IκBζ Is a Transcriptional Key Regulator of CCL2/MCP-1 Dominic G. Hildebrand, Eva Alexander, Sebastian Hörber, Simon Lehle, Kerstin Obermayer, Niels-Arne Münck, Oliver This information is current as Rothfuss, Julia-Stefanie Frick, Masami Morimatsu, Ingo of October 1, 2021. Schmitz, Johannes Roth, Jan M. Ehrchen, Frank Essmann and Klaus Schulze-Osthoff J Immunol 2013; 190:4812-4820; Prepublished online 1 April 2013; doi: 10.4049/jimmunol.1300089 Downloaded from http://www.jimmunol.org/content/190/9/4812

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2013 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

IkBz Is a Transcriptional Key Regulator of CCL2/MCP-1

Dominic G. Hildebrand,* Eva Alexander,* Sebastian Ho¨rber,* Simon Lehle,* Kerstin Obermayer,* Niels-Arne Mu¨nck,† Oliver Rothfuss,* Julia-Stefanie Frick,‡ Masami Morimatsu,x Ingo Schmitz,{,‖ Johannes Roth,† Jan M. Ehrchen,†,# Frank Essmann,* and Klaus Schulze-Osthoff*,**

CCL2, also referred to as MCP-1, is critically involved in directing the migration of blood monocytes to sites of inflammation. Consequently, excessive CCL2 secretion has been linked to many inflammatory diseases, whereas a lack of expression severely impairs immune responsiveness. We demonstrate that IkBz, an atypical IkB family member and transcriptional coactivator required for the selective expression of a subset of NF-kB target , is a key activator of the Ccl2 . IkBz-deficient macro- phages exhibited impaired secretion of CCL2 when challenged with diverse inflammatory stimuli, such as LPS or peptidoglycan. These findings were reflected at the level of Ccl2 gene expression, which was tightly coupled to the presence of IkBz. Moreover, mechanistic insights acquired by chromatin immunoprecipitation demonstrate that IkBz is directly recruited to the proximal Downloaded from promoter region of the Ccl2 gene and is required for transcription-enhancing histone H3 at lysine-4 trimethylation. Finally, IkBz-deficient mice showed significantly impaired CCL2 secretion and monocyte infiltration in an experimental model of perito- nitis. Together, these findings suggest a distinguished role of IkBz in mediating the targeted recruitment of monocytes in response to local inflammatory events. The Journal of Immunology, 2013, 190: 4812–4820.

esident tissue macrophages (Mw) make up the first de- pivotal role is emphasized by observations in many (auto)inflam- http://www.jimmunol.org/ fense line against infiltrating pathogens. In these cells, matory disorders, such as rheumatoid arthritis, multiple sclerosis, R pathogen recognition triggers a rapid and extensive cas- atherosclerosis, glomerulonephritis, or inflammatory bowel dis- cade of physiological responses, including, besides others, the ease, where increased CCL2 levels have been linked to the onset or direct intruder abatement and release of proinflammatory cyto- progression of disease (3, 8, 9). Strikingly, disruption of the Ccl2 kines that initiate a systemic immune response (1–3). gene in mice causes reduced susceptibility for inflammatory dis- Among the most prominent , CCL2 (MCP-1), the first eases (4, 5, 10, 11). As a consequence, almost all major pharma- discovered member of the CC family of chemokines, mediates the ceutical companies aim at developing antagonistic small molecules targeting the most relevant CCL2 receptor CCR2 for the therapy of

