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(Journal of Leukocyte Biology. 2002;72:995-1002.)
© 2002 by Society for Leukocyte Biology

IL-12-dependent nuclear factor-{kappa}B activation leads to de novo synthesis and release of IL-8 and TNF-{alpha} in human neutrophils

Futwan Al-Mohanna, Soad Saleh, Ranjit S. Parhar and Kate Collison

Department of Biological and Medical Research, King Faisal Specialist Hospital and Research Centre, Riyadh, Saudi Arabia

Correspondence: Futwan Al-Mohanna, Biological and Medical Research Department, MBC 03, King Faisal Specialist Hospital and Research Centre, Riyadh 11211, Saudi Arabia. E-mail: Futwan{at}kfshrc.edu.sa


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The cytokine interleukin (IL)-12 plays a bridging role between innate and adaptive immunity. Here, we demonstrate that treatment of neutrophils with IL-12 leads to a transient increase in intracellular-free calcium [Ca++]i levels, which is necessary for the production of reactive oxygen metabolites (ROM). This production is associated with the activation and nuclear translocation of the transcription factor nuclear factor (NF)-{kappa}B and is inhibited in the presence of the intracellular calcium chelator 1,2-bis(O-amminophenoxy) ethane-N,N-N',N'-tetraacetic acid-acetoxymethyl ester and the ROM production inhibitor diphenyl iodonium. We show that IL-12 causes a significant increase in total mRNA levels, which appear dependent on the generated ROM. In addition IL-12 induces the de novo synthesis and production of IL-8 and tumor necrosis factor {alpha} (TNF-{alpha}) in a calcium- and ROM-dependent manner. Our data demonstrate a direct role for IL-12 in the activation of human neutrophils and suggest a ROM-dependent interplay between IL-12-induced [Ca++]i transient and the release of IL-8 and TNF-{alpha} through NF-{kappa}B activation.

Key Words: calcium • diphenylene iodonium • neutrophils


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The neutrophils are the major innate immune cells comprising approximately 60% of circulating white blood cells. They are designed to rid the body of infection through an array of functions, namely chemotaxis, phagocytosis, intracellular killing, and secretion. Each of these functions is thought to be mediated by a multitude of intracellular signals, which ensures that a specific stimulus is coupled to a specific response, i.e., stimulus-response coupling. These cells roam the vasculature with frequent transmigration into adjacent tissues through diabedesis. Because of the relatively short life span of these cells (approximately 6 h in circulation and 1–2 days in tissues), together with the paucity of protein synthesis capabilities within them, it was assumed for many years that very little nuclear activities occurred in the neutrophils.

The proinflamatory and immunoregulatory cytokine interleukin-12 (IL-12) is produced by many cell types, including human neutrophils, in response to soluble and particulate stimuli [1 2 3 4 ]. This heterodimeric cytokine induces the expansion of T helper type 1 cells, which produce IL-2 and interferon-{gamma} (IFN-{gamma}), and inhibits IL-4-producing cells [5 6 7 ]. IL-12 also stimulates the production of IFN-{gamma} and tumor necrosis factor {alpha} (TNF-{alpha}) from natural killer (NK) cells and enhances cell-mediated immunity by the increased production of opsonins such as immunoglobulin G2a isotype [8 9 10 11 ].

The transcription factor nuclear factor-{kappa}B (NF-{kappa}B) comprises a number of proteins, namely P50/105 (NF-{kappa}B1), P65 (RelA), P52/100, and RelB. These proteins homo- or heterodimerize to produce isoforms of NF-{kappa}B. The most abundant, inducible form in mammals is p50/p65, a heterodimer that is sequestered in the cytosol of unstimulated cells by its interaction with the I{kappa}B family of inhibitor proteins [12 13 14 15 ]. I{kappa}B masks nuclear localization signals (NLS) of NF-{kappa}B, thus keeping the complex in the cytosol. Upon stimulation, I{kappa}B is phosphorylated and proteolytically activated, thus unmasking NLS [16 17 18 ]. Phosphorylated NF-{kappa}B translocates to the nucleus and starts the transcription of many target genes [19 ].

We have previously reported that human neutrophils express functional receptors for IL-12, the engagement of which leads to calcium-dependent activation with the consequential production of reactive oxygen metabolites (ROM) [20 ].

