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This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Rights and Permissions Citing Articles Citing Articles via HighWire Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by Kubota, T. Articles by Koeffler, H. P. Search for Related Content PubMed PubMed Citation Articles by Kubota, T. Articles by Koeffler, H. P. Related Collections Gene Expression Phagocytes Social Bookmarking                   What's this?  Previous Article  |  Table of Contents  |  Next Article  Blood, 1 December 2000, Vol. 96, No. 12, pp. 3953-3957 PHAGOCYTES Representational difference analysis using myeloid cells from C/EBP deletional mice Tetsuya Kubota, Seiji Kawano, Doris Y. Chih, Yasuko Hisatake, Alexey M. Chumakov, Hirokuni Taguchi, and H. Phillip Koeffler From the Division of Hematology/Oncology, Cedars-Sinai Research Institute, UCLA School of Medicine, Los Angeles, CA; and Department of Medicine, Kochi Medical School, Kochi, Japan.     Abstract Top Abstract Introduction Materials and methods Results Discussion References C/EBP is a recently cloned member of the C/EBP family of transcriptional factors. Previous studies demonstrated that the expression of this gene is tightly regulated in a tissue specific manner; it is expressed exclusively in myeloid cells. C/EBP-deficient mice developed normally but failed to generate functional neutrophils and eosinophils, and these mice died of opportunistic infections suggesting that C/EBP may play a central role in myeloid differentiation. To identify myelomonocytic genes regulated by the C/EBP gene, we performed representational difference analysis (RDA), a polymerase chain reaction (PCR)-based subtractive hybridization using neutrophils and macrophages from wild-type and C/EBP knockout mice. We identified a set of differentially expressed genes, including chemokines specific to myelomonocytic cells. Several novel genes were identified that were differentially expressed in normal myelomonocytic cells. Taken together, we have found several genes whose expression might be enhanced by C/EBP. (Blood. 2000;96:3953-3957) © 2000 by The American Society of Hematology.     Introduction Top Abstract Introduction Materials and methods Results Discussion References The human CCAAT/enhancer binding protein (C/EBP) family of transcription factors includes C/EBP,1 C/EBP,2-7 C/EBP,8 C/EBP,3,9,10 C/EBP,11,12 and C/EBP.13 These proteins have highly conserved C-terminal basic amino acid-rich regions with 3' flanking leucine zipper domains that are essential for DNA binding and dimerization, but these proteins differ in their N-terminal regions.14,15 The members of this family are believed to bind the same recognition sites on DNA, having the consensus sequences 5'-ATTGCGCAAT-3'.16 Previous studies have suggested that the C/EBP proteins interact with some transcriptional factors, including c-Myb,17 cAMP response element-binding protein (CREB),18 Fos-Jun family,19 and NF-B.20 The human C/EBP gene was recently cloned and mapped to chromosome 14q11.2.11,12 Differential splicing and using alternative promoters can generate a total of 4 proteins with calculated molecular masses of 32, 30, 27, and 14 kd.12,21 The functional significance of these variants remains unclear.22 Previous studies demonstrated that the expression of this family was tightly regulated in a tissue-specific manner.23 C/EBP, C/EBP, and C/EBP are all expressed during hematopoiesis, suggesting that these proteins may regulate a variety of hematopoietic-related genes.24 C/EBP is exclusively expressed in the myeloid and T-lymphoid lineages, especially during granulocytic differentiation.11,12,21,25 A previous study using C/EBP-deletional mice demonstrated that these mice developed normally but failed to generate functional neutrophils and eosinophils, and died of opportunistic infections between 3 and 5 months after birth, suggesting that C/EBP may play a central role in