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Keywords:

  • Autoimmunity;
  • Elastase;
  • Inflammation;
  • Interleukin-18;
  • Neutrophils

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements

Interleukin-18 (IL-18), a member of the IL-1 cytokine superfamily, is an important regulator of both innate and acquired immune responses. We demonstrate here constitutive expression of IL-18 by human neutrophils. Unexpectedly, we observed that neutrophils from peripheral blood or rheumatoid synovial compartments contained not only pro and mature IL-18, but also several novel smaller-molecular-weight IL-18-derived species. Using specific protease inhibitors, and serine protease gene-targeted mice, we demonstrate that these IL-18-derived products arose through caspase-independent cleavage events mediated by the serine proteases, elastase and cathepsin G. Moreover, we report that the net effect of elastase treatment of mature recombinant IL-18 was to reduce its IFN-γ-inducing activity. Thus, human neutrophils contain IL-18 and IL-18-derived molecular species that can arise through novel enzymatic processing pathways. Through cytosolic, membrane or secretory expression of such processing enzymes, together with generation of IL-18 itself, neutrophils likely play a critical role in regulating IL-18 activities during early innate immune responses.

Abbreviations:
BzATP:

benzyl ATP

CG:

cathepsin G

fMLP:

N-formyl-methionyl-leucyl-phenylalanine

HV:

healthy volunteer

ICE:

IL-1β-converting enzyme

IL-18BP:

IL-18-binding protein

MALDI-ToF:

matrix-assisted laser desorption-ionization time-of-flight

NE:

neutrophil elastase

PR3:

proteinase 3

RA:

rheumatoid arthritis

SF:

synovial fluid

SLPI:

secretory leukocyte protease inhibitor

Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements

IL-18 is a pleiotropic cytokine known to be produced by a variety of cells including macrophages, dendritic cells, keratinocytes, osteoblasts and synovial fibroblasts 1. IL-18, like IL-1β, is produced as a 24-kDa inactive precursor (pro-IL-18) lacking a signal peptide. Original studies indicated that pro-IL-18 is processed by IL-1β-converting enzyme (ICE or Caspase 1), resulting in the generation of a biologically active 18-kDa protein 2, 3. Recently, evidence for alternate processing has emerged through proteinase 3 (PR3) 4 generating IL-18 with biological activity 5. In contrast, cleavage of pro or mature IL-18 by Caspase 3 results in the generation of biologically inactive peptides 6.

Originally described as an IFN-γ-inducing factor 7 promoting Th1 responses in synergy with IL-12, IL-18 is now ascribed a broader role in both innate and adaptive immunity. Via binding to a heterodimeric IL-18Rα/IL-18Rβ receptor, IL-18 enhances T and NK cell cytotoxicity and cytokine production, macrophage nitric oxide (NO) production and cytokine release, endothelial proliferation and chondrocyte activation 814. IL-18-deficient mice exhibit impaired Th1 responses to several bacteria and parasites 1517, and neutralization of IL-18 results in diminished responses to various infectious agents 18, 19. We recently demonstrated that IL-18 is a potent neutrophil-activating factor, leading to migration, integrin expression, enhanced respiratory burst and degranulation, and cytokine/chemokine release 20. Moreover, in vivo, IL-18-deficient mice exhibit altered responses to gram-positive bacteria and IL-18 neutralization suppresses carrageenan-induced acute inflammation 21. These data, together with the constitutive expression of pro-IL-18 in a broad range of tissues, strongly suggest an important role for IL-18 in innate immune responses.

Neutrophils rapidly accumulate and activate during inflammatory responses, producing a variety of cytokines and hydrolytic enzymes 22, 23. Azurophilic granules within neutrophils contain several serine proteases including cathepsin G (CG), elastase and PR3, which after degranulation may also be expressed at the cell surface 2426. These proteases are capable of modulating the biological activity of a variety of substrates including collagen, angiotensin 1, pro-IL-1β, IL-6, IL-8, and TNF-α 2733.

