 1–2.5 µM SB203580. The PMNs were then incubated for 15–30 min with saline or IL-18 (0.01–100 ng/ml) and then activated with 200 ng/ml PMA. The maximum rate of superoxide anion production was measured [23 24 25 , 34 , 35 ]. PMA was chosen as the activating agent, as it does not require p38 MAP kinase or changes in cytosolic Ca2+ for activation of the oxidase. BAPTA at 50 µM was used, as this concentration completely chelates the rise in cytosolic Ca2+ in response to fMLP, platelet-activating factor (PAF), or ionomycin [32 ].

Statistical analysis
The data are presented as the mean ± SEM. Statistical differences between the groups were determined by a paired ANOVA followed by a Tukey post hoc analysis for multiple comparisons.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
IL-18 priming of the PMN oxidase
Because of the structural similarity between IL-18 and IL-1ß and the ability of IL-1ß to prime PMNs in less than 60 min, we evaluated the propensity of IL-18 to prime the fMLP-activated respiratory burst of PMNs over a concentration range of 0.001–100 ng/ml and a time course of 5–60 min [17 , 22 , 36 37 38 39 ]. As shown in Figure 1 , the priming activity of IL-18 was first significant at 15 min of incubation for the 10 ng/ml and 100 ng/ml concentrations (P<0.05). At 30 min, IL-18 concentrations of 0.1–100 ng/ml primed the fMLP-activated respiratory (P<0.05); moreover, longer periods of incubation (45–60 min) yielded decreased IL-18 priming activity, which was only different from controls at the 100 ng/ml concentration (P<0.05). Regardless of the incubation time, IL-18 concentrations less than 0.1 ng/ml did not prime the fMLP-activated respiratory burst (P>0.05; Fig.1 ; results not shown). In addition, simultaneous incubation of PMNs with 1 ng/ml IL-12 and 1 ng/ml IL-18 did not augment the IL-18 priming (30 min, 10 ng/ml) of the fMLP-activated oxidase (fMLP: 1.5±0.5; IL-12/fMLP: 1.5±0.6; IL-18/fMLP: 6.2±1.4; IL-18+IL-12/fMLP: 6.00±2.1).



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  		Figure 1. Time- and concentration-dependent IL-18 priming of the fMLP-activated respiratory burst in isolated PMNs. PMNs were incubated with 0.01–100 ng/ml IL-18 for a time course of 5–60 min and activated with 1 µM fMLP, and the maximal rate of superoxide anion production was measured as the SOD-inhibitable reduction of cytochrome c. As compared with saline-treated controls activated with fMLP ({triangledown}), priming of the oxidase became significant at 15 min (100 ng/ml, {blacktriangleup}; 10 ng/ml, {triangleup}), reached a relative maximum at 30 min (100 ng/ml, {blacktriangleup}; 10 ng/ml, {triangleup}; 1 ng/ml, {square}; 0.1 ng/ml, {circ}), and decreased for all concentrations at 45 and 60 min of priming, respectively. Each time point represents a sample size of seven separate, healthy donors (*, P<0.05 as compared with buffer-treated PMNs).

 
IL-18-mediated cellular redistribution of p47phox and p67phox
PMNs were treated with 2 µg/ml LPS, 10–100 ng/ml IL-18, or saline for 15–30 min at 37°C. IL-18 and LPS caused changes in PMN morphology as shown in Figures 2A and 3A . LPS-mediated translocation of p47phox to the PMN membrane was detected by immunofluorescent image quantitation in agreement with previous results from centrifugal fractionation and immunoblotting [40 ]. The data are representative of PMN (Fig. 2A) with red staining for p47phox, green for the WGA membrane stain, blue for the bis-benzimide nuclear stain, and yellow for the colocalization of the p47phox with the membrane. Quantification of the cellular localization of the p47phox is focused on the cell membrane from the PMNs isolated from four separate, healthy donors (Fig. 2B) . At 30 min, we observed that LPS (2 µg/ml) increased p47phox staining 16 ± 3% in the membrane fraction relative to cellular content (Fig. 2) . Notably, the total cellular content had decreased 20 ± 3% by this time, presumably as a result of secretion of p47phox-containing granules (Fig. 2B) [22 ]. IL-18 also caused a similar translocation to membranes (18±3%) but without decreasing the total cellular content (Fig. 1) . Using a shorter incubation, 15 min, with IL-18 (10–100 ng/ml) or LPS did not elicit significant p47phox translocation, although there was a slight, general increase in the cell membrane (results not shown). Furthermore, 60-min IL-18 incubations (100 ng/ml) demonstrated a decreased level of p47phox in the membrane, commensurate with a decrease in IL-18 priming activity as compared with the 30-min incubations (results not shown; Fig. 2 ).