myeloid differentiation.26 C/EBP-deficient neutrophils possess distinctive defects, including a lack of secondary granule proteins, which may contribute to the loss of normal responses to inflammatory stimuli.27,28 In addition, studies have shown that retinoic acid (RA) can dramatically enhance the expression of C/EBP in acute promyelocytic leukemia (APL) cells as they are induced to differentiate, suggesting that C/EBP is a retinoic acid response gene that helps mediate terminal differentiation of APL cells.21,29,30 Meanwhile, a previous study showed that genes coding for macrophage inhibitory protein 1  (MIP-1), MIP-1, and the macrophage colony-stimulating factor (M-CSF) receptor have been implicated as transcriptional targets of C/EBP in mice.31 To identify other myelomonocytic target genes regulated by the C/EBP gene, we performed a representational difference analysis (RDA) using peritoneal lavage cells from wild-type and C/EBP knockout mice. Several differentially expressed genes, including chemokines, were identified, suggesting that C/EBP could enhance chemokine gene expression in mice.     Materials and methods Top Abstract Introduction Materials and methods Results Discussion References Mice and thioglycollate challenge C/EBP / mice were generously provided by Dr K. G. Xanthopoulos and Julie Lekstrom Himes (NIH, the former now at Aurora Bioscience, La Jolla, CA)26 and wild-type mice (129SV) were obtained from Harlan Sprague Dawley, Inc (Indianapolis, IN) and maintained in a pathogen-free condition. Their genotypes were evaluated, as previously reported.26 Twenty C/EBP / mice at 1 month of age and 20 age-matched wild-type mice received 2 mL of 4% thioglycollate broth (Sigma Chemical Co, St Louis, MO) by intraperitoneal injection, as previously reported.26 Twenty-four hours later, all mice were killed by neck dislocation, and peritoneal exudate cells were harvested by lavage with 8 mL of Hanks' balanced salt solution (Life Technologies, Inc, Gaithersburg, MD) on ice. Total cell numbers were counted and cytospins of lavage fluid cells were stained with Wright-Giemsa and the percentage of neutrophils and macrophages was determined by light microscopy. RNA preparation and complementary DNA synthesis Total RNA was isolated from the peritoneal lavage cells of 20 wild-type and 20 knockout mice using TRIzol (Life Technologies, Inc). To minimize individual variation, the RNAs from either all the wild-type mice or all the knockout mice were mixed together and used for RDA analysis. Complementary DNA (cDNA) samples of tester (containing differentially expressed transcripts in wild-type mice) and driver (containing transcripts also expressed in C/EBP-knockout mice) were synthesized and amplified in parallel using SMART PCR cDNA Synthesis Kit (CLONTECH, Palo Alto, CA), as per the manufacturer's instructions. Representational difference analysis RDA was performed using PCR-Select cDNA Subtraction Kit (CLONTECH), as described previously.32-34 Subtracted nested-polymerase chain reaction (PCR) products were cloned into a plasmid using an Original TA cloning kit (Invitrogen, Carlsbad, CA) and electroporated into competent Escherichia coli. Finally, subtracted cDNA libraries were constructed. To make 2 sets of membrane filters, E coli colonies were blotted onto nylon membranes and duplicated. Probes from either tester or driver cDNA were generated from the same subtracted nested-PCR product by separating adaptors with restriction enzyme RsaI. After probes were purified using the GENECLEAN II kit (BIO 101 Inc, La Jolla, CA), membranes containing cDNA libraries were hybridized with either tester or driver probe in a standard condition. By comparing signals between 2 blots, differentially expressed clones were picked and cultured. Plasmids were isolated with a standard method and sequenced with Thermo Sequenase II dye terminator cycle sequencing premix kit (Amersham Pharmacia Biotech, Inc, Cleveland, OH), as