In the present work, we investigate the downstream effects of IL-12 receptor engagement following calcium and ROM production. We demonstrate that IL-12 activates NF-{kappa}B through its ability to produce ROM in a calcium-dependent manner. We also show that such activation leads to the de novo transcription and translation of two key inflammatory mediators, namely IL-8 and TNF-{alpha}.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
IL-12 used in initial experiments was a kind gift from Dr. S. Wolf (Genetics Institute Inc., Cambridge, MA) and was subsequently purchased from Genetics Institute Inc. and R&D Systems (Minneapolis, MN). Fluo-3 AM, Fura-Red AM, 6-carboxy-2', 7'-dichlorodihydrofluorescein diacetate (2,7 DCFDA), ionomycin, and 1,2-bis(O-amminophenoxy) ethane-N,N-N',N'-tetraacetic acid-acetoxymethyl ester (BAPTA-AM) were purchased from Molecular Probes (Eugene, OR) and were dissolved in dimethylsulfoxide (DMSO) and delivered to the cells at final concentrations of 1 µM, 1 µM, 1 µM, 2 µM, and 10 µM, respectively, in a final DMSO concentration of 0.1%. 5–3H uridine and diphenylene iodonium (DPI) were purchased from ICN (Irvine, CA). Enzyme-linked immunosorbent assay (ELISA) kits for NF-{kappa}B were obtained from Active Motif (Carlsbad, CA). ELISA kits for quantification of IL-8 and TNF-{alpha} were purchased from R&D Systems. All other reagents were AnalaR grade and were purchased from BDH Chemicals (Poole, UK) Lipopolysaccharide was routinely tested for and found to be undetectable.

Preparation of human neutrophils
Human peripheral blood neutrophils were prepared by dextran sedimentation of heparinized whole blood obtained from healthy donors and centrifuged through Ficoll-Paque as described previously [20 ]. Contaminating red blood cells were removed by hypotonic lysis with isotonic NH4Cl. The remaining cells were suspended in Krebs-HEPES medium (pH 7.4) containing 120 mM NaCl, 1.3 mM CaCl2, 1.2 mM MgSO4, 4.8 mM KCl, 1.2 mM KH2PO4, 25 mM HEPES, and 0.1% bovine serum albumin (BSA) and were further purified through neutrophil-isolation medium (Cardinal Associates, Santa Fe, NM). Final purity and viability were between 98% and 99% as indicated by flow cytometry and trypan blue dye exclusion tests.

Measurement of calcium
Neutrophils were loaded with Fluo-3 AM (1 µM) or Fura-Red (1 µM) as described previously [20 ]. The cells were washed, placed on glass coverslips, and allowed to adhere for 15 min at room temperature. Coverslips were secured between two plates of a custom-designed coverslip holder and placed onto a heated microscope stage (33°C), and [Ca++]i images were acquired at 140 m-s intervals, on average, using a Leica DM iRBE inverted microscope attached to a true confocal system (Leica-Kaki, Riyadh, Saudi Arabia). Images were analyzed using Scanware software (Leica Microsystems, Heidleberg, Germany), and fluorescence intensity (from each cell) was transformed into absolute calcium levels as described previously [21 ].

Measurement of ROM production
The production of ROM by neutrophils was measured using luminol-dependent chemiluminescence (LDCL) as described previously [22 , 23 ]. To confirm that ROM production was a result of activation of the neutrophil reduced nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase system and not a result of effects on LDCL, oxygen consumption rates of neutrophils following IL-12 challenge were also measured using a Clark-type oxygen electrode (Oxygraph, Gilson Medical Electronics, Middleton, WI), essentially as described previously [22 ]. ROM production was also followed fluorimetrically using 6-carboxy-2,7 DCFDA, essentially as described previously [24 ]. Briefly, 106 neutrophils were incubated with 1 µM 2,7 DCFDA (room temperature for 15 min). Cells were washed and allowed to adhere to glass coverslips (10 min at room temperature). Coverslips were washed and secured between two plates of a custom-designed coverslip holder, placed onto a microscope stage, and viewed using Leica TCS confocal microscope (Leica-Kaki). Images were obtained at a temporal resolution of 140 ms/image. Fluorescence intensity was measured using the Leica Scamware software (Leica Microsystems) and was transferred to an Excel spreadsheet for further analysis.

