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Aberrant expression of neutrophil and macrophage-related genes in a murine model for human neutrophil-specific granule deficiency Adrian F. Gombart,1 Utz Krug, James O’Kelly, Eun An, Vijaya Vegesna, and H. Phillip Koeffler Cedars-Sinai Medical Center, Division of Hematology/Oncology, Burns & Allen Research Institute and David Geffen School of Medicine at University of California Los Angeles

Abstract: Neutrophil-specific granule deficiency involves inheritance of germline mutations in the CCAAT/enhancer-binding protein ␧ (C/EBPE) gene. Humans and mice lacking active C/EBP␧ suffer frequent bacterial infections as a result of functionally defective neutrophils and macrophages. We hypothesized that these defects reflected dysregulation of important immune response genes. To test this, gene expression differences of peritoneally derived neutrophils and macrophages from C/EBP␧ⴚ/ⴚ and wild-type mice were determined with DNA microarrays. Of 283 genes, 146 known genes and 21 expressed sequence tags (ESTs) were down-regulated, and 85 known genes and 31 ESTs were up-regulated in the C/EBPⴚ/ⴚ mice. These included genes involved in cell adhesion/chemotaxis, cytoskeletal organization, signal transduction, and immune/inflammatory responses. The cytokines CC chemokine ligand 4, CXC chemokine ligand 2, and interleukin (IL)-6, as well as cytokine receptors IL-8RB and granulocyte-colony stimulating factor, were down-regulated. Chromatin immunoprecipitation analysis identified binding of C/EBP␧ to their promoter regions. Increased expression for lipid metabolism genes apolipoprotein E (APOE), scavenger receptor class B-1, sorting protein-related receptor containing low-density lipoprotein receptor class A repeat 1, and APOC2 in the C/EBP␧ⴚ/ⴚ mice correlated with reduced total cholesterol levels in these mice before and after maintenance on a high-fat diet. Also, C/EBP␧-deficient macrophages showed a reduced capacity to accumulate lipids. In summary, dysregulation of numerous, novel C/EBP␧ target genes impairs innate immune response and possibly other important biological processes mediated by neutrophils and macrophages. J. Leukoc. Biol. 78: 1153–1165; 2005. Key Words: C/EBPε 䡠 SGD 䡠 DNA microarray 䡠 lipid metabolism

INTRODUCTION Neutrophils, monocytes, and tissue-based macrophages are essential cellular components of the innate immune system, 0741-5400/05/0078-1153 © Society for Leukocyte Biology

which provides the host initial defense against invading pathogens. Impairment of one or more of these components greatly affects the ability of an individual to fight infection. Neutrophil-specific granule deficiency (SGD) is a rare, hematologic disorder characterized by a lifetime of recurrent, pyogenic infections [1, 2]. The neutrophils from these individuals display abnormal nuclear morphology (pseudo-Pelger-Hue¨t anomaly), defects in chemotaxis, stimulated oxygen metabolism, and bacterial cell killing, as well as loss of some azurophilic, most specific and tertiary granule proteins. These proteins include defensins, lactoferrin (LTF), and gelatinase [1–7]. Defects in eosinophil-specific granule content, including eosinophil cationic protein, eosinophil-derived neurotoxin, and major basic protein (MBP), were noted as well [8]. Loss of a myeloid-specific transcription factor was hypothesized to be involved in the development of SGD [5] but not until the development of the CCAAT/enhancer binding protein (C/EBP)ε-deficient murine model was a candidate gene apparent [9]. Neutrophils from C/EBPε-deficient mice possessed bilobed nuclei, lacked expression of specific and tertiary granule proteins, and displayed aberrant chemotaxis, phagocytosis, respiratory burst, and bactericidal activities similar to neutrophils from individuals with SGD [9 –11]. In addition, expression of eosinophilic granule genes (eosinophil peroxidase and MBP) was impaired [12, 13]. Also, the mice were susceptible to bacterial infections [9]. The striking phenotypic similarities between the human and murine conditions suggested a loss of functional C/EBPε in SGD. Germ-line mutations in the CEBPE locus were identified in two SGD patients, explaining the genetic defect responsible for this disease [14 –16]. Although clearly an important factor in normal neutrophil differentiation and function, mounting evidence suggested that C/EBPε was involved in monocyte/macrophage function. C/EBPε is expressed at significant levels in monocytes/macrophages and related cell lines in humans and mice [17, 18]. The forced overexpression of C/EBPε in a pre-B acute lymphoblastic leukemia cell line induced expression of several cytokines expressed by macrophages [17]. Representational difference

1

Correspondence: Cedars-Sinai Medical Center, Division of Hematology/ Oncology, Davis Bldg. 5019, Los Angeles, CA 90048. E-mail: gombarta @csmc.edu Received May 11, 2005; revised June 23, 2005; accepted July 7, 2005; doi: 10.1189/jlb.0504286

Journal of Leukocyte Biology Volume 78, November 2005 1153

analysis using peritoneal neutrophils and macrophages from wild-type and C/EBPε-deficient mice identified a small set of differentially regulated genes. These included several genes specific to myelomonocytic cells [19]. Consistent with aberrant macrophage gene expression, phenotypic changes were observed in vivo. Signs of immaturity, impaired phagocytosis, and further altered myelomonocytic-specific gene expression were identified in macrophages from C/EBPε-deficient mice [18]. Recently, the role of C/EBPε in human monocyte/macrophage function was addressed. Abnormalities in monocytic cells from one SGD individual who lacked a functional C/EBPε were reported [20]. Flow cytometric analysis of peripheral blood cells revealed aberrant expression of CD45, CDllb, CD14, CD15, and CD16 on cells from the SGD individual as compared with normal controls. Also, CD14⫹ cells from this individual stained weakly for the monocyte-specific enzyme, nonspecific esterase, and electron micrographs revealed abnormal morphology [20]. Taken together, studies of C/EBPε-deficient myeloid cells from humans and mice demonstrate an essential role for C/EBPε in the normal development and function of neutrophils and macrophages. The parallels between the human and murine conditions indicate that the C/EBPε-deficient murine model will serve as an extremely powerful tool in further characterizing this rare human disease. In this study, we sought to define further the role that C/EBPε plays in mediating host immune defense by identifying possible target genes of C/EBPε in neutrophils and macrophages.