per manufacturer's protocol. Sequence data were identified via the GenBank. Virtual Northern blot and Northern blot analyses Five hundred nanograms of PCR-amplified (nonsubtracted) cDNAs were electrophoresed on agarose gels (1.2%), and Southern blotted onto nylon membranes. These filters were hybridized at 65°C for 3 hours using a Rapid-hyb buffer (Amersham Life Science) with [-32P] dCTP-labeled DNA probes, which were obtained by RDA. Filters were rinsed to a final stringency of 0.25 × SSC and exposed to a Kodak X-Omat or a Biomax film (Eastman Kodak, Rochester, NY) at 70°C with an intensifying screen. To obtain a glyceraldehyde-3-phosphate dehydrogenase (GAPDH) probe for control hybridization, murine cDNAs were amplified with specific primers for GAPDH included in the PCR-Select cDNA subtraction kit (CLONTECH), cloned into plasmid using the TA cloning method, and sequenced for integrity. Clones that showed no significant difference, as determined by virtual Northern blot analysis, were excluded for further consideration. Ten micrograms of each total RNA sample were electrophoresed on agarose formaldehyde gels and transferred onto nylon membranes. The blots were hybridized at 65°C overnight with [-32P] dCTP-labeled DNA probes generated by RDA using a standard method.32-34 Filters were rinsed to a final stringency of 0.25 × SSC and exposed to a Kodak X-Omat or a Biomax film (Eastman Kodak, Rochester, NY) at 70°C with an intensifying screen.     Results Top Abstract Introduction Materials and methods Results Discussion References Thioglycollate challenge Twenty C/EBP-knockout mice at the age of 1 month and 20 age-matched wild-type mice received thioglycollate intraperitoneally, and peritoneal lavage fluids were collected after 24-hour stimulation. Total cell numbers in the fluid were not different between control and knockout (control, [2.1 ± 0.9] × 107; knockout, [2.7 ± 2.0] × 107, respectively). In addition, the ratios of the granulocyte (G) to macrophage (M) populations showed no difference between these 2 groups (control, G:M = 65.6 ± 9.5:34.4 ± 9.5; knockout, G:M = 61.0 ± 9.2 :39.1 ± 9.2, respectively). Identification of differentially expressed genes between C/EBP +/+ and / peritoneal cells determined by representational difference analysis After RDA was used to isolate cDNA fragments that were expressed specifically in normal neutrophils/macrophages, a total of 174 clones were isolated, grown up, and sequenced. These fragments, which were theoretically differentially expressed, were sequenced and compared with known GenBank sequences via a BLAST search to determine their identity. Homology analysis revealed that these clones consisted of 33 clones with different nucleotide sequences. The abundant clones were: Early T-Lymphocyte Activation-1 protein (ETa-1) (28.2%), MIP-1 (10.2%), cathepsin L (6.3%), C10 chemokine (6.3%), and galactose/N-acetyl galactosamine (Gal/GalNAc)-specific lectin (1.7%). The other clones were not so frequent as these (data not shown). Interestingly, C/EBP was not identified. Fifteen clones disclosed no major homology to previously known sequences, suggesting that these genes might be unknown. The unknown clones 1, 2, and 3 of the 15 unknown clones showed partial homology to asialoglycoprotein receptor gene (87 of 407 bases), proapoptotic protein (siva) gene (50 of 340 bases), and pig alveolar macrophage chemotactic factor II gene (26 of 420 bases), respectively. Expression of genes in C/EBP / mice To confirm that these fragments were differentially expressed in normal cells, virtual Northern blot analyses using PCR-amplified cDNAs were initially carried out; this approach was used because the amount of RNA from peritoneal lavage cells was limited. Figure 1 shows that cathepsin L, MIP-1, monocyte chemotactic protein-3 (MCP-3), and Gal/GalNAc-specific lectin were differentially expressed in normal cells, as measured by virtual Northern blot. Cathepsin L was strongly