The Role of C/EBP Isoforms in the Control of Inflammatory and Native Immunity Functions

CAAT/enhancer-binding protein acute phase interleukin IL-6-responsive element tumor necrosis factor granulocyte colony-stimulating factor. CAAT/enhancer-binding proteins (C/EBPs)1 are a family of leucine zipper transcription factors involved in the regulation of various aspects of cellular differentiation and function in multiple tissues. Six different members of the family have been isolated and characterized (C/EBPα to ζ), all sharing a strong homology in the carboxyl-terminal domain, which carries a basic DNA-binding domain and a leucine zipper motif. The general characteristics and patterns of expression of the C/EBP family have been described in the first minireview of this series (1Lekstrom-Himes J. Xanthopoulos K.G. J. Biol. Chem. 1998; 273: 28545-28548Abstract Full Text Full Text PDF PubMed Scopus (693) Google Scholar). Here I will focus on the functions of several C/EBP family members in regulating various aspects of inflammation and immunity in the liver and in cells of the myelomonocytic lineage, in vitro as well as in vivo. Among the many liver-specific or liver-enriched genes whose expression is variably regulated by members of the C/EBP family of transcription factors are a very prominent class of genes coding for acute phase (AP) proteins. These are plasma proteins whose levels of expression are either positively or negatively regulated during the acute phase of inflammation (reviewed in Ref. 2Fey G.H. Gauldie J. Popper H. Schaffner F. Progress in Liver Diseases. 9. W. B. Saunders Co., Philadelphia1990: 89-116Google Scholar). As initially suggested by the observation that hepatocytes are able to respond to local tissue injury at distal sites, it has been demonstrated that a number of cytokines and hormones are involved in the regulation of the AP response (reviewed in Ref. 3Koj A. Gauldie J. Baumann H. Mackiewicz A. Kushner I. Baumann H. Acute Phase Proteins: Molecular Biology, Biochemistry, and Clinical Applications. CRC Press, Inc., Boca Raton, FL1993: 275-287Google Scholar). AP genes have been divided into two major classes according to the pattern of responsiveness to cytokines. For maximal induction class I genes require a combination of both interleukin (IL)-1 and IL-6, sometimes with the additional need for glucocorticoids. In contrast, class II genes are solely responsive to IL-6 and related cytokines, either alone or in combination with dexamethasone. Functional C/EBP-binding motifs (initially known as type I IL-6-responsive elements (IL-6REs)) have been characterized on the promoters of most class I genes (hemopexin, haptoglobin, α1-acid glycoprotein, serum amyloid A1, A2, and A3, complement C3, C-reactive protein), often in association with IL-1-responsive NF-κB sites (see below) (4Poli V. Cortese R. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 8202-8206Crossref PubMed Scopus (120) Google Scholar, 5Oliviero S. Cortese R. EMBO J. 1989; 8: 1145-1151Crossref PubMed Scopus (141) Google Scholar, 6Chen H.M. Liao W.S.L. J. Biol. Chem. 1993; 268: 25311-25319Abstract Full Text PDF PubMed Google Scholar, 7Juan T.S. Wilson D.R. Wilde M.D. Darlington G.J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 2584-2588Crossref PubMed Scopus (126) Google Scholar, 8Alam T. An M.R. Mifflin R.C. Hsieh C.C. Ge X. Papaconstantinou J. J. Biol. Chem. 1993; 268: 15681-15688Abstract Full Text PDF PubMed Google Scholar, 9Ray B.K. Ray A. Eur. J. Biochem. 1994; 222: 891-900Crossref PubMed Scopus (37) Google Scholar, 10Majello B. Arcone R. Toniatti C. Ciliberto G. EMBO J. 1990; 9: 457-465Crossref PubMed Scopus (145) Google Scholar, 11Ray A. Hannink M. Ray B.K. J. Biol. Chem. 1995; 270: 7365-7374Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 12Li X. Liao W.S.L. Nucleic Acids Res. 1992; 20: 4765-4772Crossref PubMed Scopus (65) Google Scholar, 13Betts J.C. Cheshire J.K. Akira S. Kishimoto T. Woo P. J. Biol. Chem. 1993; 268: 25624-25631Abstract Full Text PDF PubMed Google Scholar, 14Shimizu H. Yamamoto K. Gene (Amst.). 1994; 149: 305-310Crossref PubMed Scopus (50) Google Scholar). In contrast, in class II genes such as α2-macroglobulin mainly type II IL-6REs, which bind the cytokine-inducible transcription factors Stat3 and Stat1 (reviewed in Ref. 15Schindler C. Darnell J.J. Annu. Rev. Biochem. 