Measurements of NF-{kappa}B translocation
Translocation of NF-{kappa}B from the cytosol to the nucleus was followed using indirect immunofluorescence and confocal microscopy. Briefly, cells were allowed to adhere to glass coverslips at room temperature for 15 min before washing with Krebs-HEPES buffer to remove nonadherent cells. The coverslips were then treated with IL-12 (1 ng/ml) for 15 min at 37°C. Cells were washed, fixed [3.7% formaldehyde in phosphate-buffered saline (PBS), 15 min at room temperature], and then permeabilized (0.5% Triton-X-100 in PBS) for 5 min at room temperature. Cells were washed and probed with rabbit anti-human NF-{kappa}B/p50 (Santa Cruz Biotechnology, Santa Cruz, CA) and then viewed by indirect immunofluorescence using fluorescein isothiocyanate (FITC)-conjugated secondary antibody (Santa Cruz Biotechnology). Nuclei were labeled in situ after obtaining the NF-{kappa}B images by the addition of ethidium bromide (0.5 µg/ml) to the medium bathing the cells. All images were obtained using confocal microscopy at a full width half maximum of 2 µm.

Cell extracts preparation and electrophoretic mobility shift assay (EMSA)
Neutrophils were incubated with and without IL-12 (1–10 ng/ml) at 37°C. BAPTA treated cells were preincubated at room temperature for 30 min with BAPTA-AM (10 µM) and then incubated with IL-12 for the time indicated. As a positive control, neutrophils were also stimulated with formyl-Met-Leu-Phe (1 µM), which was previously shown to activate NF-{kappa}B [25 ]. Whole-cell extracts were prepared by pelleting the cells for 10 s and resuspending in 400 µl ice-cold lysis buffer containing 1.5 mM MgCl2, 10 mM KCl, 0.5 mM EDTA, 0.1 mM EGTA, 0.5 mM dithiothreitol (DTT), 0.2 mM phenylmethylsulfonyl fluoride, 20% glycerol, 1% (v/v) Nonidet P-40, HEPES-KOH (10 mM), pH 7.9, and protease inhibitor cocktail containing chymostatin (5 µg/ml), pepstatin A (5 µg/ml), antipain (5 µg/ml), and leupeptin (10 µg/ml). Cells were allowed to swell on ice for 10 min and then vortexed to sheer the cytoplasmic membrane. Cell debris was removed by centrifugation at 14,000 g for 10 min at 4°C. The supernatant (containing the DNA-binding proteins) was stored at -70°C. Total protein was determined using the Sigma Chemical Co. (St. Louis, MO) assay kit, according to the manufacturer’s instructions using BSA as standard.

EMSA was performed as previously described [26 ] with slight modifications. Briefly, binding reactions were performed in 20 µl binding buffer (20% glycerol, 5 mM MgCl2, 2.5 mM EDTA, 2.5 mM DTT, 250 mM NaCl, 50 mM Tris-HCl, 1.5 mM HEPES) in the presence of nonspecific blocker poly di-dc (1 µg). Extracts were then added and allowed to equilibrate for 15 min at room temperature. For supershift experiments, binding reactions were performed in the presence of specific antisera for 20 min at 4°C. Specificity was evaluated by adding 50-fold excess of a specific unlabeled, double-stranded probe. Finally 50,000 cpm/sample of an oligonucleotide containing the consensus NF-{kappa}B sequence (5'-AGTTGAGGGGACTTTCCCAGGC-3'), 32P-end-labeled, was added to the binding mixture and incubated for 30 min at room temperature. The samples were electrophoresed on 7% native gels, in 0.5x Tris-borate-EDTA (TBE) running buffer at 100 V for 3 h. Gels were vacuum-dried and subjected to autoradiography at -70°C overnight. Autoradiograms were scanned and analyzed using Foto/Analysis PC Image, Version 1.01 (Fotodyne, Inc., Hartland, WI). Confirmation of EMSA results was performed using a TransAM NF-{kappa}Bp50/NF-{kappa}Bp65 transcription factor assay kit according to the manufacturer’s recommendations (Active Motif). The mutated NF-{kappa}B sequence used was 5'-AGTTGAGGCCACTTTCCCAGGC-3'. ELISA plates were read using CERES UV 900C spectrophotometer (Bio-TEK Instruments Inc., Winooski, VT).

RNA synthesis assay
RNA synthesis was performed by measuring [5–3H] uridine incorporation into RNA as previously described [27 ]. Briefly, neutrophils (100 µl 5x106/ml) were incubated with 1 µCi [5–3H] uridine in 96-well plates in the presence and absence of various treatments for 4 h at 37°C, 5% CO2. Cells were thoroughly washed and then harvested by suction into distilled water with a multiple cell culture harvester on borosilicate glass fiber paper. The sections of the filter paper corresponding to each micro well were then punched out and placed in scintillation counting vials in the presence of aquasol-2.