MATERIALS AND METHODS Mice and sample preparation The C/EBPε wild-type (⫹/⫹) and deficient (⫺/⫺) mice (129/SvEv x NIH Black Swiss) were generously provided by Kleanthis G. Xanthopoulos (Anadys Pharmaceuticals, Inc., San Diego, CA) and Julie Lekstrom-Himes (Millennium Pharmaceuticals, Inc., Cambridge, MA). They were maintained in pathogenfree facilities. To prepare RNA for array hybridization, four age-matched (6 – 8 weeks) mice of each genotype received an intraperitoneal injection of 2 ml 4% sterile thioglycollate broth (Sigma Chemical Co., St. Louis, MO). At 24 h post-injection, all mice were killed, and peritoneal exudate cells were harvested by lavage with Hanks’ balanced salt solution (HBSS; Sigma Chemical Co.) and kept on ice. Total cell numbers were counted, and percentages of neutrophils and macrophages were determined by differential counting of Wright-Giemsa-stained cytospins (100 –200 cells per sample) using a light microscope. The preparations were composed of ⬃30% monocytes, 60% neutrophils, and 7–10% lymphocytes in the C/EBPε⫹/⫹ and C/EBPε⫺/⫺ mice. To reduce individual variation in subsequent experiments, the cells from all wild-type or all C/EBPε null mice were pooled prior to probe synthesis. To isolate macrophages and neutrophils from the peritoneal lavage for quantitative reverse transcriptase-polymerase chain reaction (QRT-PCR) analysis, thioglycollate-elicited cells were harvested, plated in tissue-culture dishes, and incubated for 2 h at 37°C. The suspension cells were removed from the plate and found to be ⬃95% neutrophils by differential counts of WrightGiemsa-stained cytospins. Total RNA and protein were isolated from cells using TRIzol reagent as described by the manufacturer (Life Technologies, Inc., Gaithersburg, MD), and DNase I was treated (Promega, Madison, WI) and purified using an RNeasy spin column (Qiagen, Valencia, CA). The quality and balance of the RNA samples were tested by electrophoresis on a denaturing agarose gel.

Hybridization and analysis of the affymetrix GeneChip murine 11K set The murine 11K set consists of two probe arrays, which allow monitoring of the relative abundance of greater than 11,000 genes selected from the UniGene

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Journal of Leukocyte Biology Volume 78, November 2005

(8/96 and Build 4.0) and TIGR (Build 1.0 ␤) databases (Affymetrix Inc., Santa Clara, CA). Biotinylated cRNAs were prepared and fragmented following the manufacturer’s protocol (Affymetrix Inc.). The murine 11K A and B arrays were prehybridized and hybridized as instructed by the manufacturer using the GeneChip Fluidics Station 400 (Affymetrix Inc.). The probed arrays were scanned with a Hewlett Packard gene array scanner. The scanned images were analyzed and compared using GeneChip 3.1 software (Affymetrix Inc.). To select genes for further analysis, the default parameters in the GeneChip 3.1 software were used to assign “increased,” “decreased,” and “no change” calls in the knockout relative to the wild-type samples. Initially, all genes that were classified as no change were excluded. From this list, all genes that were scored as absent on both arrays were excluded. Furthermore, genes with less than a twofold change in expression (increase or decrease) were excluded.

Real-time QRT-PCR analysis For cDNA synthesis, 1 ␮g DNase-treated total RNA was reverse-transcribed using Superscript II RT as described by the manufacturer (Invitrogen, Carlsbad, CA). The quantity of the cDNA was determined, and the samples were diluted to 10 ng/␮l. For QRT-PCR, 50 ng each sample was amplified in quadruplicate on an iCycler thermal cycler equipped with an optical module to measure fluorescence during each cycle (Bio-Rad, Hercules, CA). Amplification was performed with HotStar Taq DNA polymerase as described by the manufacturer (Qiagen, Chatsworth, CA). To monitor the amplification of the target gene, each reaction contained SYBR Green diluted 1:60,000 (Molecular Probes, Eugene, OR). Reactions were heated at 95°C for 15 min and then subjected to 45 cycles of 95°C, 15 s; 60°C, 15 s; 72°C, 30 s; and a fourth step, at the empirically determined Tm –2°C of the PCR product for 20 s. The intensity of fluorescence was determined during the fourth step of each cycle to minimize fluorescence from nonspecifically amplified products. Gel electrophoresis indicated that the primer pairs used in this study amplified little or no spurious products after 35 or more cycles. The primers were designed using the Primer 3 program to have a Tm of 60°C and to produce an 80- to 150-base pair product [21]. The primer sequences are available upon request. A cDNA was serially diluted to generate a standard curve for each gene. For down-regulated genes in the C/EBPε⫺/⫺ mice, the wild-type cDNA was used, and for up-regulated genes, the knockout cDNA was used. The relative fluorescence readings were plotted on a Boltzmann-Sigmoidal curve, and the V50 was determined for each sample. The V50 values were plotted on the standard curve to determine the relative expression levels for each sample. All gene expression levels were normalized using the 18S rRNA levels in each sample.

Chromatin immunoprecipitation (ChIP) Five wild-type mice were injected with 4% thioglycollate, and peritoneal cells were harvested at 24 h post-injection as described above. In addition, bone marrow cells were flushed from the femur with HBSS. Cells were resuspended in Iscove’s modified Dulbecco’s medium with 10% fetal bovine serum. Chromatin was prepared for immunoprecipitation as instructed by the manufacturer (Upstate USA, Inc., Charlottesville, VA). The sonicated chromatin was immunoprecipitated with a rabbit anti-C/EBPε antiserum [22]. As negative controls, immunoprecipitations containing no antibody or rabbit preimmune serum were included. Reactions were incubated at 4°C overnight on a rotator. Washing and preparation of the sample for PCR were performed as instructed by the manufacturer (Upstate USA, Inc.). PCR was performed as described above for the following promoters: granulocyte-colony stimulating factor receptor (CSF3R), CXC chemokine ligand 2 (CXCL2), CC chemokine ligand 4 (CCL4), interleukin (IL)-6, IL-8 receptor (IL-8R)B, CD14, osteopontin/secreted phosphoprotein 1 (SPP1), LTF, and telomerase RT (TERT). Each promoter was amplified for 35 cycles with the exception of the IL-6 promoter, which was amplified for 40 cycles. The primer sequences are available upon request.

Atherogenic diet, determination of blood cholesterol levels, and Oil-Red-O staining Mice were fed mouse chow (Ralston Purina Co., St. Louis, MO) until 12 weeks old. Subsequently, the mice were placed on an atherogenic diet for 18 weeks consisting of 75% Purina chow plus 15% fat (primarily cocoa butter), 1.25% cholesterol, and 0.5% sodium cholate (TD90221; Teklad Research Diets, Madison, WI). Plasma lipid levels were determined as described previously [23]. Results are given as mean ⫾ SE. Student’s t-test of unpaired observations

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was used to determine significance of differences between wild-type and C/EBPε null mice in lipid and lipoprotein levels. Values of P ⬍ 0.05 were considered to be significant. To detect the accumulation of lipids in peritoneal macrophages, cells were harvested by lavage from the peritoneum of mice 4 days post-thioglycollate injection. Cells (⬎95% macrophages by Wright-Giemsa staining) were attached to glass slides by cytocentrifugation, fixed in 10% formalin, stained for 30 min in Oil-Red-O, and washed in double-distilled H2O, and coverslips were attached with an aqueous mounting solution and sealed. Cells were visualized under a light microscope and photographed at 200⫻ and 1000⫻ (with oil).

RESULTS

infection are those involved in cell adhesion/chemotaxis (6%), cytoskeleton (6%), proteases and protease inhibitors, and transcriptional regulation (6%). These comprised another 18% of the genes on the list (Table 1). Of the 41 genes that are highly active in macrophages and neutrophils, 28 belong to these five categories. Finally, 4% of the 231 known genes included those involved in lipid metabolism, an important activity of macrophages (Table 1). These included increased expression of APOC2, scavenger receptor class B-1 (SCARB1), sorting protein-related receptor containing LDLR class A repeat 1 (SORL1), and APOE in the C/EBPε-deficient mice (Table 1).