1995; 64: 621-651Crossref PubMed Scopus (1661) Google Scholar), have been identified. Type II IL-6REs are also found in promoters of class I genes. The activity and/or mRNA and protein levels of various C/EBP genes are differentially modulated in response to inflammatory stimuli and to recombinant cytokines. Indeed C/EBPβ, the second C/EBP family member to be isolated, was originally identified thanks to its inducibility by IL-6 or by IL-1 in human hepatoma cells and in a glioblastoma cell line, respectively (16Poli V. Mancini F.P. Cortese R. Cell. 1990; 63: 643-654Abstract Full Text PDF PubMed Scopus (459) Google Scholar, 17Akira S. Issihiki H. Sugita T. Tanabe O. Kinoshita S. Nishio Y. Nakajima T. Hirano T. Kishimoto K. EMBO J. 1990; 9: 1897-1906Crossref PubMed Scopus (1215) Google Scholar). It has been subsequently determined by many independent studies that both C/EBPβ and -δ are strongly up-regulated at the transcriptional level by inflammatory stimuli such as turpentine oil and bacterial lipopolysaccharide and by recombinant cytokines such as IL-6, IL-1, and TNF-α (reviewed in Ref.18Akira S. Kishimoto T. Adv. Immunol. 1997; 65: 1-46Crossref PubMed Google Scholar). Conversely, C/EBPα is slightly down-regulated under the same conditions (19Alam T. An M.R. Papaconstantinou J. J. Biol. Chem. 1992; 267: 5021-5024Abstract Full Text PDF PubMed Google Scholar). Studies of the interplay of different C/EBP factors on the promoters of several AP genes in hepatocytes have shown that at steady state, the majority of DNA-protein complexes contain various forms of C/EBPα homodimers and C/EBPα/C/EBPβ heterodimers (20Ossipow V. Descombes P. Schibler U. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8219-8223Crossref PubMed Scopus (324) Google Scholar). Upon AP induction, however, the amount of complexes containing C/EBPα is dramatically reduced, replaced by C/EBPβ and C/EBPδ, although the exact composition of these complexes varies in the different experimental systems (6Chen H.M. Liao W.S.L. J. Biol. Chem. 1993; 268: 25311-25319Abstract Full Text PDF PubMed Google Scholar, 7Juan T.S. Wilson D.R. Wilde M.D. Darlington G.J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 2584-2588Crossref PubMed Scopus (126) Google Scholar, 8Alam T. An M.R. Mifflin R.C. Hsieh C.C. Ge X. Papaconstantinou J. J. Biol. Chem. 1993; 268: 15681-15688Abstract Full Text PDF PubMed Google Scholar, 9Ray B.K. Ray A. Eur. J. Biochem. 1994; 222: 891-900Crossref PubMed Scopus (37) Google Scholar, 21Ramji D.P. Vitelli A. Tronche F. Cortese R. Ciliberto G. Nucleic Acids Res. 1993; 21: 289-294Crossref PubMed Scopus (160) Google Scholar, 22Ray A. Ray B.K. Mol. Cell. Biol. 1994; 14: 4324-4332Crossref PubMed Google Scholar). Although C/EBPδ mRNA and protein levels are almost undetectable under uninduced conditions, C/EBPβ is relatively abundant in several tissues, including the liver, even before induction. C/EBPβ is known to undergo a series of post-translational modifications that modulate its activity. The first suggestion that induced post-translational modifications can increase the transcriptional activating potential of C/EBPβ came from studies on Hep3B cells, in which transfected C/EBPβ was able to induce transcription of a reporter gene much more efficiently in the presence of IL-6 (16Poli V. Mancini F.P. Cortese R. Cell. 1990; 63: 643-654Abstract Full Text PDF PubMed Scopus (459) Google Scholar). Subsequently, several phosphorylation sites have been demonstrated on this protein. Phosphorylation of serine 276 has been shown to take place in a pituitary cell line in response to intracellular Ca2+ increase via a calcium/calmodulin-dependent kinase (23Wegner M. Cao Z. Rosenfeld M.G. Science. 1992; 256: 370-373Crossref PubMed Scopus (309) Google Scholar); activation of the protein kinase C pathway causes phosphorylation of Ser-105 in HepG2 cells (24Trautwein C. Caelles C. van der Geer P. Hunter T. Karin M. Chojkier M. Nature. 1993; 364: 544-547Crossref PubMed Scopus (294) Google Scholar). Finally, activation of mitogen-activated protein kinase following induction of the Ras pathway leads to phosphorylation of Thr-235 (25Nakajima T. Kinoshita S. Sasagawa T. Sasaki K. Naruto M. Kishimoto T. Akira S. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 2207-2211Crossref PubMed Scopus (519) Google Scholar). Although all these events lead to increased transactivating potency of C/EBPβ, only the last one can be linked to IL-6 signaling. In contrast, the C/EBPδ-dependent activation of target AP genes is considered to be solely secondary to transcriptional activation of its gene. Accordingly, ectopical expression of C/EBPδ in hepatoma cells is sufficient to transactivate responsive promoters in a cytokine-independent manner (21Ramji D.P. Vitelli A. Tronche F. Cortese R. Ciliberto G. Nucleic Acids Res. 1993; 21: 289-294Crossref PubMed Scopus (160) Google Scholar). Far from being liver-specific, induction of both C/EBPβ and -δ levels by inflammatory stimuli occurs in most tissues analyzed, thus suggesting a more general role of these two factors in inflammation (17Akira S. Issihiki H. Sugita T. Tanabe O. Kinoshita S. Nishio Y. Nakajima T. Hirano T. Kishimoto K. EMBO J. 1990; 9: 1897-1906Crossref PubMed Scopus (1215) Google Scholar, 26Kinoshita S. Akira S. Kishimoto T. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 1473-1476Crossref PubMed Scopus (262) Google Scholar). Notably, expression of various C/EBP isoforms is differentially induced during macrophage and/or granulocyte differentiation. C/EBP-binding motifs have been identified in the functional regulatory regions of various genes expressed by cells of the myelomonocytic lineages, including those encoding the inflammatory cytokines IL-6, IL-1β, and TNF-α (17Akira S. Issihiki H. Sugita T. Tanabe O. Kinoshita S. Nishio Y. Nakajima T. Hirano T. Kishimoto K. EMBO J. 1990; 9: 1897-1906Crossref PubMed Scopus (1215) Google Scholar, 27Wedel A. Sukski G. Ziegler-Heitbrock H.W. Cytokine. 1996; 8: 335-341Crossref PubMed Scopus (40) Google Scholar, 28Pope R.M. J. Clin. Invest. 1994; 94: 1449-1455Crossref PubMed Scopus (167) Google Scholar, 29Shirakawa F. Saito K. Bonagura C.A. Galson D.L. Fenton M.J. Webb A.C. Auron P.E. Mol. Cell. Biol. 1993; 13: 1332-1344Crossref PubMed Google Scholar), other cytokines such as IL-8 and IL-12 (30Matsusaka T. Fujikawa K. Nishio Y. Mukaida N. Matsushima K. Kishimoto T. Akira S. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 10193-10197Crossref PubMed Scopus (883) Google Scholar, 31Plevy S.E. Gemberling J.H.M. Hsu S. Dorner A.J. Smale S.T. Mol. Cell. Biol. 1997; 17: 4572-4588Crossref PubMed Scopus (273) Google Scholar), genes encoding proteins important for macrophagic or granulocytic functions such as inducible nitric oxide synthase (32Lowenstein C.J. Alley E.W. Raval P. Snowman A.M. Snyder S.M. Russel S.W. Murphy W.J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 9730-9734Crossref PubMed Scopus (1008) Google Scholar), lysozyme (33Goethe R. Loc P.V. J. Biol. Chem. 1994; 269: 31302-31309Abstract Full Text PDF PubMed Google Scholar), myeloperoxidase (34Ford A.M. Bennett C.A. Healy L.E. Towatari M. Greaves M.F. Enver T. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 10838-10843Crossref PubMed Scopus (122) Google Scholar), and neutrophil elastase (35Oelgeschlager M. Nuchprayoon I. Luscher B. Friedman A.D. Mol. Cell. Biol. 1996; 16: 4717-4725Crossref PubMed Scopus (206) Google Scholar), the gene encoding the granulocyte colony-stimulating factor (G-CSF) (36Dunn S.M. Coles L.S. Lang R.K. Gerondakis S. Vada M.A. Shannon M.F. Blood. 1994; 83: 2469-2479Crossref PubMed Google Scholar), and the macrophage, granulocyte, and granulocyte-macrophage receptor genes (37Zhang D.E. Hohaus S. Voso M.T. Chen H.M. Smith L.T. Hetherington C.J. Tenen D.G. Curr. Top. Microbiol. Immunol. 1996; 211: 137-147PubMed Google Scholar). Interestingly, C/EBP sites are also present on several viral promoters (38Bowers W.J. Baglia L.A. Ruddel A. J. Virol. 1996; 70: 3051-3059Crossref PubMed Google Scholar, 39Ohno H. Kaneko S. Kobayashi K. Murakami S. J. Med. Virol. 1997; 52: 413-418Crossref PubMed Scopus (30) Google Scholar, 40Kyo S. Inique M. Yukihiro N. Nakanishi K. Akira S. Inoue H. Yutsudo M. Tanizawa O. Hakura A. J. Virol. 1993; 67: 1058-1066Crossref PubMed Google Scholar, 41Bauknecht T. See R.H. Shi Y. J. Virol. 1996; 70: 7695-7705Crossref PubMed Google Scholar, 42Tesmer V.M. Rajadhyaksha A. Babin J. Bina M. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 7298-7302Crossref PubMed Scopus (91) Google Scholar, 43Ruocco M.R. Chen X. Ambrosino C. Dragonetti E. Liu W. Mallardo M. Falco G.D. Palmieri C. Franzoso G. Quinto I. Venuta S. Scala G. J. Biol. Chem. 1996; 271: 22479-22486Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). Of particular relevance in this context is the finding that C/EBP sites are specifically required for replication of the human immunodeficiency virus-1 in macrophages but not in CD4+ cells (44Henderson A.J. Calame K.L. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 8714-8719Crossref PubMed Scopus (112) Google Scholar). However, due to the complexity of the C/EBP family and to the co-expression of several family members in the same cell, the relative role of each C/EBP isoform in the regulation of all these genes is still unclear. Studies of different promoters have assigned a predominant role for the induction of particular genes to either C/EBPβ or C/EBPδ (6Chen H.M. Liao W.S.L. J. Biol. Chem. 1993; 268: 25311-25319Abstract Full Text PDF PubMed Google Scholar, 7Juan T.S. Wilson D.R. Wilde M.D. Darlington G.J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 2584-2588Crossref PubMed Scopus (126) Google Scholar, 8Alam T. An M.R. Mifflin R.C. Hsieh C.C. Ge X. Papaconstantinou J. J. Biol. Chem. 1993; 268: 15681-15688Abstract Full Text PDF PubMed Google Scholar, 9Ray B.K. Ray A. Eur. J. Biochem. 