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CHEMOKINES
From the Klinik und Poliklinik für
Hautkrankheiten and the Institut für Medizinische Strahlenkunde
und Zellforschung, University of Würzburg, Würzburg,
Germany.
The cytokine-induced C-C chemokine monocyte chemoattractant
protein-1 (MCP-1) is an important regulator of leukocyte recruitment to
sites of inflammatory challenge. Here, it is demonstrated that the
widely distributed contact hapten NiCl2, like tumor
necrosis factor Endothelial cells, strategically located between
blood and tissue compartments, play an important role for initiation
and regulation of inflammatory events. Targeted by cytokines, such as
tumor necrosis factor (TNF)- Induced expression of MCP-1 is strongly dependent on activation of the
transcription factor NF- Cytokines and reagents
Cells and cell culture
DNA constructs MCP-1 promoter luciferase constructs containing the proximal promoter (between 107 and +60) and distal enhancer region (between 2742 and 2513) of MCP-1 (pGLM-ENH) as well as different NF- B binding-site mutants of pGLM-ENH were kindly provided by T. Yoshimura (Laboratory of Immunobiology, National Cancer Institute,
Frederick, MD) and have been previously described.10 The
3xNF-B-tk or the 5xGal4 promoter constructs contain 3 or 5 copies
of a NF-B- or Gal4-binding motif cloned upstream of a minimal
promoter-driven luciferase gene. These plasmids as well as constructs
expressing kinase-inactive IKK , the transcriptional coactivator p300
(CMVp300), or -Gal were obtained from T. Wirth, University of
Würzburg, Germany. The construct Gal4p65 expressing the
DNA-binding domain of the yeast Gal4 protein N-terminally fused to
full-length p65 (amino acids 1 through 551) was described
previously27 and provided by R. Schreck, University of
Würzburg. The GFPS65T expression vector pGreenLantern was
purchased from Life Technologies (Karlsruhe, Germany).
In situ hybridization In situ hybridization experiments were performed as previously described.26 Briefly, stimulated cells were sedimented onto lysine-coated glass slides, fixed in paraformaldehyde, acetylated, dehydrated, and air-dried. Thereafter, cells were overlaid with the hybridization solution containing 35S-labeled antisense and, for controls, sense probes of MCP-1. After hybridization nonhybridized probes were removed by several high-stringency washes. To further minimize nonspecific background, noncomplementary nonhybridized single-stranded probes were digested with RNase A and RNase T1. Slides were then dipped into Kodak NTB-2 solution, exposed for autoradiography, and finally analyzed by means of a Zeiss Axiophot microscope (Zeiss, Oberkochen, Germany).Enzyme-linked immunosorbent assay Supernatants of HDMECs and HUVECs were collected after exposure to NiCl2 or TNF , centrifuged at 13 000g to
remove cellular debris, and analyzed for MCP-1 synthesis by a sandwich
enzyme-linked immunosorbent assay (ELISA) as
described.28
Monocyte preparation and chemotaxis assays Peripheral blood monocytes were obtained from healthy volunteers by gradient density centrifugation and used in chemotaxis assays employing Nucleopore 48-well chemotaxis chamber plates (Costar, Bodenheim, Germany) as described earlier.29 Endothelial cell supernatant obtained after stimulation was studied for monocyte chemoattractive activity in triplicate. In some experiments, chemotaxis was assayed in the presence of neutralizing polyclonal goat immunoglobulin (Ig)-G against recombinant human MCP-1 or macrophage inflammatory protein-1 (MIP-1) (R&D Systems), which had
been added to the endothelial cell supernatant in the lower well of the
chemotaxis chamber.
