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The Journal of Immunology, 1999, 162: 503-509.
Copyright © 1999 by The American Association of Immunologists

Characterization of Human Endotoxin Lipopolysaccharide Receptor CD14 Expression in Transgenic Mice1

Christopher J. Hetherington*, Paul D. Kingsley{dagger}, Francesco Crocicchio*, Pu Zhang*, Michael S. Rabin*, James Palis{dagger} and Dong-Er Zhang2,*

* Department of Medicine, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA 02115; and {dagger} Department of Pediatrics and Cancer Center, University of Rochester, Rochester, NY 14642


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
CD14 is a major receptor for the bacterial endotoxin LPS. Since CD14 is specifically and highly expressed on the surface of monocytic cells, it has been used as a monocyte/macrophage differentiation marker. To identify elements that are critical for the direction of the tissue-specific expression of CD14, an 80-kb genomic DNA fragment containing the coding region of the CD14 gene, as well as a considerable amount of both upstream and downstream sequence, was used to generate transgenic mice. The analysis of mice from six different founder lines demonstrated that this genomic DNA fragment was sufficient to direct human CD14 gene expression in a monocyte-specific manner among hematopoietic cells. Furthermore, the data lead us to a new finding that CD14 is highly expressed in the human liver, a primary organ involved in the acute phase response. These transgenic mice provide a useful model to analyze the biological function of human CD14.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Endotoxin LPS is the major mediator of shock induced by Gram-negative bacteria (1). This endotoxin triggers the production of inflammatory mediators, including IL-6 and TNF-{alpha}, that can lead to fatal septic shock. CD14, a phosphatidylinositol-linked glycoprotein, is a major cellular receptor for the complex of LPS and plasma LPS-binding protein and plays an important role in the signal transduction causing endotoxin shock (2). LPS-binding protein is an acute phase protein produced by liver hepatocytes that catalyzes the binding of LPS and CD14 (3, 4). Besides its important biological function, previous studies using mAbs have shown that CD14 is highly expressed on the surface of monocytes/macrophages and strongly up-regulated during the differentiation of monocytic precursor cells into monocytic cells (5, 6, 7). Therefore, it has been used as a differentiation marker for this cell lineage. Monocytes/macrophages, found in peripheral blood and most tissues of the human body, are involved in the regulation of specific immune responses in addition to nonspecific defense against infection and malignancy (8). CD14 serves as an excellent model for the study of monocytic gene regulation. Understanding the regulation of tissue-specific gene expression will deepen our knowledge of monocyte/macrophage lineage differentiation and the development of acute myeloid leukemia. We have previously reported that tissue-specific CD14 expression is regulated at the level of transcription (9, 10). To further investigate the mechanism of this gene’s regulation, we placed a P1 phagemid clone containing 80 kb of human CD14 genomic sequence into transgenic mice. The results indicate that this 80-kb genomic fragment contains critical regulatory elements that enhance the tissue-specific expression of CD14. Furthermore, the data reveal that human CD14 is also highly expressed in the liver, a key organ in acute phase protein synthesis.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
P1 phagemid preparation and generation of transgenic mice

Two oligonucleotides, 5'-CCTGAGTCATCAGGACAC-3' and 5'-CGATAAGTCTTCCGAACCTC-3', which correlate to bp -227 to -211 and bp +83 to +98 of the human CD14 gene (10), were sent to Genome Systems (St. Louis, MO) for PCR screening of a human genomic DNA library constructed in the bacteriophage P1 (11). Three positive clones were received. Further analysis of these clones by restriction enzyme digestion and oligonucleotide hybridization revealed that one of the three clones contains ~40 kb of sequence both upstream and downstream from the CD14 coding region. This clone was used in our transgenic studies.

Phagemid DNA was prepared for microinjection by alkaline lysis and phenol/chloroform extraction followed by gentle treatment using a GeneClean Kit (Bio 101, Vista, CA). The DNA was then dialyzed against microinjection buffer (10 mM Tris (pH 8.0) and 0.1 mM EDTA) on a 0.025 µm membrane (type VS; Millipore, Bedford, MA) and filtered through a Spin-X column (Costar, Cambridge, MA). Transgenic mice were produced in the transgenic facility of Beth Israel Deaconess Medical Center using zygotes from FVB/N mice.

