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Department of Biological and Medical Research, King Faisal Specialist Hospital and Research Center, Riyadh, Saudi Arabia
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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from NK and
T cells (3). IL-12 and IL-12-induced IFN- prime CD4+ T
cells, resulting in differentiation and proliferation of Th1 cells (4).
Three classes of receptor (IL-12R) with different affinities for IL-12
have been identified on T lymphoblast cell membranes (5, 6, 7). Their
engagement has been associated with phosphorylation of two members of
the Janus kinase (JAK)3
family, namely JAK2 and TYK2 tyrosine kinases (8), and three STATs,
namely STAT1, -3, and -4 (9). Neutrophils, the predominant circulating leukocytes, play a central role in the innate defense against invading micro-organisms. Neutrophil production of IL-12 is well established, and based on their sheer numbers, neutrophils are considered the major producer of IL-12 in vivo (10, 11). Although the immunoregulatory role of IL-12 is well characterized, its direct effect(s) on innate immune cells such as neutrophils has yet to be defined.
In this paper we show the direct binding of IL-12 to human neutrophils. This binding causes a transient calcium rise and is accompanied by actin polymerization, tyrosine phosphorylation, and increased production of reactive oxygen metabolites (ROM). Our data demonstrate the existence of IL-12R on human neutrophils and directly implicate IL-12 in calcium-dependent activation of neutrophils.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Preparation of human neutrophils
Human peripheral blood neutrophils were prepared by dextran sedimentation of heparinized whole blood obtained from healthy donors and were centrifuged through Ficoll-Paque as described previously (12). Contaminating RBC 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% BSA and were further purified through neutrophil isolation medium (Cardinal Associates, Santa Fe, NM). Final purity and viability were both between 98 and 99% as indicated by flow cytometry and trypan blue dye exclusion tests.
Radioiodination and binding of IL-12
Preservative-free IL-12 was iodinated as described previously (13). The labeled protein was isolated on a Sephadex G-25 column, and its specific activity was determined (2.25 µCi/µg of protein). For binding experiments, 2.5 x 105 neutrophils were seeded in 96-well, flat-bottom, Microtest III tissue culture plates (Becton Dickinson, Mountain View, CA) and allowed to adhere for 30 min at room temperature. Twenty microliters of radiolabeled IL-12 were added to each well in a total volume of 250 µl of cell suspension, and the cells were kept on ice for 1 h and washed thoroughly to remove any unbound radioactivity. The cells were harvested, and the radioactivity associated with them was measured in a gamma counter (CliniGamma 1272, LKB-Wallac, Turku, Finland) and expressed as a percentage of the total added radioactivity obtained from 20 µl of labeled protein. Specific binding was measured as the cell-associated radioactivity due to [125I]IL-12 alone minus that with [125I]IL-12 in the presence of a 50-fold molar excess of unlabeled IL-12.
Detection of IL-12R by indirect immunofluorescence
Expression of IL-12R on neutrophils was investigated by FACS analysis (Becton Dickinson). Freshly isolated human neutrophils (2.5 x 105 cells in 250 µl) were incubated with anti-IL-12R Ab alone (10 ng/ml for 45 min on ice), with carrier-free human IL-12 (100 ng/ml, 30 min on ice) then with anti-IL-12R Ab as described above, or with nonimmune rabbit serum (nonspecific binding). Cells were pelleted, washed, and incubated with FITC-labeled secondary Ab (Pierce, Rockford, IL) at a 1/50 dilution for 30 min on ice. Cells were pelleted, washed with PBS (three times), and resuspended in 500 µl of ice-cold PBS. Fluorescence analysis was performed using FACScan (Becton Dickinson).