Immunoprecipitation, immune-complex kinase assay, and Western blotting Endothelial cells were lysed with 20 mM Tris pH 7.4, 137 mM NaCl, 10% glycerol, 1% Triton X-100, 2 mM EDTA, 50 mM sodium--glycerophosphate, 20 mM sodium pyrophosphate, 1 mM Pefabloc
(Merck), 5 µg/mL aprotinin, 5 µg/mL leupeptin, and 5 mM
benzamidine (TLB buffer) at 4°C for 30 minutes. Cell lysates were
incubated with protein A agarose (Roche Molecular Biochemicals,
Mannheim, Germany) and 1 µg/mL rabbit antiserum against MAP
kinase-activated protein (MAPKAP) kinases 2 and
3,30 p38 (C-20), or IKK (H-470, Santa Cruz
Biotechnology, Heidelberg, Germany) for 2 hours at 4°C. After
washing in TLB buffer supplemented with 500 mM NaCl and, thereafter,
kinase buffer (25 mM Hepes pH 7.5, 10 mM MgCl2, 25 mM
sodium--glycerophosphate supplemented with 5 mM benzamidine, 1 mM
sodium orthovanadate, and 0.5 mM dithiothreitol), samples were
incubated with 3pK K73M (K > M), Hsp27, or GST-IB as
substrates for p38, MAPKAP kinases 2/3, or IKK in the presence of
100 µM unlabeled adenosine triphosphate (ATP), 5 µCi
[ 32P]-ATP, and kinase buffer for 15 minutes at 30°C.
Samples were subsequently subjected to sodium dodecyl
sulfate-polyacrylamide gel electrophoresis, blotted, and visualized by
autoradiography or detected by a BioImaging Analyzer BAS 2000 (Fuji;
via Raytest, Straubenhardt, Germany). Western blot analysis was
performed to confirm equal loading of p38, MAPKAP kinases 2/3, and
IKK proteins. IB and IB expression was detected in crude
Laemmli lysates with rabbit anti-IB (#9242, New England Biolabs,
Schwalbach, Germany) or anti-IB antisera (M-121, Santa Cruz Biotechnology).
Northern blot HUVECs were stimulated with NiCl2 or TNF in the
absence or presence of pharmacological inhibitors as indicated. Total
cellular RNA was isolated by means of a Qiagen RNeasy kit (Hilden,
Germany). Denaturated total RNA (10 µg) was separated on
agarose/formaldehyde gels and transferred to Hybond N+ membranes
(Amersham Pharmacia Biotech, Freiburg, Germany). Filters were
UV-cross-linked and subsequently hybridized with a human MCP-1
complementary DNA probe31 labeled with
[32P]-deoxycytidine triphosphate (dCTP) by means of a
random primed DNA-labeling kit (Roche Molecular Biochemicals).
Autoradiography was performed at 80°C with the use of Amersham
Hyperfilm. For control of RNA loading of lanes, blots were
densitometrically analyzed for 28S ribosomal RNA
(rRNA) amounts.
Transient transfection and reporter gene assays Endothelial cells were cultured to 50% to 60% confluence in 6-well plates prior to transient transfection according to a diethylaminoethyl (DEAE)-dextran protocol.32 Cells were incubated with 1 µg reporter construct and 250 µg DEAE-dextran (Amersham Pharmacia Biotech) in 1 mM Hepes/phosphate-buffered saline (PBS) in a final volume of 1 mL for 30 minutes at 37°C. Thereafter, 1.5 mL EGM medium containing 0.15 mM chloroquine was added to each well, and cells were incubated for another 2.5 hours. Medium was then removed and cells were treated with 10% dimethyl sulfoxide in EGM medium for 2.5 minutes. Endothelial cells were subsequently cultured for 36 hours in EGM medium and finally stimulated with NiCl2 or other reagents for the time intervals indicated. Cells of each well were harvested in lysis buffer (50 mM sodium 2-[-morpholino]ethanesulfonic acid, 50 mM Tris-HCl pH 7.8, 10 mM dithiothreitol, and 2% Triton X-100) for 30 minutes at 4°C. We added 50 µL of precleared cell extracts to 50 µL luciferase assay buffer containing 125 mM sodium 2-(N-morpholino)ethanesulfonic acid, 125 mM Tris-HCl pH 7.8, 25 mM magnesium acetate, and 2 mg/mL ATP). After addition of 50 µL of 1 mM D-luciferin (AppliChem, Darmstadt, Germany), luminescence was detected by means of an LB96P luminometer (Berthold, Bad Wildbach, Germany). Induced luciferase activities were normalized on the basis of protein contents and are expressed as "fold" stimulation compared with unstimulated controls.Flow cytometry MCP-1 expression by HUVECs cotransfected with a vector expressing dominant-negative IKK and GFPS65T was detected as
follows. At 36 hours after transfection, cells were exposed to
NiCl2 or TNF for the time intervals indicated. We added
2 µM monensin in order to avoid secretion of the chemokine via the
Golgi pathway. Cells were subsequently harvested, washed, fixed with
4% paraformaldehyde in PBS at 4°C for 20 minutes, and then incubated
with a monoclonal antibody against MCP-1 (mouse IgG1, clone 5D3-F7) or
corresponding isotype control monoclonal antibody (Becton Dickinson,
Heidelberg, Germany) that had been diluted in permeabilization buffer
containing 1% fetal calf serum, 0.1% saponin, and PBS. Thereafter,
cells were successively stained with biotin-SP-conjugated
goat-antimouse IgG F(ab')2 and streptavidin-Cy-chrome
(Becton Dickinson). Fluorescence was determined with a FACScalibur
(Becton Dickinson). Only cells that expressed GFPS65T (detected in the
FL-1 channel) were considered for detection of MCP-1 expression
(measured in the FL-3 channel). Nonviable cells were excluded by means
of forward scatter and side scatter parameters.