chemotactic recruitment of circulating monocytes to inflammatory by guest on October 1, 2021 sites (4–7). Even though further signal molecules, such as CCL7 chronic inflammation (12, 13). and CCL13, exhibit overlapping functions, CCL2 seems to be Under noninflammatory conditions, the Ccl2 locusistranscribed the only nonredundant monocyte-specific chemokine (3, 5, 8). Its at low level, but rapid induction of gene expression occurs on ex- posure of cells to various proinflammatory stimuli (14, 15). The differential Ccl2 gene expression is controlled at the promoter level *Interfaculty Institute for Biochemistry, Eberhard Karls University, D-72076 Tu¨bingen, Germany; †Institute of Immunology, University of Mu¨nster, D-48149 Mu¨nster, Germany; by a complex network of transcriptional regulators, including NF- ‡Institute for Medical Microbiology and Hygiene, University Hospital Tu¨bingen, D-72076 x kB, C/EBP, AP-1, and Sp-1 (16). Despite such complexity, in- Tu¨bingen, Germany; Institute for Genetic Medicine, Hokkaido University, Sap- poro 060-8638, Japan; {Systems-Oriented Immunology and Inflammation Research, flammation-linked, high-level expression appears to be dominantly Helmholtz Center for Infection Research, D-38124 Braunschweig, Germany; ‖Insti- regulated by NF-kB (16–19). Recent research has shed new # tute for Molecular and Clinical Immunology, D-39120 Magdeburg, Germany; De- light on the mechanisms underlying transcriptional activation by NF- partment of Dermatology, University of Mu¨nster, D-48149 Mu¨nster, Germany; and **German Cancer Consortium (DKTK) and German Cancer Research Center (DKFZ), kB, which can regulate hundreds of different target genes. Current D-69120 Heidelberg, Germany efforts focus on understanding of how only certain subsets of NF-kB Received for publication January 9, 2013. Accepted for publication March 1, 2013. target genes are selectively induced depending on the cell type, a This work was supported by the Deutsche Forschungsgemeinschaft (SFB685, SFB773, given stimulus, and environmental parameters (20–22). Such insights and GRK1302) and Bundesministerium fu¨r Bildung und Forschung (AID-NET). may open up novel therapeutic strategies aiming to specifically D.G.H., F.E., J.M.E., J.-S.F., J.R., and K.S.-O. designed research; D.G.H., E.A., S.H., modulate NF-kB responses in chronic inflammation (20). S.L., N.-A.M., K.O., and O.R. performed research; I.S. and M.M. contributed new Despite the presence of high-affinity binding sites, only a fraction reagents and tools; D.G.H., N.-A.M., J.M.E., and O.R. analyzed data; D.G.H., F.E., and K.S.-O. wrote the manuscript. of NF-kB target genes is generally activated in response to an in- Address correspondence and reprint requests to Prof. Klaus Schulze-Osthoff, Inter- flammatory stimulus. It was suggested that inflammatory genes can faculty Institute for Biochemistry, Eberhard Karls University, D-72076 Tu¨bingen, be categorized in two groups, based on their kinetics of induction Germany. E-mail address: [email protected] and the requirement of synthesis. Whereas primary response The online version of this article contains supplemental material. genes are rapidly induced, the expression of secondary target genes Abbreviations used in this article: BMMw, bone marrow–derived macrophage; ChIP, is delayed and requires the prior synthesis of additional NF-kB chromatin immunoprecipitation; H3K4, H3 at lysine-4; H3K4m3, trimethylation of histone H3 at lysine-4; Mw, macrophage; MEF, mouse embryonic fibroblast; PGN, coregulators. In this context, IkBz,anatypicalIkB protein, has been peptidoglycan; PMw, peritoneal macrophage; qPCR, quantitative PCR; qPCR-ChIP, recently identified and implicated in differential NF-kB target gene quantitative PCR-coupled chromatin immunoprecipitation; qRT-PCR, quantitative expression in Mw (23–25), even though its physiological function RT-PCR; Zym, zymosan A. remains largely unknown. The IkBz-coding Nfkbiz gene is rapidly Copyright Ó 2013 by The American Association of Immunologists, Inc. 0022-1767/13/$16.00 induced as a primary NF-kB response gene by various inflamma- www.jimmunol.org/cgi/doi/10.4049/jimmunol.1300089 The Journal of Immunology 4813 tory stimuli. Upon de novo synthesis, protein interactions with p50 centrifugation. After resuspension, 3 3 106 cells/ml were seeded in un- NF-kB homodimers seem to mediate the recruitment of IkBz to coated tissue culture flasks and cultured under low oxygen conditions (5% target promoters (23, 26). IkBz is believed to act as a transcriptional CO2,5%O2)inMw medium supplemented with murine M-CSF (30 ng/ml; Immunotools). After 7 d of differentiation, cells were washed twice with coactivator required for the expression of a subset of NF-kBsec- PBS. Adherent cells were scraped off and cultured in 96-well plates (2 3 ondary response genes, presumably by facilitating transcription- 105 cells/cm2) under low oxygen conditions. enhancing nucleosome remodeling (21, 27). Activation and proinflammatory stimulation of PMw In this article, we identify the murine Ccl2 gene as a novel target and BMMw of IkBz-regulated gene expression and show that IkBz is critical forCCL2secretioninMw. Furthermore, in vivo evidence in Isolated PMw and differentiated BMMw were incubated in the presence of a model of experimental peritonitis suggests that this regulatory 20 ng/ml murine IL-4 (Immunotools) or 30 ng/ml murine IFN-g (Immu- notools) for 24 h to achieve alternative or classical Mw activation. For mechanism is relevant in systemic inflammation and necessary for proinflammatory stimulation, media of activated and nonactivated cells were the proper recruitment of monocytes to sites of inflammation. supplemented with 25 ng/ml murine IL-1b or TNF-a (Immunotools), 1 mg/ ml LPS (Escherichia coli serotype O111:B4; Sigma), 1 mg/ml peptidoglycan (PGN; Bacillus subtilis; Sigma), or 1 mg/ml zymosan A (Zym; Sigma). Materials and Methods Animals Generation of Raw264.7/TetOn-IkBz cells Nfkbiz ko/ko (28) and littermate control mice were used at 6–18 wk of age. Raw264.7 cells were genetically modified to allow inducible expres- Mouse work was conducted in accordance with the German law guidelines sion of the canonical IkBz isoform (IkBz(L); GenBank accession no. of animal care, as permitted by regional authorities (“Regierungspra¨sidium NM_001159394.1) using the pINDUCER system (29). In brief, IkBz(L) Tubingen,” application no. H6/12). was amplified from murine cDNA applying the primers 59-CACCAT-

¨ Downloaded from GATCGTGGACAAGCTGCTG-39 and 59-CTAGTATGGTGGTGCTCG- Culture of peritoneal and bone marrow Mw CTG-39. The amplicon was cloned into pENTR-D-TOPO using TOPO cloning (Invitrogen), and the coding sequence was transferred to pIN- Female C57BL/6 mice were euthanized by CO2 asphyxiation. For isolation DUCER20 using gateway technology (Invitrogen). Upon sequence verifi- of peritoneal Mw (PMw), the abdominal skin was removed, a catheter (24 cation, lentivirus was generated using the Lenti-X system (Clontech). After gauge) was inserted into the peritoneal cavity, and 10 ml ice-cold PBS was lentiviral transduction, Raw264.7 cells were cultured in the presence of injected. After massage of the peritoneum, peritoneal fluids were aspirated, 500 mg/ml G418 for selection of Raw264.7/TetOn-IkBz cells. centrifuged at 500 3 g for 5 min, and yielded PMw were resuspended in 0.5–1.5 ml Mw medium containing DMEM/Hams F12, 10% FCS, and Culture and stimulation of Raw264.7 cells and mouse http://www.jimmunol.org/ MycoZapPlusCL antibiotics (Lonza). Next, cells were seeded in 96-well embryonic fibroblasts plates (100 ml cell suspension/well) and cultivated at 37˚C for 2 h. Finally, adherent cells were washed four times with culture medium to acquire Mouse embryonic fibroblasts (MEFs) were isolated at embryonic day 10.5 purified PMw. For isolation of bone marrow–derived Mw (BMMw), femur (E10.5) according to standard procedures (30). MEFs and Raw264.7 cells and tibia were separated and flushed with PBS, and cells were pelleted by were cultured under low oxygen and standard conditions, respectively. The by guest on October 1, 2021