In recent studies, we have identified the expression and functional importance of IL-18 in chronic inflammation using rheumatoid arthritis (RA) synovial tissues and murine arthritis model systems 34, 35. Given the foregoing data suggesting a critical role for IL-18 in innate responses, we have now addressed the possibility that effector functions of IL-18 in these models could be regulated in part through neutrophil function. In the present report, we show that IL-18 is itself a product of human neutrophils. Unexpectedly, we have detected not only pro and mature IL-18 species in human neutrophils, but also smaller-molecular-weight moieties derived from IL-18 through alternative enzymatic processing. Thus, neutrophil activation may contribute not only to local IL-18 expression but could also modify IL-18 released by adjacent cell lineages and therefore influence its role on subsequent effector function in developing immune responses.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements

Human neutrophils constitutively express IL-18

As neutrophils are responsive to IL-18 20, we first sought evidence of an autocrine loop whereby IL-18 could promote neutrophil activation. RT-PCR detected IL-18 mRNA in highly purified neutrophils derived from peripheral blood (PB) of healthy volunteers (HV) (Fig. 1A). Intracellular IL-18 protein expression was also detected by FACS analysis (Fig. 1B). The specificity of IL-18 staining was confirmed using an isotype-matched irrelevant control antibody (Fig. 1B). To confirm that IL-18 expression was maintained in the context of prior activation in vivo, highly purified PB and synovial fluid (SF)-derived neutrophils from RA patients were examined. IL-18 was again detected by RT-PCR and FACS analysis (Fig. 1A and data not shown). Finally, as IL-1β and IL-18 release by human monocytes may be mediated via P2X7 receptors 36, 37, we sought evidence for a similar pathway for IL-18 release by neutrophils, detecting such using an ELISA specific for mature (18-kDa) IL-18. Whereas resting neutrophils or those stimulated with 10–6 M N-formyl-methionyl-leucyl-phenylalanine (fMLP) for 20 min failed to release detectable levels of mature IL-18, neutrophils cultured with the P2X7 agonist, benzyl ATP (BzATP), were found to secrete considerably greater levels of mature IL-18 (Fig. 1C). These data clearly document that IL-18 synthesis, expression and release is a feature of mature human neutrophils.

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Figure 1. Neutrophils constitutively express IL-18. (A) Total RNA extracted from representative HV PB- (lane 1), RA patient PB- (lanes 2, 4) and SF-derived (lanes 3, 5) neutrophils was reverse transcribed and amplified with IL-18- and β-actin-specific primers. (B) Intracellular expression of neutrophil IL-18 protein was analyzed by FACS analysis where PMN were gated on their characteristic forward/side scatter profiles. Panels represent staining of HV PB with an irrelevant isotype-matched control antibody (left) and constitutive IL-18 staining (right). The example shown is representative of four HV PB and four RA SF samples examined. (C) Neutrophils (2 × 107/mL) were incubated for 20 min in serum-free Hank's Hepes medium alone, with fMLP (10–6 M) or BzATP (500 µM). Release of mature IL-18 was subsequently assayed in cell-free supernatants by ELISA. Results are expressed as means ± SEM (n = 4 HV PB).

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Neutrophils contain multiple molecular speciesof IL-18

To confirm that human neutrophils contained pro and mature forms of IL-18, we probed PB- (HV or RA) and RA SF-derived neutrophil total protein extracts with monoclonal anti-IL-18 antibodies. We detected the pro form as well as the mature form of IL-18 as expected (Fig. 2 and data not shown) in all samples examined. However, in addition, we consistently detected smaller-molecular-weight species that specifically bound anti-IL-18 antibody, including a predominant form with a molecular weight of approximately 14.3 kDa and a less prominent form of approximately 6 kDa. As neutrophil lysates were prepared in the presence of a cocktail of protease inhibitors, these smaller-molecular-weight species recognized by the anti-IL-18 antibody are likely specific and not the result of inappropriate neutrophil lysis. Moreover, these appearances were not disease specific since similar moieties were detected in neutrophils derived from ascites of patients with chronic alcoholic hepatitis (data not shown). Thus, neutrophils are apparently capable of IL-18 processing to generate moieties distinct from pro and mature IL-18.

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Figure 2. Neutrophils express a number of IL-18-specific products. (A) Examples shown are of neutrophil total protein extracts freshly isolated from the PB of three individual HV donors, fractionated on 12% SDS-PAGE gels and probed with a monoclonal anti-human IL-18 antibody as described in Materials and methods. rIL-18 used as a positive control for IL-18 detection runs at approximately 21 kDa. (B) Densitometry values for each IL-18-specific band depicted in (A).