Detection of IL-8 and TNF-{alpha} by reverse transcriptase-polymerase chain reaction (RT-PCR)
Total RNA was isolated from 5 x 106 neutrophils (>98% purity) by the guanidine isothiocyanate method using TRI REAGENT (Sigma Chemical Co.), according to the manufacturer’s instructions. cDNA was synthesized from the total RNA using avian myloblastosis virus-RT (Promega, Madison, WI) according to the manufacturer’s protocol. PCR amplification of human IL-8, human TNF-{alpha}, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was carried out using specific primers that yield 335 bp, 548 bp, and 399 bp products for IL-8, TNF-{alpha}, and GAPDH, respectively. PCR was performed using ReadymixTM TAQ with MgCl2 (Sigma Chemical Co.) on PTC-200 Peltier thermal cycler (MJ Research Inc., Las Vegas, NV). The primers for the examined cytokines were as follows: IL-8 upstream primer, 5'-GGA CAA GAG CCA GGA AAC C-3', downstream primer, 5'-CTT CAA AAA CTT CTC CAC AAC C-3'; TNF-{alpha} upstream primer, 5'-CTT CTG CCT GCT GCA CTT TGG A-3', downstream primer, 5'-TCC CAA AGT AGT CCT GCC CAG A-3'; GAPDH upstream primer, 5'-TGG AAA GCT GTG TGA TG-3', downstream primer, 5'-TCC ACC ACC CTG TTG CTG TAG C-3'. Thirty-five cycles of denaturing for 1 min at 94°C, annealing for 1 min at 58°C, and extension for 1 min at 72°C were used for amplification. PCR products were electrophoresed on 2% agarose gels and stained with ethidium bromide.

Quantification of IL-8 and TNF-{alpha}
Quantification of IL-8 and TNF-{alpha} was done by ELISA. Briefly, samples of human neutrophil suspension in Krebs-HEPES medium (5x106 cells/ml) were incubated with the IL-12 (1 ng/ml) for various time intervals at 37°C and 5% CO2. Cells were pelleted for 10 s, and supernatants were collected for ELISA analysis. ELISA was performed using paired capture and biotinylated detection of antibodies from R&D Systems, according to the manufacturer’s instructions. Standard curves were constructed using serial dilutions. ELISA plates were read using CERES UV 900C spectrophotometer (Bio-TEK Instruments).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Effect of IL-12 on calcium and ROM in human neutrophils
Resting, human naïve neutrophils exhibited a calcium level of 87 ± 5 nM. This was increased to 770 ± 30 (n=31) by treatment with IL-12 (1 ng/ml). The response to IL-12 was asynchronous and heterogenous (Fig. 1a ), and the number of neutrophils responding to IL-12 depended on the concentration used. The calcium change seems to be mediated by release from intracellular store(s), as revealed by confocal microscopy of fluo-3-loaded neutrophils stimulated by IL-12 (1 ng/ml) in the absence of extracellular calcium and the presence of 1 mM extracellular EGTA (Fig. 1b) . As calcium is associated with the activation of the neutrophil oxidase system, we investigated the effect of IL-12 on the neutrophils ability to produce ROM. In a series of experiments, we found that IL-12 induced ROM (Fig. 1c) . This activation was inhibited in the presence of the intracellular calcium challator BAPTA-AM (10 µM), the neutrophil oxidase system inhibitor DPI (10 µM), and antibodies to the IL-12 receptor (200 ng/ml; Fig. 1c ).



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  		Figure 1. Activation of human neutrophils by IL-12. (a) Intracellular calcium maps following challenge of human neutrophils with IL-12 at various time intervals as indicated by the numbers under each frame in a population of cells under normal concentration of extracellular calcium and (b) in the absence of extracellular calcium and presence of 1 mM EGTA. The color bar indicates changes in intensity such that cold colors indicate low intensity, and warm colors indicate high intensity. (c) Luminol-dependent chemiluminescence responses following IL-12 treatment under various conditions as indicated on each trace and at the concentrations indicated in the text. Arrows indicate the time of addition of IL-12.