Identification of differentially regulated genes

Confirmation of differential gene expression

For all the experiments described herein, the peritoneal lavage samples were harvested 24 h after a thioglycollate-induced peritonitis. This treatment activates the cells as compared with untreated, resident peritoneal cells. Morphologically, the preparations for array hybridization were composed of ⬃30% monocytes, 60% neutrophils, and 7–10% lymphocytes in the C/EBPε⫹/⫹ and C/EBPε⫺/⫺ mice, as determined by differential counts of cytospins stained with Wright-Giemsa. This cellular composition was consistent with prior studies and would allow us to identify monocytic and granulocytic gene expression differences [10, 19]. To minimize variation among individual mice, the samples were pooled according to the mouse genotype, and the chip set was hybridized. A total of 283 differentially regulated genes were identified. Of these, 231 were known and 52 were expressed sequence tags (ESTs; Tables 1 and 2). The genes were grouped using the Gene Ontology Consortium database according to a known or putative biological role (Table 1); however, a number of these genes may be classified under one or more categories, as they possess multiple biological functions [24]. For example, APOE, which is up-regulated in the knockout mouse, is primarily involved in lipid metabolism but also is implicated in defense-immune responses [25]. For simplicity, each gene was placed in only one category. Of the 231 known genes, 41 were reported previously as highly expressed in macrophages and/or neutrophils [26 –33]. These genes are differentially regulated during differentiation or in response to cytokines, bacterial infection, or LPS (Table 1, bold text). In addition, 22 of the 231 known genes have C/EBP family members implicated in their regulation or possess C/EBP sites in their promoters as determined by searches of the PubMed database (Table 1). Of these 22 genes, 12 were present in the aforementioned group of 41 genes expressed in macrophages and/or neutrophils, suggesting that C/EBPε may be important for their transcriptional regulation. The immune/inflammatory and signal transduction categories comprised 25% (59 of 231) of the known genes. These included eight down-regulated cytokines/chemokines (CCL2, CCL7, CSF1, CCL4, CXCL2, IL-1B, IL-1RN and IL-6), 10 down-regulated receptors (IL-1R1 and -2, Csf2rb1, IL-8RB, IL-10RA, CCR1, CCR3, CCR7, IL-1R2, and CSF3R), and four up-regulated receptors (IFNGR1, CCR2, TNF-R1, and -2) in the myeloid cells of C/EBPε⫺/⫺ mice as compared with those from the wild-type mice (Table 1). Other categories of genes important in the response of neutrophils and/or macrophages to

To confirm the differential expression for some of the genes in Table 1, we performed real-time QRT-PCR on cDNAs synthesized from the same RNA samples used for the DNA array. Also, cDNAs prepared from RNA isolated from purified, peritoneal macrophages or neutrophils, as described in Materials and Methods, were analyzed. Of 22 genes that were examined, expression of 17 (77%) agreed with the array results (Table 1). Those that did not included CDllc, retinoid X receptor-␤, LDLR class A, early growth response-2, and cyclin D2. These genes showed a less-than-twofold difference between the wildtype and knockout mice and were removed from the final list. Previously, expression of CD14 was shown to decrease twofold in C/EBPε⫺/⫺ macrophages [18]. As a positive control, we verified that this also occurred in the peritoneal lavage samples used in this study. Consistent with the previous results, CD14 decreased 1.9-fold in the C/EBP⫺/⫺ sample according to the array but was excluded from the final list because of the 2.0-fold cutoff. Although the trend of up- or down-regulation was verified, the fold-change was estimated inaccurately by the array. The altered transcript levels for five genes were examined in isolated macrophages. The increased expression of the lipid metabolism genes APOE and APOC2 was approximately threefold for each in the purified C/EBPε⫺/⫺ macrophages (Fig. 1). The transcription factors IDB1 and MAD1 were decreased five- and twofold in C/EBPε⫺/⫺ macrophages, respectively (Fig. 1). The apoptosis inhibitor API6 or apoptosis inhibitor expressed by macrophages was decreased by threefold in macrophages from the C/EBPε⫺/⫺ mice (Fig. 1). It is interesting that the expression of CSF3R (G-CSFR) mRNA was reduced by fivefold in the peritoneal cells from the C/EBPε⫺/⫺ mice (Table 1). This gene has been described as a target of C/EBP␣ [34]. Western blot analysis of peritoneal lavage cells showed a clear reduction of the G-CSFR protein in the C/EBPε-deficient (⫺/⫺) cells as compared with the wildtype (⫹/⫹) cells (Fig. 2). The knockout sample was overloaded slightly as indicated by ␤-actin, but the level of GCSFR protein was significantly lower than in the wild-type. No reduction was observed in protein lysates from fresh bone marrow cells (Fig. 2). Western blot analysis with an anti-C/ EBPε antibody demonstrates the presence of the protein in the wild-type but not the knockout samples (Fig. 2). The data suggest that C/EBPε is important for maintaining wild-type levels of expression of G-CSFR in the activated peritoneal cells.

Gombart et al. Gene expression of C/EBP␧-deficient myeloid cells

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TABLE 1.

Biological process Apoptosis 1 2 3 4 Cell adhesion/Chemotaxis 5 6 7 8 9 10 11 12 13 14 15 16 17 18 Cell cycle/division 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 Cytoskeleton/nuclear matrix 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 DNA/RNA synthesis and processing 51 52 53 54 55 56 57 58 59 1156

Differential Expression of Genes in C/EBPε–/– Murine Peritoneal Lavage Cells Fold change

C/EBP site

Array

RT-PCR

N N N N

–3.7 2.6 3.8 9.3

–20

N Yes Yes N N N N N N N N Yes N N

Gene symbol

Aliases

API6 BIRC1 TIA1 PRKCH

apoptosis inhibitor expressed by macrophage (M␾; AIM) neuronal apoptosis inhibitory protein (naip) cytotoxic granule-associated RNA-binding protein 1 phorbol ester receptor/protein kinase, nPKC ␩

–14.2 –6.7 –3.5 –3 –2.7 –2.4 –2.1 –2.1 –2 –2 2.3 2.4 2.9 3.1

CSPG2 COL1A2 SPP1 COL3A1 CDH1 DAB2 CDH6 COL5A1 SELL CD164 SEMA4D LSP1 SEMA3E ITGB5

PG-M core protein; chondroitin sulfate proteoglycan 2 pro-␣-2(I) collagen osteopontin collagen ␣-I E-cadherin disabled homolog 2 (Drosophila) K-cadherin/cadherin-6 collagen ␣1(VI)-collagen L-selectin, CD62L cell-surface sialomucin MGC-24 semaphorin 4D lymphocyte-specific protein-1 semaphorin 3e integrin ␤-5