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Enzymatic processing of IL-18 by neutrophils

We next sought to identify the enzymatic activities mediating these effects in neutrophils using recombinant IL-18 as bait. Since neutrophil lysates contained IL-18 a priori (Fig. 2), we were unable to explore the capacity of cytosolic neutrophil extracts to cleave rIL-18. We utilized instead a cell-free system. Neutrophils rapidly degranulate ex vivo. PB neutrophils were therefore cultured for 20 min in the absence of exogenous stimuli and the resultant cell-free supernatant was incubated with rIL-18. These supernatants effectively cleaved rIL-18 into a number of distinct moieties (Fig. 3) closely resembling those observed in native neutrophils ex vivo (Fig. 2). Prior incubation of culture supernatants with increasing concentrations of the broad-spectrum caspase inhibitor Z-VAD failed to prevent the characteristic cleavage of rIL-18 by culture supernatants, suggesting that caspase-independent pathways operated in this process. In addition, neutrophil supernatants in the absence of rIL-18 failed to bind anti-IL-18 antibodies, highlighting that the IL-18 products detected in the above experiments resulted from cleavage of rIL-18 and were not a result of pre-existing IL-18 (data not shown).

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Figure 3. Extracellular, caspase-independent IL-18 processing by neutrophil culture supernatants. rIL-18 (50 ng) was incubated in 50 µL of HV PB neutrophil-derived culture supernatants for 1 h at 30°C. Samples were subsequently probed for IL-18-specific bands by Western blotting as described in Materials and methods. rIL-18 used as positive controls for IL-18 detection runs at approximately 21 kDa. The example shown is representative of five individual experiments.

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Processing of IL-18 by neutrophil elastase and CG

Using defined inhibitors, we next identified candidate pathways mediating IL-18 processing. Cleavage was abolished in the presence of PMSF, a serine protease inhibitor, but was unaffected by aprotinin, leupeptin or EDTA, inhibitors of trypsin, chymotrypsin, and metalloproteases, respectively (Fig. 4A and data not shown). Secretory leukocyte protease inhibitor (SLPI), an inhibitor of neutrophil elastase (NE) and CG, but not PR3, inhibited IL-18 processing in a dose-dependent manner (Fig. 4A). Patterns of cleavage and inhibition were observed in culture supernatants isolated from both RA SF- and HV PB-derived neutrophils, suggesting that this is not a disease-specific event (data not shown). When rIL-18 was incubated with purified NE, cleavage similar to that obtained with neutrophil supernatants was observed. Purified CG yielded a similar cleavage pattern to that produced by NE; in contrast, PR3 failed to process rIL-18 in this manner, even at high concentrations (Fig. 4B). We confirmed these observations using NE, CG and NE/CG gene-targeted mice. Culture supernatants derived from wild-type neutrophils processed human rIL-18 into moieties similar to those generated by human neutrophil-derived culture supernatants (Fig. 4C). However, IL-18 processing by supernatants from either NE or CG gene-targeted mice was clearly reduced, with a virtual absence of rIL-18 processing by neutrophil supernatants derived from double gene-targeted animals. Collectively, these data strongly suggest that elastase- and CG-dependent pathways could operate to process IL-18 at neutrophil-rich inflammatory sites.

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Figure 4. Serine protease involvement in neutrophil extracellular IL-18 processing. (A) SF neutrophil culture supernatants were pre-incubated in the absence or presence of PMSF (2 mM), aprotinin (2 µg/mL), leupeptin (2 µg/mL) (A) or increasing concentrations of SLPI (B) for 45 min at 30°C prior to addition of rIL-18 (50 ng) and a further 1 h of incubation. Samples were subsequently probed for IL-18-specific bands by Western blotting as described in Materials and methods. (B) rIL-18 (50 ng) was incubated with HV PB neutrophil culture supernatant or increasing concentrations of purified NE, CG (A) or PR3 (B) for 1 h at 30°C. IL-18 processing was then examined as previously described. Each example shown is a representative of three individual experiments. (C) Neutrophil culture supernatants from wild-type (WT), NE–, CG– or NE–/CG– mice were incubated with rIL-18 for 1 h at 30°C. IL-18 processing was then examined as previously described. Results shown are representative of four individual animals per group examined.