 
NF-{kappa}B activation following IL-12 treatment
As the transcription factor NF-{kappa}B is intimately involved in regulating many proinflammatory mediators, we tested the possibility of its involvement in IL-12 signaling in human neutrophils. Using indirect immunofluorescence and confocal microscopy, we found that resting neutrophils displayed diffused cytosolic fluorescence with a dark, nonstained central nuclear area when probed with anti-NF-{kappa}B/p50 antibodies (Fig. 2a ). However, 15 min after treatment with IL-12 (1 ng/ml), this pattern was changed, and a more pronounced fluorescence appeared in the nuclear area, suggesting translocation of this protein to the nuclear milieu. No translocation was observed in control experiments using nonimmune rabbit serum. EMSA revealed the existence of two major bands associated with the shifted consensus oligonucleotide sequence for NF-{kappa}B, displaying increased intensity with time following treatment of naïve neutrophils with IL-12 (1 ng/ml; Fig. 2b ). No such increase was observed in the presence of excess unlabeled probes. As the p50 homodimer and NF-{kappa}Bp50/p65 (NF-{kappa}B1/RelA) heterodimers are the most common found in the NF-{kappa}B signaling pathway, we tested the possibility of involvement of p50 and p65 in IL-12 signaling. Using an ELISA-based kit to detect and quantify NF-{kappa}B activation, we found that IL-12-treated neutrophils exhibited a significant increase in NF-{kappa}B/p50 activity that was inhibited in the presence of the intracellular calcium chelator BAPTA-AM (Fig. 3a ). In contrast, no significant increase in NF-{kappa}B/p65 activity was observed following IL-12 treatment. The involvement of NF-{kappa}B/p50 was further confirmed in supershift experiments with antibody to the NF-{kappa}B/p50 and NF-{kappa}B/p65 (Fig. 3c) . We found that antibody NF-{kappa}B/p50 supershifted both bands, whereas the antibody to NF-{kappa}B/p65 failed to shift either band. Although EMSA and ELISA experiments demonstrated that prior treatment of neutrophils with the intracellular calcium chelator BAPTA-AM inhibited the IL-12-induced NF-{kappa}B activation (Fig. 3a and 3b ), the possibility existed that signals downstream from calcium might be responsible for the observed NF-{kappa}B activation. The most likely candidate is ROM, especially as NF-{kappa}B activation has been linked to ROM production in other cell types [14 , 28 , 29 ]. For this, we tested the effect of inhibiting the neutrophil’s nonmitochondrial oxidase system, the major producer of ROM, on the IL-12-induced, NF-{kappa}B activation. In a series of experiments, we treated naïve neutrophils with DPI (10 µM) prior to exposure to IL-12. Under such conditions, DPI abolished or significantly reduced the NF-{kappa}B activation as detected by EMSA and ELISA (Fig. 3a and 3b ). The possibility that DPI itself might inhibit the IL-12-induced calcium rise was addressed by simultaneously measuring calcium homeostasis and ROM production using Fura-Red and 2,7 DCFDA as calcium and ROM indicators, respectively. In dually labeled cells, treatment with IL-12 (1 ng/ml) caused a transient decrease in Fura-Red fluorescence with a concomitant increase in 2,7 DCFDA fluorescence (Fig. 4a ), a result that was consistent with a transient increase in intracellular-free calcium levels and ROM production. The IL-12-induced Fura-Red intensity changes consisted of an initial decrease followed by a recovery phase that was consistent with a transient increase in intracellular-free calcium levels. Treatment of the cells with DPI prior to IL-12 challenge caused a sustained decrease in the fluorescence intensity of Fura-Red and 2,7 DCFDA (Fig. 4b) . This is consistent with an increase in calcium but not ROM production. It is noteworthy, however, that the kinetics of IL-12-induced calcium change was modified by DPI such that sustained rather than transient changes were observed (Fig. 4b) .



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  		Figure 2. Activation of NF-{kappa}B following treatment of human neutrophils with IL-12. (a) Translocation of NF-{kappa}B to neutrophils nuclei 15 min after IL-12 challenge. Confocal micrographs of human neutrophils using rabbit polyclonal antibodies to human NF-{kappa}B/p50 (Santa Cruz Biotechnology) or nonimmune rabbit serum in an indirect immunofluorescence using antirabbit-FITC-labeled secondary antibody (Santa Cruz Biotechnology; i and iii, respectively). Nuclei were stained positive after addition of ethidium bromide (0.5 µg/ml) at the end of the experiment (ii and iv). (b) Time-dependent activation of NF-{kappa}B as revealed by EMSA. SP and SP1 indicate the the presence of 50-fold excess of a specific and nonspecific, unlabeled, double-stranded probe, respectively.

