International Immunology Advance Access originally published online on October 11, 2006
International Immunology 2006 18(12):1663-1670; doi:10.1093/intimm/dxl100
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An invertebrate TNF functional analogue activates macrophages via lectin–saccharide interaction with ion channels
ilerová1
1 Department of Immunology, Institute of Microbiology, Academy of Sciences of the Czech Republic, Víde
ská 1083, 142 20 Prague 4, Czech Republic
2 Laboratory of Cellular and Molecular Immunology, Department of Cellular and Molecular Interactions, ‘Vlaams Interuniversitair Instituut voor Biotechnologie, Vrije Universiteit Brussel', Brussels, Belgium
3 Department for Molecular Biomedical Research, ‘Vlaams Interuniversitair Instituut voor Biotechnologie’, Ghent, Belgium
Correspondence to: M. Bilej; E-mail: mbilej{at}biomed.cas.cz
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Abstract |
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The invertebrate pattern-recognition protein named coelomic cytolytic factor (CCF) and the mammalian cytokine tumor necrosis factor (TNF) share functional analogies that are based on a similar saccharide recognition specificity. In particular, CCF and TNF have been shown to interact with ion channels on the surface of vertebrate cells via N,N'-diacetylchitobiose lectin-like activity. In the present study, we show that CCF-induced membrane depolarization results in the release of TNF, IL-6 and nitric oxide (NO) by macrophages via nuclear factor-
B signaling. Interestingly, our data suggest that TNF contributes, through lectin–saccharide interaction, to the secretion of IL-6 and NO induced by CCF. This experimental non-physiological setting based on the interaction of an invertebrate defense lectin with vertebrate cells involved in the innate immune response may have highlighted an evolutionarily ancient mechanism of macrophage activation in vertebrates.
Keywords: invertebrate cytokine, membrane depolarization, NF-B, pattern-recognition protein, TNF
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Introduction |
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Coelomic fluid of common brandling earthworms Eisenia fetida (Oligochaeta, Annelida) contains numerous molecules with different activities, including anti-bacterial, hemolytic and cytolytic [for review see (1)]. Previously we have isolated from the coelomic fluid a cytolytic factor named coelomic cytolytic factor (CCF) (GenBank accession no. AF030028) acting in earthworm defense as a pattern-recognition molecule (2, 3). Upon binding microbial pathogen-associated molecular patterns, namely the O-antigen of LPS, ß-1,3-glucans, peptidoglycan constituents or N,N'-diacetylchitobiose, CCF triggers the prophenoloxidase-based anti-microbial defense mechanism in the earthworm coelomic fuid. In addition, CCF was shown to exhibit tumor necrosis factor (TNF)-like features. Indeed, CCF lyses TNF-sensitive tumor cell lines (4). CCF expression is up-regulated in macrophage-like coelomocytes upon LPS stimulation (5, 6) while TNF is produced by macrophages (7). Moreover, both TNF and CCF were suggested to interact with various pathogens via saccharide recognition (2, 8), for review see Lucas et al. (9), and to lyse African and American trypanosomes via a similar lectin-like activity with N,N'-diacetylchitobiose specificity (10–12). Interestingly, the functional similarity of CCF and TNF is not based on a structural homology but rather represents a convergence of function based on a similar lectin-like activity (11, 13).
More recently, the capacity of the N,N'-diacetylchitobiose lectin-like domain of TNF to induce a pH-dependent increase of membrane conductance resulting in membrane depolarization has been demonstrated in primary lung microvascular endothelial cells and in peritoneal macrophages (14). Since the insertion of TNF into the cell membrane is not sufficient to change the conductance, it was suggested that the membrane depolarization is due to the interaction of TNF with amiloride-sensitive, most likely sodium ion channels (15, 16). Similarly, CCF was found to activate amiloride-sensitive ion channels in lung endothelial cells and peritoneal macrophages in a TNF receptor (TNFR)-independent manner via its N,N'-diacetylchitobiose lectin-like domain (17). In the present study, we investigated whether the interaction of CCF lectin-like domain with the membrane of non-elicited adherent peritoneal cells (PECs), mainly macrophages, affects the activation of the cells. We show that CCF-induced membrane depolarization via N,N'-diacetylchitobiose lectin-like activity results in the release of TNF, IL-6 and NO by macrophages via nuclear factor-B (NF-B) signaling. Further, we propose that the lectin-like activity of the invertebrate functional analogue of TNF divulges an ancient mechanism of macrophage activation in vertebrates.