Detection of IL-12R ß1 chains by RT-PCR
Quantitative extraction of total RNA from 5 x 106 neutrophils (>98% purity) or an equivalent number of mononuclear cells was performed using Tri-Reagent (MRC, Cincinnati, OH) according to the manufacturer’s instructions. RT-PCR was used for semiquantitative analysis of transcript levels. cDNA was synthesized from the total RNA representative of neutrophils (2.5 x 105) or PBMCs (2.5, 0.125, 0.25, and 0.5 x 105), using AMV reverse transcriptase (Promega, Madison, WI) according to the manufacturer’s protocol. Sense and antisense primers for human IL-12R ß1 chain were 5'-AGCTTCCAGAAGGCTGTCAAGG-3' (U03187; nucleotides 861–882) and 5'-GCTGCCATTCAATGCAATACGTC-3' (U03187; reverse complement of nucleotides 1159–1181), respectively. The total cDNA of each sample was amplified by PCR in a final volume of 50 µl containing 100 ng of each primer, 100 mM dNTPs, 50 mM KCl, 10 mM Tris-HCl (pH 8.3), 1.5 mM MgCl2, and 1 U of Taq polymerase (Pharmacia, Uppsala, Sweden). Thirty-five cycles of denaturing for 1 min at 94°C, annealing for 1 min at 55°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.
Measurement of calcium
Neutrophils were loaded with fura-2/AM as described previously (14). The cells were washed, placed on glass coverslips, and allowed to adhere for 15 min at room temperature. Coverslips with adherent cells were rinsed with either Krebs-HEPES or modified Krebs-HEPES in which 1.3 mM CaCl2 was substituted with 1 mM EGTA (calcium-free Krebs-HEPES). Coverslips were secured between two plates of a custom-designed coverslip holder and placed onto a heated microscope stage (33°C), and [Ca2+]i measurements were performed on individual cells using the IonVision dual excitation system (ImproVision, Coventry, U.K.) as described previously (15).
Confocal calcium measurements were performed essentially as described previously (16). Images were acquired at 140-ms intervals, on the average, using a Leica DM iRBE inverted microscope attached to TCS confocal system (Leica-Kaki, Riyadh, Saudi Arabia). Images were converted into PICT format and analyzed by IonVision software (ImproVision). Each image was divided (pixel by pixel) by an averaged image acquired before challenge with IL-12. The changes in calcium in divided images were calculated as described previously (16).
Gel electrophoresis and Western blotting
Unless otherwise stated, 10% SDS-PAGE was used throughout. SDS-PAGE and Western blotting were performed as described previously (17, 18).
Actin polymerization measurements
The extent of actin polymerization was measured by the increase in binding sites for rhodamine phallacidin as described previously (19, 20). Briefly, neutrophils (107/ml) were incubated at 37°C for 10 min in a stirred temperature-controlled chamber. Samples (100 µl) were drawn, and cells were fixed in 3.7% formaldehyde in PBS for 15 min at room temperature. IL-12 (1 ng/ml) was added to the cells, and 100-µl samples were drawn at different time intervals and fixed as described above. Fixed samples were washed and permeabilized in 0.5% Triton X-100 in PBS for 5 min, washed three times with PBS, and then stained with rhodamine phallacidin (0.33 µM) for 1 h at room temperature. The fluorescence intensity of washed cells was measured using flow cytometry (FACScan, Becton Dickinson). Actin polymerization was also measured by SDS-PAGE and immunoblotting of the actin associated with the Triton X-100-insoluble cytoskeleton. SDS-PAGE of actin was performed essentially as described previously (21). Separated protein bands were electrotransferred onto nitrocellulose membrane (Bio-Rad, Hercules, CA), and blots were washed twice with double-distilled water, preblocked with 3% nonfat milk in PBS for 1 h at room temperature, and rinsed with double-distilled water. Incubation with primary anti-actin Ab (4°C, overnight) was followed by two rinses in double-distilled water and three washes in PBS containing 0.02% Tween-20. Incubation with secondary horseradish peroxidase-conjugated Ab (1 h, 4°C) was followed by washing as described above and detection using enhanced chemiluminescence (ECL, Amersham, Aylesbury, U.K.).