expressed in the wild-type; however, the difference between wild-type and knockout mice was modest. MIP-1 was also strongly expressed in the wild-type and weakly expressed in the knockout mice. MCP-3 was modestly expressed in the wild-type and very weakly expressed in the knockout mice. Gal/GalNAc-specific lectin was not expressed in the knockout mice, as determined by virtual Northern blot. Interestingly, unknown clones 1, 2, and 3 were almost exclusively expressed in the wild-type mice. Unexpectedly, another 26 clones, including ETa-1, C10 chemokine, and some unknown genes, did not show a significant difference between wild-type and knockout mice, as examined by virtual Northern blot. Thus, these clones were excluded for further consideration. Northern blot analysis using total RNA from peritoneal cells of wild-type and knockout mice and using cathepsin L, MIP-1, and MCP-3 cDNAs as probes confirmed that these genes were differentially expressed in wild-type mice, as measured by the "real" Northern blot (Figure 2). These results are summarized in Table 1. View larger version (41K): [in this window] [in a new window]   Figure 1. Virtual Northern blot analysis. Total RNAs were extracted from cells in the peritoneal lavage fluid. cDNAs were synthesized and polyclonally amplified. Five hundred nanograms of PCR-amplified (nonsubtracted) cDNAs were electrophoresed on agarose gels (1.2%), and Southern blotted onto nylon membranes. These filters were hybridized at 65°C for 3 hours using Rapid-hyb buffer (Amersham Life Science) with [-32P] dCTP-labeled cDNA probes. Filters were rinsed to a final stringency of 0.25 × SSC and exposed to a Kodak X-Omat or a Biomax film at 70°C with an intensifying screen. Probes used in this study were generated from the cDNA library made by RDA. GAPDH was used as a control of expression. Size markers on the left expressed in kilobase. GenBank accession nos. BE846999, BE8470001, and BE847000 for unknown #1, #2, and #3, respectively. View larger version (55K): [in this window] [in a new window]   Figure 2. Northern blot analysis. Ten micrograms of total RNA sample were electrophoresed on agarose formaldehyde gels and transferred onto nylon membranes. The blots were hybridized with [-32P] dCTP-labeled cDNA probes at 65°C overnight using a standard method. Filters were rinsed to a final stringency of 0.25 × SSC and exposed to a Kodak X-Omat or a Biomax film at -70 °C with an intensifying screen. After sequential stripping of the probes by a standard method, the blot was rehybridized with a GAPDH probe as a control.                                View this table: [in this window] [in a new window]   Table 1. Genes differentially expressed in wild-type mice compared with C/EBP-deletional mice     Discussion Top Abstract Introduction Materials and methods Results Discussion References The expression of the C/EBP gene is regulated in a strictly lineage-specific manner. It is exclusively expressed in myeloid cells.11,12,25 Expression is highest around the promyelocyte stage of development but C/EBP is detectable in monocytes/macrophages and related cell lines, as well as granulocytes in mice.31 C/EBP-deficient mice develop functionally defective neutrophils.27,28 Previous studies revealed that C/EBP could transactivate the expression of neutrophil-mediated genes, including lactoferrin and gelatinase.27,28 Another study showed that C/EBP could enhance the expression of the M-CSF receptor as well as chemokines, suggesting that macrophage-related genes may also be targets for transcriptional regulation by C/EBP in mice.31 To identify other potential targets of C/EBP, we performed PCR-based RDA analysis using peritoneal cells from C/EBP knockout mice compared with those from wild-type animals. One hundred and seventy-four clones were isolated; sequencing showed that 33 independent genes were identified. However, virtual Northern blot analysis revealed that only 7 of the 33 