FIGURE 1. LPS-triggered Ccl2 expression is impaired in IkBz-deficient PMF.(A)NonactivatedPMF from Nfkbizwt/wt and Nfkbizko/ko mice were stimulated with 1 mg/ml LPS for 4 h or left untreated. Total RNA was isolated and subjected to microarray analysis to identify novel putative targets of IkBz-dependent gene regulation. The heat map illustrates an exemplary subset of acquired data with representative members of the following three gene clusters: 1) putative IkBz-dependent target genes, 2) housekeeping genes, and 3) NF-kB target genes supposedly independent of IkBz. Fluorescence intensities of each gene were rescaled on a linear basis to fit into the range of 0–1 relative units and were color converted as indicated. Each experimental group comprised n = 3 animals. (B) Nfkbizwt/wt and Nfkbizko/ko PMF, classically activated with IFN-g, were cultured in the presence of 1 mg/ml LPS for indicated periods. Total RNA was subjected to qRT-PCR to determine levels of Nfkbiz, Tnfa, Ccl2,andIl6 expression. Values are mean 6 SEM from four to six experiments. (C) Nfkbizwt/wt and Nfkbizko/ko PMF, classically activated with IFN-g, were cultured in the presence of 1 mg/ml LPS for 24 h or left untreated (Ctrl). Culture supernatants were assayed for levels of CCL2, IL-6, and TNF-a. levels are given as amounts of cytokine per total protein. Values are mean 6 SEM from six experiments. Asterisks indicate statistical significance in a t test comparing wt and Nfkbizko/ko cells: *p , 0.05, **p , 0.005. 4814 CRITICAL ROLE OF IkBz IN MCP-1/CCL2 EXPRESSION latter additionally received 0.5 mg/ml G418 to maintain the transduced cell database (GEO accession no. GSE43075; http://www.ncbi.nlm.nih.gov/ population. Cells were seeded at a density of 105 cells/cm2 24 h before the geo/query/acc.cgi?token=vhghhumuiqkawje&acc=GSE43075). experiments. Optionally, 2 mg/ml doxycycline (Sigma) was added to in- duce ectopic IkBz expression. Subsequently, cells were cultured in the Immunoblotting presence of 1 mg/ml LPS for proinflammatory stimulation. Immunoblotting was performed as described previously (32). Anti–b-actin Quantitative RT-PCR mAb was purchased from Sigma (clone AC-74; 1:2000). Polyclonal rabbit anti-IkBz Ab was raised against a combination of peptides corresponding Whole-cell RNA was isolated using the RNeasy mini kit (Qiagen) and to short sequences of murine IkBz(L) (N-PGSPGSDSSDFSSTSSVSSC-C, reverse-transcribed (QuantiTect kit; Qiagen) as per manufacturer’s instruc- N-QIRRILKGKSIQQRAPPY-C), affinity-purified with these peptides, and tions. Quantitative PCR (qPCR) was performed in a LightCycler 480 II used at a concentration of 0.5 mg/ml. (Roche) using the Maxima Hot Start Taq DNA polymerase (Thermo) in the manufacturer’s two-step cycling protocol (384-well plates; 10 ml reaction). Chromatin immunoprecipitation Gapdh 7 Transcripts were analyzed applying the following primer pairs: A total of 10 cells/sample were stimulated with 1 mg/ml LPS for indicated 9 9 9 (5 -ACCACAGTCCATGCCATCAC-3 ,5-CACCACCCTGTTGCTGTAG- periods. Chromatin immunoprecipitation (ChIP) was performed with Dia- CC-39), Il6 (59-AGTTGCCTTCTTGGGACTGA-39,59-TCCACGATTTC- genode’s Red ChIP kit using polyclonal rabbit anti-IkBz or anti-histone H3 CCAGAGAAC-39), Ccl2 (59-GAGGAAGGCCAGCCCAGCAC-39,59-TG- (trimethyl K4; Abcam) Abs. ChIP efficiencies were determined by qPCR on 9 Nfkbiz 9 GGGCGTTAACTGCATCTGGC-3 ), (5 -TATCGGGTGACACAG- a LightCycler 480 II (Roche) using the Maxima Hot Start Taq DNA Poly- 9 9 9 Elam1 9 TTGGA-3 ,5-TGAATGGACTTCCCCTTCAG-3 ), (5 -CTCACT- merase in the manufacturer’s two-step cycling protocol (96-well plates; 9 9 9 Tnfa CCTGACATCGTCCTC-3 ,5-ACGTTGTAAGAAGGCACATGG-3 ), 20 ml volume). The following primer pairs spanning indicated promoter 9 9 9 9 (5 -CCTCAGCCTCTTCTCCTTCCT-3 ,5-GGTGTGGGTGAGGAGCA-3 ). regions of the murine Ccl2 gene were used: Ccl2 (59-TACCAAAT- Quantification of reverse-transcribed mRNA was performed using the -190/-3 TCCAACCCACAGTTTCTC-3, 59-CTCTGCACTTCTGGCTGCTCT- second derivate maximum-based “Advanced relative quantification al- G-59), Ccl2-1379/-1245 (59-GCCATCACAATGCTGCTAAGGA-39,59-