 


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  		Figure 3. Activation of NF-{kappa}B by treatment of neutrophils with IL-12. (a) ELISA quantitation of NF-{kappa}B/p50 (dark histograms) and NF-{kappa}B/p65 (light histograms)-dependent activation. Each data point represents the mean ± SEM of triplicate samples obtained from the same donor. The data are representative of at least four different experiments with blood obtained from four different individuals. (b) EMSA showing inhibition of NF-{kappa}B activation by pretreatment of cells with BAPTA-AM (10 µM) and DPI (10 µM). Each treatment consists of three lanes showing NF-{kappa}B status in resting or stimulated cells (left lane), in the presence of 50-fold excess of cold, unlabeled probe (middle lane), and in the presence of 50-fold excess of cold, unlabeled, nonspecific probe (right lane). (c) EMSA showing supershift of IL-12-induced NF-{kappa}B complexes in the presence of antibody to NF-{kappa}B/p50 (p50), antibody to NF-{kappa}B/p65 (p65), and nonimmune serum (NIS).

 


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  		Figure 4. Dual measurements of intracellular calcium and intracellular ROM in neutrophils following activation by IL-12. The arrowhead indicates time of addition of IL-12. Neutrophils were labeled with Fura-Red (solid squares) and 2,7 DCFDA (solid circles) as indicated in Materials and Methods. Cells were observed using confocal microscopy. Images were analyzed using Scanware software, and intensity changes are displayed. (a) Control experiment; (b) similar experiment performed in cells treated with DPI (10 µM) prior to addition of stimulus. The data are from one representative cell from one individual representative of at least 20 different fields obtained from four different donors.

 
Effect of IL-12 on total RNA synthesis
As RNA expression pattern in neutrophils is dependent on the state of cellular activation [30 , 31 ], we tested the effect of IL-12 on total RNA synthesis as estimated by [5–3H] uridine incorporation. We found that RNA synthesis was stimulated in the presence of IL-12 (1 ng/ml; P<0.0001; Fig. 5a ). This effect was totally abolished in the presence of actinomycin D (5 µg/ml). It is interesting that inhibition of the IL-12-induced intracellular calcium transient by the intracellular calcium chelator BAPTA-AM inhibited RNA synthesis significantly (P=0.0006). Similarly, DPI at the concentration used (10 µM) also inhibited total RNA synthesis (P=0.0012). This effect was significantly inhibited in the presence of antibodies to the IL-12 receptor (P=0.0006).



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  		Figure 5. Effect of IL-12 on mRNA synthesis. (a) Total RNA synthesis as measured by [5–3H] uridine incorporation in unstimulated neutrophils (Cells) and in the presence of actinomycin D (cells+AD), after treatment with IL-12 in the absence (IL-12) and presence of actinomycin D (IL-12+AD), in the presence of antibody to IL-12 receptor (IL-12+antiIL-12R), in the presence of BAPTA (IL-12+BAPTA), and in the presence of DPI (IL-12+DPI). (b) RT-PCR of TNF-{alpha} and GAPDH transcripts from quiescent and IL-12-treated neutrophils at various time intervals and (c) RT-PCR of IL-8 and GAPDH transcripts from quiescent and IL-12-treated neutrophils at various time intervals. Occurrence of TNF-{alpha} and IL-8 amplicons was inhibited in the presence of BAPTA or DPI. Time intervals are in min; o/n is overnight. The data are representative of at least five different experiments with cells isolated from five different donors.