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Methods |
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Animals and reagents
Female wild-type C57BL/6 mice (8- to 10-weeks old) were purchased from Harlan. tnf knock-out C57BL/6 mice were a kind gift from K. Sekikawa (National Institute of Animal Health, Tsukuba City, Japan). tnfr1 and tnfr2 knock-out C57BL/6 mice were a kind gift of H. Bluethmann (Hoffman-La Roche). All knock-out mice were bred at our animal facility and were kept in filter-top cages. The mice care was in accordance with institutional guidelines.
N,N'-diacetylchitobiose, amiloride, benzamil and phenamil were obtained from Sigma. Bis-oxonol [bis-(1,3-dibutylbarbituric acid)-trimethine oxonol, DiBAC4] was purchased from Molecular Probes.
Recombinant CCF (CCF) was expressed as described (2) and further purified to homogeneity on an anti-CCF-1 mAb column (4). CCF was re-suspended in PBS (pH 8.0) and LPS contamination was excluded using QCL LAL test (Bio-Whittaker Europe, Verviers, Belgium).
Cells
Resident peritoneal cells were collected in 0.34 M sucrose and washed in complete medium (RPMI 1640 medium supplemented with 10% heat-inactivated FCS, 5 x 10–5 M 2-mercaptoethanol, 2 mM L-glutamine, 100 IU ml–1 penicillin, 100 µg ml–1 streptomycin and 0.1 mM non-essential amino acids; all from Invitrogen Life Technologies). Adherent cells were recovered after plastic adherence as described previously (18). Collected cells [peritoneal exudate cells (PECs)] had a viability >95%, as determined by trypan blue exclusion, and were F4/80+ from 75%, as determined by flow cytometry analysis (data not shown).
Quantification of cytokines and nitrite
PECs (2 x 105 per 200 µl) from individual mice were cultured in triplicate in humidified atmosphere containing 5% CO2 in complete medium in the absence or presence of CCF at the indicated concentration for 24 h at 37°C. Cytokines were quantified using sandwich ELISAs for TNF and IL-6, performed as recommended by the supplier (R&D Systems and PharMingen, respectively). NO in culture supernatants was estimated by quantifying NO2 using Griess reagent as described previously (19). When required, PECs were incubated with amiloride, benzamil or phenamil at the indicated concentrations for 30 min before CCF activation. Alternatively, CCF was pre-incubated with the indicated concentrations of N,N'-diacetylchitobiose before addition to the cell cultures.
Analysis of membrane depolarization
For fluorescence microscopy, PECs (106 ml–1) were adhered overnight on microscope cover slips and extensively washed (1% BSA in PBS). Cells were then incubated with 100 µl serum-free RPMI 1640 alone or 100 µl CCF (10 µg ml–1) in serum-free RPMI 1640 for 1 h at room temperature and pulsed with bis-oxonol at a final concentration of 1 µM for a further 30 min. For inhibition experiments, cells were incubated with 100 µM amiloride for 30 min before CCF activation. Alternatively, CCF was pre-incubated with N,N'-diacetylchitobiose (5 µg ml–1) before addition to the cell cultures. Samples were washed three times with 3 ml 1% BSA in PBS and examined under an Olympus Provis (Olympus Optical) fluorescence microscope.
For flow cytometric analysis, PECs were seeded in polystyrene FACS tubes, washed and then treated with CCF, inhibitors and bis-oxonol as described above. After the final washing, cells were re-suspended in 200 µl of FACS buffer and analyzed immediately using a Becton Dickinson FACSVantage SE system. Relative fluorescence unit values were calculated by dividing the percentage of positive cells in treated samples by that in untreated samples.