gorithm” of Roche’s LightCycler 480 Software (V1.5). Downloaded from TCGTGGAGACAATGACTGTAAAGG-39), Ccl2-2491/-2303 (59-CCTT- Affymetrix analysis TGTTGAGTCATTTCAGATTCTCC-39,59-TCTGTCAACAAAGGA- TGTTCTTCCC-39). Primers covering portions of Aktb (59-TCGAT- Total RNA was isolated from nonactivated PMF, which were either left ATCCACGTGACATCCA-39,59-GCAGCATTTTTTTACCCCCTC-39) untreated or stimulated with 1 mg/ml LPS for 4 h, and processed for hy- and Gapdh (59-TACTAGCGGTTTTACGGGCG-39,59-TCGAACAG- bridization on Affymetrix MoGene 1.0 ST arrays according to the manu- GAGGAGCAGAGAGCGA-39) genes were used as negative controls. facturer’s instructions. Single-strand cDNA was prepared using the Specificities of the primers were verified and PCR efficiencies were deter- Ambion WT Expression kit and labeled using the GeneChip WT Terminal mined. Analysis was performed according to manufacturer’s standard pro- http://www.jimmunol.org/ Labeling kit. Hybridization, scanning, and generation of probe cell in- cedures. In brief, treatment-specific ChIP efficiencies, expressed as percentage tensity files were performed on an Affymetrix GeneChip fluidics station of input, were first determined via the formula: Eff = d21*(1+E)(CpINPUT-CpIP), 450 and GeneChip Scanner 3000 7G using Affymetrix Expression Console where Eff represents immunoprecipitation- and primer-specific efficiencies, 1.1 software. Identification of LPS-regulated genes and comparison of d is dilution factor of respective input DNA, E is respective primer efficiency, LPS-induced gene expression in wild-type (wt) and knockout cells were CpINPUT is Cp value of respective input sample, and CpIP is immunopre- performed as described previously (31). Data are published in the GEO cipitation-specific Cp value. Next, treatment-specific relative occupan- by guest on October 1, 2021

FIGURE 2. The impact of IkBz on Ccl2 gene expression is conserved among murine cell lines and cell types. (A) Raw264.7 cells (Ctrl) were modified to enable the inducible doxycycline-dependent expression of the canonical isoform of IkBz [IkBz(L)], yielding the cell line Raw264.7/TetOn-IkBz (assigned IkBz). Both cell lines were cultured in presence or absence of 2 mg/ml doxycycline for 24 h to verify the induction of IkBz by immunoblot analysis using Abs specific for IkBz and b-actin (upper panel). Moreover, Raw264.7/TetOn-IkBz Mw were incubated in the presence of indicated increasing amounts of doxycycline for 24 h to induce IkBz in a dose-dependent manner (lower panel). Afterward, total RNA was isolated and subjected to qRT-PCR analysis of Ccl2, Il6, Nfkbiz, Nfkbia, and Elam1 levels (expressed as relative units [RU]; expression levels from cells treated with 2 mg/ml doxycycline were defined as 1 RU). In addition, supernatants were assayed in triplicate for secreted amounts of CCL2 (secretion of untreated cells was defined as 1 RU). Representative data sets of qRT-PCR and immunoblot analyses or means 6 SD of the cytokine measurements are shown for three experiments. (B and C) BMMF (nonactivated, B) or MEFs (C) from Nfkbizwt/wt and Nfkbizko/ko animals were cultured in the presence of 1 mg/ml LPS for 4 h. Total RNA was subjected to qRT-PCR analysis to determine levels of Nfkbiz, Tnfa, Ccl2, and Il6 expression. Values are mean 6 SEM for four (BMMF) or eight (MEFs) experiments, respectively. Asterisks indicate statistical significance comparing Nfkbizwt/wt and Nfkbizko/ko cells. *p , 0.05, **p , 0.005. The Journal of Immunology 4815 cies, which define the specific ratio of a distinct signal over the background at the indicated dilutions was added: rat anti-mouse F4/80:PE (A3-1; 1:20; and are expressed as “Fold Ctrl,” were determined using the equation: AbD), rat anti-mouse CD19:FITC (AbD; 6D5; 1:20), rat anti-mouse Gr-1: O=EffLocus,Treatment/(EffGapdh,Treatment +EffAktb,Treatment)*2, where O is Pacific Blue (RB6-8C5; 1:50; BioLegend), rat anti-mouse CD11b:allo- treatment- and -specific relative occupancy and EffLocus,Treatment is phycocyanin (M1/70; 1:50; Becton Dickinson), hamster anti-mouse CD11c: locus- and treatment-specific ChIP efficiency. allophycocyanin-Cy7 (HL3; 1:50; Becton Dickinson), and rat anti-mouse CD117:PE-Cy5 (2B8; 1:50; Becton Dickinson). After 20 min, cells were Quantification of cytokines pelleted and resuspended in FACS Lysing solution (Becton Dickinson) to Concentrations of CCL2, TNF-a, and IL-6 in peritoneal fluids and culture lyse RBCs, washed twice in FACS buffer, counted, and analyzed on an supernatants were determined by Cytometric Bead Arrays (Becton- LSRII flow cytometer (Becton Dickinson). BD CompBead (Becton Dickinson) standard operating procedures were used to calculate color compensa- Dickinson). Before stimulation of cells in 96-well plates, the culture me- tion parameters. Data were acquired using the WEASEL software (The dium was exchanged with 200 ml fresh medium per well. In case of BMMw, cytokine concentrations in culture supernatants were directly Walter and Eliza Hall Institute of Medical Research, Melbourne, Australia) compared and expressed as cytokine amounts per volume. Because of and evaluated applying the gating scheme outlined in Supplemental Fig. 1 donor mouse-specific differences in peritoneal cell counts, cytokine con- to determine the relative proportion of respective cell types. centrations in supernatants of PMw were normalized to the protein content Statistical analysis of cell lysates. To this end, 50 ml of 0.2 M NaOH was added to each well after aspiration of the supernatants, and protein concentrations in lysates Values are given as mean 6 SD or mean 6 SEM. For statistical com- were measured with the BCA Protein Assay (Thermo). parisons, hypotheses were tested using unpaired standard t tests. Asterisks indicate statistical significance (*p , 0.05, **p , 0.005, ***p , 0.0005). Experimental peritonitis Male Nfkbiz2/2 and control mice were used at 6–18 wk of age. Animals Results received an i.p. injection of 500 ml PBS containing 40 mg Zym (Sigma) per Identification of Ccl2 as a potential target gene of IkBz Downloaded from gram body weight. Mice were sacrificed 4 h later by CO2 asphyxiation. Subsequently, peritoneal cavity fluids were harvested and analyzed for Even though IkBz (Nfkbiz) was shown to be expressed in the cytokine amounts. In addition, peritoneal cells were FACS-phenotyped as monocyte cell lineage upon proinflammatory stimulation, its described previously (32). After centrifugation (500 3 g, 5 min), peritoneal physiological relevance remains largely unclear. To elucidate cells were resuspended in 10 ml FACS buffer (PBS containing 2% FCS), novel aspects of IkBz function, we focused on its impact on Mw repelleted by centrifugation (500 3 g, 5 min), and resuspended in FACS ko/ko buffer supplemented with Mouse SeroBlock FcR (1:100; AbD) to block Mw physiology. Initially, PMF and BMMF from wt and Nfkbiz FcRs CD16 and CD32. After 10 min of incubation on ice, an equivalent mice were assayed for basic cell-type features, such as phagocy- http://www.jimmunol.org/ volume of FACS buffer containing the following fluorochrome-labeled Abs tosis, oxidative burst, or chemotactic responsiveness, which did by guest on October 1, 2021