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  		Figure 2. IL-18-mediated translocation of p47phox. PMNs were incubated with saline (Control), IL-18, or LPS for 30 min at 37°C. The PMNs were triple stained with bis-benzimide (blue, nuclear), WGA (green, membrane), and p47phox (red). (A) Representative 100x images of stained PMNs ± the green channel (WGA). IL-18 and LPS caused translocation of the p47phox to the cell membrane, visualized as an increase in a yellow color in the membrane. (B) Data from the PMNs of four separate, healthy donors depicting IL-18- and LPS-induced increases in membrane content of p47phox (integrated intensities as determined by digital-image processing) relative to the content in the same cell, i.e., the cellular translocation of p47phox and the loss of total cellular p47phox in the LPS-treated PMNs. *, Statistical differences (P<0.05) between control and treated PMNs [control (Ctrl), n=32 cells; LPS, n=28 cells; IL-18, n=29 cells].

 


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  		Figure 3. IL-18-mediated changes in the cellular location of p67phox. PMNs were incubated with saline (Control), IL-18, or LPS for 30 min at 37°C. The PMNs were triply stained with bis-benzimide (blue, nuclear), WGA (green, membrane), and p67phox (red). (A) Representative 100;tx images of stained PMNs ± the green channel (WGA). LPS but not IL-18 caused translocation of the p67phox to the cell membrane, visualized as an increase in a yellow color in the membrane. (B) Data from the PMNs of four separate, healthy donors depicting LPS-induced increases in membrane content of p67phox (integrated intensities as determined by digital image processing) relative to the content in the same cell, i.e., the cellular translocation. In addition, demonstrated is the loss of total cellular p67phox in LPS-treated PMNs but a relative increase in cellular content of p67phox in response to IL-18 (B). *, Statistical differences (P<0.05) between control and treated PMNs [control (Ctrl), n=24 cells; LPS, n=16 cells; IL-18, n=16 cells].

 
Figure 3 depicts the identical results for the p67phox protein, except that the red color is p67phox and the yellow color demonstrates the area of translocation of p67phox, to the cell membrane. As seen in Figure 3A , LPS and IL-18 caused changes in PMN morphology. However, contrary to previous results at 60 min of LPS incubation, the ratio of p67phox staining in the membrane versus total cellular p67phox was significantly enhanced by a 30-min incubation with LPS (P<0.05; Fig. 3 ) [40 ]. Similar to p47phox, translocation of p67phox to the membrane occurred despite a 24% decrease in the total p67phox content in each PMN (Fig. 3B) . It is important to note that the integrated intensity over the entire cell (the region of interest) is independent of changes in cell area. In contrast to the tandem translocation of p47phox and p67phox by LPS, IL-18 did not cause a specific enrichment of p67phox p67phox in the PMN membranes by 30 min, but did appear to increase the total p67phox content per cell by 33 ± 5% (Fig. 3) .

Surface expression of CD11b and the fMLPr and changes in PMN size and complexity
IL-18 elicited changes in CD11b surface expression in a concentration- and time-dependent fashion, paralleling its priming activity (Fig. 1 , Fig. 4 ). At 15 min, IL-18 (10 and 100 ng/ml) significantly augmented CD11b surface expression as compared with controls (Fig. 4 ; P<0.05). IL-18 concentrations of 0.1–100 ng/ml significantly increased the surface expression of CD11b at 30 min, but only the highest concentration of IL-18 increased CD11b expression at 60 min (P<0.05, n=8; Fig. 4 ). IL-18 concentrations of 0.001–0.01 ng/ml did not affect the surface expression of CD11b at any of the incubation times tested (results not shown).