Southern blot analysis

Mouse genomic DNA was isolated from mouse tails as previously described (12). The DNA samples were digested with EcoRI, electrophoresed on a 1% agarose gel, and transferred to positively charged Biotrans nylon membrane (ICN, Costa Mesa, CA). Human CD14 cDNA radiolabeled with [32P]dCTP was used as a probe to hybridize with the mouse tail DNA samples. The transgene copy number was estimated by comparing the mouse tail DNA samples with human genomic DNA using LKB Ultroscan XL Enhanced Laser Densitometer (Pharmacia, Uppsala, Sweden).

Isolation of RNA and Northern blot hybridization

Peritoneal macrophages were harvested from the abdominal cavity of mice 48–72 h after the i.p. injection of thioglycolate (0.1 g/ml, 1.5 ml/mouse). Total RNA from various tissues from the injected mice, as well as from cell lines, was prepared by guanidine isothiocyanate extraction followed by purification on a cesium chloride gradient. The purified RNA samples were denatured in a formamide/formaldehyde solution followed by electrophoresis on a 1% agarose gel containing 0.22 M formaldehyde. The RNA was then transferred to positively charged Biotrans Plus nylon membrane (ICN). The human tissue RNA dot blot was from Clontech (Palo Alto, CA). Murine and human CD14 cDNA was radiolabeled with [32P]dCTP using random priming and was subsequently used to probe the RNA blots. The hybridization was conducted at 65°C for 16 h in a buffer containing 7% SDS and 1% BSA in 0.5 M sodium phosphate (pH 7.2). The membranes were then washed twice for 25 min at 65°C in 0.2x SSC and 0.1% SDS. Autoradiography was performed at -80°C with either Kodak XAR-5 film or Kodak BioMax MS film. The level of expression was calculated using an LKB Ultroscan XL Enhanced Laser Densitometer (Pharmacia, Uppsala, Sweden).

FACS analysis

FACS reactions were performed as previously described (9). Phycoerythrin-conjugated Ab against human CD14, FITC-conjugated Ab against murine F4/80 or Mac-1, and their respective isotype controls were purchased from PharMingen (San Diego, CA). FACS analysis was performed on a FACScan flow cytometer (Becton Dickinson, Franklin Lakes, NJ).

In situ hybridization

Sample preparation and in situ hybridization were previously described (13). A fragment of human CD14 cDNA containing bp +75 to +309 was amplified by PCR. The ends of the product were filled in using Klenow fragment, and the resultant product was cloned into the SmaI site of pBluescriptII KS-. An antisense riboprobe was prepared by digesting the above construct with EcoRI and using T3 RNA polymerase to synthesize the riboprobe. A sense riboprobe was prepared by digesting the above construct with BamHI and using T7 RNA polymerase to synthesize the riboprobe.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
P1 clones with human CD14 gene

As previously reported, the human CD14 gene consists of two exons separated by an 88-bp intron (14). We have cloned a 5.7-kb EcoRI fragment from a human chromosome 5 library, which contains ~1.5 kb of the CD14 coding region and 4.2 kb of upstream sequence (Fig. 1Go). Transient transfection analysis has demonstrated that this upstream fragment has monocyte-specific promoter activity and that the transcription factor Sp1 plays a critical role for directing CD14 expression (9, 10). To further study whether this 5.7-kb DNA fragment is sufficient to direct monocyte-specific CD14 expression in vivo, we generated transgenic mice that contain this DNA fragment. Mice from five different founder lines did not show any evidence of human CD14 expression (data not shown). These results indicate that although the proximal promoter has tissue-specific activity, other regulatory elements are required for chromosomal CD14 expression. To search for the other important elements, we analyzed P1 phagemids. Of three phagemid clones that contained human CD14, one consisted of ~80-kb human genomic DNA sequence, with ~40 kb upstream and 40 kb downstream DNA sequence from the CD14 gene (Fig. 1Go). We decided to further analyze CD14 expression by using this P1 clone in transgenic mice.



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  		FIGURE 1. Schematic diagram of the P1 phagemid clone which contains the human CD14 gene. The P1 clone contains ~40 kb of sequence both upstream and downstream from the transcription initiation site. The Sp6 and T7 sites are contained within the P1 vector very close to the cloning site. A close-up diagram is shown for a 5.7-kb EcoRI fragment that contains the proximal promoter and the entire CD14 gene. This fragment has ~4.2 kb of upstream sequence that contains three Sp1 binding sites at 110 bp, 85 bp, and 60 bp upstream from the transcription initiation site.