Measurement of tyrosine phosphorylation
For phosphotyrosine and STAT Western blotting, the protocol used was essentially that supplied by the Ab manufacturer. Briefly, cells were lysed in a medium containing 20 mM Tris-HCl (pH 7.4); 1% Nonidet P-40; 1 mM sodium orthovanadate; 150 mM NaCl; 10 mM EGTA; 1 µg/ml each of aprotinin, leupeptin, pepstatin A, and chymostatin; and 1 mM PMSF. The lysates were separated by SDS-PAGE and transferred onto nitrocellulose. Blots were washed and blocked with 4% BSA in PBS for 1 h at room temperature. Primary Abs were incubated at 4°C for 18 h at a concentration of 1 µg/ml. Secondary horseradish peroxidase-conjugated Abs were incubated with the blots for 2 h at the concentrations recommended by the manufacturer.
Immunoprecipitation
Cell lysates were prepared from 107 cells/ml in ice-cold cell lysis buffer. Debris was pelleted (15,000 x g), and lysates were precleared using protein A/G plus agarose conjugates (20 µl of a 40% slurry). Supernatant was incubated with 5 µg/ml of the designated primary Ab or serum at 4°C for 4 h, then with protein A/G plus agarose (30 µl of 40% slurry) at 4°C overnight. Immunoprecipitates were collected, washed, pelleted, and resuspended in electrophoresis sample buffer containing 20% glycerol, 8% SDS, 125 mM Tris-HCl, 10% ß-ME, 1 mM sodium orthovanadate, and 0.05% bromophenol blue. Finally, the samples were subjected to SDS-PAGE as described above.
Measurement of ROM production
The production of ROM by neutrophils was measured using luminol-dependent chemiluminescence (LDCL) as described previously (22, 23, 24). To confirm that ROM production was due to activation of the neutrophil NADPH oxidase system and not to effects on LDCL, oxygen consumption rates of neutrophils following IL-12 challenge were also measured using a Clark-type oxygen electrode (Oxygraph, Gilson, Denver, CO) essentially as described previously (25).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Incubation of freshly prepared human neutrophils with
[125I]IL-12 at 4°C resulted in a time-dependent
specific binding (difference between binding observed with
[125I]IL-12 and that in the presence of a 50-fold excess
of unlabeled IL-12) of the cytokine that reached a maximum within
1 h (Fig. 1
A). This
binding was concentration dependent, with half-maximum receptor
occupancy occurring at 280 ± 80 pM (Fig. 1B). The
kinetics of this binding fit a one-binding site model with a maximum of
1.7 ± 0.25 x 105 binding sites/neutrophil.
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C). RT-PCR revealed the
presence of IL-12R-ß1 mRNA in human neutrophils (Fig. 1D).
Since the neutrophil preparations are typically 98 to 99% pure (as
detected by FACS analysis) with 1 to 2% contamination of other cell
types, namely PBMC, the possibility existed that the IL-12R amplicons
revealed by RT-PCR were a product of the contaminating PBMC rather than
of neutrophils. This was tested by running RT-PCR of purified
neutrophils (2.5 x 105 cells; Fig. 1D, lane 1) and various numbers of PBMC prepared
from the same blood donor. Figure 1D, lanes 2,
3, 4, and 5, shows the IL-12R ß1
amplicons obtained from 2.5 x 105, 1.25 x
104, 2.5 x 104, and 5 x
104 PBMC, respectively. Even with 5 x 104
PBMC equivalent (Fig. 1D, lane 5), the amount of
IL-12R mRNA amplicons was less than that obtained from routinely
prepared neutrophils (Fig. 1D, lane 1),
suggesting that the signal in lane 1 is predominantly
derived from mRNA of neutrophil origin. IL-12 evokes a transient rise in [Ca2+]i
Quiescent human neutrophils exhibit a resting calcium level of
81 ± 7 nM. Upon stimulation with IL-12, a transient rise in