N N Yes N N N Yes N N N N N N N N N

–8.2 –7.6 –5.3 –3.8 –3.5 –2.9 –2.6 –2.5 –2.5 –2.5 –2.1 –2 2.3 2.8 3.4 2.4

BTG3 GOS2 CCND1 SEI1 CDK2 MAPK1 CDKN1A MCM7 CCNB1 MCM6 PA2G4 CCNA2 STYX GAS2 NIN SEPTIN6

␤ cell translocation gene 3 G0/G1 switch-2-like protein cyclin D1 p34SEI-1, CDK4-binding protein cyclin-dependent kinase 2 mitogen-activated protein kinase 1, (p42)/ERK2 p21/WAF1 mCDC47 cyclin B1 mini-chromosome maintenance-deficient 6 (Mcmd6) p38-2G4; proliferation-associated protein 1 cyclin A2 phospho-Ser/Thr/Tyr interaction protein growth arrest-specific 2 ninein septin 6

N N N N N N N N N N N N N N N N

–4.9 –4 –3.4 –3.1 –3.1 –2.4 –2.3 –2.2 –2.1 –2.1 –2 2 2.3 2.3 4.6 5.2

LMNB1 MYO1B ITSN1 MACS MYRL2 TUBB5 SNL TUBB5 MLP RABGGTB TUBA2 TC10 DCTN5 MYOZ DDEF1 MARK3

lamin B myosin I intersectin 1 myristoylated alanine-rich C-kinase substrate (MARCKS) myosin regulatory light chain 2 ␤-tubulin (isotype M␤ 5) fascin (Fscn1) tubulin, ␤ 5 MARCKS-like protein RAB geranylgeranyl transferase ␤ subunit tubulin ␣ 2 ras-like protein dynactin 5 myozenin SrcSH3-binding protein ELKL motif kinase 2 long form (EMK2)

N N N N N N N N N

–6.7 –2.6 –2.4 –2.2 –2.2 –2.1 –2.1 –2.1 –2

HIST2 SMN1 H2A.1 POLA SNRPD2 HMG17 HRMT1L2 SFRS3 FBL

histone H2A (CD-1) survival motor neuron replication-dependent histone H2A.1 gene DNA polymerase ␣ catalytic subunit p180 snRNP SM D2 HMG-17 chromosomal protein hnRNP methyltransferase-like 2 srp20 gene; splicing factor fibrillarin

4



TABLE 1.

Biological process 60 61 62 63 64 65 Immune/inflammatory response 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 Lipid metabolism 98 99 100 101 102 103 104 105 106 107 Metabolism 108 109 110 111 112 113 114 115 116 117 118

Fold change

(Continued)

C/EBP site

Array

N N N N N N

–2 –2 2 2.3 2.3 3

SNRPD1 NOLA1 SNRP70 ORC2L PRP8 DBY

small nuclear ribonucleoprotein D1 nucleolar protein family A, member 1 U1 snRNP 70 kDa polypeptide A origin recognition complex PRP8/U5 snRNP-specific protein (220 kD) DBY RNA helicase; DEAD-box protein

N Yes Yes N Yes N N N N N N N N N Yes N N N N N Yes Yes N N N N N N Yes N N N

–8.3 –7.2 –7 –5.3 –5 –4.8 –4.1 –3.8 –3.7 –3.7 –3.6 –3.5 –3.5 –3.5 –3.3 –2.6 –2.6 –2.6 –2.5 –2.4 –2.2 2 2.1 2.1 2.4 3 3.1 3.2 4.4 4.8 6.1 8.6

CCL7 CCL4 CRP CCR3 CCL2 IL1RL1 MRC1 IFI202A IL8RB CXCL2 IRG1 TGTP CD24 PF4 IL6 CCR7 CCR1 FCGRT LY6G GBP1 IL1B CCR2 SLC11A1 TNFAIP2 CD48 IFNGR1 LY78 TNFRSF1B LBP LY9 TCRG-V4 SEPP1

monocyte chemoattractant protein (MCP)-3 macrophage inflammatory protein (MIP)-1␤ c-reactive protein MIP1-␣ receptor-like 2 MCP-1 ST2L protein; highly similar to IL-1 receptor type 1 macrophage mannose receptor 202 interferon (IFN)-activatable protein IL-8 receptor MIP-2/(GRO2) immune-responsive gene 1 T cell-specific protein with GTP-binding motif CD24a platelet factor 4 IL-6 G protein-coupled receptor; MIP-3␤ receptor MIP-1␣ receptor fcrn gene for Fc receptor Ly-6G.1 neurotoxin homologue M␾ activation-associated GTP-binding protein gene (mag-1) IL-1␤ MCP-1 receptor/CC chemokine receptor 2 (CCR2) solute carrier family 11, member 1 tumor necrosis factor ␣ (TNF-␣)-induced protein 2 BCM1 antigen; (Blast-1/OX45) IFN-␥ receptor RP105, Toll-like receptor binds lipopolysaccharide (LPS) TNFRII LPS-binding protein Ly-9 antigen T cell receptor ␥-chain selenoprotein P, plasma, 1

N N N N Yes N Yes Yes N N

–10.9 –7.5 –3.7 –2.5 –2.2 –2 3.2 10.1 14.6 60.1

APOC1 SCD2 LPIN2 MSR1 FABP4 BAAT SORL1 APOE SCARB1 APOC2

apolipoprotein (apo)-C1 stearoyl-CoA desaturase 2 lipin 2 scavenger receptor fatty acid-binding protein 4 (aP2) bile acid CoA: amino acid N-acyltransferase low-density lipoprotein receptor (LDLR) class of proteins apo-E scavenger receptor class B type I (mSR-BI) apo-C2

N N N Yes N N

–12.2 –10.7 –9.6 –6.4 –6.2 –3.2

GCNT1 RRM2 IMPDH ARG1 PTGIS ATP50

Yes N N N N

–3.2 –3 –2.7 –2.4 –2.3

␤-1,6-N-acetylglucosaminyltransferase ribonucleotide reductase M2 subunit type 1 inosine monophosphate dehydrogenase arginase prostacyclin synthase adenosine 5⬘-triphosphate (ATP) synthase oligomycin sensitivity conferral protein ␤-1-globin uridine-cytidine kinase 2 chloride intracellular channel 1 solute carrier family 2, member 1 solute carrier family 16, member 1

RT-PCR

–4.3 –2.1

4.4 3.5 5.3

1.5 1.4

4.5 4.3 7

–1.7

Gene symbol

HBB UCK2 CLIC1 SLC2A1 SLC16A1

Aliases


1157

TABLE 1.