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Sequence identity of IL-18 processed moieties

Matrix-assisted laser desorption-ionization time-of-flight (Maldi-ToF) mass spectrometry was employed to characterize fragments generated by NE cleavage of mature IL-18 (Fig. 5A). Comparison of the masses of the protein fragments observed in the Maldi-ToF spectra with the sequence of mature IL-18 using BioLynx was used to indicate where cleavage may have occurred, by seeking fragments that would give rise to the observed mass. The mass accuracy of Maldi-ToF was insufficient to unequivocally assign cleavage sites, and in some cases a number of sites were identified as being possible from this analysis. To rank-order these, we used the following criteria: (1) the known specificity of NE (Ala/Val); (2) where no peptide of appropriate mass could be determined with this cleavage, allowing for cleavage at Leu or Ile, which had been demonstrated to be possible in an associated MS/MS study on one fragment (data not shown); (3) the mass error between the predicted mass and the observed mass; and (4) peptides resulting from cleavage at common sites. In some cases there were two sequences that could fit the data. The best-fit data are shown in Fig. 5B and correspond to five of the following nine identified fragments (numbering for the mature IL-18 construct): 26–72 (m/z 5366), 113–159 (m/z 5546), 18–68 (m/z 5918), 29–81 (m/z 5895), 69–142 (m/z 8600), 1–98(m/z 11 358), 29–128 (m/z 11 417), 18–143 (m/z 14 614) and 29–155 (m/z 14 672).

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Figure 5. MALDI-ToF mass spectra from sinapinic acid matrix of rIL-18. (A) rIL-18 (1 µg) was incubated in the presence of 100 ng/mL purified human NE for 1 h at 30°C. Cleavage products were subsequently separated and analyzed at (1) high and (2) medium mass. Spectra are externally calibrated and Gaussian-smoothed. (B) Predicted elastase cleavage sites of mature rIL-18 generating fragments from residues 1–98 (dark), 18–68 (light), 18–143 (dotted), 69–142 (dashed) and 113–159 (crossed). Residues important for IL-18 bioactivity and IL-18BP binding are shown in grey.

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IL-18 processing diminishes IL-18-inducedIFN-γ production

Finally, we investigated the net biological significance of this processing pathway using functionally active rIL-18. To determine the effect of prior elastase processing on net rIL-18 bioactivity, native or elastase-cleaved rIL-18 was added to KG-1 cells. Native rIL-18 induced higher IFN-γ (403 ± 90 pg/mL) secretion than did elastase-cleaved rIL-18 (168 ± 63 pg/mL) (Fig. 6). Indeed, elastase processing of IL-18 led to a complete loss of its IFN-γ-inducing ability. To confirm that this loss of IFN-γ was a result of IL-18 processing, and not direct degradation by NE, recombinant IFN-γ was treated with 500 ng/mL NE for 1 h or overnight prior to quantitation by IFN-γ ELISA. No significant differences in levels of rIFN-γ were observed between untreated and NE-treated samples (data not shown). Finally, exposure of KG-1 cells to the NE preparation without IL-18 had no effect on spontaneous IFN-γ release (data not shown).

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Figure 6. Elastase IL-18 processing results in diminished IFN-γ production. TNF-α-pretreated KG-1 cells (106/mL) were cultured with a final concentration of 100 ng/mL rIL-18 pretreated or not with 500 ng/mL purified NE (1 h at 30°C) for 24 h at 37°C, and subsequent IFN-γ production was assessed by ELISA. Results shown are means ± SEM of four PB HV donors.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements

IL-18 likely plays an important role in early phases of inflammatory and innate immune responses. IL-18 mRNA and pro-IL-18 are constitutively expressed in a wide variety of tissues, indicating the capacity for rapid cytokine expression upon tissue insult 1. Here, we show that human neutrophils not only constitutively express pro and mature IL-18, but also express novel IL-18-derived moieties. Moreover, we show that generation of such moieties may be subject to novel caspase-independent processing pathways. IL-18 is a known enhancer of neutrophil effector function 20 that, when introduced into murine models, promotes neutrophil-predominated local inflammation 38, 39. In contrast, systemic inflammation following LPS injection is suppressed by anti-IL-18 antibodies, with concordant reduction in tissue neutrophil invasion to liver or lung 40. That IL-18 is itself a product of neutrophil activation indicates that some of the foregoing observations subserve in part an IL-18-dependent autocrine loop for neutrophil activation.