[Ca2+]i was evoked (Fig. 2), reaching a maximum of 663 ± 119
nM. The calcium rise was both asynchronous and heterogeneous, with a
dose-dependent increase in the number of cells displaying a calcium
transient that was at least twofold higher than the resting levels. At
IL-12 concentrations of 0.02, 0.1, and 1 ng/ml, the percentages of
cells exhibiting a calcium transient were 32, 56, and 85%,
respectively. Removal of the extracellular calcium reduced the extent
of the IL-12-induced calcium rise. However, the rate of decay of the
calcium transient was higher in the absence of extracellular calcium
than in normal extracellular calcium concentrations (Fig. 3, a and b),
suggesting an extracellular calcium influx component to the
intracellular transient. Calcium influx was tested directly by using
Mn2+ as the surrogate ion following IL-12 challenge. In a
series of experiments, the fluorescence of fura-2-labeled neutrophils
was monitored using two excitation wavelengths of 340 nm (calcium
sensitive) and 360 nm (calcium insensitive). Under such conditions
IL-12 caused a sharp rise in fura-2 emission due to the 340-nm
excitation wavelength and a concomitant decrease in emission due to the
360-nm excitation (Fig. 3c). The former result suggests the
release of calcium from an internal store(s), and the latter result
suggests a direct influx of calcium from the extracellular medium. The
ability of IL-12 to cause calcium influx was confirmed by the partial
inhibition of IL-12-induced calcium transients in the presence of
Cd2+ and Ni2+ (data not shown). Depletion of
the [Ca2+]i store(s) by pretreatment of
neutrophils with thapsigargin caused a rapid increase in
[Ca2+]i, reaching a sustained level of
407 ± 50 nM. Further addition of IL-12 failed to induce any
significant calcium rise (Fig. 3d). The IL-12-induced
calcium rise was also inhibited by pretreatment of neutrophils with
pertussis toxin (1 µg/ml), suggesting the involvement of
heterotrimeric G proteins (Fig. 3e). The IL-12-sensitive
calcium store(s) was visualized directly using fast (temporal
resolution of 140 ms/frame) confocal laser scanning microscopy of
fluo-3/AM-labeled neutrophils. Treatment of these cells with IL-12
revealed a small, rapid, and diffused increase in cytosolic calcium
followed by larger increases within punctate areas with the eventual
global rise throughout the cytosol (Fig. 3f).
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One of the earliest events following neutrophil activation is a
rapid and transient increase in actin polymerization (20). Using flow
cytometry to measure the binding of rhodamine phallacidin to F-actin,
IL-12 (1 ng/ml) caused a rapid and transient increase in the number of
binding sites for rhodamine-phallacidin (Fig. 4A). This is consistent with a
transient rise in actin polymerization, which reached a maximum within
60 s and decayed slowly to prestimulatory levels. This rise was
only partially affected by inhibition of the calcium transient with
bis(2-aminophenoxy)ethane-N,N,N',N'-tetra-acetate
acetoxymethyl ester (BAPTA-AM) or by inhibition of tyrosine
phosphorylation by the phosphotyrosine inhibitor genistein (Fig. 4A). To further confirm the effect of IL-12 on actin
polymerization we measured the amount of actin associated with the
Triton X-100 insoluble cytoskeleton. In a series of experiments
neutrophils were treated with IL-12 (1 ng/ml), and the actin associated
with the Triton-insoluble cytoskeleton was run on SDS-PAGE followed by
Western blotting and probing with anti-actin mAb. Under such
conditions a transient rise in the amount of actin associated with the
cytoskeleton was observed, reaching a maximum at about 60 s (Fig. 4B).