RNA isolation, cDNA synthesis and PCR
Total RNA was prepared from PECs by using 1 ml of Trizol reagent according to the manufacturer's protocol (Invitrogen). DNAse I-treated total RNA (2 µg) was reverse transcribed with Oligo(dT)12–18 (Invitrogen) and SUPERSCRIPTTM II Rnase H– Reverse Transcriptase (Invitrogen). Resulting cDNAs were used in PCR with specific primers for Na+/H+ exchanger (NHE-1, GenBank accession no. U51112)—sense primer 5'-CTT CTG TTG GCC AGT TCT AC-3' and anti-sense primer 5'-TAC ATG GTT GTC GAT GTC AC-3'. PCRs were performed with cDNA templates (0.5 µl of the RT reaction product), to which 2 U of Taq polymerase (Invitrogen), 0.2 mM deoxynucleoside triphosphate, 1x company-supplied buffer, 1.5 mM MgCl2 and 0.4 µM primer pairs were added. PCR conditions were 94°C for 3 min, followed by 35 cycles of 94°C for 30 s, 60°C for 40 s, 72°C for 90 s and 72°C for 7 min.
The identity of PCR products was confirmed by sequencing.
Statistical analysis
All comparisons were tested for statistical significance (P < 0.05) via the unpaired t-test, using GraphPad Prism 3.0 software. Data were expressed as the mean ± SD of three individual mice and are representative of at least three independent experiments.
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Results |
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The effect of CCF on cytokine and NO production by PECs
The in vitro activation of non-elicited adherent PECs (75% F4/80+ cells, data not shown) isolated from wild-type and TNFR (1 and 2) knock-out mice with CCF resulted in the production of TNF in a dose-dependent manner in the range of 0.5–5 µg ml–1 (Fig. 1). A higher CCF concentration did not further enhance the cytokine release. The differences in TNF production by PECs isolated from wild-type and TNFR knock-out mice were negligible, thus excluding the autocrine effect of TNF via surface TNFRs. Expectedly, PECs from TNF knock-out mice did not produce TNF upon activation with CCF. CCF triggered similar production of IL-6 and of NO in PECs from wild-type and TNFR knock-out mice (Fig. 1). IL-6 and NO production in PECs from TNF knock-out mice was <40% of the value obtained in PECs from wild-type or TNFR knock-out mice suggesting the cumulative effect of CCF and TNF on IL-6 and NO release.
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Increased TNF, IL-6 and NO production by PECs was likely not due to LPS contamination since CCF samples were below the detection limit of LAL test. Moreover, comparable cytokine and NO secretion levels were observed with CCF samples treated or not treated with polymixin. Finally, the digestion of CCF with pronase abolished its ability to induce cytokine and NO release (data not shown).
The effect of ion channel inhibitors on CCF-mediated PECs activation
In agreement with our previous patch-clamp experiments (17), CCF caused a membrane depolarization in PECs that was evidenced using the fluorescent anionic dye bis-oxonol in fluorescence microscopy and flow cytometry. As shown in Fig. 2, CCF-induced membrane depolarization was inhibited by amiloride and by N,N'-diacetylchitobiose, confirming a lectin-like saccharide interaction of CCF with a sodium channel or a sodium channel-associated structure. Similar data were observed in PECs from TNFR1 knock-out, TNFR2 knock-out and TNF knock-out mice (data not shown).
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To further address the role of sodium channels in the induction of TNF, IL-6 and NO production by CCF-activated macrophages, three amiloride-related molecules were tested. Amiloride is a potent inhibitor of epithelial sodium channels as well as Na+/H+ exchangers. Yet, benzamil and phenamil are amiloride derivatives that have higher specificity for the cell type and the type of ion channel, inhibiting both ion-gated sodium channels and Na+/H+ exchanger. The three inhibitors did not exert any toxic activity on PECs up to a concentration of 200 µM as assessed by the MTT test (data not shown). On this basis, PECs from the four mouse strains (wild-type, TNFR1 knock-out, TNFR2 knock-out and TNF knock-out) were stimulated with the sub-optimal CCF concentration of 0.5 µg ml–1 in the presence of inhibitors in the range of 6.25–100 µM. Phenamil was the most efficient inhibitor in the four cell types, causing already 50% inhibition of TNF, IL-6 and NO secretion at the lowest concentration tested (6.25 µM; Fig. 3). Amiloride and benzamil, given in the concentration of 25–50 µM, caused ~50–60% inhibition. Yet, amiloride, the inhibitor of epithelial sodium channels and Na+/H+ exchangers, was a less potent inhibitor of CCF-mediated macrophage activation as compared with its derivatives with specificity for ion-gated sodium channels or Na+/H+ exchanger. Furthermore, PCR analysis using specific primers followed by sequencing of the amplified products revealed the expression of
, ß and 
subunits of epithelial sodium channel (GenBank accession nos NM_011324, NM_011325, NM_011326, respectively) in cells of mouse lung and kidney but not in PECs (data not shown). On the other hand, when specific primers for Na+/H+ exchanger (NHE-1, GenBank accession no. U51112) were tested, gene expression was detected in the absence of CCF stimulation in PECs from all mouse strains used in the present study (Fig. 4). These data indicate that a Na+/H+ exchanger could be involved in CCF-mediated activation of macrophage, resulting in the secretion of TNF, IL-6 and NO.