Enzymatic pathways implicated thus far in processing IL-18 into biologically active moieties include Caspase-1 and PR3 2, 5, whereas processing by Caspase-3 6 generates biologically inactive IL-18. Our data using human neutrophil-derived culture supernatants, purified NE, CG and PR3, in addition to gene-targeted mice, collectively demonstrate that neutrophils can preferentially utilize the serine proteases elastase and CG to extracellularly process IL-18. Our observations extend a number of previous studies. In these NE/CG and PR3 processing of pro-IL-1β and pro-IL-18 generates biologically active proteins 41. In contrast, IL-8 and TNF-α processing by NE and CG diminishes bioactivity 31, 32, 42. Finally, in concordance with our observations, non-mammalian species process IL-18 through a cysteine protease-dependent mechanism into biologically inactive moieties 43.

Through IL-18 expression, neutrophils likely occupy an important role in the transition from innate to adaptive immune responses. IL-18 regulation in the extracellular compartment is mediated in part through the IL-18-binding protein (IL-18BP) 44. Our data suggest that soluble or membrane-bound protease activities comprise an additional regulatory functional loop commensurate with the critical role played by IL-18 in adaptive immune responses. It is noteworthy that IL-18 enhances NE release 45. Reports that PBMC secrete pro-IL-18 46 raise the possibility that during cognate cell-cell interactions extracellular processing of IL-18 may occur 4. We report that the net effect of elastase treatment of mature rIL-18 was to reduce its IFN-γ-inducing activity. Elastase had no direct effect on spontaneous KG-1 cell IFN-γ release (ELISA-detectable IFN-γ; data not shown), and IFN-γ has previously been shown to be resistant to elastase-mediated cleavage 47. Taken together, these data suggest that this phenomenon likely results from a loss of Glu-42 (Glu-12 in the mature IL-18 sequence) from IL-18 following elastase processing (Fig. 5), as this residue has been identified as critical for IL-18 bioactivity 48. At present, we propose that these elastase-generated fragments are incapable of acting as agonists on the IL-18R complex, but we cannot exclude their possible receptor binding and function as partial antagonists in this system. We note, however, that an 11.4-kDa product (positions 1–98 in the mature IL-18 sequence) retains this residue and the generation of a functional product cannot be ruled out. Moreover, as Glu-42, in conjunction with Lys-89 (Lys-59 in the mature IL-18 sequence), is implicated in IL-18BP interactions 49 and as this residue is retained in several defined processing products, it is possible that some decoy action on IL-18BP effects could operate. Detailed analyses are now required to formally evaluate the functional contribution of the products predicted on our analyses to be present in an inflammatory environment in which neutrophils are processing IL-18-derived molecules either in an autocrine manner or indeed from adjacent dendritic cells or macrophages. Nevertheless, we believe that our data uncover an important enzyme-dependent regulatory pathway that could operate in the protease-rich environment of an acute inflammatory response characteristic of a neutrophil infiltrate.

Culture supernatants derived from neutrophils of oral cavity cancer patients are known to contain IL-18 50; however, mechanisms regulating IL-18 release remain poorly understood. BzATP, a P2X7 receptor agonist, significantly augments IL-1β and IL-18 release from LPS-stimulated human blood cultures 51. Neutrophils express P2X7 receptors 52, and mature IL-18 release in response to ATP in vitro suggests that a similar P2X7-mediated cytokine release may operate in neutrophils as in monocytes. In inflammatory diseases such as RA where neutrophils abound in SF, in conjunction with high levels of active elastase 5355, neutrophil IL-18 production and processing is likely to be biologically significant.

In summary, we have shown that neutrophils may be a rich source of IL-18 in an inflammatory environment and that via at least serine proteases they may modify the local bioactivity of this cytokine. One intriguing possibility is that early neutrophil activation may critically regulate the ability of IL-18 to contribute to the phenotype of ensuing adaptive immune responses (Fig. 7). Further evaluation of IL-18 production and function utilizing recombinant IL-18 processing products and appropriately gene-targeted murine models will be required to address this possibility.