 
Tissue-specific expression of CD14 in transgenic mice

To analyze human CD14 gene expression in transgenic mice, we first studied murine CD14 expression. As shown in Fig. 2Go, among the various tissues examined, murine CD14 is strongly and specifically expressed in peritoneal macrophages. Furthermore, murine CD14 cDNA and human CD14 cDNA did not show any cross-hybridization when they were used as probes in Northern blot hybridizations (Fig. 3Go). A sequence comparison of the murine CD14 cDNA and the human CD14 cDNA shows that there is only a 67% similarity between the two (data not shown). The significant difference between the two genes explains the lack of cross-hybridization between human and murine samples. These results demonstrate that we can directly use human CD14 cDNA as a probe to analyze whether transgenic mice carrying the human CD14 gene express human CD14 in a tissue-specific manner. Six independent transgenic founder lines were generated using a P1 phagemid containing 80 kb of human genomic DNA sequence. The first four lines did not have germline transmission of the transgene. Therefore, these founder mice were used directly to analyze CD14 expression. Although there was no germline transmission, the P1 CD14 DNA fragment was present in the cells from different tissues of founder 1, as shown in Fig. 4Go. Northern blot analysis of RNA prepared from different tissues showed that human CD14 is strongly expressed in peritoneal macrophages (Fig. 4Go). Surprisingly, a high level of human CD14 expression was also detected in the liver. The other three nongermline transmitted founders showed a similar pattern of human CD14 expression (data not shown). Founder 5 and founder 6 had germline transmission of the transgene. To analyze human CD14 expression, peritoneal macrophages from wild-type control mice and from founder lines 5 and 6 were tested by FACS analysis with specific mAbs against human CD14 and the murine myeloid cell marker Mac-1. As shown in Fig. 5Go, there is a significant increase in the number of cells from human CD14 transgenic mice that stain positive with the human CD14 Ab relative to wild-type controls. To study the tissue specificity of human CD14 expression directed by DNA sequences in the 80-kb genomic fragment, RNA was harvested from different tissues of mice from founder 5 and founder 6. Fig. 6Go, A and B, show the Northern blot hybridization results for founder 5 and founder 6, respectively. Human CD14 cDNA was used as a probe in the analysis. Peritoneal macrophages from the wild-type control mice did not give any positive signal with the human CD14 probe. Peritoneal macrophages from both founder 5 and founder 6 showed a strong positive signal for human CD14 expression. The spleen and the thymus are enriched with cells of hematopoietic origin, mainly B cells and T cells, respectively. As shown in Fig. 6Go, there was no detectable human CD14 expression in either the spleens or the thymi of these transgenic mice. These results indicate that the 80-kb genomic sequence can direct human CD14 expression specifically in monocytic cells during hematopoiesis. Among the other tissues tested, human CD14 was highly expressed in the liver only. To verify the correlation between the copy number of human CD14 fragments and the level of human CD14 expression in the transgenic mice, we analyzed their DNA and peritoneal macrophage RNA using Southern blot and Northern blot hybridizations, respectively. As shown in Table IGo, although all six lines of transgenic mice had significant amounts of human CD14 expression, there was not a strict correlation between the copy number and the expression level. Therefore, the data from six independent founder lines showed that the 80 kb of genomic sequence surrounding the human CD14 gene was sufficient to direct the monocyte-specific expression of CD14 in vivo, but not in a copy number-dependent manner. In addition, this sequence also activated human CD14 expression in mouse liver.



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  		FIGURE 2. Northern blot analysis of murine CD14 expression in murine tissues. Total RNA was isolated from various tissues of wild-type FVB mice, and 10 µg of each RNA sample was electrophoresed on a 1% agarose gel containing 0.22 M formaldehyde. The gel was then transferred to a nylon membrane and probed with murine CD14 cDNA. There is a low level of CD14 expression in some of the tissues, but the peritoneal macrophages show a high level of tissue-specific expression of murine CD14. The ethidium bromide staining of the 18S ribosomal RNA is presented to show the loading of the RNA samples.