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Since IL-12-mediated responses in a variety of cell types have
been associated with tyrosine phosphorylation (8, 26, 27), the
possibility existed that similar effects may be seen with human
neutrophils. IL-12 elicited an increase in tyrosine phosphorylation of
a number of proteins compared with that in unstimulated control cells
(Fig. 5A). Five major bands of
relative molecular masses of 78, 75, 52, 36, and 26 kDa and several
minor bands were detected with antiphosphotyrosine Abs following IL-12
challenge. The occurrence of these bands followed similar kinetics,
with maximal phosphorylation occurring within 1 to 5 min of stimulation
before decaying to prestimulatory levels within 20 min. Interestingly,
the phosphotyrosine inhibitor genistein (20 ng/ml) attenuated the
phosphorylation state of some, but not all, bands induced by IL-12
(Fig. 5B). Since the IL-12-induced calcium transient
preceded tyrosine phosphorylation, the possibility existed that the
calcium transient was a prerequisite for the phosphorylation step.
IL-12-induced tyrosine phosphorylation was inhibited in neutrophils
treated with the calcium chelator BAPTA-AM before IL-12 encounter (Fig. 5B).
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The effect of IL-12 on neutrophil function was tested by
monitoring the production of ROM. In a series of experiments human
neutrophils were treated with increasing concentrations of IL-12, and
ROM production was measured by LDCL. IL-12 was found to induce LDCL in
a concentration-dependent manner (Fig. 6a). This was confirmed by the
increased oxygen consumption rate in neutrophils exposed to IL-12 using
a Clark-type oxygen electrode (Fig. 6a, inset).
IL-12-induced ROM production was inhibited in the presence of
neutralizing Ab to IL-12 (Fig. 6b). Since neutrophil ROM
production is intimately linked to both the
[Ca2+]i transient and actin polymerization
(14, 28, 29), we investigated the effects of inhibition of
IL-12-induced calcium transients and IL-12-induced actin polymerization
on ROM production. Whereas inhibition of the
[Ca2+]i transient by preincubation with
BAPTA-AM totally abolished ROM production (Fig. 6c),
inhibition of actin polymerization by cytochalasin B enhanced ROM
production (Fig. 6d). This enhancement was also abolished by
inhibition of the IL-12-induced calcium rise with BAPTA-AM. Similarly,
genistein caused inhibition of IL-12-induced ROM production (Fig. 6e), suggesting an intimate correlation between tyrosine
phosphorylation and IL-12-induced oxidase activation. The potency of
IL-12 to induce LDCL was compared with that of other known neutrophil
activators, namely the chemokine IL-8 and the chemotactic peptide FMLP.
IL-12 was several orders of magnitude more potent than either IL-8 or
FMLP, with half-maximum responses (K1/2)
occurring at 6.0 ± 1.4 pM, 2.0 ± 0.9 nM, and 0.24 ±
0.06 µM for IL-12, IL-8, and FMLP, respectively.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and TNF-
from NK and
Th1 cells, both of which are potent regulators of phagocytic cell
function, and further enhances cell-mediated immunity by activation of
macrophages and production of opsonins such as IgG2a isotype (38, 39, 40, 41).
IL-12 also induces the expansion of Th1 cells, which produce more IL-2
and IFN-, and inhibits IL-4-producing cells (1, 42, 43). It has been
proposed that immune responses are regulated by the balance between
IL-12, which favors Th1 responses, and IL-4, which favors Th2 responses
(43). IL-12 involvement in the activation of professional phagocytes
such as macrophages and neutrophils has been suggested (44, 45), and
directional migration of human neutrophils in response to IL-12 has
been reported (45). In addition, IL-12 administration to mice is known
to cause a decrease in the number of circulating leukocytes/neutrophils
(46, 47), whereas administration of IL-12 together with
Candida albicans causes an impaired neutrophil
response (47).
In the present work we report that human neutrophils express functional
IL-12Rs, as illustrated by 1) the presence of IL-12R ß1 mRNA (Fig. 1D), 2) the expression of IL-12R on the neutrophil membrane
(detected by anti-IL-12R Ab) and the ability of IL-12 to attenuate
the binding of anti-IL-12R Ab to neutrophils (Fig. 1C),
3) the specific binding of IL-12 to neutrophils (Fig. 1B),
and 4) the ability of IL-12 to activate human neutrophils. IL-12
specifically binds and activates human neutrophils in a
concentration-dependent manner. The Kd for
binding was 280 ± 80 pM. Three binding sites for IL-12 exist on T
lymphoblast cell membranes; high, medium, and low affinity binding
sites (5, 6). Because of the low Kd for binding,
the neutrophil membrane is likely to have the medium to low affinity
receptors. It is noteworthy, however, that the difference between the
reported kilodaltons may be due to differences in the specific activity
associated with the recombinant proteins.