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  		Figure 4. IL-18 enhancement of CD11b surface expression on PMNs. PMNs were treated for 5–60 min with IL-18 (0.01–100 ng/ml) or buffer control. Following incubation, the PMNs were rapidly cooled to 4°C, incubated with a PE-labeled mAb to CD11b, and fixed with paraformaldehyde. The amount of surface CD11b was measured by flow cytometry (*, P<0.05 as compared with buffer-treated PMNs). The data in this figure represent the results from six different donors.

 
Recent reports have documented that chemoattractants increase the surface expression of the fMLPr [41 , 42 ]. As IL-18 increased CD11b expression, we investigated its effects on the surface expression of the fMLPr. By digital microscopy, IL-18 caused a concentration-dependent increase in the surface expression of the fMLPr (1–100 ng/ml) at 15 min (Fig. 5 ) and 30 min of incubation (results not shown). The change in fMLPr expression was then quantified by flow cytometry and demonstrated that surface expression of the fMLPr was significantly increased at concentrations of IL-18 from 1–100 ng/ml at 30 min of incubation as compared with saline-treated controls (Table 1 ). The observed increases in fMLPr expression in response to IL-18 were similar to the increased expression in response to 2 µM PAF, the positive control (Table 1) . Lesser concentrations of IL-18 (0.01–0.1 ng/ml) increased the surface expression of the fMLPr (P<0.05 as compared with controls).



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  		Figure 5. Digital microscopy of IL-18-induced changes in fMLPr expression in PMNs. Isolated PMNs were pretreated with 0.5% DMSO or 2.5 µM SB203580, a specific p38 MAP kinase inhibitor for 15 min at 37°C in low light. The PMNs were then incubated with differing concentrations of IL-18 (0.01–100 ng/ml) for 15 and 30 min at 37°C and were then immediately cooled to 4°C, washed, and incubated for 30 min with a PE-labeled mAb to CD11b at 4°C. The PMNs were then visualized by digital microscopy at 100x. The figure depicts IL-18-mediated changes in the fMLP at 15 min. The figure is representative of three separate experiments from different donors.

 

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  		Table 1. IL-18 Induced Changes in fMLPr Surface Expression and PMN Size and Complexity

 
Agents that prime or activate PMNs cause changes in PMN size (forward scatter) and cellular complexity (side scatter) [29 , 30 ]. IL-18 caused an increase in PMN size but not complexity over a range of concentrations from 0.1–10 ng/ml as compared with saline-treated controls using a reaction time of 30 min (P<0.05, n=6; Table 1 ). Such changes in PMN size were also demonstrated in PMNs treated for 5 min with 1 µM fMLP or 200 ng/ml PMA (Table 1) . IL-18 (0.1–10 ng/ml) did not affect the cellular complexity of PMNs over the observed reaction times, similar to PMA and fMLP (Table 1) .

Release of elastase from PMNs exposed to IL-18
A previous report documented that incubation of PMNs with IL-18 caused release of the contents from specific granules [22 ]. Because of the importance of the proteases from azurophilic granules, especially elastase, in human disease, we investigated if IL-18 directly caused the release of elastase from isolated PMNs [3 , 31 ]. At concentrations of 0.1–10 ng/ml, IL-18 directly caused elastase release from isolated PMNs as compared with vehicle-treated controls (P<0.05), and the amount of elastase released by IL-18 was similar to fMLP-activated PMNs (Table 2 ). Concentrations of IL-18 less than 0.1 ng/ml did not yield significant elastase release, and IL-18 did not augment fMLP-mediated release of elastase from PMNs.


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  		Table 2. IL-18-Mediated Elastase Release from Isolated PMNs

 
Cytosolic calcium influx following exposure to IL-18
As rapid changes in cytosolic Ca2+ are required for PAF and fMLP stimulation of PMNs, the role of cytosolic Ca2+ in IL-18-mediated stimulation of PMNs was evaluated. Indo-1-loaded PMNs were incubated with IL-18, and the changes in cytosolic Ca2+ concentration were measured over real time (Fig. 6 ) [32 ]. PMNs stimulated with IL-18 showed a rapid, cytosolic Ca2+ response at concentrations (0.1–10 ng/ml) that primed the PMN oxidase and increased CD11b expression. Concentrations of IL-18 less than 0.1 ng/ml, which did not prime the PMN oxidase, did not elicit a detectable change in cytosolic Ca2+ concentration as compared with the baseline cytosolic Ca2+ concentration in buffer-treated PMNs (0.01 ng/ml IL-18: 65±9 nM vs. controls: 68±10 nM; Fig. 6 ). It is also important to note that IL-18 at 10 ng/ml did not increase the peak level of cytosolic Ca2+ but rather caused a prolonged cytosolic Ca2+ flux (Fig. 6) .