 
Effect of IL-12 on IL-8 and TNF-{alpha} production
As neutrophils are among the first cells intercepting an invading microorganism, the possibility existed that activation of these cells may lead to amplification of the inflammatory signal so as to recruit more neutrophils and other cell types to the site of inflammation. Therefore, we tested the ability of neutrophils to produce inflammatory mediators following IL-12 challenge. In a series of experiments, we measured the effect of IL-12 on de novo synthesis of TNF-{alpha} and the chemokine IL-8. Using RT-PCR, we found that IL-12 caused the transient increase in the mRNA levels of TNF-{alpha} and IL-8, as indicated by the increased mRNA amplicons for both of these mediators (Fig. 5b) . This increase reached a maximum within 30 min before decaying to prestimulatory levels. Occurrence of these amplicons was inhibited by pretreatment of the cells with the intracellular calcium chelator BAPTA-AM and ROM inhibitor DPI (Fig. 5b and 5c ). Expression and release of these two mediators were subsequently measured using ELISA. In a series of experiments, we found that treatment of neutrophils with IL-12 (1 ng/ml) caused a time-dependent increase in the IL-8 levels, reaching a maximum within 3–4 h, and remained elevated 18 h post-exposure to IL-12 (Fig. 6a ). The level of release varied between neutrophil donors, and the maximum amount was released by 106 neutrophils ranging from 52 pg to 1735 pg (median=198 pg/106 cells). Similarly, the release of TNF-{alpha} was time-dependent, reaching a maximum within 3–4 h, and remained elevated at 18 h post-exposure of neutrophil to IL-12 (Fig. 6b) . Like IL-8, the amount of released TNF-{alpha} varied between donors and ranged from 62 pg to 117 pg (median=85 pg per 106 cells). The release of these two mediators was inhibited by BAPTA-AM and by DPI (Fig. 6c and 6d ).



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  		Figure 6. Induction of TNF-{alpha} and IL-8 release by IL-12. (a) Time course of TNF-{alpha} released from 106 neutrophils as measured by ELISA in quiescent (0) and at 5 min, 10 min, 30 min, 60 min, 4 h, and 18 h. (b) Time course of IL-8 released from 106 neutrophils as measured by ELISA in quiescent (0) and at 10 min, 30 min, 60 min, 4 h, and 18 h. The survival rate of neutrophils after 18 h was assessed by trypan blue exclusion test and found to be more than 90%. Striped histogram is control, and solid histogram is IL-12-treated cells. Each data point is the mean ± SEM from a representative experiment of at least five different experiments with cells isolated from five different donors. (c and d) Effect of DPI and BAPTA on the IL-12-induced TNF-{alpha} and IL-8 release. Each histogram is the mean ± SEM of a representative experiment performed in triplicate with neutrophils isolated from one donor. The data are representative of at least six different experiments performed with neutrophils isolated from six individuals.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
IL-12 is a 75-kDa heterodimeric cytokine, known to act as a bridge between the innate and adaptive immunity with a major role in infection [7 ]. It is produced primarily by phagocytic cells including neutrophils [5 , 32 , 33 ]. IL-12 has been shown to act on various immune cells, including T and B lymphocytes and peripheral blood neutrophils [1 , 20 ]. Its biological activities include enhancement of NK and cytolytic T lymphocyte functions and induction of cytokine production, particularly IFN-{gamma}, from NK and T cells [34 35 36 ].

The neutrophils are terminally differentiated cells of the innate-immune system with the primary function of ridding the body of infection. They are key mediators in the inflammatory responses and are considered the major producers of IL-12 [2 , 33 ]. Receptors for IL-12 are abundant on the neutrophil membranes [20 ]. Engagement of such receptors leads to cytoskeletal rearrangements, transient increase in intracellular calcium levels, tyrosine phosphorylation, ROM production, and enhanced phagocytosis [20 ]. Recent studies have shown that various soluble and particulate stimuli induce neutrophils to transcribe and translate genes that encode inflammatory mediators [37 , 38 ]. The mechanism(s) involved in gene transcription in neutrophils have not been fully elucidated. Transcription factors of the signal transducer and activator of transcription family, AP1, GATA1 and -2, and NF-{kappa}B [14 , 39 40 41 42 43 44 ], have all been ascribed to neutrophil gene expression. Of these, NF-{kappa}B stands out as a major player because of its activation by ROM, which is liberally produced following neutrophil stimulation by a variety of soluble and particulate stimuli. NF-{kappa}B is an inducible transcription factor that is involved in nucleocytoplasmic signaling. The potential for neutrophils to use NF-{kappa}B as a transcription factor has been described [14 , 45 ] together with the cell’s ability to de novo synthesize RNA and proteins following activation [37 , 38 , 46 ].

NF-{kappa}B was shown to regulate the expression of a wide variety of cellular genes including cytokines, inducible nitric oxide synthase, cell adhesion molecules, and proinflammatory mediators [28 , 29 , 46 47 48 ]. In addition, NF-{kappa}B has been implicated in the pathophysiology of many diseases such as cancer, rheumatoid arthritis, and inflammatory diseases [49 , 50 ].