Electrophoretic mobility shift assay AB-specific probe33 was labeled in a reaction
mixture containing 200 ng double-stranded DNA probe,
[-32P]-dCTP, 1 mM deoxyadenosine triphosphate, 1 mM
deoxyguanosine triphosphate, 1 mM thymidine 5'-triphosphate, 500 mM
Tris-HCl pH 7.5, 100 mM MgCl2, and 2 U Klenow fragment.
After 30 minutes' incubation at 37°C, oligonucleotides were
separated on a G-25 Sephadex spin column (Roche Molecular Biochemicals)
and finally resuspended in Tris-EDTA (30 000 cpm/µL). For
the typical binding reactions, 5 µg of nuclear lysates were incubated
on ice for 5 minutes in the absence or presence of competitor DNA in an
18-mL reaction mixture containing 25 mM Tris pH 7.5, 1 mM EDTA, 0.5 mM
DDT, 100 mM KCl, 0.1% (vol/vol) NP40, 1 µg (wt/vol) bovine serum
albumin, 10% (vol/vol) glycerol, and 0.5 µg (wt/vol) poly(dI-dC); 60 000 cpm of labeled oligonucleotide was added, and the mixture was
incubated for 15 minutes at 25°C. The samples were loaded on a 5%
nondenaturing polyacrylamide gel equilibrated with 0.5 × TBE
(tris borate-EDTA) and electrophoresed for 2.5 hours at 180 V. Gels
were dried and DNA-protein complexes were visualized by autoradiography.
NiCl2 induces endothelial MCP-1 mRNA and protein synthesis NiCl2, a widely distributed contact sensitizer, is known to activate vascular endothelium (see Grabbe and Schwarz25 for a review). To investigate its effects on endothelial chemokine production, primary HUVECs (Figure 1A) or HDMECs (Figure 1B) were exposed to different concentrations of NiCl2, which leads to secretion of MCP-1 in a concentration-dependent manner. Significant amounts of MCP-1 could be already detected 120 minutes after stimulation of HUVECs (Figure 1C) and of HDMECs (data not shown) with NiCl2. Like TNF, NiCl2 induces a strong up-regulation of MCP-1
message in both cell types (Figure 1D [inset] and data not shown), as evaluated at a single-cell level by in situ hybridization. To exclude
the possibility that MCP-1 release into the medium is simply due to
toxic effects of NiCl2, we determined cell integrity via
detection of lactate dehydrogenase activity in the supernatants. Concentrations of this obligate intracellular enzyme did not
significantly differ in supernatants of cells exposed to control medium
or NiCl2 (data not shown). To analyze the functionality of
NiCl2-induced chemoattractive activity, we performed
chemotaxis assays employing peripheral blood monocytes. Conditioned
medium of HUVECs (Figure 1D) or HDMECs (Figure 1E) exposed to
NiCl2 for 8 hours showed strong chemotactic activity toward
monocytes as compared with supernatant obtained from nonstimulated
cells. Addition of a neutralizing antiserum against human MCP-1
effectively blocked chemotaxis of monocytes, thus identifying MCP-1 as
the principal monocyte-attractive activity produced by endothelial
cells upon stimulation with NiCl2 (Figure 1E). A
species-matched neutralizing antiserum against the chemokine MIP-1,
which is not inducible in endothelial cells,26 did not
show a significant inhibitory effect.