FIGURE 3. Mechanistic analysis of IkBz-dependent effects on Ccl2 gene regulation. (A) Schematic illustration of the murine Ccl2 promoter (National Center for Biotechnology Information gene ID: 20291). Gray areas indicate transcribed gene portions (light gray: intronic regions; dark gray: exons). Black ovals mark positions of NF-kB binding sites kb1, kb2, and kb3. Dashed lines highlight promoter portions known to be relevant for Ccl2 gene regulation, and assigned numbers indicate relative promoter positions. (B) Chromatin from LPS-treated Raw264.7 cells (2 h) and untreated control cells was analyzed in qPCR-ChIP assays using IkBz-specific Ab and primers spanning indicated genomic regions of the Ccl2 locus or portions of Actb and Gapdh genes, coding for b-actin and GAPDH, which served as control genes not targeted by IkBz. Values are mean 6 SD from three experiments. ChIP analyses with isotype control Abs were performed (data not shown) to exclude experimental artifacts. (C) Chromatin from LPS-treated Nfkbizwt/wt and Nfkbizko/ko nonactivated BMMF (5 h), as well as untreated control cells (Ctrl), was subjected to qPCR-ChIP assays applying H3K4m3-specific Ab. Primer Ccl2 (I) was used to determine the degree of H3K4 trimethylation of the Ccl2 promoter. Values are mean 6 SD from three experiments. ChIP analyses with isotype control Abs were performed (data not shown) to exclude experimental artifacts. 4816 CRITICAL ROLE OF IkBz IN MCP-1/CCL2 EXPRESSION not exhibit any phenotypical differences between both genotypes of IkBz, was expressed at comparable levels in wt and Nfkbizko/ko (data not shown). Next, global gene expression patterns of unstim- PMF. In contrast, regardless of LPS stimulation, the mRNA levels ulated and LPS-challenged nonactivated PMF were compared with of Ccl2 were significantly lower in IkBz-deficient PMF and, respect to Nfkbiz-specific differences using cDNA microarray anal- moreover, exhibited altered induction kinetics with mRNA levels ysis (GEO accession no. GSE43075). In accordance with the peaking earlier compared with wt cells. Similar characteristics function of IkBz as a transcriptional coactivator (23), numerous were observed for Il6 that served as control for IkBz-targeted gene LPS-inducible genes were expressed to a lower extent in Nfkbizko/ko regulation. Consistently, CCL2 levels in supernatants of Nfkbizwt/wt PMF, including Lcn2, Edn2, Il6, Il12b, Csf2, and Csf3 (Fig. 1A). PMF were ∼6-fold higher than in knockout supernatants, whereas At the same time, inducibility of NF-kB target genes supposedly amounts of TNF-a did not differ (Fig. 1C). not regulated by IkBz, including Cxcl1 (27), Cxcl2 (27), and Tnfa To determine whether this dependency of Ccl2 expression on the (23), did not significantly differ between both genotypes. Ac- Nfkbizko/ko-genotype is conserved among Mw, we included the Mw- cordingly, the approach seemed to allow the identification of like Raw264.7 cell line and BMMF into our analysis. Raw264.7 several new putative target genes of IkBz (e.g., Ccl17, Cxcl5, and cells were genetically modified to allow the doxycycline-inducible Lif) exhibiting lower expression in stimulated knockout PMF. Our expression of the canonical isoform of IkBz (IkBz(L);Fig.2A,upper special interest hereafter focused on the Ccl2 locus, because CCL2 panel). In Raw264.7/TetOn-IkBz cells, doxycycline treatment in- secretion is essential for monocyte recruitment during the onset of duced Nfkbiz mRNA levels tightly correlating with increased mRNA inflammation (4) and is, therefore, directly linked to the guardian levels of Ccl2 (maximum: ∼30-fold) and Il6,whereastheIkBz-in- function of Mw in peripheral tissues. dependent NF-kB target genes Elam1 and Nfkbia were not expressed to a higher degree (Fig. 2A, lower panel). This regulation of Ccl2 k z Downloaded from I B expression correlates with Ccl2 expression in was also reflected at the level of CCL2 secretion, which was ∼5-fold LPS-stimulated cell lines and types induced upon doxycycline-triggered IkBz expression (Fig. 2A, lower Previous analyses on Nfkbiz-dependent gene expression had been panel). performed in classically (IFN-g) activated PMF (23). To confirm Similar to PMF,BMMF from wt and Nfkbizko/ko mice were Ccl2 as an IkBz target gene, we therefore determined the kinetics stimulated with LPS and compared with respect to mRNA ex- of induction of Ccl2 mRNA levels and CCL2 cytokine levels in pression of Ccl2, Il6, and Tnfa (Fig. 2B). The inducibility of Ccl2 IFN-g–activated PMF in response to LPS by quantitative RT-PCR and Il6 was massively impaired in Nfkbizko/ko BMMF, whereas http://www.jimmunol.org/ (qRT-PCR; Fig. 1B) and cytometric bead assay assays (Fig. 1C), Tnfa expression did not significantly differ in both genotypes. respectively. TNF-a, which is supposedly regulated independently Finally, MEFs isolated from Nfkbizko/ko mice revealed a similar by guest on October 1, 2021