1994; 222: 891-900Crossref PubMed Scopus (37) Google Scholar, 21Ramji D.P. Vitelli A. Tronche F. Cortese R. Ciliberto G. Nucleic Acids Res. 1993; 21: 289-294Crossref PubMed Scopus (160) Google Scholar, 22Ray A. Ray B.K. Mol. Cell. Biol. 1994; 14: 4324-4332Crossref PubMed Google Scholar). Although this may be partly because of different experimental conditions, it is also likely to reflect physiological differences due to promoter context, to the relative affinity of specific sites for C/EBP proteins, and to the interactions with other transcriptional activators or co-activators. It is also interesting to note that different promoters all containing C/EBP binding sites can undergo completely divergent regulation. For example, the negative AP reactant mouse albumin gene carries various high affinity C/EBP motifs in its promoter, but its expression is very high at steady state and is down-regulated by IL-6 (45Geiger T. Andus T. Klapproth J. Hirano T. Kishimoto T. Heinrich P.C. Eur. J. Immunol. 1988; 18: 717-721Crossref PubMed Scopus (361) Google Scholar); in contrast the human C-reactive protein promoter, with two weak C/EBP sites, is poorly expressed at the steady state but is stimulated by IL-6 (10Majello B. Arcone R. Toniatti C. Ciliberto G. EMBO J. 1990; 9: 457-465Crossref PubMed Scopus (145) Google Scholar). Clearly, the specific composition of these two promoters and the participation of different non-C/EBP factors in their complex regulation can partially explain this apparent paradox. On the other hand, the specific transactivating capacities of the different C/EBP polypeptides are also likely to play an important role. C/EBPα is for example a stronger transcriptional activator than C/EBPβ (16Poli V. Mancini F.P. Cortese R. Cell. 1990; 63: 643-654Abstract Full Text PDF PubMed Scopus (459) Google Scholar); moreover, both C/EBPα and C/EBPβ mRNAs can give origin to truncated forms that can act either as inhibitors or as weak activators (20Ossipow V. Descombes P. Schibler U. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8219-8223Crossref PubMed Scopus (324) Google Scholar, 46Descombes P. Schibler U. Cell. 1991; 67: 569-579Abstract Full Text PDF PubMed Scopus (863) Google Scholar). In vivo, many different combinations in either homodimeric or heterodimeric forms are possible, giving rise to multiple polypeptides with distinct physiological properties whose differential occupancy of promoters may be dictated by their relative abundance and affinity and by protein-protein interactions with other factors binding to adjacent sites. A number of different transcription factors have been reported to be able to physically and functionally interact with C/EBP members and in particular with C/EBPβ. Among those, the interactions with members of the NF-κB family of transcription factors (47Stein B. Cogswell P.C. Baldwin Jr., A.S. Mol. Cell. Biol. 1993; 13: 3964-3974Crossref PubMed Google Scholar, 48LeClair K.P. Blanar M.A. Sharp P.A. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 8145-8149Crossref PubMed Scopus (272) Google Scholar) are particularly intriguing because they link the pathways of two major mediators of inflammation, IL-1 and IL-6. Interestingly, adjacent C/EBP and NF-κB motifs are found in the promoters of many AP class I genes that require both IL-1 and IL-6 for their induction, as well as those of several cytokine genes, suggesting that cooperative interaction between the two families of transcription factors may represent a general mechanism of coordinating transcriptional responses to selected stimuli. Indeed, synergistic activation by C/EBP and NF-κB members has been demonstrated for the genes encoding the acute phase proteins serum amyloid A1, A2, A3, and α1-acid glycoprotein, as well as the cytokines IL-6, IL-8, and IL-12 and the G-CSF (11Ray A. Hannink M. Ray B.K. J. Biol. Chem. 1995; 270: 7365-7374Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 12Li X. Liao W.S.L. Nucleic Acids Res. 1992; 20: 4765-4772Crossref PubMed Scopus (65) Google Scholar, 13Betts J.C. Cheshire J.K. Akira S. Kishimoto T. Woo P. J. Biol. Chem. 1993; 268: 25624-25631Abstract Full Text PDF PubMed Google Scholar, 30Matsusaka T. Fujikawa K. Nishio Y. Mukaida N. Matsushima K. Kishimoto T. Akira S. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 10193-10197Crossref PubMed Scopus (883) Google Scholar,31Plevy S.E. Gemberling J.H.M. Hsu S. Dorner A.J. Smale S.T. Mol. Cell. Biol. 1997; 17: 4572-4588Crossref PubMed Scopus (273) Google Scholar, 36Dunn S.M. Coles L.S. Lang R.K. Gerondakis S. Vada M.A. Shannon M.F. Blood. 1994; 83: 2469-2479Crossref PubMed Google Scholar, 49Lee Y.M. Miau L.H. Chang C.J. Lee S.C. Mol. Cell. Biol. 1996; 16: 4257-4263Crossref PubMed Google Scholar, 50Vietor I. Oliveira I.C. Vilcek J. J. Biol. Chem. 1996; 271: 5595-5602Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). Cooperativity between the two families of transcription factors has also been demonstrated in the case of the human immunodeficiency virus long terminal repeat (43Ruocco M.R. Chen X. Ambrosino C. Dragonetti E. Liu W. Mallardo M. Falco G.D. Palmieri C. Franzoso G. Quinto I. Venuta S. Scala G. J. Biol. Chem. 1996; 271: 22479-22486Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). C/EBP and NF-κB interactions have also been shown to lead to antagonistic effects (47Stein B. Cogswell P.C. Baldwin Jr., A.S. Mol. Cell. Biol. 1993; 13: 3964-3974Crossref PubMed Google Scholar, 51Brasier A.R. Ron D. Tate J.E. Habener J.F. EMBO J. 1990; 9: 3933-3944Crossref PubMed Scopus (130) Google Scholar), again suggesting that promoter architecture and specific cell type are likely to play a major role. Although the mechanisms responsible for cooperative effects have not yet been entirely clarified, productive interaction requires the integrity of both the NF-κB rel homology domain and the C/EBP leucine zipper motif (48LeClair K.P. Blanar M.A. Sharp P.A. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 8145-8149Crossref PubMed Scopus (272) Google Scholar). Increased affinity of C/EBP and NF-κB for their respective sites has been demonstrated (43Ruocco M.R. Chen X. Ambrosino C. Dragonetti E. Liu W. Mallardo M. Falco G.D. Palmieri C. Franzoso G. Quinto I. Venuta S. Scala G. J. Biol. Chem. 1996; 271: 22479-22486Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 47Stein B. Cogswell P.C. Baldwin Jr., A.S. Mol. Cell. Biol. 1993; 13: 3964-3974Crossref PubMed Google Scholar), and DNA-protein complexes containing both proteins have been detected using both NF-κB and C/EBP sites (11Ray A. Hannink M. Ray B.K. J. Biol. Chem. 1995; 270: 7365-7374Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 43Ruocco M.R. Chen X. Ambrosino C. Dragonetti E. Liu W. Mallardo M. Falco G.D. Palmieri C. Franzoso G. Quinto I. Venuta S. Scala G. J. Biol. Chem. 1996; 271: 22479-22486Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 50Vietor I. Oliveira I.C. Vilcek J. J. Biol. Chem. 1996; 271: 5595-5602Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). The availability of mouse strains in which the genes coding for different C/EBP members have been inactivated provided a model to test the relative importance of their different functions in the inflammatory pathway as well as in numerous other systems. Sometimes in apparent contrast with expectations it has certainly taught us a lesson about the dramatic oversimplification of extrapolating from a tissue culture plate to a living organism. Analysis of AP mRNAs in C/EBPβ-deficient mice has shown that not all AP genes thought to require C/EBPβ for their induction are in fact equally regulated by this factor (52Cappelletti M. Alonzi T. Fattori E. Libert C. Poli V. Cell Death Differ. 1996; 3: 29-35PubMed Google Scholar); indeed, induction of both hemopexin and haptoglobin mRNAs was normal in the C/EBPβ−/− mice, whereas induction of serum amyloid A and P mRNAs was reduced and induction of C3 was totally impaired, at least at the protein level. The most prominent role of C/EBPβ in the transcriptional regulation of the serum amyloid A, serum amyloid P, and α1-acid glycoprotein genes appeared to be the maintenance of the induced state rather than the initial induction; after an initial accumulation comparable with that of the wild type mice, the mRNA levels for these three genes started to decrease in the C/EBPβ−/− mice, dropping at almost background levels by 24 h, the time point at which the wild type mice reached (in contrast) their peak. Because induction of both C/EBPβ and C/EBPδ mRNAs occurs relatively late after the inflammatory stimulus, 2V. Poli, unpublished observation. it is likely that transcription factors like NF-κB and Stat3, whose activation is much faster but transient, are responsible for the first burst of induction, being partly replaced after a few hours by C/EBP members. In agreement with this idea, it has been recently demonstrated that Stat3 is involved in the IL-6-induced up-regulation of both C/EBPδ and C/EBPβ gene promoters (53Cantwell C.A. Sterneck E. Johnson P.F. Mol. Cell. Biol. 