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  		Figure 6. IL-18-mediated changes in cytosolic Ca2+. PMNs were loaded with 5 µM indo-1-AM for 10 min at 37°C and washed with fresh, warm KRPD buffer, and the changes in cytosolic Ca2+ in response to IL-18 (0.01–10 ng/ml) were measured in a dual-wavelength spectrofluorimeter. The data represent one of three identical experiments.

 
Activation of the p38 MAP kinase in PMNs stimulated with IL-18
As p38 MAP kinase mediates the actions of LPS, tumor necrosis factor {alpha} (TNF-{alpha}), and other receptor-linked PMN priming agents, we examined IL-18-induced activation of p38 MAP kinase [32 , 43 ]. Previous results from this and other laboratories have demonstrated that dual phosphorylation (Thr 180/Tyr 182) is required for p38 MAP kinase activity, and detection of the dual phosphorylated form of p38 MAP kinase correlates with enzyme activity [32 , 43 44 45 ]. Therefore, in these experiments, p38 MAP kinase activation was investigated by immunoblotting the separated proteins from whole cell extracts of PMNs incubated with 1 ng/ml IL-18 for 15 s–15 min with an antibody to activated p38 MAP kinase. IL-18 activated p38 MAP kinase at 5 min, which became maximal at 10 min, and a mild decrease was at 15 min (Fig. 7A ). This pattern of p38 MAP kinase activation was identical for all IL-18 concentrations tested that primed the PMN oxidase (0.1–100 ng/ml). The effect of IL-18 on p38 MAP kinase activation was concentration-dependent with a significant activation at 0.1 ng/ml and 1 ng/ml; although a band of activity could be visualized at 10 pg/ml, the densitometry was not statistically different from buffer-treated controls (results not shown; Fig. 7B ). Higher concentrations of IL-18 (10 and 100 ng/ml) also caused activation of p38 MAP kinase at identical time points (results not shown).



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  		Figure 7. IL-18-induced p38 MAP kinase activation and the effect of cytosolic Ca2+ chelation. (A) PMNs were treated with 1.0 ng/ml IL-18 or buffer control for 15–900 s. The proteins from the cell lysates were separated by 10% SDS-polyacrylamide gel electrophoresis (PAGE) and were Western blotted with an antibody to active, dual-phosphorylated (Tyr180/Thr182) p38 MAP kinase. The Western blot represents one of three separate experiments. (B) PMNs were treated with IL-18 in concentrations 0.001–1 ng/ml or buffer for 10 min. The proteins from the cell lysates were separated by 10% SDS-PAGE and immunoblotted with an antibody to activated, dual-phosphorylated (Tyr180/Thr182) p38 MAP kinase. The Western blot represents one of four separate experiments. (C) PMNs were pretreated with DMSO or 50 µM BAPTA (B) for 15 min at 37°C and then incubated with IL-18 (0.1–1.0 ng/ml) for 10 min. The PMNs were extracted, and the proteins were separated by SDS-PAGE. The proteins were transferred to nitrocellulose and immunoblotted with an antibody to active, dual-phosphorylated (Tyr180/Thr182) p38 MAP kinase. These data are representative of two identical experiments.

 
To determine the role of cytosolic Ca2+ in IL-18-mediated p38 MAP kinase activation, PMNs were incubated with 50 µM BAPTA, which rapidly chelates cytosolic Ca2+, rendering it biologically unavailable [32 ]. Preincubation of PMNs with BAPTA resulted in inhibition of IL-18 activation of p38 MAP kinase (Fig. 7C) . This inhibition by BAPTA was also observed for higher concentrations of IL-18 (10–100 ng/ml; results not shown).