Induction of p38 MAP kinase by NiCl2 One of the major signaling pathways initiated by environmental stress stimuli results in activation of p38 MAP kinase. We therefore investigated whether NiCl2 mediates its effects via activation of p38. HUVECs were exposed to NiCl2 or TNF
for the time intervals indicated. Subsequently, activity of endogenous p38 was assessed in immune-complex kinase assays (Figure
2). We detected a sustained activation of
p38 by NiCl2 that steadily increases during the observation
period. TNF also strongly activated p38, albeit with different
kinetics: p38 activity was maximal 10 minutes after TNF stimulation
and steadily declined thereafter.
p38- and PC-PLC-dependent signal transduction pathways contribute to NiCl2-induced MCP-1 expression NiCl2 shares some pro-inflammatory properties with TNF, eg, up-regulation of MCP-1 (see above) or induction of
E-selectin and VCAM-123; however, the signaling mechanisms
induced by this compound are not well defined. We therefore analyzed
whether both stimuli use common pathways of intracellular signaling
that finally result in endothelial activation as reflected by, eg,
activation of NF-B.24 At present, it is not fully
understood how these different signaling pathways interfere with each
other. To address this question, we studied the impact of p38 as well
as of PC-PLC on NiCl2-induced activation of endothelium.
PC-PLC has earlier been implicated in TNF-induced activation of
NF-B14 although this role has been challenged after
the detection of intact NF-B signaling in cells derived from
acidic sphingomyelinase-deficient mice.34
We found that p38 or PC-PLC activities were blocked by the
pharmacological inhibitors SB202190 or D609, respectively. Endothelial cells were exposed to 1.5 mM NiCl2; 30 minutes prior to
stimulation, SB202190 (Figure 3A,C) or
D609 (Figure 3B,D) was added to the culture medium with increasing
concentrations. SB202190 partially blocked NiCl2-induced
MCP-1 mRNA and protein synthesis (Figure 3A,C). Addition of D609
resulted in a potent dose-dependent inhibition, which was maximal at
levels above 6.25 µg/mL D609 (Figure 3B,D).
To evaluate whether the SB202190- and D609-sensitive pathways act
independently of each other, the activation of the p38 substrates MAPKAP-kinase 2 and 3 in the presence of the inhibitors was analyzed. SB202190 dose-dependently blocked MAPKAP-kinase activation by NiCl2, TNF
NiCl2-induced transactivation of the MCP-1 promoter
and an artificial NF- B-binding
sites involved in the transcriptional control of the MCP-1
gene.10 To address the question of whether p38- and
PC-PLC-dependent pathways might interfere with NF-B activation, we
also studied effects of SB202190 and D609 on an NF-B-dependent
promoter construct. Again, NiCl2-induced NF-B-dependent
luciferase expression was decreased by approximately 50% when p38 was
inhibited by SB202190, whereas D609 completely abolished
NiCl2-induced NF-B-dependent reporter gene expression (Figure 5C-D). Thus, NF-B-mediated transcription appears to be a
target for p38- and PC-PLC-dependent signaling pathways. Mutation of
one or both of the 2 NF-B-binding sites of the MCP-1 promoter resulted in complete loss of promoter-inducibility, indicating that
NiCl2 induces signals that require intact NF-B-binding
sites (data not shown).
NiCl2 induces activation of IKK B-dependent gene expression, we studied its influence on IKK,
the major IB-phosphorylating enzyme during inflammatory
activation (see Karin11 for a review). IKK activity was
determined by immune-complex kinase assay after stimulation of
endothelial cells with various concentrations of NiCl2 or
TNF. NiCl2 dose-dependently induced IKK activation as
visualized by phosphorylation of GST-IB (Figure 6A). In contrast to the rapid activation
of IKK by TNF, NiCl2 induced a delayed but steady
increase of activity (Figure 6B). The kinetics of induced IKK
activity were then compared with the kinetics of IB degradation.
