Journal of Leukocyte Biology BioLegend: Treg, Th17, Stem Cell
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Originally published online as doi:10.1189/jlb.0504286 on October 4, 2005

Published online before print October 4, 2005
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(Journal of Leukocyte Biology. 2005;78:1153-1165.)
© 2005 by Society for Leukocyte Biology

Aberrant expression of neutrophil and macrophage-related genes in a murine model for human neutrophil-specific granule deficiency

Adrian F. Gombart1, 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

1Correspondence: Cedars-Sinai Medical Center, Division of Hematology/Oncology, Davis Bldg. 5019, Los Angeles, CA 90048. E-mail: gombarta{at}csmc.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Neutrophil-specific granule deficiency involves inheritance of germline mutations in the CCAAT/enhancer-binding protein {epsilon} (C/EBPE) gene. Humans and mice lacking active C/EBP{epsilon} 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{epsilon}–/– 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/EBP–/– 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{epsilon} 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{epsilon}–/– mice correlated with reduced total cholesterol levels in these mice before and after maintenance on a high-fat diet. Also, C/EBP{epsilon}-deficient macrophages showed a reduced capacity to accumulate lipids. In summary, dysregulation of numerous, novel C/EBP{epsilon} target genes impairs innate immune response and possibly other important biological processes mediated by neutrophils and macrophages.

Key Words: C/EBP{epsilon} • SGD • DNA microarray • lipid metabolism


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Neutrophils, monocytes, and tissue-based macrophages are essential cellular components of the innate immune system, 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-Huët 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 2 3 4 5 6 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){epsilon}-deficient murine model was a candidate gene apparent [9 ]. Neutrophils from C/EBP{epsilon}-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 10 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/EBP{epsilon} in SGD. Germ-line mutations in the CEBPE locus were identified in two SGD patients, explaining the genetic defect responsible for this disease [14 15 16 ].

Although clearly an important factor in normal neutrophil differentiation and function, mounting evidence suggested that C/EBP{epsilon} was involved in monocyte/macrophage function. C/EBP{epsilon} is expressed at significant levels in monocytes/macrophages and related cell lines in humans and mice [17 , 18 ]. The forced overexpression of C/EBP{epsilon} in a pre-B acute lymphoblastic leukemia cell line induced expression of several cytokines expressed by macrophages [17 ]. Representational difference analysis using peritoneal neutrophils and macrophages from wild-type and C/EBP{epsilon}-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/EBP{epsilon}-deficient mice [18 ].

Recently, the role of C/EBP{epsilon} in human monocyte/macrophage function was addressed. Abnormalities in monocytic cells from one SGD individual who lacked a functional C/EBP{epsilon} 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/EBP{epsilon}-deficient myeloid cells from humans and mice demonstrate an essential role for C/EBP{epsilon} in the normal development and function of neutrophils and macrophages. The parallels between the human and murine conditions indicate that the C/EBP{epsilon}-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/EBP{epsilon} plays in mediating host immune defense by identifying possible target genes of C/EBP{epsilon} in neutrophils and macrophages.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Mice and sample preparation
The C/EBP{epsilon} 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 pathogen-free 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/EBP{epsilon}+/+ and C/EBP{epsilon}–/– mice. To reduce individual variation in subsequent experiments, the cells from all wild-type or all C/EBP{epsilon} 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 Wright-Giemsa-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 (8/96 and Build 4.0) and TIGR (Build 1.0 ß) 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 µg 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/µl. 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/EBP{epsilon}–/– 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/EBP{epsilon} 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 was used to determine significance of differences between wild-type and C/EBP{epsilon} 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 200x and 1000x (with oil).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Identification of differentially regulated genes
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/EBP{epsilon}+/+ and C/EBP{epsilon}–/– 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.


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  		Table 1. Differential Expression of Genes in C/EBP{epsilon}–/– Murine Peritoneal Lavage Cells

 

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  		Table 2. Differential Expression of ESTs in C/EBP{epsilon}–/– Murine Peritoneal Lavage Cells

 
Of the 231 known genes, 41 were reported previously as highly expressed in macrophages and/or neutrophils [26 27 28 29 30 31 32 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/EBP{epsilon} 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/EBP{epsilon}–/– 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 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/EBP{epsilon}-deficient mice (Table 1) .

Confirmation of differential gene expression
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-ß, LDLR class A, early growth response-2, and cyclin D2. These genes showed a less-than-twofold difference between the wild-type and knockout mice and were removed from the final list. Previously, expression of CD14 was shown to decrease twofold in C/EBP{epsilon}–/– 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/EBP{epsilon}–/– macrophages (Fig. 1 ). The transcription factors IDB1 and MAD1 were decreased five- and twofold in C/EBP{epsilon}–/– macrophages, respectively (Fig. 1) . The apoptosis inhibitor API6 or apoptosis inhibitor expressed by macrophages was decreased by threefold in macrophages from the C/EBP{epsilon}–/– mice (Fig. 1) .