Whereas IL-12-induced responses in a variety of cell types are known to be mediated via the JAK/STAT signaling pathway (8, 9, 27), the existence of other signaling pathways for IL-12 has not been reported. Here we demonstrate that treatment of neutrophils with IL-12 causes a concentration-dependent and transient rise in intraneutrophil-free calcium. This rise is due to the release of [Ca2+]i from an intracellular membrane-enclosed store(s) and its influx from the extracellular medium. Evidence for this derives from the fact that 1) removal of extracellular calcium reduces the amplitude of the IL-12-induced calcium transient; and 2) experiments in which calcium was substituted for the surrogate ion Mn2+ revealed a decrease in fura-2 fluorescence due to the calcium-independent excitation wavelength of 360 nm, suggesting the influx of Mn2+. The calcium rise was inhibited by treatment of neutrophils with pertussis toxin (1 µg/ml) before IL-12 challenge, suggesting the involvement of heterotrimeric G proteins. The ability of IL-12 to release calcium from an intracellular store(s) was further confirmed using confocal laser scanning microscopy at temporal and spatial resolutions of 140 ms and 3 µm, respectively. The fast temporal acquisition was needed to directly demonstrate the IL-12-induced release of the [Ca2+]i store. Challenging fluo-3/AM-loaded neutrophils with IL-12 revealed a small global rise in cytosolic calcium followed by a localized and punctate rise within the cytosol, suggesting areas of [Ca2+]i storage. The source of the initial small global rise before emptying of the calcium store(s) by IL-12 has yet to be determined.
Since calcium transients are intimately linked to the tyrosine phosphorylation signaling pathway, we tested the effect of IL-12 on phosphotyrosine levels. We found that IL-12 treatment induced phosphorylation of a number of proteins.
Neutrophil activation is normally coupled to transient actin polymerization in response to many soluble stimuli (28, 29). Here we demonstrate that IL-12 causes actin polymerization, as detected by increased binding sites for rhodamine phallacidin and by an increase in the amount of actin associated with the Triton X-100-insoluble cytoskeleton. Following IL-12 treatment, a biphasic actin response occurred, with polymerization reaching a maximum within 60 s, followed by a slow depolymerization. F-actin levels were still significantly higher than the resting levels after 5 min of IL-12 challenge. Inhibition of the IL-12-induced calcium rise by BAPTA-AM had no apparent effect on the extent or the kinetics of actin polymerization. Furthermore, inhibition of tyrosine phosphorylation had no significant effect on actin polymerization. It is therefore reasonable to speculate that at least two independent intracellular signals occur following IL-12R engagement on the neutrophil membrane: one for calcium transients and another for actin polymerization. It is noteworthy that both calcium changes and actin polymerization exhibited slow kinetics, which may suggest an indirect effect of IL-12 on the neutrophils, perhaps through the generation of secondary signals induced by IL-12.
The involvement of calcium transients in activation of the neutrophil oxidase system to produce ROM is well established. We tested the hypothesis that the IL-12-induced calcium rise was necessary for ROM production by neutrophils. In a series of experiments we demonstrated that IL-12 caused ROM production in a dose-dependent manner. The K1/2 for ROM production was 6.0 ± 1.4 pM, suggesting that only a relatively small number of the total IL-12Rs need to be occupied to evoke ROM response. Using pulse binding techniques, Painter et al. (48) found that for [Ca2+]i, cAMP, and membrane potential responses, <5% of the total FMLP receptors need to be occupied. For superoxide production, however, the receptor occupancy needed was around 30%. Sklar et al. (49) reported that for the initial phase of actin polymerization in response to FMLP, only 100 receptors/neutrophil (of 50,000) are required. Based on the K1/2 for ROM and the Kd for binding, it is estimated that <2% of human IL-12R need to be engaged to evoke 50% of the maximum IL-12-induced ROM production.