genes had a differential expression pattern, indicating that the number of false-positive genes using PCR-based RDA analysis is relatively high. The extremely abundant genes contained in the tester cDNA could remain after subtractive hybridization, and these clones would be included in the cDNA library. We have found that 4 known genes (cathepsin L, MIP-1, MCP-3, Gal/GalNAc-specific lectin) plus 3 unidentified genes were differentially expressed in wild-type mice using the virtual Northern blot, suggesting that C/EBP may enhance their expression. The results of the Northern blot analysis paralleled the virtual Northern blot data. The 4 known genes are all expressed in macrophages and are important to their catabolism and/or inflammatory activities. Interestingly, C/EBP was not detected by RDA, suggesting that the expression level of C/EBP was much lower than that of chemokines in activated macrophages. Because C/EBP is preferentially expressed in promyelocytes and myelocytes, these macrophage-related transcripts detected by subtraction may reflect secondary events due to C/EBP deficiency. Cathepsin L (also called macrophage cysteine proteinase [MCP], major excreted protein [MEP]) is a member of the papain superfamily of lysosomal cysteine proteinase with a major role in intracellular protein catabolism.35 Cathepsin L has the greatest collagenolytic and elastinolytic activity in vitro of any of the cathepsins. It is expressed in various cell types, including macrophages, fibroblasts, and also malignant cells.35,36 MIP-1 and MCP-3 are 2 members of the C-C-chemokine family. Chemokines have been subdivided, based on the position of their conserved cysteine residues, into the C-X-C and the C-C groups, the latter includes MIP-1, MIP-1, MIP-1, RANTES, MCP-1, MCP-2, MCP-3, and C10.37,38 These chemokines are homologous to one another. Not only do they mediate chemotaxis, they also modulate a variety of functional properties of leukocytes, including the activation of the contractile cytoskeleton, the transient rise in intracellular Ca++ concentration, exocytosis, and the expression of adhesion molecules. The MIP-1 can activate both neutrophils and monocytes/ macrophages.39,40 The MCP-3 can also act on macrophages, activated T lymphocytes, eosinophils, and basophils.41,42 These chemokines appear to regulate inflammation by the selective recruitment and activation of leukocyte populations. We have found that consistent with decreased expression of these chemokines, as also previously reported,27 neutrophil migration was impaired in granulocytes lacking C/EBP. When 2-month-old C/EBP knockout mice were used for thioglycollate stimulation, much fewer (less than 1 of 10) leukocytes appeared in the peritoneal lavages, compared with numbers found in the wild-type mice at the same age (data not shown). Our results suggest that the reduced expression of these chemokines in C/EBP knockout mice may partly contribute to diminished ability to migrate and impaired functions of macrophages and neutrophils. Gal/GalNAc-specific lectin (also called asialoglycoprotein-binding protein [ASGP-BP]) is expressed on macrophages and is inducible with thioglycollate injection.43,44 Nonstimulated macrophages showed negligible levels of expression.44 This protein is located on the cell surface with its COOH terminus on the extracellular side. It is homologous to the hepatic asialoglycoprotein receptor (rat hepatic lectin).43 Gal/GalNAc-specific lectin is responsible for carbohydrate-mediated endocytosis and plays an important role in cell-to-cell adhesion. Our results suggest that the absence of the Gal/GalNAc-specific lectin may contribute to the impaired ability of neutrophils and macrophages to migrate correctly in the C/EBP knockout mice. Experimentally induced overexpression of C/EBP in a murine macrophage cell line enhanced the expression of MCP-1 as well as MIP-1 and MIP-1, suggesting that these genes may be targets of regulation