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The effect of N,N'-diacetylchitobiose on CCF-mediated PECs activation
Since CCF interacts with ion channels via its lectin-like domain (Fig. 2), the inhibitory capacity of N,N'-diacetylchitobiose on PECs activation was tested in the range of 25–100 µg ml–1 as described above for ion channel inhibitors. It was found that this saccharide efficiently inhibits cytokine and NO production in CCF-activated PECs from wild-type as well as TNFR1 knock-out, TNFR2 knock-out and TNF knock-out mouse strains (Fig. 5).
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The effect of signaling pathway inhibitors on CCF-mediated PECs activation
Three different inhibitors were tested in order to identify the intracellular signaling pathway involved in CCF-mediated PECs activation. PD 98,059 is a specific inhibitor of the activation of mitogen-activated protein kinase kinase (MAPKK), SB 203580 inhibits the activation of p38 mitogen-activated protein kinase (MAPK) and MAPKAP kinase-2, but not JNK or p42 MAPK, and finally MG132 blocks I-
B degradation and thus NF-B activation. The inhibitors did not exert toxic activity on PECs up to a concentration of 200 µM for PD 98,059 and SB 203580 or up to 20 µM for MG132 as assessed by MTT test (data not shown). As shown in Fig. 6, the inhibition of MAPKK or of p38 MAPK and MAPKAP kinase-2 had no significant effect on PECs activation triggered by CCF in the four mouse strains used in the present study. In contrast, the inhibition of NF-B activation already reduced the cytokine and NO produced by PECs from the four mouse strains by as much as 50–60% at concentration of MG132 as low as 1.25 µM.
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Discussion |
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The invertebrate pattern-recognition protein CCF displays functional analogy with the mammalian cytokine TNF that is based on a similar lectin-like N,N'-diacetylchitobiose specificity [for review see (13)]. Beside cytolytic and trypanolytic activities (4, 11), the ability of CCF to interact with ion channels on mammalian cell surface has been demonstrated using the whole-cell patch-clamp technique (17). Here we show that the membrane depolarization caused by CCF via a lectin–saccharide interaction results in the activation of non-elicited adherent peritoneal cells leading to the production of cytokines and NO. The composition of PECs (75% F4/80+ cells) and our unpublished observation that CCF triggers membrane depolarization and TNF, IL-6 and NO production in the 2C11-12 macrophage cell line (20) support the idea that CCF is mainly interacting with macrophages. CCF-induced depolarization is not due to the interaction of autocrine TNF with TNFRs since the depolarization was not affected in PECs from TNFR1 and TNFR2 knock-out mice, and as reported previously in microvascular endothelial cells that do not produce TNF (17).