Biological process 119 120 121 122 123 Mitochondrial 124 125 126 127 128 129 130 131 Oncogenesis 132 133 134 135 136 137 Proliferation 138 139 140 141 142 Proteases and protease inhibitors 143 144 145 146 147 148 149 150 151 152 Protein synthesis/metabolism/ modification 153 154 155 156 157 158 159 160 161 162 163 164 165 166 167 168 169 170 171 172 173 Signal transduction 174 175

1158

C/EBP site

Fold change Array

RT-PCR

(Continued)

Gene symbol

Aliases

N N Yes N N

2 4.5 6.4 3.5 3.5

GLA ATP1A1 HSD11B1 NARS CALM2

galactosidase, ␣ Na⫹/K⫹ transporting a1 polypeptide 11␤-hydroxysteroid dehydrogenase 1 asparaginyl-tRNA synthetase calmodulin 2

N N N N N N N N

–6.4 –6.1 –4.1 –2.7 –2.3 –2.3 –2 2

ALAS2 DHODH C1QBP TIMM8A GPD1 IDH2 SLC25A4 CYP2a4

aminolevulinate synthase dihydroorotate dehydrogenase complement component deafness dystonia protein 1 (DDP1) glycerol 3-phosphate dehydrogenase 1 isocitrate dehydrogenase 2 solute carrier family 25, member 4 cytochrome P450, 2a4

N N N N N N

–6.4 –2.2 –2 2.1 2.3 4.6

PIM1 ARHB PTEN RAB10 RAB5B PTTG1

proviral integration site 1 rhoB gene PTEN RAB10, member RAS oncogene family RAB5B, member RAS oncogene family pituitary tumor-transforming gene protein

N N N N N

–12.1 –2.9 –2.9 –2.2 –2

STMN1 MKI67 NAP1L1 PCNA GPC4

stathmin; leukemia-associated phosphoprotein-18 (LAP-18) Ki-67 nucleosome assembly protein 1-like 1 proliferating cell nuclear antigen K-glypican/glypican 4

N N N N N N N N N N

–8.8 –7.8 –6.2 –5.8 –5.4 –4.1 –2.7 –2 3.1 22.2

KLK8 SERPINB1 TIMP1 REN CTSE ADAM8 SERPINB2 SERPINE1 TIMP2 CTSC

neuropsin leukocyte elastase inhibitor; ELANH2 tissue inhibitor of metalloproteinases, type 1 renin procathepsin E MS2/ADAM 8/mCD156 plasminogen activator inhibitor 2 (PAI-2) PAI-1 tissue inhibitor of metalloproteinases, type 2 cathepsin C

N N N N N N N N N N N N N N N N N N N N N

–5.4 –3.2 –3.1 –2.6 –2.3 –2.2 –2.1 –2.1 –2.1 –2.1 –2.1 –2 –2 2.2 2.2 2.6 2.6 3.5 4.8 4.9 6.3

GTPBP2 CCT6A DNPEP NOLC1 HSP86 EIF5a SET MRPS7 PSMA4 HSP105 HSPB1 SERPINH1 ERP70 EEF1A1 STXBP3 SEC23A ASM3A GNAI2 EIF2S3 PIGF ARF3

GTP-binding protein 2 chaperonin containing TCP-1, ␨ subunit (CCTZ) aspartyl aminopeptidase 140 kDa nucleolar phosphoprotein heat shock protein 86 (HSP86); chaperone eukaryotic translation initiation factor 5A SET translocation ribosomal protein S7, mitochondrial proteasome (prosome, macropain) subunit, ␣ type 4 HSP-105 kDa ␣ small HSP25 47-kDa HSP47 protein disulfide isomerase-related protein eukaryotic translation elongation factor 1 ␣1 syntaxin-binding protein 3 protein transport protein SEC23 homolog isoform A acid sphingomyelinase-like phosphodiesterase 3a inhibitory G protein of adenylate cyclase, ␣ chain 2 Eif2s3y eukaryotic translation initiation factor 2 phosphatidylinositol glycan class F ADP-ribosylation factor 3

N N

–6.1 –5.3

CSF1 IL1R2

macrophage (m)-CSF Il-1R2, type II IL-1 receptor



TABLE 1.

Biological process 176 177 178 179 180 181 182 183 184 185 186 187 188 189 190 191 192 193 194 195 196 197 198 199 200 Transcriptional regulation 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 Others 216 217 218 219 220 222 222 223 224 225 226 227 228 229 230 231

Fold change

C/EBP site

Array

N N N N N N N Yes N N N N N N Yes N N N N N N N N N N

–5.2 –4.9 –4.8 –4 –2.8 –2.5 –2.4 –2.3 –2.1 –2.1 –2 –2 2 2.1 2.1 2.1 2.5 2.8 3 3.7 3.8 3.9 4.8 4.9 10.2

Yes Yes N N N N N N N N N N N N N

–35 –7.1 –3.7 –3.1 –2.6 –2.6 2.1 2.1 2.2 2.4 2.5 3.3 3.2 5.1 6.3

N N N N N N N N N N N N N N N N

–4.5 –3.2 –3.1 –2.3 –2.2 –2.2 –2 2 2 2.1 2.2 2.2 2.3 2.6 3.5 8.5

RT-PCR

–5.3

–5 –3.8

(Continued) Gene symbol

Aliases

GABRG2 WNT10A BMP2 POR1 TNFSF11 IL1RN IL11RA CSF3R IFRD1 TULP4 RANBP1 BASP1 CALML4 RRAS CSF2RB2 IL10RA TM7SF1 GNAO ATP2A3 TLE4 CSNK2b JAK3 YWHAB PLD2 SHC1

GABA-A receptor ␥-2 subunit Wnt10a bone morphogenic protein-2 partner of RAC-1 (arfaptin-2) receptor activator of nuclear factor-␬ B ligand (RANKL) IL-1R antagonist protein ETL-2; novel putative type-1 cytokine receptor granulocyte (G)-CSF receptor Ifrd1, IFN-related developmental regulator 1 tubby-like protein 4 RAN-binding protein 1 brain abundant, membrane-attached signal protein 1 calmodulin-like 4 Harvey rat sarcoma oncogene, subgroup R IL-3 receptor IL-10 receptor ␣ putative seven pass transmembrane protein guanine nucleotide-binding protein, ␣ o ATPase, Ca⫹⫹ transporting, ubiquitous transducin-like enhancer of split 4 casein kinase II, ␤ subunit JAK3 14-3-3 protein ␤ phosphatidylcholine-specific phospholipase D2 src homology 2 domain-containing transforming protein C1

MAD IDB1 CBX3 MYC GATA6 GTF2I NFE2L2 HDAC5 LYL1 CCRN4L BRPF1 CNOT2 ZNF216 IRF3 FOXP1

MAX dimerization partner inhibitor of DNA-binding 1 chromobox homolog 3 (Drosophila HP1 ␥) c-myc GATA-binding protein 6 general transcription factor II-I (TFII-I) p45 NF-E2-related factor 2 (NRF2) histone deacetylase 5 lymphoblastic leukemia carbon catabolite repression 4 protein homolog bromodomain and PHD finger containing, 1 CCR4-NOT transcription complex, subunit 2 zinc finger protein ZNF216 IFN regulatory factor 3 forkhead box P1

BCAS2 FANCG FLNB FGL2 FIN16 LY6A HIG1 OVCOV1 TRIM12 QKI HPS HBA1 LST1 NLI-1 RAP140 VWF

breast carcinoma-amplified sequence 2 Fanconi anemia, complementation group G filamin, ␤ fibrinogen-like protein, prothrombinase fibroblast growth factor-inducible gene 16 lymphocyte differentiation antigen (Ly-6.2); Sca1 hypoxia-induced gene 1 ovarian cancer overexpressed 1 tripartite motif protein 12 quaking type I Hermansky-Pudlak syndrome protein/pale ear hemoglobin ␣, adult chain 1 lymphocyte-specific transcript 1 nuclear LIM interactor-interacting factor 1 retinoblastoma-associated protein 140 Von Willebrand factor homolog

Minus and plus refer to fold decrease or increase in expression, respectively, in the C/EBPε–/– compared with the wild-type myeloid cells. Gene name in bold type indicate gene identified previously as differentially regulated in macrophages, neutrophils, or both. Yes ⫽ C/EBP family member implicated in regulation of expression; N ⫽ no C/EBP family members implicated in regulation of expression.