FIGURE 4. Ccl2 gene expression and CCL2 secretion are generally impaired in Nfkbizko/ko PMF in re- sponse to proinflammatory stimu- lation. wt and Nfkbizko/ko PMF, classically activated with IFN-g, were cultured with 1 mg/ml PGN (A), 1 mg/ml Zym (B), 25 ng/ml IL- 1b (C), or 25 ng/ml TNF-a (D). Total RNA was isolated after the indicated time points and subjected to qRT-PCR analysis to determine levels of Nfkbiz, Tnfa, Ccl2,andIl6 mRNA expression. Values are mean 6 SEM from five to six experiments. (E) Culture super- natants were assayed for levels of CCL2, IL-6, and TNF-a after 24 h. Cytokine levels are given as amount of cytokine per total protein. Values are mean 6 SEM for six experiments. Asterisks indicate statistical signifi- cance in a t test comparing Nfkbizwt/wt and Nfkbizko/ko cells: *p , 0.05, **p , 0.005, ***p , 0.0005. BD, Below limit of detection; ND, not determined. The Journal of Immunology 4817 pattern of gene regulation and showed significantly lower levels of Actb) in unstimulated cells exhibiting no or minimal IkBz ex- Ccl2 mRNA after LPS stimulation as compared with Nfkbizwt/wt pression. In contrast, the occupancy of the proximal promoter cells (Fig. 2C). region was ∼2.5-fold higher compared with the ones of control loci in the presence of LPS-induced IkBz. Importantly, no en- k z I B targets the proximal promoter region of the Ccl2 genomic richment was detected at the distal enhancer region in these locus samples, indicating that IkBz was specifically recruited to the Because our data clearly indicated an influence of IkBz on Ccl2 proximal Ccl2 promoter region. transcription, we next investigated a direct interaction of IkBz The trimethylation of histone H3 at lysine-4 (H3K4m3), a his- with the Ccl2 promoter. Two distinct promoter portions have been tone signature of open chromatin, is linked to promoter regions shown to be relevant in Ccl2 gene regulation: a proximal promoter with active transcription. We therefore investigated H3K4m3 oc- region and a distal enhancer portion, both of which harbor NF- cupancy of the proximal Ccl2 promoter region. qPCR-ChIP anal- kB–binding sites that may serve as anchor points for the recruit- ysis revealed increased H3K4 trimethylation after LPS stimulation ment of IkBz (Fig. 3A). of BMMF from Nfkbizwt/wt mice, which was absent in non- We first performed qPCR-coupled ChIP (qPCR-ChIP) analysis stimulated cells (Fig. 3C). Interestingly, almost no binding of in LPS-stimulated and untreated Raw264.7 cells (Fig. 3B). The H3K4m3 was found after LPS treatment of Nfkbizko/ko BMMF, relative occupancy of the Ccl2 locus was not increased using an indicating that the transcription-enhancing H3K4 trimethylation at IkBz-specific Ab compared with negative control loci (Gapdh, the Ccl2 promoter depends on IkBz. Downloaded from http://www.jimmunol.org/ by guest on October 1, 2021

FIGURE 5. Nfkbizko/ko Mw exhibit impaired CCL2 secretion independent of their state of activation. Differentially activated Nfkbizwt/wt and Nfkbizko/ko PMF (A) or BMMF (B) were cultured in the presence of 1 mg/ml LPS, 25 ng/ml IL-1b, 25 ng/ml TNF-a,1mg/ml PGN, 1 mg/ml Zym, or left untreated (Ctrl) for 24 h. Starting 24 h before stimulation, cells received 30 ng/ml IFN-g or 20 ng/ml IL-4 to induce classical and alternative activation, respectively. Naive Mw were left untreated. Culture supernatants were assayed for levels of CCL2, IL-6, and TNF-a. Cytokine levels are given as amounts of cytokine per total protein (PMF) or per volume of supernatant (BMMF). Values are mean 6 SEM from six experiments. Asterisks indicate statistical significance in a t test comparing Nfkbizwt/wt and Nfkbizko/ko cells: *p , 0.05, **p , 0.005. BD, Below limit of detection; ND, not determined. 4818 CRITICAL ROLE OF IkBz IN MCP-1/CCL2 EXPRESSION