1998; 18: 2108-2117Crossref PubMed Google Scholar). This suggests a sequential model in which the induction of inflammatory cytokines such as IL-1 and IL-6 in response to inflammatory stimuli would first trigger the activation of pre-existing, inactive forms of Stat3 and NF-κB (illustrated in the upper part of Fig. 1, early inflammation). These factors would in turn initiate the activation of both class I and class II genes although at the same time Stat3 would induce new synthesis of both C/EBPδ and C/EBPβ. The pre-existing population of C/EBPβ molecules, activated by phosphorylation, would also participate in this initial induction of acute phase genes by interacting with NF-κB. However, the main role played by C/EBP factors in the induction of AP genes is linked to the synthesis of new C/EBPβ and δ polypeptides, which will substitute for the early factors NF-κB and Stat3, thus allowing the activated status of AP genes to be maintained (lower part of Fig. 1, late inflammation). According to this model, C/EBPδ is likely to act even at a later stage in the induction of AP genes, and its induction, which appears to be normal in C/EBPβ-deficient mice,2 is likely to partially compensate for the absence of C/EBPβ. Late induction of AP genes should therefore be severely impaired in mice lacking both C/EBPβ and -δ. This double mutant mouse strain has been recently generated (54Tanaka T. Yoshida N. Kishimoto T. Akira S. EMBO J. 1997; 16: 7432-7443Crossref PubMed Scopus (648) Google Scholar), but no information is available about regulation of the AP response. C/EBPα-deficient mice die shortly after birth (55Wang N.-D. Finegold M.J. Bradley A. Ou C.N. Abdelsayed S.V. Wilde M.D. Taylor L.R. Wilson D.R. Darlington G.J. Science. 1995; 269: 1108-1112Crossref PubMed Scopus (842) Google Scholar), preventing an analysis of the liver AP response. Although liver-specific inactivation of the C/EBPα gene was recently described (56Lee Y.-H. Sauer B. Johnson P.F. Gonzalez F.J. Mol. Cell. Biol. 1997; 17: 6014-6022Crossref PubMed Google Scholar), no information is available on the regulation of the AP response in these mice. More puzzling are the results of the analysis of cytokine gene expression in the different C/EBP mutant mice. Induction of serum IL-6 was unchanged in C/EBPβ-deficient mice, whereas serum TNF-α induction was impaired, indirectly suggesting a more important role for the factor in the control of the TNF-α than of the IL-6 gene (52Cappelletti M. Alonzi T. Fattori E. Libert C. Poli V. Cell Death Differ. 1996; 3: 29-35PubMed Google Scholar). Among all the cytokines thought to be regulated by C/EBPβ, only G-CSF mRNA induction is impaired in peritoneal macrophages from C/EBPβ−/− mice (57Tanaka T. Akira S. Yoshida K. Umemoto M. Yoneda Y. Shirafuji N. Fujiwara H.F. Suematsu S. Yoshida N. Kishimoto T. Cell. 1995; 80: 353-361Abstract Full Text PDF PubMed Scopus (472) Google Scholar). Notably, IL-6 serum levels were elevated in aging C/EBPβ−/− mice (58Screpanti I. Musiani P. Bellavia D. Cappelletti M. Aiello F.B. Maroder M. Frati L. Modesti A. Gulino A. Poli V. J. Exp. Med. 1996; 184: 1561-1566Crossref PubMed Scopus (57) Google Scholar, 59Screpanti I. Romani L. Musiani P. Modesti A. Fattori E. Lazzaro D. Sellitto C. Scarpa S. Bellavia D. Lattanzio G. Bistoni F. Frati L. Cortese R. Gulino A. Ciliberto G. Costantini F. Poli V. EMBO J. 1995; 14: 1932-1941Crossref PubMed Scopus (376) Google Scholar), further suggesting that this factor is totally dispensable for IL-6 gene activity. No defects in cytokine production have been detected in macrophages from C/EBPδ-deficient mice. 3S. Akira, personal communication.Interestingly, although cytokine gene expression has not been analyzed in C/EBPα-deficient mice, G-CSF receptor mRNA is almost undetectable in their liver (60Zhang D.-E. Zhang P. Wang N.-D. Hetherington C.J. Darlington G.J. Tenen D.G. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 569-574Crossref PubMed Scopus (764) Google Scholar). In contrast, inactivation of the gene encoding C/EBPε, a recently isolated family member specifically expressed in cells of the myelomonocytic lineages (61Yamanaka R. Kim G.-D. Radomska H.S. Lekstrom-Himes J. Smith L.T. Antonson P. Tenen D.G. Xanthopoulos K.G. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 6462-6467Crossref PubMed Scopus (154) Google Scholar, 62Morosetti R. Park D.J. Chumakov A.M. Grillier I. Shiohara M. Gombart A.F. Nakamaki T. Weinberg K. Koeffler H.P. Blood. 1997; 90: 2591-2600Crossref PubMed Google Scholar), results in decreased levels of several cytokine mRNAs (interferon-γ, TNF-α, IL-2, IL-4, and IL-12 p40) in the spleen (63Yamanaka R. Barlow C. Lekstrom-Himes J. Castilla L.H. Liu P.P. Eckhaus M. Decker T. Wynshaw-Boris A. Xanthopoulos K.G. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 13187-13192Crossref PubMed Scopus (312) Google Scholar). Unfortunately, neither the inducibility of these cytokines nor the levels of IL-6, IL-1β, G-CSF, granulocyte-macrophage CSF, or macrophage CSF have been analyzed yet in the C/EBPε mice. Perhaps the most striking picture emerging from the analysis of different knock-out mice in the field of inflammation and immunity is that of a coordinated role of different C/EBP family members in regulating the differentiation and function of cells of the myelomonocytic lineage. Several lines of evidence suggested an important role of the various C/EBP isoforms in myelomonocytic cells. The expression of C/EBPα, -β, and -δ is differentially regulated in myelomonocytic cell lines (64Natsuka S. Akira S. Nishio Y. Hashimoto S. Sugit T. Isshiki H. Kishimoto T. Blood. 1992; 79: 460-466Crossref PubMed Google Scholar, 65Scott L.M. Civin C.I. Rorth P. Friedman A.D. Blood. 1992; 80: 1725-1735Crossref PubMed Google Scholar).In vivo, C/EBPα expression is relatively high in immature granulocytic cells but is down-regulated in most mature granulocytes (65Scott L.M. Civin C.I. Rorth P. Friedman A.D. Blood. 1992; 80: 1725-1735Crossref PubMed Google Scholar), and C/EBPε is preferentially expressed during granulocytic differentiation both in cell lines and in human primary CD34+ cells (61Yamanaka R. Kim G.-D. Radomska H.S. Lekstrom-Himes J. Smith L.T. Antonson P. Tenen D.G. Xanthopoulos K.G. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 6462-6467Crossref PubMed Scopus (154) Google Scholar, 62Morosetti R. Park D.J. Chumakov A.M. Grillier I. Shiohara M. Gombart A.F. Nakamaki T. Weinberg K. Koeffler H.P. Blood. 1997; 90: 2591-2600Crossref PubMed Google Scholar). Moreover, as already mentioned, C/EBP family members can specifically transactivate the promoters of several myeloid-specific genes, and the chicken homologue of C/EBPβ, NF-M, is a myeloid-specific factor mediating eosinophils differentiation (66Muller C. Kowen-Leutz E. Grieser-Ade S. Graf T. Leutz A. EMBO J. 1995; 14: 6127-6135Crossref PubMed Scopus (81) Google Scholar). Remarkably in agreement with these findings, mice in which the genes encoding C/EBPα, -β, and -ε have been inactivated show specific defects in either macrophagic (C/EBPβ) or granulocytic (C/EBPα and C/EBPε) differentiation and/or functions (see below). In contrast, no abnormality was detected in C/EBPδ-deficient mice. C/EBPβ appears to play an important role in determining activation and/or terminal differentiation of macrophages. Indeed, C/EBPβ-deficient mice developed a lymphoproliferative disorder that can be linked to defects in macrophagic activation, as suggested by defective nitric oxide (NO) production by splenic macrophages and by the observation that no active IL-12, normally produced by activated macrophages, could be detected in the serum of the mutant mice infected with Candida albicans (58Screpanti I. Musiani P. Bellavia D. Cappelletti M. Aiello F.B. Maroder M. Frati L. Modesti A. Gulino A. Poli V. J. Exp. Med. 1996; 184: 1561-1566Crossref PubMed Scopus (57) Google Scholar, 59Screpanti I. Romani L. Musiani P. Modesti A. Fattori E. Lazzaro D. Sellitto C. Scarpa S. Bellavia D. Lattanzio G. Bistoni F. Frati L. Cortese R. Gulino A. Ciliberto G. Costantini F. Poli V. EMBO J. 1995; 14: 1932-1941Crossref PubMed Scopus (376) Google Scholar). Macrophages from C/EBPβ-deficient mice were also defective in intracellular killing of Listeria monocytogenes and displayed impaired tumoricidal and tumoristatic activity (57Tanaka T. Akira S. Yoshida K. Umemoto M. Yoneda Y. Shirafuji N. Fujiwara H.F. Suematsu S. Yoshida N. Kishimoto T. Cell. 1995; 80: 353-361Abstract Full Text PDF PubMed Scopus (472) Google Scholar). The peritoneal macrophages used for these studies were able to produce normal amounts of NO, which is thought to play an important role in the elimination of intracellular bacteria and parasites (67Nathan C.F. Hibbs J.M. Curr. Opin. Immunol. 1991; 3: 65-70Crossref PubMed Scopus (1330) Google Scholar), thus suggesting that a NO-independent, C/EBPβ-dependent pathway may be involved in Listeria killing and tumoricidal activity. Mainly as a result of these specific defects in macrophage functions, the C/EBPβ−/− mice are extremely susceptible to infections with microorganisms such as C. albicans and Listeria. No specific defects related to granulocytic differentiation or activation have been detected in these mice, although no direct tests have been performed. In contrast, C/EBPα and C/EBPε seem to play a crucial role in specific stages of granulocytic differentiation. C/EBPα-deficient mice (55Wang N.