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  		Figure 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{phi}). The y-axis represents relative values for the pairs [wild-type (wt) or knockout (ko)] normalized to 18S.

 
It is interesting that the expression of CSF3R (G-CSFR) mRNA was reduced by fivefold in the peritoneal cells from the C/EBP{epsilon}–/– mice (Table 1) . This gene has been described as a target of C/EBP{alpha} [34 ]. Western blot analysis of peritoneal lavage cells showed a clear reduction of the G-CSFR protein in the C/EBP{epsilon}-deficient (–/–) cells as compared with the wild-type (+/+) cells (Fig. 2 ). The knockout sample was overloaded slightly as indicated by ß-actin, but the level of G-CSFR 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/EBP{epsilon} antibody demonstrates the presence of the protein in the wild-type but not the knockout samples (Fig. 2) . The data suggest that C/EBP{epsilon} is important for maintaining wild-type levels of expression of G-CSFR in the activated peritoneal cells.



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  		Figure 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 µg 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-G-CSFR, anti ß-actin, or anti-C/EBP{epsilon} antibody (Santa Cruz Biotechnology, CA) was performed. The arrow indicates the location of the G-CSFR protein. *, Locations of the ß-actin and C/EBP{epsilon} proteins. The numbers at the left of the panel indicate the position of the molecular weight markers.

 
Identification of C/EBP{epsilon} target genes by ChIP assay
The differentially regulated genes identified by the array may be direct targets of C/EBP{epsilon} (i.e., C/EBP{epsilon} binds to their promoters and activates or represses transcription). Alternatively, their altered expression may be secondary to the loss of C/EBP{epsilon} target genes. Regarding the first possibility, we identified 22 genes by literature searches, which are regulated by C/EBP family members or possess C/EBP-binding sites in their promoters (Table 1) . The C/EBP{alpha} or -ß transcription factors are implicated in the regulation of the genes encoding the CSF3R, CXCL2, CCL4, IL-6, CD14, and SPP1 (osteopontin) proteins [17 , 36 37 38 ]. To determine if C/EBP{epsilon} 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 ). 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/EBP{epsilon} ({epsilon}), or anti-C/EBP{alpha} ({alpha}; Fig. 3 ). The promoter sequences were amplified by PCR.



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  		Figure 3. Identification of direct targets of C/EBP{epsilon} 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/EBP{epsilon} ({epsilon}), or anti-C/EBP{alpha} ({alpha}) 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/EBP{epsilon} 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.

 
The promoters of LTF and TERT were included as positive and negative controls, respectively. The LTF gene is regulated by C/EBP{alpha} and C/EBP{epsilon}, 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{alpha} (faint) and C/EBP{epsilon} 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/EBP{epsilon} to the promoter of a known target gene, LTF, in a tissue where it is transcribed actively.

Specific binding of C/EBP{epsilon} 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/EBP{epsilon} was detected for all except the IL-6 promoter (Fig. 3) . Binding of C/EBP{alpha} 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{alpha} 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/EBP{epsilon} to the promoter regions of a majority of the selected genes. In addition, the results implicate C/EBP{epsilon} as an important regulator of their expression in vivo.

Altered cholesterol levels in the blood of C/EBP{epsilon}-deficient mice
The dysregulation of numerous lipid metabolism genes led us to hypothesize that levels of lipids in the blood of C/EBP{epsilon}–/– mice may, in turn, be altered. To test this hypothesis, we placed C/EBP{epsilon} 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/EBP{epsilon} 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. 4A and 4B ). 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/EBP{epsilon} 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/EBP{epsilon}-deficient mice.



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  		Figure 4. Lipid levels are altered in the blood of C/EBP{epsilon}-deficient mice. C/EBP{epsilon} 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.

 
Altered lipid accumulation by C/EBP{epsilon}–/– macrophages
To determine if the C/EBP{epsilon}–/– 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 Oil-Red-O staining was observed in the C/EBP{epsilon}–/– 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/EBP{epsilon}–/– sample (10.8±0.7%). The lipid droplets were fewer in number and in size in the C/EBP{epsilon}-deficient murine macrophages. Extraction of the Oil-Red-O by isopropanol from equal numbers of cells revealed two to three times less lipid in the C/EBP{epsilon}–/– macrophages as compared with the wild-type (Fig. 6 ). These data indicate the C/EBP{epsilon}–/– macrophages accumulate lower levels of lipids than the wild-type macrophages.