Role of the proinflammatory stimulus and Mw polarization on IkBz-induced cytokine expression Expression of IkBz is not restricted to LPS stimulation but can be induced in response to several activators of NF-kB, and especially of TLR/IL-1 signaling (23, 24). To investigate whether expression of Ccl2 and CCL2 secretion were generally impaired in IkBz- deficient cells, we analyzed the kinetics of Nfkbiz and Ccl2 in- duction in IFN-g–activated PMF in response to several proin- flammatory stimuli, including PGN, Zym, IL-1b,andTNF-a. PGN (Fig. 4A) and Zym (Fig. 4B) strongly induced Nfkbiz, as well as Il6 and Ccl2. The mRNA induction of both cytokines, but not of Tnfa, was significantly reduced in Nfkbizko/ko cells when compared with wt PMF. Induction of Nfkbiz and the cytokines was signif- icantly lower in response to TNF-a (Fig. 4C) and IL-1b (Fig. 4D), but even under these conditions an impaired Ccl2 expression was observed in IkBz-deficient cells. Consistent results were obtained at the level of cytokine secretion, when culture supernatants of cells were assayed after 24 h of stimulation (Fig. 4E).

The previous experiments used PMw that were classically ac- Downloaded from tivated with IFN-g, which induces Mw M1 polarization. Naive FIGURE 6. Zym-induced experimental peritonitis. (A) Schematic out- PMF and PMF activated with IL-4 to induce alternative (M2) line of the experiment. Mice were i.p. injected with 500 ml PBS in the polarization were assayed for cytokine secretion after 24 h of absence (Ctrl) or presence of 40 mg Zym/g body weight. Animals were stimulation with LPS, PGN, IL-1b, and TNF-a, respectively, to sacrificed 4 h later after the onset of inflammation to analyze peritoneal cavity fluids. (B) Levels of CCL2, IL-6, and TNF-a were measured and are investigate whether gene-regulatory effects of IkBz are dependent given as amounts per cavity. Values are mean 6 SEM from five experi- on Mw polarization (Fig. 5A). The same experiments were also ments. (C) Quanta of monocyte infiltration into the peritoneal cavity were http://www.jimmunol.org/ performed with differentially activated BMMF (Fig. 5B). More- determined by counting and FACS phenotyping of peritoneal cells. Values over, stimulation of PMF with Zym was performed, as that trigger are mean 6 SEM from five experiments. Asterisks indicate statistical was later used for in vivo experiments. Interestingly, whereas IL-6 significance comparing Nfkbizwt/wt and Nfkbizko/ko mice: *p , 0.05. BD, production strictly depended on IkBz in classically (IFN-g) acti- Below detection limit. vated PMF (Fig. 4E), in naive and IL-4–primed PMF, IL-6 se- cretion occurred largely independently of IkBz (Fig. 5A). Unlike PMF, BMMF revealed a strict IkBz dependence of IL-6 secretion Discussion after stimulation with LPS and PGN, regardless of their polari- Growing evidence suggests that the induction of NF-kB–regulated genes is not solely defined by the nuclear translocation of NF-kB, zation status (Fig. 5B). With respect to CCL2, cytokine secretion by guest on October 1, 2021 was strongly impaired in Nfkbizko/ko PMF, as well as in BMMF, but that each NF-kB target gene has an individual expression regardless of whether experiments were conducted in naive, profile regarding kinetics, cell type, stimulus specificity, or re- classically activated, or alternatively activated cells. Secretion of quirement of cofactors, which is thought to ensure the selectivity of an immune response. A comparison of global gene expres- TNF-a, which is not directly regulated by IkBz, was not impaired wt/wt ko/ko but generally even slightly increased in the absence of IkBz. Al- sion patterns of LPS-stimulated Nfkbiz and Nfkbiz PMF together, these results demonstrate that CCL2 secretion, if trig- identified, among other genes, Ccl2 as a strongly inducible target gered by a proinflammatory stimulus, is strictly dependent on of IkBz-mediated transcription. To verify this finding, we chal- IkBz in both PMF and BMMF in each state of activation. In lenged PMF and BMMF with various proinflammatory activators for the analysis of Ccl2 gene expression and CCL2 secretion. Our contrast, for other cytokines such as IL-6, the requirement of IkBz ko/ko is influenced by the Mw type and their activation status. experiments consistently revealed that Nfkbiz Mw exhibited profoundly impaired Ccl2 expression and CCL2 secretion, in In vivo evidence in a model of experimental peritonitis particular, when stimulated with TLR ligands. At the level of gene CCL2 has originally been identified to be necessary for the in- transcription, similar observations were made in untreated cells or flammatory recruitment of monocytes in several models of exper- after stimulation with IL-1b or TNF-a, which elicit only weak imental peritonitis (4–6). Thus, to investigate whether IkBz is also inflammatory responses. These findings were supported by the required for the regulation of Ccl2/CCL2, we used an experimental observation that the inducible expression of ectopic IkBz in peritonitis model in IkBz-proficient and -deficient mice (Fig. 6A). Raw264.7 cells tightly correlated with increasing Ccl2 mRNA This seemed reasonable, because the induction of endogenous levels. Thus, our results in knockout and overexpression models CCL2 in this system is highly Mw dependent (6). To induce sys- suggest that transcriptional regulation of Ccl2 directly depends temic inflammation, we injected mice with Zym into the peritoneal on IkBz. cavity, and 4 h later, levels of TNF-a, IL-6, and CCL2, as well as IkBz has been shown to exert its gene-activating effects largely quantities of monocyte infiltration, were determined. FACS phe- on so-called secondary response genes. In contrast with primary notyping procedures applied for that