That ROM induction was due to activation of the neutrophil oxidase system was confirmed by the increased rates of oxygen consumption following neutrophil challenge with IL-12. ROM production was inhibited in cells pretreated with the [Ca2+]i chelator BAPTA/AM. Similarly, the tyrosine kinase inhibitor genistein inhibited the IL-12-induced ROM production. The question therefore arose as to whether genistein also inhibited the IL-12-induced calcium transient. Genistein alone had no apparent effect on resting neutrophil calcium (data not shown). Furthermore, pretreatment of neutrophils with genistein at concentrations known to inhibit tyrosine phosphorylation had no apparent effect on the IL-12-induced calcium transient.
Since actin polymerization occurs within the time of [Ca2+]i changes, the possibility of a direct role for actin polymerization in the activation of the neutrophil oxidase system has been suggested (29). It is thought that the degree of ROM production is inversely proportional to the amount of F-actin within an activated neutrophil (29). Our present findings show that inhibition of actin polymerization enhances IL-12-induced ROM production and also confirm the role of F-actin in oxidase activation.
In this paper we demonstrate that human neutrophils express functional IL-12Rs that upon engagement by IL-12 activate the cells via a calcium-dependent mechanism(s). This activation leads to increased ROM production. Human IL-12R can exist as human IL-12Rß1 and human IL-12ß2, each independently exhibiting low affinity binding to IL-12, and/or a human IL-12ß1/ß2 complex, exhibiting high affinity binding (50, 51, 52, 53, 54). Neither human IL-12Rß1 nor human IL-12Rß2 contains the seven-span membrane motif characteristic of G protein-dependent and pertussis toxin-sensitive receptor signaling, yet their engagement by IL-12 exhibits both G protein-dependent and pertussis toxin-sensitive signaling. This apparent anomaly is by no means restricted to human IL-12R. Hepatocyte growth factor induces phospholipid signaling and calcium changes in a pertussis toxin-sensitive fashion (55). Furthermore, epidermal growth factor has been shown to stimulate inositol 1,4,5-trisphosphate production and [Ca2+]i, which is inhibited by pretreatment with pertussis toxin (56). Neither the hepatocyte growth factor receptors nor the epidermal growth factor receptors exhibit the seven-transmembrane motif. The membrane events leading to stimulus-response coupling for this class of receptors have yet to be defined. Although neutrophil migration in response to IL-12 has previously been demonstrated (45), to our knowledge our work is the first to report a direct effect of IL-12 on calcium homeostasis, actin polymerization, and ROM production in phagocytic cells. The existence of a calcium signaling pathway in IL-12-mediated responses has many implications, especially with respect to the JAK/STAT signaling pathway. It is noteworthy that the calcium ionophore ionomycin "interrupts" JAK/STAT signaling in mononuclear cells following IL-6 stimulation (57). A similar mechanism may exist in human neutrophils following the IL-12-induced rise in calcium.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Futwan Al-Mohanna, Biological and Medical Research Department, MBC 03, King Faisal Specialist Hospital and Research Center, Riyadh 11211, Saudi Arabia. E-mail address:
3 Abbreviations used in this paper: JAK, Janus kinase; ROM, reactive oxygen metabolite; [Ca2+]i, intracellular free Ca2+; LDCL, luminol-dependent chemiluminescence; F-actin, filamentous actin; K1/2, half-maximum response; BAPTA-AM, bis(2-aminophenoxy)ethane-N,N,N',N'-tetra-acetate acetoxymethyl ester.
Received for publication December 8, 1997. Accepted for publication June 1, 1998.