 


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  		FIGURE 3. Human CD14 and murine CD14 do not cross-hybridize in Northern blot analyses. Total RNA was isolated from wild-type murine kidney, wild-type murine peritoneal macrophages, and human monocytes, as well as from various cell lines: RAW 264.7 (a murine monocytic-macrophage cell line), HeLa (a human cervical carcinoma cell line), and THP-1 (a human monocytic cell line) treated with vitamin D3. The RNA samples were electrophoresed on a 1% agarose gel containing 0.22 M formaldehyde and transferred to a nylon membrane. The membrane was then sequentially probed with murine CD14 cDNA and human CD14 cDNA. The murine CD14 probe hybridizes specifically with RNA from peritoneal macrophages and the murine monocytic cell line. The human CD14 probe hybridizes specifically with RNA from human macrophages and the human monocytic cell line. The ethidium bromide staining of the 18S ribosomal RNA is presented to show the loading of the RNA samples.

 


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  		FIGURE 4. Southern and Northern blot analyses of various tissues from founder 1. Founder 1 was euthanized, and various tissue samples were collected. Genomic DNA and total RNA were isolated from these tissue samples. The DNA and RNA samples (10 µg each) were then electrophoresed on a 1% agarose and a 1% agarose gel containing 0.22 M formaldehyde, respectively. The gels were transferred to nylon membranes and were probed with human CD14 cDNA (Southern blot and Northern blot analyses) and murine CD14 (Southern blot analysis only). The human CD14 transgene and the endogenous murine CD14 gene are present in all of the samples (top panels). Human CD14 is strongly expressed in the macrophages and liver of the transgenic mouse (bottom panels). The ethidium bromide staining of the 18S ribosomal RNA is presented to show the loading of the RNA samples.

 


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  		FIGURE 5. Flow cytometry analysis of peritoneal cells from founder lines 5 and 6. Cells obtained from the peritoneal cavities of wild-type and trangenic mice stimulated by i.p. thioglycolate injection were stained with Abs against murine Mac-1 (a mature monocyte/granulocyte surface marker) and human CD14. The cells from founders 5 and 6 stain positively for human CD14, while the wild-type cells do not. All three samples show positive staining for murine Mac-1.

 


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  		FIGURE 6. Northern blot analyses of human CD14 in various tissues from founder lines 5 (A) and 6 (B). Total RNA was isolated from various tissues from transgenic mice in founder lines 5 and 6, and 10 µg of each RNA sample was electrophoresed on a 1% agarose gel containing 0.22 M formaldehyde. The gel was then transferred to a nylon membrane and probed with human CD14 cDNA. Both of the founder lines exhibit the same pattern of expression in the liver and the macrophages. The ethidium bromide staining of the 18S ribosomal RNA is presented to show the loading of the RNA samples.

 

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  		Table I. Comparison of the relative expression level of CD14 with the copy number of the transgene in various founder lines1

 
CD14 is highly expressed in human liver

As shown in Fig. 2Go, murine CD14 expression was not detectable in the liver. Therefore, it was quite surprising to find a high level of human CD14 expression in the livers of the transgenic mice. The results implied that this 80-kb human CD14 genomic sequence contains regulatory elements necessary to direct liver-specific expression. To gain more knowledge about CD14 expression in different human tissues, human CD14 cDNA was used as a probe to hybridize with a dot blot containing RNAs prepared from a variety of human tissues. As shown in Fig. 7Go, the highest level of CD14 expression was detected in the liver. A relatively high level of expression was also detected in the RNA prepared from lung, placenta, and peripheral leukocytes, which include monocytes and granulocytes. Furthermore, RNA samples prepared from normal human liver were analyzed by Northern blot hybridization. As shown in Fig. 8GoA, a message that is the correct size for CD14 can be detected in human liver. Hepatocytes are the major cell population in the liver. To demonstrate that the high level of CD14 expression seen in the liver is due to its expression not only in Kupffer cells (liver macrophages) but also in hepatocytes, we analyzed CD14 expression in human hepatocyte cell lines using Northern blot hybridization. As shown in Fig. 8GoB, CD14 expression was detected in two human hepatocyte cell lines (HepG2 and Hep3B) and a monocytic cell line (THP-1), but not in HeLa cells. The evidence for hepatocyte expression was further supported by in situ hybridization analysis. As shown in Fig. 9Go, human CD14 transcripts were expressed at high levels throughout the parenchyma of the liver and not just in Kupffer cells.



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  		FIGURE 7. Northern blot analysis of a dot blot containing RNA samples from various human tissues. A dot blot containing various human RNA samples was probed with human CD14 cDNA. There are four tissues that strongly express CD14. The liver has the highest level of expression followed by the lung and the placenta. The peripheral leukocytes, which contain macrophages and granulocytes, also show a fairly high level of expression.