-D. Finegold M.J. Bradley A. Ou C.N. Abdelsayed S.V. Wilde M.D. Taylor L.R. Wilson D.R. Darlington G.J. Science. 1995; 269: 1108-1112Crossref PubMed Scopus (842) Google Scholar) were strikingly found to totally and selectively lack mature granulocytes (60Zhang D.-E. Zhang P. Wang N.-D. Hetherington C.J. Darlington G.J. Tenen D.G. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 569-574Crossref PubMed Scopus (764) Google Scholar), probably as a result of a specific maturational block of myeloid precursors toward mature neutrophils and eosinophils. This defect appears to be intrinsic to the hematopoietic precursor cells, and the loss of granulocytic maturation correlated with loss of expression of G-CSF receptor in the liver. However, the observation that mice in which the gene encoding G-CSF receptor has been inactivated still can form mature neutrophils (68Liu F. Wu H.Y. Wesselschmidt R. Kornaga T. Link D.C. Immunity. 1996; 5: 491-501Abstract Full Text Full Text PDF PubMed Scopus (405) Google Scholar) suggests that other still unidentified critical targets of C/EBPα must exist that make this factor essential for granulocytic differentiation. In agreement with these findings, it has been recently shown that conditional expression of C/EBPα in bipotential myeloid progenitors is sufficient to trigger neutrophilic differentiation (69Radomska H.S. Huettner C.S. Zhang P. Cheng T. Scadden D.T. Tenen D.G. Mol. Cell. Biol. 1998; 18: 4301-4314Crossref PubMed Scopus (412) Google Scholar). Unfortunately, analysis of the immune functions of the C/EBPα−/− mice was not possible because the mutation caused perinatal death (55Wang N.-D. Finegold M.J. Bradley A. Ou C.N. Abdelsayed S.V. Wilde M.D. Taylor L.R. Wilson D.R. Darlington G.J. Science. 1995; 269: 1108-1112Crossref PubMed Scopus (842) Google Scholar). In contrast to the complete maturational block toward granulocytic cells observed in the C/EBPα−/− mice, mice deficient in C/EBPε displayed only slightly reduced numbers of eosinophils and high numbers of granulocytes, which were, however, morphologically atypical and not fully functional, as suggested by a defective oxidative burst (63Yamanaka R. Barlow C. Lekstrom-Himes J. Castilla L.H. Liu P.P. Eckhaus M. Decker T. Wynshaw-Boris A. Xanthopoulos K.G. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 13187-13192Crossref PubMed Scopus (312) Google Scholar). In agreement with defective granulocytic function, these mice succumbed to opportunistic infections between 2 and 5 months of age, presenting various infection-related tissue lesions. In conclusion, although C/EBPα appears to act as a main differentiative switch toward the granulocytic lineage, the main function played by C/EBPε in regulating granulocytic functions appears to be specular to the function of C/EBPβ in macrophages, that is the specification of specialized functions and of terminal differentiation. As has often been the case with gene-targeting experiments, the generation and analysis of single C/EBP knock-out mice, although confirming the importance of different family members in regulating various aspects of inflammation and immunity, have on the other hand revealed new layers of complexity that will require more systematic studies of gene expression in the different C/EBP-deficient strains and in multiple mutant strains lacking more than one family member. However, it has already clearly emerged that the relative role of different C/EBP isoforms in regulating the expression of distinct classes of genes may vary according not only to the cell type but also to the quality and quantity of the signal, being determined by specific promoter architecture and by interactions with other transcription factors. Moreover, because members of the C/EBP family do play important roles in inflammation by regulating the functions of both cytokine-producing effector cells (macrophages and granulocytes) and target cells (hepatocytes), the cell specificity of the observed phenotypes is not always clear. The tissue-specific inactivation of different C/EBP genes either in the liver on in different myelomonocytic lineages would allow a more precise dissection of their specific functions in each cell type. Finally, it appears that the main function of C/EBP factors in myelomonocytic cells is that of determining differentiation and expression of specialized functions, thus suggesting that C/EBPα, -β, and -ε-deficient mice will be precious tools to study the regulation of myeloid cells differentiation at the transcriptional level.