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  L. F. Olsen, U. Kummer, A. L. Kindzelskii, and H. R. Petty A Model of the Oscillatory Metabolism of Activated Neutrophils Biophys. J., January 1, 2003; 84(1): 69 - 81. [Abstract] [Full Text] [PDF]
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 J. E. A. Portielje, C. H. J. Lamers, W. H. J. Kruit, A. Sparreboom, R. L. H. Bolhuis, G. Stoter, C. Huber, and J. W. Gratama Repeated Administrations of Interleukin (IL)-12 Are Associated with Persistently Elevated Plasma Levels of IL-10 and Declining IFN-{gamma}, Tumor Necrosis Factor-{alpha}, IL-6, and IL-8 Responses Clin. Cancer Res., January 1, 2003; 9(1): 76 - 83. [Abstract] [Full Text] [PDF]
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 F. Al-Mohanna, S. Saleh, R. S. Parhar, and K. Collison IL-12-dependent nuclear factor-{kappa}B activation leads to de novo synthesis and release of IL-8 and TNF-{alpha} in human neutrophils J. Leukoc. Biol., November 1, 2002; 72(5): 995 - 1002. [Abstract] [Full Text] [PDF]
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T. H. Wyman, C. A. Dinarello, A. Banerjee, F. Gamboni-Robertson, A. A. Hiester, K. M. England, M. Kelher, and C. C. Silliman Physiological levels of interleukin-18 stimulate multiple neutrophil functions through p38 MAP kinase activation J. Leukoc. Biol., August 1, 2002; 72(2): 401 - 409. [Abstract] [Full Text] [PDF]
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K. S. Collison, R. S. Parhar, S. S. Saleh, B. F. Meyer, A. A. Kwaasi, M. M. Hammami, A. M. Schmidt, D. M. Stern, and F. A. Al-Mohanna RAGE-mediated neutrophil dysfunction is evoked by advanced glycation end products (AGEs) J. Leukoc. Biol., March 1, 2002; 71(3): 433 - 444. [Abstract] [Full Text] [PDF]
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F. Ethuin, C. Delarche, S. Benslama, M.-A. Gougerot-Pocidalo, L. Jacob, and S. Chollet-Martin Interleukin-12 increases interleukin 8 production and release by human polymorphonuclear neutrophils J. Leukoc. Biol., September 1, 2001; 70(3): 439 - 446. [Abstract] [Full Text] [PDF]
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 D. A. Knight, P. G. Holt, and P. D. Sly Lipopolysaccharide Inhibits the Late-Phase Response to Allergen by Altering Nitric Oxide Synthase Activity and Interleukin-10 Am. J. Respir. Cell Mol. Biol., May 1, 2001; 24(5): 640 - 646. [Abstract] [Full Text]
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L. Fantuzzi, P. Puddu, B. Varano, M. Del Cornò, F. Belardelli, and S. Gessani IFN-{alpha} and IL-18 exert opposite regulatory effects on the IL-12 receptor expression and IL-12-induced IFN-{gamma} production in mouse macrophages: novel pathways in the regulation of the inflammatory response of macrophages J. Leukoc. Biol., November 1, 2000; 68(5): 707 - 714. [Abstract] [Full Text]
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 E. Di Carlo, A. Comes, S. Basso, A. De Ambrosis, R. Meazza, P. Musiani, K. Moelling, A. Albini, and S. Ferrini The Combined Action of IL-15 and IL-12 Gene Transfer Can Induce Tumor Cell Rejection Without T and NK Cell Involvement J. Immunol., September 15, 2000; 165(6): 3111 - 3118. [Abstract] [Full Text] [PDF]
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H. Nagayama, K. Sato, H. Kawasaki, M. Enomoto, C. Morimoto, K. Tadokoro, T. Juji, S. Asano, and T. A. Takahashi IL-12 Responsiveness and Expression of IL-12 Receptor in Human Peripheral Blood Monocyte-Derived Dendritic Cells J. Immunol., July 1, 2000; 165(1): 59 - 66. [Abstract] [Full Text] [PDF]
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