 


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  		FIGURE 8. Expression of human CD14 in hepatocytes. Total RNA was isolated from two human liver samples as well as from several cell lines: HepG2 and Hep3B (human hepatocytic cell lines), HeLa (a human cervical carcinoma cell line), and THP-1 (a human monocytic cell line) with and without vitamin D3 treatment. A, Northern blot analysis of human liver RNA and total RNA from HepG2 cells and THP-1 cells using human CD14 cDNA as a probe. There is CD14 expression in all of the samples, although HepG2 has a lower level of expression than the other samples. B, Northern blot analysis of human cell lines using human CD14 cDNA as a probe. The two hepatocytic cell lines (HepG2 and Hep3B) and the monocytic cell line (THP-1) show expression of CD14. There is no CD14 expression in HeLa cells.

 


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  		FIGURE 9. In situ hybridization of liver from transgenic mice and wild-type mice with human CD14 antisense and sense probes. The wild-type liver with the antisense probe gives no signal above background. The transgenic liver shows human CD14 transcripts present in a uniform distribution when probed with the antisense probe. The transgenic liver with the sense probe gives no signal above background. The size bar represents 0.1 mm.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Pluripotent hematopoietic stem cells proliferate and differentiate toward cells of different lineages. During this process, the expression of lineage-specific genes is activated in each particular lineage. The aim of this study was to analyze the important regulatory elements that direct gene expression in monocytic cells using CD14 as a model. We studied the proximal upstream sequence of the CD14 gene in transient transfection assays with luciferase as a reporter. The results demonstrate that this upstream sequence contains monocyte-specific promoter activity (10). However, further analyses in transgenic mice indicate that this sequence is not sufficient to direct CD14 expression. As demonstrated in other systems, a transcriptionally active gene is often regulated by multiple cis elements (15, 16). These elements can be present at a considerable distance from the transcription initiation site. Therefore, we expanded our analysis of CD14 gene expression by including more upstream and downstream sequences. We isolated 80 kb of genomic sequence containing the CD14 gene and used it in transgenic mouse analysis. Data from six independent transgenic founder lines suggested that the 80-kb genomic sequence has sufficient regulatory elements to direct CD14 expression in a monocyte-specific manner among hematopoietic cells. These results significantly restrict our search for regulatory elements to this 80-kb fragment of DNA. Distal regulatory elements such as locus control regions, enhancers, matrix attachment regions, and insulators have been previously reported (17, 18, 19, 20). Some of these sequences enhance promoter activity in both transient and stable transfection analyses as well as in studies with transgenic mice. The other elements are more likely to control chromatin structure, keeping it in an active open form (15). An open chromatin structure is thought to make the region more accessible for the interaction of transcription factors with regulatory elements. Therefore, such cis elements may allow transcription of the gene to occur by keeping the DNA in an open active form, but do not directly enhance the level of gene expression. Our data demonstrate that the 80 kb of genomic DNA sequence surrounding the human CD14 gene contains the important regulatory elements necessary for directing tissue-specific expression of CD14. As shown in Table IGo, we did not find a strict correlation between copy number of the gene and the level of expression. This finding indicated that there are multiple regulatory elements that control CD14 expression. It is possible that the 80-kb region does not contain the element needed to define the boundary of the active domain of CD14 but has the critical element for directing gene expression. Further studies within this 80-kb sequence are required to elucidate the mechanism that regulates CD14 gene expression.