The effect of p38 MAP kinase inhibition on IL-18-mediated priming of the PMN oxidase and changes in fMLPr expression
As IL-18 caused activation of p38 MAP kinase, we tested the ability of the specific p38 MAP kinase inhibitor SB203580 (2.5 µM) on IL-18 priming of the respiratory burst and IL-18-mediated increases in fMLPr surface expression [34 , 35 , 46 ]. In the oxidase experiments, 200 ng/ml PMA was used as the activator of the oxidase, as PMA-mediated oxidase assembly does not require activation of p38 MAP kinase [32 ]. IL-18 primed the PMA-activated respiratory burst by 1.3- to 2.0-fold as compared with saline-primed, PMA-activated PMNs (n=15, P<0.05; Table 3 ). Pretreatment of PMNs with SB203580 (2.5 µM) inhibited IL-18 priming (10–100 ng/ml) at 15 min by 82±7% and 100±10% (n=6 for all groups; P<0.05; results not shown), and at 30 min, SB203580 inhibited IL-18 priming (0.1–100 ng/ml) of PMA activation by 88 ± 12% to 97 ± 4% (n=5 for all groups; P<0.05). In analogous experiments, SB203580 (2.5 µM) inhibited IL-18-mediated up-regulation ofthe fMLPr, visually (Fig. 5) and by flow cytometry (54±8% to 73±9%) at the 10 ng/ml and 100 ng/ml IL-18 concentrations, respectively.


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  		Table 3. SB203580 and BAPTA Inhibition of IL-18 Priming of the PMA-Activated Respiratory Burst

 
Lastly, because BAPTA chelation of cytosolic Ca2+ inhibited p38 MAP kinase activation, we tested the ability of 50 µM BAPTA to inhibit IL-18 priming of the PMA-activated respiratory burst (Table 3) . BAPTA pretreatment inhibited IL-18 priming in PMNs by 95 ± 6% to 99 ± 4% at the 10–100 ng/ml concentrations (n=5, P<0.05), but inhibition at the 1 ng/ml concentration only approached significance (n=6, P=0.09; Table 3 ).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Initially, the cellular effects of IL-18 were documented in studies using mononuclear leukocytes, specifically the synergistic role of IL-18 with IL-12 for interferon-{gamma} (IFN-{gamma}) production in natural killer cells and T lymphocytes [14 , 15 , 17 18 19 , 21 , 39 ]. These studies focused on IL-18 induction of IFN-{gamma} and its role in host defense [14 , 15 , 17 18 19 , 21 , 39 ]. Moreover, IL-18 appeared to effectively stimulate lymphocytes in the picomolar range, as a single agent or as a cofactor [14 , 15 , 17 18 19 , 21 , 39 ]. In addition, bacterial infections, especially Mycobacterium tuberculosis, resulted in significant increases of IL-18 in vivo [19 ]. From animal data and clinical cases of sepsis and acute lung injury, increased levels of IL-18 appeared to correlate with tissue injury/inflammation [7 , 8 , 13 , 20 , 47 ].

The data presented in this report arose from the hypothesis that physiologic concentrations of IL-18 would stimulate PMNs to facilitate their emigration from the vasculature and augment their microbicidal activity. Distinct from previous work, this report documented that IL-18 caused translocation of the cytosolic protein p47phox to the cell membrane, which appeared to correlate with the observed time course of IL-18-priming activity. Furthermore, LPS also caused similar translocation of the p47phox to the membrane, despite the observed loss of p47phox. This loss of p47phox was presumably a result of membrane secretion and was supported by the loss of WGA staining in LPS-treated PMNs (data not shown). However, LPS also caused translocation of p67phox to the PMN membrane contrary to a previous report [40 ]. Such differences may have been a result of the distinct methods used. First, the present study used LPS concentrations of 2 µg/ml, tenfold higher than the previous study. Second, the present study used an LPS incubation time of 30 min not 60–70 min as used by the previous study. Finally, the present study used fixed, porated PMNs for digital microscopy, whereas the previous studies used immunoblots of subcellular fractions separated by gradient centrifugation [40 ]. Digital microscopy is a well-accepted method of quantifying the cellular locality and content of a number of proteins, requires fewer cells for study, and may be more desirable for some studies because it does not destroy the cellular architecture [26 27 28 ]. Despite the observed differences, both studies implicated a role for LPS-mediated translocation of p47phox to the membrane; moreover, the current study demonstrated an identical role of p47phox translocation in IL-18 priming of the NADPH oxidase [40 ].