In the present manuscript, we show that following IL-12 receptor engagement, the neutrophils mobilize their intracellular calcium transiently. This mobilization is necessary for ROM production. Evidence for this was drawn from the finding that chelation of intracellular calcium by BAPTA-AM totally abolishes the IL-12-induced ROM production. Nucleocytoplasmic interactions following IL-12 receptor engagement were tested by investigation of NF-{kappa}B activation. We found that IL-12 caused the translocation of NF-{kappa}B from the cytosol to the nucleus within 15 min of IL-12 treatment, suggesting phosphorylation and activation of NF-{kappa}B. That this translocation leads to nuclear activity was demonstrated by EMSA and supershift analysis, which revealed two major bands that were supershifted by antibodies to NF-{kappa}B/p50. Whether these bands corresponded to p50 homodimers or p50/p65 heterodimers was ascertained by using an ELISA-based kit to detect NF-{kappa}B activation. We found that the IL-12 caused a transient increase in the activation of NF-{kappa}B/p50 but not NF-{kappa}B/p65, suggesting lack of involvement of the p50/p65 heterdimeric pathway in the IL-12-induced NF-{kappa}B activation. EMSA and ELISA analyses demonstrated inhibition of NF-{kappa}B activation in the presence of the calcium chelator BAPTA-AM. Two possibilities therefore existed: a direct role of calcium in inhibiting NF-{kappa}B activity or an indirect role through calcium-induced ROM production. Repeating the experiment in the presence of the NADPH-oxidase inhibitor, DPI tested these possibilities. Under such conditions, DPI inhibited the NF-{kappa}B mobility, suggesting that under conditions in which the IL-12-induced calcium transient was kept relatively intact, NF-{kappa}B activity was inhibited by the inhibitor of ROM production. It is noteworthy, however, that using EMSA, BAPTA-AM and DPI inhibited IL-12-induced NF-{kappa}B activation to a level that is considerably lower than that observed in unstimulated cells. This suggests that the neutrophils were already activated during their isolation procedure, hence the residual NF-{kappa}B activation and/or that unstimulated neutrophils do exhibit small ROM-induced NF-{kappa}B activity. The former seems unlikely, as no other indications of neutrophil activation (such as high-resting calcium level and cell aggregation) were observed over the time scale of these experiments. The latter seems likely, as resting neutrophils still need to maintain a steady state activity that may involve nucleocytoplasmic interactions through NF-{kappa}B, which in itself suggests a low-level production of ROM in the apparent absence of incoming signals. However, it is interesting that although NF-{kappa}B/p65 was not activated by IL-12 receptor engagement, resting activity was significantly reduced in the presence of BAPTA-AM or DPI, suggesting that residual, resting nucleocytoplasmic interactions in neutrophils may involve NF-{kappa}B/p65.

The central role for oxidative stress in the activation of NF-{kappa}B has been challenged by Bowie and O’Neill [51 ], who argued that such a role was invariably cell-specific and often open to reinterpretation. In our manuscript, we demonstrate that inhibition of oxidative stress by DPI was sufficient to inhibit NF-{kappa}B activation, even under conditions where a major signal-transducing pathway, i.e., calcium, was still intact, and that the calcium signal alone was not sufficient to activate this transcription factor.

Total mRNA content following IL-12 treatment was found to be dependent on ROM production. The ability of neutrophils to transcribe genes was not realized until very recently. It was generally thought that these terminally differentiated cells have very little nuclear activity aside from that what is needed for general housekeeping maintenance. In the present manuscript, we demonstrate that two proinflammatory cytokines, namely IL-8 and TNF-{alpha}, are induced at the message and protein levels by IL-12 in a NF-{kappa}B-dependent manner. The variations in the amount of released IL-8 and TNF-{alpha} in response to IL-12 may be related to the donors’ genetic makeup.

It is known that activated neutrophils produce many inflammatory mediators, which have a direct impact on many immune cells including the neutrophils themselves. This dual autocrine/paracrine signaling might serve to spatially limit the inflammatory response by immobilizing the neutrophils to the specific site of inflammation and amplifying the response so as to recruit new cells into that site.

Judging by the increased total mRNA levels following IL-12 receptor engagement, it is likely that many more proinflammatory mediators are also induced.


   ACKNOWLEDGEMENTS
 
The authors are indebted to Drs. A. A. Kwaasi, B. F. Meyer, and K. S. A. Khabar for critical discussions and Dr. S. Wolf for the kind gift of IL-12 and critical review of the manuscript.

Received March 10, 2002; revised June 8, 2002; accepted July 12, 2002.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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