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Figure 7. Cartoon depicting neutrophil IL-18 production, processing and possible implications thereof. Neutrophil IL-18 production at early inflammatory sites potentially acts as a source of autocrine activation, increases local IL-18 expression and may influence the generation of developing effector T cells. Through expression of serine proteases (potential effects of elastase processing of IL-18 shown in grey), neutrophils may not only modify the local bioactivity of this cytokine but also IL-18 released by adjacent cell lineages, thereby influencing its role on subsequent effector function in ensuing adaptive immune responses.

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Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements

Isolation of human neutrophils and generation ofcell-free culture supernatants

To heparinized whole blood from HV or RA patients, 1/10 (vol/vol) of 5% dextran was added and incubated at 37°C for 30 min to allow erythrocyte sedimentation. The buffy coat was subsequently layered onto lymphoprep (Sigma, Poole, UK) and centrifuged at 800 × g for 15 min. Following hypotonic lysis, polymorphonuclear neutrophils (PMN) were recovered from cell pellets, washed twice in serum-free Hank's Hepes medium (Life Technologies, Paisley, UK) and adjusted to a cell concentration of 107 cells/mL. Subsequent to a 20-min incubation in 24-well plates at 37°C, cultures were spun at 800 × g for 5 min, cell-free supernatants were removed, 0.2-micron filtered, aliquoted and frozen at –20°C until required. Neutrophils from RA SF were isolated by density gradients, but in the absence of dextran sedimentation. Neutrophils isolated were routinely >95% pure, as determined by FACS analysis. Samples were obtained following approval from the Glasgow Royal Infirmary Ethical Committee (Glasgow, UK). RA patients met ACR diagnostic criteria 56.

IL-18 RT-PCR

Neutrophils isolated as above and used for the generation of RNA were routinely >95% pure, as determined by FACS analysis. RNA extracted from PB or SF neutrophils using TRIzol Reagent (Life Technologies) was reverse transcribed to cDNA using SuperScript II (Life Technologies) according to the manufacturer's recommendations. PCR for IL-18 was performed using the following primers: IL-18 sense 5′-ATCAGGATCCTTTGGCAAGCTTGAATCTAAATTATC-3′, antisense 5′-ATAGGTCGACTTCGTTTTGAACAGTGAACATTATAG-3′. PCR products were visualized by electrophoresis on 1% agarose gels stained with ethidium bromide.

FACS analysis

Neutrophil intracellular IL-18 expression was determined in permeabilized PB or SF neutrophils by FACS analysis (Becton Dickinson, Cowley, UK) using a murine anti-human IL-18 monoclonal antibody (MAB 318; R&D Systems, Abingdon, UK) as the primary identifying antibody, followed by detection with a PE-conjugated goat anti-mouse antibody (Serotec, Oxford, UK). To verify the specificity of IL-18 staining, in parallel experiments neutrophils were also stained with an isotype-matched irrelevant control antibody. Neutrophils were gated on their characteristic forward/side scatter profiles.

ELISA

Neutrophils isolated and used in ELISA experiments were routinely >95% pure. Neutrophils (2 × 107/mL) in serum-free Hank's Hepes medium were cultured for 20 min at 37°C in the absence or presence of 10–6 M fMLP or 500 µM 2′3′-O-(4-benzoyl)-ATP (BzATP; Sigma, Poole, UK). Cell-free supernatants were removed and mature IL-18 levels quantitated by ELISA following the manufacturer's recommendations (Biosource International, CA). In direct communications with Biosource International it was confirmed that this ELISA had less than 1% recognition of pro-IL-18 and was therefore deemed specific for mature IL-18. IFN-γ production by IL-18-stimulated KG-1 cells was similarly measured by ELISA (Biosource International).

Western blotting

After removal of culture supernatants, neutrophil cell pellets (107 cells/mL) were lysed in buffer containing 50 mM Tris-HCL pH 8.0, 150 mM NaCl, 1 mM EDTA, 1% NP40, 0.05% SDS, 1 mM DTT, 1 mM PMSF, 1 µg/mL aprotinin, 1 µg/mL leupeptin and 1 µg/mL pepstatin A (all Sigma). Lysates were subsequently freeze-thawed three times, centrifuged at 12 000 × g for 15 min at 4°C, and supernatants containing total proteins were fractionated on 12% SDS-polyacrylamide gels, followed by transfer (semi-dry) to nitrocellulose membranes at 16 V for 25 min. Blots were blocked in 5% milk powder/0.05% Tween-20 for 1 h at room temperature prior to overnight incubation at 4°C with 1 µg/mL anti-IL-18 monoclonal antibody (MAB 318; R&D Systems). After washing, blots were incubated for 45 min at room temperature with horseradish peroxidase-conjugated sheep anti-mouse Ig antibody, and IL-18-specific bands were detected by enhanced chemiluminescence (both from Amersham Biosciences, Chalfont St. Giles, UK).