purpose (33) are outlined in NF-kB target genes that are immediately induced in the absence Supplemental Fig. 1. As expected, Zym-treated Nfkbizko/ko mice of new protein synthesis, secondary response genes require the exhibited lower levels of CCL2 (∼50%) compared with equally prior synthesis and coactivator function of IkBz, which itself is treated wt animals (Fig. 6B). Similar results were observed for IL-6 a primary NF-kB target gene (21, 23, 27). Consequently, these (∼30% of Nfkbizwt/wt mice), whereas levels of TNF-a were ∼5-fold genes exhibit a delayed kinetics of gene induction. Consistent with higher in IkBz-deficient animals. With respect to the physiology this model, the kinetics of gene induction obtained in LPS- of inflammation, the number of infiltrating monocytes was inter- stimulated Raw264.7 cells and PMF could clearly discriminate estingly ∼3-fold higher in wt animals (Fig. 6C). between Nfkbiz and Tnfa as IkBz-independent primary response The Journal of Immunology 4819 genes on one side, and Il6 and Ccl2 as IkBz-dependent secondary influence homeostasis and trafficking of monocytes. IL-6, for ex- response genes on the other hand. Maximal expression of Il6 and ample, has been shown to orchestrate the switch from neutrophil Ccl2 was delayed but prolonged in Nfkbizwt/wt cells. In contrast, in to monocyte recruitment during acute inflammation. In that con- the absence of IkBz, Ccl2 and Il6 expression were considerably text, the observation that TNF-a levels in peritoneal cavity fluids impaired and not sustained, but rather resembled Tnfa and Nfkbiz were dramatically higher in case of Nfkbizko/ko mice upon i.p. in the kinetics of gene induction (Fig. 1B). In addition, our ChIP injection of Zym is interesting, because Nfkbizwt/wt and Nfkbizko/ko analyses revealed that IkBz was directly recruited to the proximal PMF did not exhibit such differences in vitro with respect to TNF- region of the Ccl2 promoter, which harbors a single NF-kB a secretion. Similar, but so far unexplained, effects have been binding site. The recruitment of IkBz was associated with histone observed previously (23) and might be secondary because of the H3K4 trimethylation of the proximal promoter region as a marker downregulation of IkBz-dependent genes regulating the expres- of active transcription. Interestingly, in the absence of IkBz, H3K4 sion of TNF-a in certain cell types. Differences in gene expression trimethylation did not occur, which, together with other lines of of additional cell type contributing to the outcome of the systemic evidence, suggests that chromatin remodeling events are essential peritonitis model might therefore explain this phenomenon. Thus, for IkBz action. the ability of IkBz to concertedly regulate various cytokines and CCL2 expression is essentially required for monocyte recruit- physiological functions might distinguish IkBz as a key tran- ment to sites of inflammation and has therefore been implicated in scriptional regulator of certain inflammatory processes (34). (auto)inflammatory diseases, as well as in the course of immune Moreover, other biological processes, such as TH2 polarization defense against pathogens. This ambivalence of beneficial and (35), bone morphogenesis (16), or tumor progression (9, 16, 36) pathogenic effects of CCL2 underscores the requirement of a tight have been shown to be regulated by CCL2 and could be controlled regulation of proinflammatory factors for an appropriate immune by IkBz. Downloaded from response. To investigate the role of IkBz-mediated CCL2 secretion In summary, we have uncovered an essential regulatory mech- in vivo, we chose a model of experimental peritonitis in which anism of Ccl2 gene regulation in Mw. We demonstrate that IkBz, CCL2 secretion is predominantly driven by PMF (6). Our results presumably by the p50 NF-kB–mediated recruitment to the demonstrate that knockout animals indeed exhibit a decreased proximal Ccl2 promoter and subsequent histone modification, capacity to secrete CCL2, as well as IL-6, and thus provide further enables transcription of the Ccl2 locus, thus allowing production evidence for the tight control of both cytokines by IkBz. Inter- of high levels of CCL2 in the course of proinflammatory pro- http://www.jimmunol.org/ estingly, whereas IL-6 and CCL2 expression were strongly re- cesses. As indicated by observations in an in vivo model of sys- duced in IkBz-deficient mice, TNF-a levels were not decreased temic inflammation, this IkBz-dependent mechanism may be but even increased in comparison with wt animals. Similar find- crucial for Mw physiology, for example, in the course of Mw- ings were observed in PGN-stimulated BMMF and PMF from mediated effector cell recruitment. Nfkbizko/ko animals and might be caused by the fact that IkBz can additionally function as a specific inhibitor of particular target Disclosures genes (23). The authors have no financial conflicts of interest. Although cells of the monocyte/Mw lineage are the major source by guest on October 1, 2021 of CCL2, several other cell types, including fibroblasts and epi- thelial and endothelial cells, can express CCL2 (16). Further in- References vestigations are necessary to verify whether the strict control of 1. Soehnlein, O., and L. Lindbom. 2010. Phagocyte partnership during the onset CCL2 by IkBz is also relevant in these cell types. Our exemplary and resolution of inflammation. Nat. Rev. Immunol. 10: 427–439. ko/ko 2. Luster, A. D. 1998. Chemokines—chemotactic cytokines that mediate inflam- investigation of wt and Nfkbiz MEFs indicates that the de- mation. N. Engl. J. Med. 338: 436–445. scribed mechanism is not restricted to Mw. Interestingly, however, 3. Charo, I. F., and R. M. 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