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TABLE 2.

Differential Expression of ESTs in C/EBPε–/– Murine Peritoneal Lavage Cells

EST

GenBank ID

Fold change

EST

GenBank ID

Fold change

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26

aa395999 aa002925 aa175185 AA269614 aa512139 C79700 AA190042 aa259683 aa204106 W47946 aa590750 W29450 w81812 aa537958 aa004011 w70453 aa574948 aa266529 aa273803 w40995 aa407875 aa268226 c76023 C78676 AA033393 AA178588

–56.1 –6.6 –6.1 –4.8 –3.7 –3.4 –3.3 –3.3 –3 –2.9 –2.7 –2.6 –2.6 –2.3 –2.2 –2.2 –2.1 –2 –2 –2 –2 2 2 2 2.1 2.1

27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52

aa182228 AA184228 D18395 U20780 AA104542 AA616077 aa408298 W50866 D19480 AA110491 aa183623 AA389977 aa238331 aa174777 C80410 AA399835 aa673574 W83326 aa419890 aa174755 aa409826 AA013976 AA166246 aa275273 AA168762 aa615883

2.1 2.1 2.1 2.2 2.3 2.3 2.4 2.4 2.5 2.6 2.6 2.6 2.8 3.1 3.3 3.4 3.5 3.6 3.9 4 4.2 4.6 5.8 6.5 6.9 20.8

Minus and plus refer to fold decrease or increase in expression, respectively, in the C/EBPε–/– compared with the wild-type myeloid cells.

Fig. 2. Reduced expression of G-CSFR protein in peritoneal lavage cells. Protein lysates were prepared from peritoneal lavage cells (PL) 24 h after injection with thioglycollate or bone marrow (BM). Approximately 30 ␮g total protein was electrophoresed on a 4 –20% gradient polyacrylamide-sodium dodecyl sulfate gel and blotted onto a nitrocellulose membrane. Detection was performed as described previously [35]. Western blot analysis with anti-GCSFR, anti ␤-actin, or anti-C/EBPε antibody (Santa Cruz Biotechnology, CA) was performed. The arrow indicates the location of the G-CSFR protein. *, Locations of the ␤-actin and C/EBPε proteins. The numbers at the left of the panel indicate the position of the molecular weight markers.

C/EBP family members or possess C/EBP-binding sites in their promoters (Table 1). The C/EBP␣ or -␤ transcription factors are implicated in the regulation of the genes encoding the CSF3R, CXCL2, CCL4, IL-6, CD14, and SPP1 (osteopontin) proteins [17, 36 –38]. To determine if C/EBPε was binding to the promoters of these genes in vivo, we performed ChIP analysis. In addition, we tested the IL-8RB (CXCR2) gene, which has not been identified as a C/EBP target (Fig. 3).

Identification of C/EBP␧ target genes by ChIP assay The differentially regulated genes identified by the array may be direct targets of C/EBPε (i.e., C/EBPε binds to their promoters and activates or represses transcription). Alternatively, their altered expression may be secondary to the loss of C/EBPε target genes. Regarding the first possibility, we identified 22 genes by literature searches, which are regulated by

Fig. 1. Select genes display differential gene expression in purified macrophages. QRT-PCR was performed for five genes on cDNAs generated from total RNA isolated from macrophages (M␾). The y-axis represents relative values for the pairs [wild-type (wt) or knockout (ko)] normalized to 18S.

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Fig. 3. Identification of direct targets of C/EBPε by ChIP analysis. Sonicated chromatin was prepared from total cells from the bone marrow (BM) or peritoneal cavity (LAV) of wild-type mice. The chromatin was immunoprecipitated with no antiserum (N), preimmune serum (P), anti-C/EBPε (ε), or antiC/EBP␣ (␣) antibody. The samples were subjected to PCR using primers to the indicated promoters and analyzed on a 2% agarose gel. The reverse image of the original ethidium bromide-stained gels is displayed. *, Known target genes of C/EBP family members, and the LTF gene is a known target of C/EBPε and serves as positive control for the ChIP assay. The TERT gene is not a known target of C/EBP family members and serves as a negative control for the ChIP assay. The input chromatin (I) was included as a positive control for PCR.


Peritoneal macrophages and neutrophils were harvested 24 h after thioglycollate injection from wild-type mice. The femurs from these same mice were flushed to obtain bone marrow cells. Sonicated chromatin was prepared and immunoprecipitated with no antibody (N), preimmune serum (P), anti-C/EBPε (ε), or anti-C/EBP␣ (␣; Fig. 3). The promoter sequences were amplified by PCR. The promoters of LTF and TERT were included as positive and negative controls, respectively. The LTF gene is regulated by C/EBP␣ and C/EBPε, and the LTF mRNA is highly expressed in cells of the bone marrow and not in peripheral blood neutrophils such as those recruited to the peritoneum by thioglycollate [10, 39, 40]. TERT is not a known target gene for C/EBP family members. Specific binding of the C/EBP␣ (faint) and C/EBPε proteins to the LTF promoter in bone marrow, but not lavage cells, was observed (Fig. 3). In contrast, binding to the TERT promoter was not detected in either population of cells (Fig. 3). Taken together, these controls demonstrated specific binding of C/EBPε to the promoter of a known target gene, LTF, in a tissue where it is transcribed actively. Specific binding of C/EBPε was detected for the CSF3R, CXCL2, CCL4, IL-6, CXCR2, CD14, and SPP1 promoters in the bone marrow cells (Fig. 3, BM). In the myeloid cells isolated by peritoneal lavage (LAV), specific binding by C/EBPε was detected for all except the IL-6 promoter (Fig. 3). Binding of C/EBP␣ was detected for the CSF3R, CXCL2, IL-6, and the CXCR2 promoters in bone marrow cells but not the CCL4, CXCR2, CD14, or SPP1 promoters (Fig. 3). In peritoneal lavage cells, binding of C/EBP␣ was detected for the CSF3R, CXCL2, and CCL4 promoters, but not the CXCR2, CD14, or SPP1 promoters (Fig. 3). These results demonstrate specific binding of C/EBPε to the promoter regions of a majority of the selected genes. In addition, the results implicate C/EBPε as an important regulator of their expression in vivo.