Under both conditions, IB degradation parallels IKK activity:
TNF was found to induce IB degradation within 5 minutes while
NiCl2-induced degradation is retarded (Figure 6C). Similar
degradation kinetics were observed for IB (data not shown), which
has recently been demonstrated to be functional for endothelial cell
activation.35 To determine the impact of IKK on
NiCl2-induced MCP-1 expression, we studied the effect of a
dominant-negative IKK mutant. MCP-1 expression in cells transiently
transfected with vector or dominant-negative IKK was determined by
flow cytometry as described in "Materials and methods." Only cells
expressing cotransfected GFPS65T to allow identification of cells
transfected with the inactive kinase mutant were analyzed. Figure
7 demonstrates that dominant-negative
IKK partially inhibits NiCl2- as well as TNF-induced
MCP-1 synthesis. Similar results could be obtained when IB
phosphorylation is blocked by the pharmacological inhibitor Bay11-0782
or when degradation of IB is blocked by the proteasome inhibitor
N-acetyl-leu-leu-norleucinal (ALLN, calpain inhibitor I; data not
shown).36
When these findings are taken together, induction of MCP-1 synthesis by
NiCl2 requires activation of IKK
Inhibition of p38 or PC-PLC signaling does not block either basal or induced transcriptional activity of nuclear Gal4p65 To analyze the transactivating capacity of nuclear p65, we overexpressed the factor that results in a nuclear accumulation. The p65 was expressed as a Gal4-DNA-binding domain (Gal4BD) fusion protein, which allows a selective assay for the transactivating features of transfected p65 by recruiting the factor to an artificial 5xGal4-binding site promoter luciferase construct.27 In the absence of the Gal4BD fusion protein, the 5xGal4 promoter is completely silent, no matter whether cells were stimulated or not (Figure 9A, lanes 1 and 2). Cotransfection of Gal4p65 results in a strong induction of promoter activity (Figure 9A, lane 3), which was further enhanced after stimulation of cells with TNF (Figure 9A, lane 4). This indicates
that TNF-induced signaling events result in an enhanced
transactivating capacity of p65, presumably by inducing a modification
of the transcription factor. This modification is most likely a
phosphorylation event, since it was reported earlier that TNF
induces phosphorylation of p65 resulting in an enhanced transcriptional
activity.37 However, stimulus-induced promoter activity
indicative of p65 modification was not blocked by either SB202190
(Figure 9B, lane 3) or D609 (Figure 9B, lane 6), suggesting that the
pathways leading to an enhanced p65 transactivating capacity are not
dependent on p38 or PC-PLC.
SB202190 blocks stimulus-induced coactivator function of p300 It was recently reported that phosphorylation of p65 results in an enhanced transactivating capacity by regulating association of transcriptional coactivators of the CBP/p300 family. We therefore cotransfected a plasmid expressing p300 to analyze its effects on the MCP-1 promoter. The p300 cotransfection resulted in a strong transcriptional activation of the MCP-1 promoter, which was further enhanced after treatment of cells with TNF (Figure 9C-D).
Interestingly, D609 treatment of nonstimulated cells slightly enhanced
p300-induced promoter activity (Figure 9D, lane 5) whereas in
stimulated cells D609 minimally reduced p300 coactivation (Figure 9D,
lane 6). However, this inhibition does not fully explain the inhibitory effect of D609, which at the same concentration resulted in a complete
loss of promoter activity in the absence of p300 (Figure 5B,D). This
was different in the presence of SB202190. Although the basal
coactivating capacity of p300 was only marginally affected (Figure 9C,
lane 5), the inhibitor completely blocked TNF-induced, p300-dependent transcriptional coactivation (Figure 9C, lane 6). Essentially similar results were seen in the case of NiCl2
(Figure 9C, lanes 7-12). This observation is compatible with the model in which the p38 MAP kinase pathway regulates stimulus-induced coactivator functions of such factors as CBP/p300, presumably by
facilitating the proper formation of the transcriptional complex.