by C/EBP.31 However, our RDA analysis did not detect these genes. To evaluate the difference of expression between knockout and wild-type mice, virtual Northern blots were hybridized with 32P-labeled MCP-1, MIP-1, and MIP-1. No significant differences were noted in the expression of these 3 genes in the macrophages and neutrophils of wild-type and C/EBP-deletional mice (data not shown). The experimental approach between our studies and those that overexpressed C/EBP in the macrophage cell line are very different. Taken together, expression of MCP-1, MIP-1, and MIP-1 does not require C/EBP, although overexpression of this transcription factor may enhance their levels. This study has found 3 previously unidentified, unknown genes that have prominent differences in expression between C/EBP knockout and wild-type mice. These genes have some homologies to known, macrophage-related genes. Characterization of these genes is ongoing. Taken together, we have found several genes that were differentially expressed in macrophages and granulocytes of wild-type but not C/EBP-deletional mice, suggesting that C/EBP may enhance their expression. Although we do not know whether C/EBP can directly or indirectly enhance the expression of these genes, our results expand the potential targets of C/EBP. As C/EBP is expressed mostly during granulocytic differentiation, C/EBP may be enhancing these macrophage-related genes indirectly. Further studies. including reporter assays, will be needed to appreciate fully the role of C/EBP in control of these genes.     Acknowledgments We thank Dr A. Fritz Gombart and Dr Hiroshi Kawabata (Cedars-Sinai Medical Center/UCLA School of Medicine) for their generous technical assistance. We are grateful to Kim Burgin for her excellent secretarial help.     Footnotes Submitted May 8, 2000; accepted July 21, 2000. Supported in part by National Institutes of Health and US Army grants as well as the Parker Hughes Fund, C. and H. Koeffler Fund, Horn Foundation, and the Lymphoma Foundation of America. The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734. Reprints: H. Phillip Koeffler, Division of Hematology/Oncology, B208, Cedars-Sinai Medical Center, UCLA School of Medicine, 8700 Beverly Blvd, Los Angeles, CA 90048.     References Top Abstract Introduction Materials and methods Results Discussion References 1. Landschulz WH, Johnson PF, Adashi EY, Graves BJ, McKnight SL. Isolation of a recombinant copy of the gene encoding C/EBP. Genes Dev. 1988;2:786-800[Abstract/Free Full Text]. 2. Akira S, Isshiki H, Suguita T, et al. A nuclear factor for IL-6 expression (NF-IL6) is a member of a C/EBP family. EMBO J. 1990;9:1897-1906[Medline] [Order article via Infotrieve]. 3. Cao Z, Umek RM, McKnight SL. Regulated expression of three C/EBP isoforms during adipose conversion of 3T3-L1 cells. Genes Dev. 1991;5:1538-1552[Abstract/Free Full Text]. 4. Chang CJ, Chen TT, Lei HY, Chen DS, Lee SC. Molecular cloning of a transcription factor, AGP/EBP, that belongs to members of the C/EBP family. Mol Cell Biol. 1990;10:6642-6653[Abstract/Free Full Text]. 5. Descombes P, Schibler U. A liver-enriched transcriptional activator protein, LAP, and a transcriptional inhibitory protein, LIP, are translated from the same mRNA. Cell. 1991;67:569-579[Medline] [Order article via Infotrieve]. 6. Katz S, Kowenz-Leutz E, Muller C, Meese K, Ness SA, Leutz A. The NF-M transcription factor is related to C/EBP beta and plays a role in signal transduction, differentiation and leukemogenesis of avian myelomonocytic cells. EMBO J. 1993;12:1321-1332[Medline] [Order article via Infotrieve]. 7. Poli V, Mancini FP, Cortese R. IL-6DBP, a nuclear protein involved in interleukin-6 signal transduction, defines a new family of leucine zipper proteins related to C/EBP. Cell. 1990;63:643-653[Medline] [Order article via Infotrieve]. 