Altered cholesterol levels in the blood of C/EBP␧-deficient mice The dysregulation of numerous lipid metabolism genes led us to hypothesize that levels of lipids in the blood of C/EBPε⫺/⫺ mice may, in turn, be altered. To test this hypothesis, we placed C/EBPε wild-type and deficient mice on a high-fat diet for 18 weeks. The levels of free fatty acids (FFA), unesterified cholesterol (UEC), high-density lipoprotein (HDL), and total cholesterol (TC) in the plasma were measured at the start and end of the experiment to determine if the loss of C/EBPε affected any general aspects of lipid metabolism. Student’s t-tests were performed to determine if observed differences between the two populations were significant. At the start (8 weeks of age), the knockout mice showed significantly lower levels of TC (approximately twofold) and HDL (approximately twofold) than the wild-type mice (Fig. 4, A and B). Levels of FFA and UEC were not significantly different between the wild-type and knockout (data not shown). At the end of 18 weeks on the high-fat diet, the levels of HDL in the wild-type decreased but did not change for the knockout; therefore, the difference in HDL levels between the wild-type and knockout mice at the end of the high-fat diet was not significant (Fig. 4B). In contrast, the TC levels showed a statistically significant increase in the wild-type but not the

knockout mice after 18 weeks on the diet (Fig. 4A). It is interesting that TC was significantly higher (approximately twofold) in the wild-type than the knockout mice at the end of the diet (Fig. 4A). Taken together, the data suggest that loss of C/EBPε alters expression of genes in the macrophages, which are involved in the regulation of lipid metabolism, and this, in turn, affected cholesterol levels in the blood of the C/EBPεdeficient mice.

Altered lipid accumulation by C/EBP␧⫺/⫺ macrophages To determine if the C/EBPε⫺/⫺ macrophages show defects in lipid metabolism, we isolated thioglycollate-elicited macrophages 4 days post-injection. The cells were deposited on slides by cytocentrifugation, stained for Oil-Red-O, and observed by light microscopy. A consistent decrease in OilRed-O staining was observed in the C/EBPε⫺/⫺ macrophages (Fig. 5). Differential counts indicated that there were two times more Oil-Red-O-positive macrophages in the wild-type (23.5⫾2.8%) sample than the C/EBPε⫺/⫺ sample (10.8⫾0.7%). The lipid droplets were fewer in number and in size in the C/EBPε-deficient murine macrophages. Extraction of the OilRed-O by isopropanol from equal numbers of cells revealed two to three times less lipid in the C/EBPε⫺/⫺ macrophages as compared with the wild-type (Fig. 6). These data indicate the C/EBPε⫺/⫺ macrophages accumulate lower levels of lipids than the wild-type macrophages.

DISCUSSION SGD is an extremely rare disease, making it difficult to study in humans; however, the C/EBPε-deficient mouse provides a convenient model system to elucidate the disease and the underlying changes in gene expression, which produce the complex, phenotypic changes that occur with the loss of C/EBPε [16]. In this study, additional, differentially expressed

Fig. 4. Lipid levels are altered in the blood of C/EBPε-deficient mice. C/EBPε wild-type (WT) and deficient (KO) mice were placed on a high-fat diet for 18 weeks. The levels of TC (A) and HDL (B) in the blood were measured at the start (WT, n⫽17; KO, n⫽12) and end (WT, n⫽17; KO, n⫽8) of the experiment. The levels indicated on the y-axis are in mg/dL. Statistically significant differences are indicated by the horizontal line.


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Fig. 5. Altered lipid accumulation by C/EBPε⫺/⫺ macrophages. Peritoneal macrophages from three wild-type (WT) and three C/EBPε⫺/⫺ (KO) mice were harvested 4 days post-injection, deposited on slides by cytocentrifugation, and stained with Oil-Red-O. Fields with comparable numbers of cells were visulalized at 200⫻ and 1000⫻ (same field as 200⫻ with oil immersion) and photographed.

genes involved in neutrophil and macrophage function, including cell adhesion/chemotaxis, cytoskeleton/nuclear matrix, signal transduction, lipid metabolism, and immune/inflammatory response, were identified. These changes result from the genes being direct targets of C/EBPε or are secondary to the loss of C/EBPε. ChIP assays demonstrated that seven genes from our list are likely direct targets of C/EBPε. The genes selected for ChIP analysis were known C/EBP␤ or -␣ target genes; therefore, it will be interesting to determine how C/EBPε participates in their regulation. For the LTF gene, a prior study showed that C/EBP␣ binds to the LTF promoter when the gene is inactive, and this shifts to increased binding of C/EBPε when the gene is actively transcribed [39]. It is hypothesized that C/EBP␣ suppresses expression of the LTF gene, and C/EBPε activates the gene during differentiation [39]. Although C/EBP␣ is a potent, transcriptional activator, it can repress gene expression in particular cell types and activate it in others [41]. We hypothesize that C/EBP␣ and C/EBPε play a role in regulating a number of the genes, which we examined by ChIP analysis. C/EBP␣ is expressed earlier in myelopoiesis than C/EBPε and may repress target genes that are activated when C/EBPε expression occurs at about the promyelocyte stage of differentiation. As C/EBP␣, -␤, -␦, and -ε are coexpressed during myeloid differentiation, a combination of DNA array, QRT-PCR, ChIP, and genetic manipulation will be necessary to elucidate the regulation of C/EBP target genes during myelopoiesis. The binding of multiple C/EBP family members may be important for the temporal expression of a gene during differentiation. Humans and mice lacking functional C/EBPε suffer from increased susceptibility to bacterial infection. This correlates with the aberrant expression of 35 defense-immune response genes in the C/EBPε-deficient mice, which were identified in prior studies. Of these 35 genes, 19 were identified in perito1162


neal-derived cells isolated in a manner similar to that used in this study. Of these 19 genes, nine were identified in this study, and six agreed with the previous reports. The six genes were IL-1RN, IL-6, CCL7, SELL, CD14, and SERPINB2. This study significantly expands the number of potentially dysregulated C/EBPε target genes that contribute to the various neutrophil and macrophage defects observed in humans with SGD and mice lacking functional C/EBPε. Morphologically, patients with SGD and mice lacking C/EBPε display defective maturation of their granulocytic compartment. This impaired differentiation may involve dysregulation of several genes including CSF3R and MAD (Table 1). The Mad protein is required for normal terminal granulocytic differentiation [42] and was recently identified as a transcriptional target of C/EBPε [43]. The reduced expression of Mad in myeloid cells from C/EBPε⫺/⫺ mice may contribute, in part, to their impaired granulocytic differentiation. The cytokine CSF3 (G-CSF) mediates granulocytic differentiation of myeloid cells and mediates this activity via its receptor. The CSF3R decreased by two- to fivefold at the mRNA and protein levels (Table 1, Fig. 1). The C/EBPε and -␣ proteins (Fig. 3) bind to the CSF3R promoter in bone marrow and peritoneal-derived myeloid cells, suggesting that they regulate its expression in these cells. Although C/EBP␣ is considered the major transcriptional regulator of CSF3R, myeloid cells from C/EBP␣deficient mice still possess the ability to differentiate into neutrophils [44, 45], concomitant with induction of CSF3R expression via a C/EBP␣-independent pathway [45]. Our findings highlight an important role for C/EBPε in regulating in vivo expression of the G-CSFR gene. Consistent with this, activation of the CSF3R gene by the acute myeloid leukemia 1-ETO fusion protein was mediated by C/EBPε [46].