Bloc et al. (17) have suggested that CCF interacts with amiloride-sensitive ion channels on the surface of lung microvascular endothelial cells. Yet, the exact type of these channels was not determined. Amiloride is described as a potent inhibitor of epithelial sodium channels. Inhibition experiments using the amiloride derivatives benzamil and phenamil indicate that other sodium channels, most likely ion-gated sodium channels or the Na+/H+ exchanger are involved in the interaction of CCF with murine macrophages. Moreover, PCR analysis evidencing the Na+/H+ exchanger expression in peritoneal macrophages, but not epithelial sodium channels expression, further supports this possibility. Finally, additional experiments with inhibitors of other types of ion channels [namely, quinine, paxilline, tetraethylammonium for potassium channels, ouabain for Na+, K+ ATPase, 5-nitro-2(3-phenylpropylamino)benzoic acid for chloride channels] rule out the direct involvement of potassium and chloride ions in CCF-mediated activation of macrophages (data not shown). Although we provide evidence that CCF interacts directly with the macrophage surface through its N,N'-diacetylchitobiose lectin-like domain, we cannot conclude whether CCF interacts directly with the saccharide moiety of an endogenous ion channel or rather with an as yet unknown receptor which in turn can activate an ion channel downstream of CCF binding.
CCF triggers the release of TNF, IL-6 and NO by macrophages likely by interacting with cell-surface ion channels considering that ion channel inhibitors completely abrogate the expression/production of these immune mediators as revealed by real-time PCR (data not shown). Moreover, this macrophage activation potential of CCF partially depends on the production of TNF since the levels of IL-6 and NO are significantly impaired in the absence of TNF, that is, in TNF knock-out mice. Furthermore, the secretion of IL-6 and NO induced by CCF in TNF knock-out mice is completely inhibited by N,N'-diacetylchitobiose. Although contention still exists as to whether or not the receptor-binding domains of TNF are involved in membrane depolarization (14, 16, 21), our data suggest that the autocrine effect of TNF is mediated mainly by its lectin-like domain in this experimental set-up. Accordingly, since CCF-mediated cytokine and NO production is similar in wild-type and in TNFR1 or TNFR2 knock-out mice, the interaction of TNF with TNFR may be less important in CCF-activated macrophages.
The induction of cytokine and NO secretion by macrophages activated by CCF depends on NF-B activation, but not on MAPKK activation. Moreover, while the p38 activation pathway contributes to the activation of cells by inflammatory molecules (22), it does not seem to be involved in CCF-induced intracellular signaling. Thus, the signaling pathway triggered by CCF in macrophages converges downstream of p38 with the one of inflammatory cytokines including TNF. This again suggests that the autocrine effect of TNF on CCF-induced macrophage activation occurs via its lectin-like domain.
In summary, we propose a model of macrophage activation (Fig. 7) where CCF binds via its N,N'-diacetylchitobiose domain to an Na+/H+ exchanger or an Na+/H+ exchanger-associated molecule (a). The resulting membrane depolarization leads to NF-B activation (b) and subsequent production of TNF (c), IL-6 and NO. In turn (the secreted), TNF interacts mainly via its N,N'-diacetylchitobiose lectin-like domain with the ion channel/ion channel-related structure (d), or ‘classically’ via its receptor-binding site with TNFR1 or TNFR2 on the macrophage surface (e) thus boosting the activation signal provided by the lectin domains of CCF and TNF (f). This artificial setting of macrophage activation triggered by an invertebrate defense molecule mimicking the lectin-like activity of TNF may reveal an ancient mechanism of cell activation that has evolved in parallel with the receptor-based network currently prevailing in vertebrates.
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Acknowledgements |
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This work, supported by the Czech Science Foundation (310/02/1437, 310/04/0806 and 310/03/H147), an Institutional Research Concept (AV0Z50200510) and a bilateral international scientific and technological cooperation grant from the Ministry of the Flemish Community (BOF-BWS 03/06), was performed within the frames of an Interuniversity Attraction Pole Program. R.Vd.B. is supported by a grant from the Institute for Promotion of Innovation by Science and Technology in Flanders (IWT—Vlaanderen). Authors are grateful to Rudolf Lucas for helpful discussion.
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Abbreviations |
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| CCF, coelomic cytolytic factor |
| MAPKK, mitogen-activated protein kinase kinase |
| MAPK, mitogen-activated protein kinase |
| NF-B, nuclear factor-B |
| NO, nitric oxide |
| PECs, peritoneal exudate cells |
| TNF, tumor necrosis factor |
| TNFR, TNF receptor |
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Notes |
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Transmitting editor: A. Falus
Received 17 March 2006, accepted 11 September 2006.
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References |
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