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  		Figure 5. Altered lipid accumulation by C/EBP{epsilon}–/– macrophages. Peritoneal macrophages from three wild-type (WT) and three C/EBP{epsilon}–/– (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 200x and 1000x (same field as 200x with oil immersion) and photographed.

 


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  		Figure 6. Altered lipid accumulation by C/EBP{epsilon}–/– macrophages. Peritoneal macrophages from two wild-type (WT) and three C/EBP{epsilon}–/– (KO) mice were harvested 4 days post-injection. Cells were fixed and stained for Oil-Red-O. The lipid content was extracted from 5 x 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.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
SGD is an extremely rare disease, making it difficult to study in humans; however, the C/EBP{epsilon}-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/EBP{epsilon} [16 ]. In this study, additional, differentially expressed 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/EBP{epsilon} or are secondary to the loss of C/EBP{epsilon}. ChIP assays demonstrated that seven genes from our list are likely direct targets of C/EBP{epsilon}. The genes selected for ChIP analysis were known C/EBPß or -{alpha} target genes; therefore, it will be interesting to determine how C/EBP{epsilon} participates in their regulation. For the LTF gene, a prior study showed that C/EBP{alpha} binds to the LTF promoter when the gene is inactive, and this shifts to increased binding of C/EBP{epsilon} when the gene is actively transcribed [39 ]. It is hypothesized that C/EBP{alpha} suppresses expression of the LTF gene, and C/EBP{epsilon} activates the gene during differentiation [39 ]. Although C/EBP{alpha} 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{alpha} and C/EBP{epsilon} play a role in regulating a number of the genes, which we examined by ChIP analysis. C/EBP{alpha} is expressed earlier in myelopoiesis than C/EBP{epsilon} and may repress target genes that are activated when C/EBP{epsilon} expression occurs at about the promyelocyte stage of differentiation. As C/EBP{alpha}, -ß, -{delta}, and -{epsilon} 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/EBP{epsilon} suffer from increased susceptibility to bacterial infection. This correlates with the aberrant expression of 35 defense-immune response genes in the C/EBP{epsilon}-deficient mice, which were identified in prior studies. Of these 35 genes, 19 were identified in peritoneal-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/EBP{epsilon} target genes that contribute to the various neutrophil and macrophage defects observed in humans with SGD and mice lacking functional C/EBP{epsilon}.

Morphologically, patients with SGD and mice lacking C/EBP{epsilon} 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/EBP{epsilon} [43 ]. The reduced expression of Mad in myeloid cells from C/EBP{epsilon}–/– 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/EBP{epsilon} and -{alpha} 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{alpha} is considered the major transcriptional regulator of CSF3R, myeloid cells from C/EBP{alpha}-deficient mice still possess the ability to differentiate into neutrophils [44 , 45 ], concomitant with induction of CSF3R expression via a C/EBP{alpha}-independent pathway [45 ]. Our findings highlight an important role for C/EBP{epsilon} 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/EBP{epsilon} [46 ].

The reduced expression of CSF3R may contribute to the delay in migration of neutrophils to the peritoneum of C/EBP{epsilon}-deficient mice [11 , 47 ]. In addition, the increased expression of leukocyte-specific protein 1 (LSP-1) and the reduction of L-selectin (SELL), IL-8RB, and CXCL2 may be important. LSP-1-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/EBP{epsilon}–/– 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/EBP{epsilon}-deficient neutrophils. It is interesting that the forced overexpression of C/EBP{epsilon} 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 51 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/EBP{epsilon}–/– mice [11 , 18 ].

It is unexpected that this study implicated C/EBP{epsilon} in the regulation of numerous lipid metabolic genes, a quite intriguing and novel role for it. This correlated with a reduced level of TC in the plasma of the C/EBP{epsilon}-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/EBP{epsilon}-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/EBP{epsilon}-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/EBP{epsilon}-deficient mice may explain their reduced TC levels.



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  		Figure 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/EBP{epsilon}-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.

 
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/EBP{epsilon}-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/EBP{epsilon}-deficient mice, but the results were not statistically significant (data not shown). Further studies are required to determine the possible protective effects of C/EBP{epsilon} deficiency.

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/EBP{epsilon} 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/EBP{epsilon}, which affects the immune functions of the macrophage so dramatically, affects lipid metabolism as well. A working hypothesis is that C/EBP{epsilon} 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/EBP{epsilon}-expressing cell lines display LPS-inducible expression of IL-6 and CCL2 [17 ]. Regulation of C/EBP{epsilon} activity may provide new approaches to reduce macrophage foam cell formation and the subsequent inflammatory responses that contribute to athersclerosis.


   ACKNOWLEDGEMENTS
 
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.

Received May 11, 2005; revised June 23, 2005; accepted July 7, 2005.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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