8. Roman C, Platero JS, Shuman JD, Calame K. Ig/EBP-1: a ubiquitously expressed immunoglobulin enhancer binding protein that is similar to C/EBP and heterodimerizes with C/EBP. Genes Dev. 1990;4:1404-1415[Abstract/Free Full Text]. 9. Kinoshita S, Akira S, Kishimoto T. A member of the C/EBP family, NF-IL6 beta, forms a heterodimer and transcriptionally synergizes with NF-IL6. Proc Natl Acad Sci U S A. 1992;89:1473-1476[Abstract/Free Full Text]. 10. Williams SC, Cantwell CA, Johnson PF. A family of C/EBP-related proteins capable of forming covalently linked leucine zipper dimers in vitro. Genes Dev. 1991;5:1553-1567[Abstract/Free Full Text]. 11. Antonson P, Stellan B, Yamanaka R, Xanthopoulos KG. A novel human CCAAT/enhancer binding protein gene, C/EBP, is expressed in cells of lymphoid and myeloid lineages and is located on chromosome 14q11.2 close to the T-cell receptor alpha/delta locus. Genomics. 1996;35:30-38[Medline] [Order article via Infotrieve]. 12. Chumakov AM, Grillier I, Chumakova E, Chih D, Slater J, Koeffler HP. Cloning of the novel human myeloid-cell-specific C/EBP- transcription factor. Mol Cell Biol. 1997;17:1375-1386[Abstract]. 13. Ron D, Habener JF. CHOP, a novel developmentally regulated nuclear protein that dimerizes with transcription factors C/EBP and LAP and functions as a dominant-negative inhibitor of gene transcription. Genes Dev. 1992;6:439-453[Abstract/Free Full Text]. 14. Landschulz WH, Johnson PF, McKnight SL. The DNA binding domain of the rat liver nuclear protein C/EBP is bibpartite. Science. 1989;243:1681-1688[Abstract/Free Full Text]. 15. Xanthopoulos KG, Mirkovitch J. Gene regulation in rodent hepatocytes during development, differentiation and disease. Eur J Biochem. 1993;216:353-360[Medline] [Order article via Infotrieve]. 16. Koldin B, Suckow M, Seydel A, Wilken-Bergmann BV, Muller-Hill B. A comparison of the different DNA binding specificities of the bzip proteins C/EBP and GCN4. Nucleic Acids Res. 1995;23:4162-4169[Abstract/Free Full Text]. 17. Mink S, Kerber U, Klempnauer KH. Interaction of C/EBP and v-Myb is required for synergistic activation of the mim-1 gene. Mol Cell Biol. 1996;16:1316-1325[Abstract]. 18. LeClair KP, Blanar MA, Sharp PA. The p50 subunit of NF-B associates with the NF-IL6 transcription factor. Proc Natl Acad Sci U S A. 1992;89:8145-8149[Abstract/Free Full Text]. 19. Hsu W, Kerppola TK, Chen PL, Curran T, Chen-Kiang S. Fos and Jun repress transcription activation by NF-IL6 through association at the basic zipper region. Mol Cell Biol. 1994;14:268-276[Abstract/Free Full Text]. 20. Ray A, Hannink M, Ray BK. Concerted participation of NF-B and C/EBP heterodimer in lipopolysaccharide induction of serum amyloid A gene expression in liver. J Biol Chem. 1995;270:7365-7374[Abstract/Free Full Text]. 21. Yamanaka R, Kim G-D, Radomska HS, et al. CCAAT/enhancer binding protein  is preferentially up-regulated during granulocytic differentiation and its functional versatility is determined by alternative use of promoters and differential splicing. Proc Natl Acad Sci U S A. 1997;94:6462-6467[Abstract/Free Full Text]. 22. Verbeek W, Gombert AF, Chumakov AM, Müller C, Friedman AD, Koeffler HP. C/EBP directly interacts with the DNA binding domain of c-myb and cooperatively activates transcription of myeloid promoters. Blood. 1999;93:3327-3337[Abstract/Free Full Text]. 23. Lekstrom-Himes J, Xanthopoulos KG. Biological role of the CCAAT/enhancer binding protein family of transcription factors. J Biol Chem. 1998;273:28545-28548[Abstract/Free Full Text]. 24. Scott LM, Civin CI, Rorth P, Friedman AD. A novel temporal expression pattern of three C/EBP family members in differentiating myelomonocytic cells. Blood. 1992;180:1725-1735. 25. Morosetti R, Park DJ, Chumakov AM, et al. A novel, myeloid transcription factor, C/EBP, is upregulated during granulocytic, but not monocytic, differentiation. Blood. 1997;90:2591-2600[Abstract/Free Full Text]. 