The molecular mechanisms of NF- Although several important components of NiCl2-induced
signaling were identified here, the initial signaling events generated by this agent are not yet clear. One clue might be that the transition metals Ni2+ and Co2+ induce formation of
reactive oxygen intermediates according to the Fenton
reaction.38-40 These intermediates are used as
second-messenger molecules, which integrate a diverse variety of
stimuli into the NF- Another mechanism of signal transmission induced by environmental
stimuli involves activation of the p38 MAP kinase module. As outlined
in this study, NiCl2 can be added to the list of
environmental factors, such as heat shock, osmotic stress, and UV
light, that activate p38. The role of reactive oxygen intermediates for
p38 activation is not clear. They may weakly activate p38; however, antioxidants have been shown not to influence42 or even
induce p38 activation16 but also not to inhibit
it.43 It is striking that NiCl2-induced
activation of both p38 (shown in this study) and NF- Another pathway activated by NiCl2 and TNF The p38 MAP kinase does not modulate activity of IKK The impact of accessory pathways supporting NF-
We thank Martina Gropengiesser, Heide Häfner, Sybille Schmid, and Atiye Toksoy for excellent technical assistance; Peifeng Chen for her help in performing EMSA assays; Teizo Yoshimura for providing MCP-1 promoter luciferase constructs; and Bernd Baumann, Stephan Feller, Alex McLellan, Conny Seitz, and Thomas Wirth for critically reading the manuscript and helpful discussions.
Submitted January 13, 2000; accepted September 6, 2000.
Supported by grants GO 811/1-1 and LU 477/2-4 from the Deutsche Forschungsgemeinschaft and from the Fonds der Chemischen Industrie (M.G. and S.L.) and by grant 95.064.2 from the W.-Sander-Stiftung (R.G.).
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Matthias Goebeler, Department of Dermatology, University of Würzburg, Josef-Schneider-Str. 2, 97080 Würzburg, Germany; e-mail: goebeler-m.derma{at}mail.uni-wuerzburg.de; and Dr. Stephan Ludwig, Institut für Medizinische Strahlenkunde und Zellforschung, University of Würzburg, Versbacher Str. 5, 97078 Würzburg, Germany; e-mail: s.ludwig{at}mail.uni-wuerzburg.de.
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E. Caselli, S. Fiorentini, C. Amici, D. Di Luca, A. Caruso, and M. G. Santoro Human herpesvirus 8 acute infection of endothelial cells induces monocyte chemoattractant protein 1-dependent capillary-like structure formation: role of the IKK/NF-{kappa}B pathway Blood, April 1, 2007; 109(7): 2718 - 2726. [Abstract] [Full Text] [PDF]
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H. Satonaka, E. Suzuki, H. Nishimatsu, S. Oba, R. Takeda, A. Goto, M. Omata, T. Fujita, R. Nagai, and Y. Hirata Calcineurin Promotes the Expression of Monocyte Chemoattractant Protein-1 in Vascular Myocytes and Mediates Vascular Inflammation Circ. Res., March 19, 2004; 94(5): 693 - 700. [Abstract] [Full Text] [PDF]
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L. Burysek, T. Syrovets, and T. Simmet The Serine Protease Plasmin Triggers Expression of MCP-1 and CD40 in Human Primary Monocytes via Activation of p38 MAPK and Janus Kinase (JAK)/STAT Signaling Pathways J. Biol. Chem., August 30, 2002; 277(36): 33509 - 33517. [Abstract] [Full Text] [PDF]
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H. Akiba, J. Kehren, M.-T. Ducluzeau, M. Krasteva, F. Horand, D. Kaiserlian, F. Kaneko, and J.-F. Nicolas Skin Inflammation During Contact Hypersensitivity Is Mediated by Early Recruitment of CD8+ T Cytotoxic 1 Cells Inducing Keratinocyte Apoptosis J. Immunol., March 15, 2002; 168(6): 3079 - 3087. [Abstract] [Full Text] [PDF]
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| Copyright © 2001 by American Society of Hematology Online ISSN: 1528-0020 | |||||||||
B-dependent
transcription of the monocyte chemoattractant protein-1 gene in primary
endothelial cells
Top
Abstract
Introduction
-dependent pathways (see Kolesnick and
Krönke