Recombinant IL-18 in vitro cleavage assays

Recombinant human mature IL-18 (rIL-18) (50 ng; generated in our laboratory as previously described) 34 was incubated in 50 µL of neutrophil-derived culture supernatants or in the presence of the commercially available proteases: NE, CG (both from Calbiochem, Oxford, UK) and PR3 (EPC, MO), in phenol red-free Hank's Hepes medium for 1 h at 30°C. The reaction was terminated by addition of denaturing sample buffer, followed by fractionation of the samples on 12% SDS-polyacrylamide gels and probing for IL-18-specific bands by Western blotting, as described. For inhibition assays, prior to the addition of rIL-18, neutrophil culture supernatants or purified commercial proteases were incubated in the presence of specific inhibitors: SLPI (R&D Systems), PMSF, EDTA, aprotinin or leupeptin, for 45 minutes at 30°C.

Generation of neutrophil culture supernatants fromNE/CG gene-targeted mice

Thioglycollate-induced neutrophils were generated by intraperitoneal injection of 3% thioglycollate broth (BD Biosciences, UK) into 129SV wild-type (WT), NE knockout (NE–), CG knockout (CG–) or double knockout (NE–/CG–) mice. After 4 h, four animals per group were CO2 asphyxiated and peritoneal exudate cells recovered by peritoneal lavage with PBS/0.5 mM EDTA. Cell concentrations were adjusted to 5 × 107 cells/mL in phenol red-free Hank's Hepes medium, and cell-free culture supernatants were generated as described for human neutrophils.

Maldi-ToF mass spectrometry

rIL-18 (1 µg) was incubated in the presence of 100 ng/mL purified human NE for 1 h at 30°C. Samples were subsequently desalted using a C-18 Zip-Tip (Millipore Corporation, Billerica, USA) using the standard method, with extensive washing with 0.1% trifluoroacetic acid in water. Samples were eluted from the tip directly onto the target plate in 1 µL matrix solution, which consisted of 10 mg/mL sinapinic acid in 50% acetonitrile in water containing 0.1% trifluoroacetic acid. MALDI-ToF spectra were collected on a Voyager DE-Pro (Applied Biosystems, Foster City, USA) in delayed extraction linear mode with a 25 kV extraction potential, 95% grid ratio and 0.05% guide wire potential in two mass ranges: 2000–10 000 Da using a 200 µs delay, and 6000–40 000 Da using a 300 µs delay. Spectra are a sum of 200 laser shots taken from the same point on the target plate. Spectra were Gaussian-smoothed using a 15-point window, then calibrated using a close external standard containing myoglobin and bovine insulin. The estimated mass error is 0.2%. Masses were fitted to potential sequences using the BioLynx tool of MassLynx 2.5 (Waters-Micromass) using a window of approximately 0.5% of the mass of the ion.

KG-1 bioassay

KG-1 human leukemia cells were maintained in complete RPMI 1640 medium supplemented with 10% fetal bovine serum, 50 IU/mL penicillin, and 50 µg/mL streptomycin at 37°C under a 5% CO2 atmosphere until required. For use in bioassays, KG-1 cells (1 × 106/mL) were stimulated overnight at 37°C with 20 ng/mL TNF-α (R&D Systems), washed three times and plated at 106 cells/mL in 100-µL volumes. Thereafter, 100 µL of either growth medium, untreated rIL-18 (200 ng/mL) or rIL-18 (200 ng/mL) pretreated with 500 ng/mL elastase at 30°C for 1 h was added, giving a final IL-18 concentration of 100 ng/mL. After 24 h, the culture supernatants removed were examined for IL-18-induced IFN-γ production by ELISA (as described).

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements

This worked was funded by research grants provided by the ARC and Wellcome Trust.

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