The data presented support the hypothesis that IL-18 affected the following PMN functions: oxidase activation, chemotaxis, and azurophilic granule release at concentrations found in septic patients. Furthermore, IL-18 caused changes in PMN size but not cellular complexity, similar to PMA and fMLP. IL-18 also primed the oxidase and stimulated PMN chemotaxis (CD11b expression) in a shorter time course than previously reported (15–30 min vs. 60 min). IL-18 priming of the NADPH oxidase and stimulation of other PMN functions (15–30 min) were similar to other physiologic priming agents, including IL-1ß (5–30 min), LPS (20–60 min), and TNF-{alpha} (5–60 min) [36 , 38 , 48 49 50 ]. However, IL-18 activation of PMNs differed from TNF-{alpha} and IL-1ß by its ability to cause a modest release of elastase [36 , 38 , 48 49 50 ]. Common PMN activation profiles by IL-18 and IL-1ß would be expected as a result of their structural similarities, for both cytokines are primarily all ß-pleated, sheet-folded molecules and, more importantly, because of the structural similarities of the receptors, which have a common, intracellular domain [39 , 51 , 52 ]. Such comparable activity for IL-18 and IL-1ß has previously been reported in lymphocytes [39 ]. In addition, unlike lymphocytes, coincubation of PMNs with IL-12 did not potentiate the effects of IL-18 [53 ]. These results were surprising, as granulocytes express the IL-12 receptor, and IL-12 has been reported to affect PMN proinflammatory function [54 , 55 ]. Thus, it appeared that the observed effects on PMNs were a result of IL-18 alone and not secondary to synergistic action with IL-12.

From investigation of IL-18 signaling, we demonstrated that IL-18 activated p38 MAP kinase in a Ca2++-dependent manner. This p38 MAP kinase activation was directly influenced by the IL-18 concentration and the incubation time, with maximal activity occurring at 10 min. Previous work from this laboratory has demonstrated that dual phosphorylation of p38 MAP kinase signifies p38 MAP kinase activation, further supported by specific in vitro phosphorylation of activating transcription factor-2 [32 ]. In addition, the inhibition of active p38 MAP kinase by SB 203580 or the inhibition of Ca2+-dependent MAP kinase activation with BAPTA directly influenced PMN function. As compared with TNF-{alpha} and LPS, both of which require p38 MAP kinase for maximal priming activity, IL-18 activated p38 MAP kinase earlier than LPS (5–10 min vs. 20 min) and was similar to TNF-{alpha} (10 min) [43 , 45 ]. Investigation of IL-18 signal transduction also demonstrated that IL-18 directly caused changes in cytosolic Ca2+ similar to IL-1ß [37 ]. This increase in cytosolic Ca2+ was required for p38 MAP kinase activation, and such Ca2+-dependent activation of p38 MAP kinase has been shown for other receptor-linked chemoattractants [32 ]. Furthermore, only those concentrations of IL-18 that affected PMN physiology elicited significant activation of p38 MAP kinase.

In conclusion, at physiologic concentrations, IL-18 stimulated PMNs through Ca2+-dependent activation of p38 MAP kinase. Increased levels of IL-18 have been associated with models of traumatic injury, human sepsis, and clinical acute lung injury, a syndrome of PMN-mediated organ injury [8 , 13 , 47 ]. Thus, IL-18 may be involved in syndromes of PMN-elicited organ injury; however, further studies are required to delineate the role of this cytokine in normal PMN physiology and syndromes of PMN-mediated organ injury [56 57 58 59 ].


   ACKNOWLEDGEMENTS
 
This work was supported by Bonfils Blood Center and National Institute of Health grants NHLBI 59355 and 2PG50 GM49222 (C. C. S.) and AI-15614 (C. A. D.). The authors acknowledge Jerry Niedzinski from Laboratories at Bonfils and Patrick Murphy from Information Services, Bonfils Blood Center, for their technical assistance.

Received August 27, 2001; revised March 27, 2002; accepted March 29, 2002.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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