26. Yamanaka R, Barlow C, Lekstrom-Himes J, et al. Impaired granulopoiesis, myelodysplasia, and early lethality in CCAAT/enhancer binding protein -deficient mice. Proc Natl Acad Sci U S A. 1997;94:13187-13192[Abstract/Free Full Text]. 27. Lekstrom-Himes J, Xanthopoulos KG. CCAAT/enhancer binding protein  is critical for effective neutrophil-mediated response to inflammatory challenge. Blood. 1999;93:3096-3105[Abstract/Free Full Text]. 28. Lekstrom-Himes J, Dorman SE, Kopar P, Holland SM, Gallin JI. Neutrophil-specific granule deficiency results from a novel mutation with loss of function of the transcription factor CCAAT/enhancer binding protein . J Exp Med. 1999;189:1847-1852[Abstract/Free Full Text]. 29. Chih DY, Chumakov AM, Park DJ, Silla AG, Koeffler HP. Modulation of mRNA expression of a novel human myeloid-selective CCAAT/enhancer binding protein gene (C/EBP). Blood. 1997;90:2987-2994[Abstract/Free Full Text]. 30. Park DJ, Chumakov AM, Vuong PT, et al. CCAAT/enhancer binding protein  is a potential retinoid target gene in acute promyelocytic leukemia treatment. J Clin Invest. 1999;103:1399-1408[Medline] [Order article via Infotrieve]. 31. Williams SC, Du Y, Schwartz RC, et al. C/EBP is a myeloid-specific activator of cytokine, chemokine, and macrophage-colony-stimulating factor receptor genes. J Biol Chem. 1998;273:13493-13501[Abstract/Free Full Text]. 32. Diatchenko L, Lau YFC, Campbell AP, et al. Suppression subtractive hybridization: a method for generating differentially regulated or tissue-specific cDNA probes and libraries. Proc Natl Acad Sci U S A. 1996;93:6025-6030[Abstract/Free Full Text]. 33. Akopyants NS, Fradkov A, Diatchenko L, et al. PCR-based subtractive hybridization and differences in gene content among strains of Helicobacter pylori. Proc Natl Acad Sci U S A. 1998;95:13108-13113[Abstract/Free Full Text]. 34. Sagerström CG, Sun BI, Sive HL. Subtractive cloning: past, present, and future. Annu Rev Biochem. 1997;66:751-783[Medline] [Order article via Infotrieve]. 35. Joseph LJ, Chang LC, Stamenkovich D, Sukhatme V. Complete nucleotide and deduced amino acid sequences of human and murine preprocathepsin L: an abundant transcript induced by transformation of fibroblasts. J Clin Invest. 1988;81:1621-1629. 36. Ishidoh K, Kominami E. Gene regulation and extracellular functions of procathepsin L. Biol Chem. 1998;379:131-135. 37. Baggiolini M. Chemokines and leukocyte traffic. Nature. 1998;392:565-568[Medline] [Order article via Infotrieve]. 38. Baggiolini M, Dewald B, Moser B. Human chemokines: an update. Annu Rev Immunol. 1997;15:675-705[Medline] [Order article via Infotrieve]. 39. Poltorak AN, Bazzoni F, Smirnova II, et al. MIP-1: molecular cloning, expression, and biological activities of a novel CC chemokine that is constitutively secreted in vivo. J Inflamm. 1995;45:207-219[Medline] [Order article via Infotrieve]. 40. Mohamadzadeh M, Poltorak AN, Bergstresser PR, Beutler B, Takashima A. Dendritic cells produce macrophage inflammatory protein-1, a new member of the CC chemokine family. J Immunol. 1996;156:3102-3106[Abstract]. 41. Loetscher P, Seitz M, Clark-Lewis I, Baggiolini M, Moser B. Monocyte chemotactic proteins MCP-1, MCP-2, and MCP-3 are major attractants for human CD4+ and CD8+ T lymphocytes. FASEB J. 1994;8:1055-1060[Abstract]. 42. Noso N, Proost P, Van Damme J, Schröder JM. Human monocyte chemotactic proteins-2 and 3 (MCP-2 and MCP-3) attract human eosinophils and desensitize the chemotactic responses towards RANTES. Biochem Biophys Res Commun. 1994;200:1470-1476[Medline] [Order article via Infotrieve]. 43. Ii M, Kurata H, Itoh N, Yamashita I, Kawasaki T. Molecular cloning and sequence analysis of cDNA encoding the macrophage lectin specific for galactose and N-acetylgalactosamine. J Biol Chem. 1990;265:11295-11298[Abstract/Free Full Text]. 44. Sato M, Kawakami K, Osawa T, Toyoshima S. Molecular cloning and expression of cDNA encoding a galactose/N-acetylgalactosamine-specific lectin on mouse tumoricidal macrophages. 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