LPS on the surface of Gram-negative bacteria is the major cause of endotoxin shock during bacterial infection (21). CD14 has been reported as a major cellular receptor for LPS on monocytic cells (2). LPS, via CD14, provokes the release of cytokines, such as TNF-{alpha}, IL-1, and IL-6, by monocytes. These cytokines trigger the systemic acute phase response including fever, neutrophilia, changes in lipid metabolism, increase in gluconeogenesis, activation of the complement and coagulation pathways, hormonal changes, and induction of acute phase protein synthesis (22). Most of the acute phase proteins are synthesized in hepatocytes and represent the major changes in plasma protein composition during the acute phase response. They are believed to play an important protective role in host defense during bacterial infection (23). Here, we report a high level of CD14 expression in normal human liver. In contrast, CD14 expression is only seen in murine liver from mice that have been treated with LPS (24). Furthermore, our transgenic mice express human CD14 in a pattern consistent with normal human expression. Hepatocytic CD14 expression may play an active role in the acute phase response. CD14 is present in both a membrane-bound form (mCD14), through a glycosyl phosphatidylinositol linkage, and a soluble form (25). The soluble form of CD14 (sCD14) can be detected in serum and urine at 2–6 µg/ml. Changes in the serum sCD14 level have been reported during injury and trauma (26). It will be important to determine whether CD14 expression in hepatocytes is regulated during the acute phase response. The function of sCD14 is not yet fully understood, but it has been reported that sCD14 promotes the response of CD14-negative cells, such as endothelial cells or epithelial cells, or CD14-positive macrophages to LPS (27, 28, 29, 30). There are also reports that sCD14 inhibits the responses of mCD14-positive cells to LPS (29, 31). Besides the function of CD14 in response to LPS, CD14 may contribute to cell-cell interactions during different immune responses (32, 33). Since these transgenic mice express CD14 in the liver, which mimics CD14 expression in human tissues, they provide an excellent model system to further study the biological function of CD14.

Murine CD14 is mainly expressed by monocytes/granulocytes, and human CD14 is highly expressed in both human liver and human monocytes/granulocytes. This difference suggests that CD14 may have one or more additional physiological functions in humans. The difference in expression between human genes and corresponding mouse genes has been reported by other groups. The murine {alpha}1-antitrypsin gene is specifically expressed in hepatocytes, while human {alpha}1-antitrypsin expression is more widespread. In addition to hepatocytes, it is also expressed in the kidneys and salivary glands as evidenced in studies with transgenic mice (34, 35). The Thy-1 gene is also expressed differently in humans and mice. Transgenic mice carrying the human Thy-1 locus reveal that the human gene is activated in murine tissues corresponding to normal sites of human Thy-1 expression, although the murine Thy-1 gene remains inactive in those tissues. In addition, the human gene is not induced in cells that express murine but not human Thy-1 (36). The species-specific CD14 expression pattern in the transgenic mice suggests that there are different cis regulatory elements present in the human and murine genes. Matsuura et al. (37) have reported an upstream region of the murine CD14 gene that is important for its promoter activity. This region is not conserved in the human CD14 promoter (10). In addition, although human and murine macrophage CSF (M-CSF) receptor gene expression patterns are very similar, transcription factors that affect their promoter activity are not the same. AML1 binds to the human M-CSF receptor promoter, in a region between the CCAAT/enhancer binding protein (C/EBP) and PU.1 binding sites, and activates the promoter (38). The murine M-CSF receptor promoter lacks the AML1 binding site in the corresponding region (our unpublished data). When a gene is expressed specifically in two different cell types, its expression is probably regulated by transcription factors commonly expressed in both tissues. We have reported that a ubiquitously expressed transcription factor, Sp1, plays a critical role in the expression of human CD14. Disruption of the Sp1 binding site at -110 bp in the CD14 promoter decreased the promoter activity to 10% of the wild-type promoter activity (10). Since CD14 is not ubiquitously expressed, Sp1 may function in combination with other tissue-specific factors that directly or indirectly interact with the CD14 promoter region or other regions important for gene expression. An interesting transcription factor that falls into this category is the C/EBP, originally cloned by analyzing the hepatocyte-specific albumin gene (39). Members of the C/EBP family are highly expressed in both hepatocytes and myeloid cells and are important regulators for many hepatocyte- and myeloid-specific genes (40, 41). It will be interesting to analyze whether C/EBP plays any role during human CD14 expression in both hepatocytes and myeloid cells.


   Acknowledgments
 
We thank Daniel Tenen for many helpful discussions and critical reading of the manuscript and Joel Lawitts, Masimo Loda, Chaker Adra, and Kathy Maltby for technical assistance.


   Footnotes
 
1 This work was supported by Grant CA/AI59589 and HL59484 from the National Institutes of Health and Grant DHP-155 from the American Cancer Society. D.-E.Z. is a Leukemia Society of America Scholar. Back

2 Address correspondence and reprint requests to Dr. Dong-Er Zhang, Harvard Institutes of Medicine, HIM 953, 77 Avenue Louis Pasteur, Boston, MA 02115. E-mail address: Back

Received for publication June 5, 1998. Accepted for publication September 22, 1998.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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