Fig. 6. Altered lipid accumulation by C/EBPε⫺/⫺ macrophages. Peritoneal macrophages from two wild-type (WT) and three C/EBPε⫺/⫺ (KO) mice were harvested 4 days post-injection. Cells were fixed and stained for Oil-Red-O. The lipid content was extracted from 5 ⫻ 105 cells using isopropanol and read by spectrophotometer at a wavelength of 510 nm to determine the amount of Oil-Red-O in the cells. The upper panel represents one experiment with one wild-type and one knockout mouse. The lower panel represents a second experiment with one wild-type and two knockout mice. OD, Optical density.


The reduced expression of CSF3R may contribute to the delay in migration of neutrophils to the peritoneum of C/EBPεdeficient mice [11, 47]. In addition, the increased expression of leukocyte-specific protein 1 (LSP-1) and the reduction of Lselectin (SELL), IL-8RB, and CXCL2 may be important. LSP1-deficient mice show enhanced chemotaxis of neutrophils into the peritoneum [48]. CXCL2 is an important neutrophil chemoattractant, and mice lacking its receptor (IL-8RB) display impaired neutrophil recruitment [49]. Also, a number of other chemoattractants and receptors for neutrophils and monocyte/ macrophages were down-regulated in the C/EBPε⫺/⫺ mice (e.g., CCL7, CCL4, IL-6, CCR1, and CCR2; Table 1). The reduced expression of these could contribute to the impaired chemotaxis of C/EBPε-deficient neutrophils. It is interesting that the forced overexpression of C/EBPε in a pre-B cell acute lymphoblastic leukemia cell line induced the expression of several of these genes [17]. The cytoskeleton plays a critical role in chemotaxis, phagocytosis, and superoxide (O2–) production. The neutrophils of SGD patients show increased ruffling, surface-to-volume ratio, and numbers of centriole-associated microtubules [3]. Therefore, the dysregulation of numerous cytoskeletal structural and regulatory proteins is intriguing (Table 1). These include MYO1B, MYRL2, MARCKS, MLP, SNL, ITSN1, RABGGTB, DDEF1, RRAS, and POR1. The decreased expression of POR1 (partner of RAC1)/Arfaptin-2 [50 –52] and the MARCKS genes (MARCKS and MLP) may play a role in the impaired generation of O2– [53] and phagocytosis [54, 55], which is observed in granulocytes and monocytes/macrophages of C/EBPε⫺/⫺ mice [11, 18]. It is unexpected that this study implicated C/EBPε in the regulation of numerous lipid metabolic genes, a quite intrigu-

ing and novel role for it. This correlated with a reduced level of TC in the plasma of the C/EBPε-knockout mice compared with their wild-type littermates before and after an atherogenic diet. More striking was the significant decrease in the accumulation of lipid droplets observed in C/EBPε-deficient peritoneal macrophages. We propose that the down-regulation of those genes involved in lipoprotein uptake [macrophage scavenger receptor 1 (MSR1)/SR-A] and accumulation of cholesterol esters (FABP4) and the concomitant up-regulation of those genes involved in the efflux of cholesterol out of the cell (APOE, APOC2, and SCARB1) and to the liver for conversion into bile acids and ultimately intestinal excretion may explain the impaired lipid accumulation observed in the macrophages and the reduced TC in the plasma of C/EBPε-deficient mice (Fig. 7) [56, 57]. The overexpression of apoE in transgenic mice reduced plasma lipoproteins [58]. Also, transplanting APOE-deficient mice with wild-type macrophages enhanced clearance of lipoproteins and normalized serum cholesterol levels, demonstrating that with only macrophages as a source of apoE, protective benefits were achieved in the APOE knockout [59]. The increase expression of the APOE gene in the C/EBPε-deficient mice may explain their reduced TC levels. Lipid-laden macrophages or “foam cells” contribute to the development of atherosclerotic plaques [60]. We hypothesized that foam cell formation may be reduced in C/EBPε-deficient mice, thus reducing lesion formation. An initial study with a limited number of mice suggested a trend toward decreased atherosclerotic plaque formation in the C/EBPε-deficient mice, but the results were not statistically significant (data not shown). Further studies are required to determine the possible protective effects of C/EBPε deficiency.

Fig. 7. Proposed model for effects of altered lipid metabolism genes on macrophage lipid accumulation. The down-regulation (black shading) of genes involved in lipoprotein uptake (MSR1/SR-A) and accumulation of cholesterol ester (CE; FABP4) and the up-regulation (gray shading) of those genes involved in the transport of cholesterol out of the cell (SORL1, APOE, APOC2, and SCARB1) may explain the reduced lipid accumulation observed in the macrophages from C/EBPε-deficient mice. Those genes in unshaded boxes are unchanged in expression [CD36 and LDLR involved in uptake; ATP-binding cassette transporter A1 (ABCA1) involved in transport out of the cell; and neutral cholesteryl ester hydrolase (NCEH) and acyl-CoA:cholesterol:acyltransferase isoform 1 (ACAT) involved in modification of CE or free cholesterol (FC), respectively]. NUC, Nucleus; ER, endoplasmic reticulum. See Table 1 for definitions of additional gene names.


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Although macrophages appear to play a protective role such as in the clearance of oxidized lipoproteins and promoting reverse cholesterol transport by efflux of cholesterol to HDL acceptors, they clearly play a key role in the development of athersclerosis [56]. The potential biological role of C/EBPε in the regulation of lipid metabolism and atherosclerosis is intriguing. As the processes that macrophages use to fend off invading microorganisms are used in the metabolism of lipoproteins and cholesterol [56], we speculate that the loss of C/EBPε, which affects the immune functions of the macrophage so dramatically, affects lipid metabolism as well. A working hypothesis is that C/EBPε may be activated by inflammation [61] and induce expression of proatheroslcerotic genes and/or inhibit expression of antiatherosclerotic genes. Consistent with this hypothesis of activation, C/EBPε-expressing cell lines display LPS-inducible expression of IL-6 and CCL2 [17]. Regulation of C/EBPε activity may provide new approaches to reduce macrophage foam cell formation and the subsequent inflammatory responses that contribute to athersclerosis.

ACKNOWLEDGMENTS This work was supported by National Institutes of Health Grant CA26038-20, Horn Foundation, Parker Hughes Trust, and C. and H. Koeffler Fund. H. P. K. holds the Mark Goodson Endowed Chair for Cancer Research and is a member of the Jonsson Cancer Center and Molecular Biology Institute of University of California Los Angeles (UCLA). A. F. G. and U. K. contributed equally to the manuscript. We thank Dr. Aldons J. Lusis (David Geffen School of Medicine at UCLA) for critically reading this manuscript, Dr. Anatole Ghanzalpour (David Geffen School of Medicine at UCLA) for performing the lipid analysis of the plasma samples, and Jonathan Frank for technical assistance.

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