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If? , m mm m III/Ill M21; "Wm-a...“ I \ * T Will/I’ll! l 3 1 HUN/111W(ll/INN!!!” 93 01029 ‘2294 This is to certify that the dissertation entitled CCAAT/ENHANCER BINDING PROTEIN;RELATED RANSCRIPTION FACTORS: REGULATORS 0F CYTOKINE XPRESSION AND MYELglIgsgytgggyTIc DIFFERENTIATION JAMES DANIEL BRETZ has been accepted towards fulfillment of the requirements for JJJD degreein MICRQBIDLOGY Major professor DaWJW4 MS U is an Affirmative Action/Equal Opportunity Institution 0- 12771 LIBRARY Michigan State University PLACE It RETURN BOX to mouthi- ehoekmnflom yourrocord. TO AVOID F INES return on or before date duo. DATE DUE DATE DUE DATE DUE IT—J:_:J ; —1 MSU is An Affirmative Action/Ecru! Opportunity Instituion m m1 CCAAT/ENHANCER BINDING PROTEIN-RELATED TRANSCRIPTION FACTORS: REGULATORS OF CYTOKINE EXPRESSION AND MYELOMONOCYTIC DIFFERENTIATION BY James Daniel Bretz A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Microbiology 1994 ABSTRACT CCAAT/ENHANCER BINDING PROTEIN-RELATED TRANSCRIPTION FACTORS: REGULATORS OF CYTOKINE EXPRESSION AND MYELOMONOCYTIC DIFFERENTIATION BY James Daniel Bretz The regulation of hematopoiesis is important for the understanding of immune system function and the mechanisms of leukemia. The myelomonocytic lineages, all derivatives of a single stem cell type, have important functions in antigen clearance, antigen presentation, and intercellular signaling of immune responses. Inhibition of differentiation of myelomonocytic cells can lead to uncontrolled growth, impaired immune function and leukemia. Transcription factors are integral to the regulation of cellular function. Transcription factors CRP2 and CRP3 are members of the C/EBP-related protein (CRP) family. CRPs have been implicated in the regulation of myelomonocytic differentiation. In this thesis I will demonstrate that lineage switch macrophages derived from lymphoid pre-B cells are capable of normal functions such as the capacity to release cytokines in response to LPS and antigen presentation. These lineage switch macrophages also gain the capacity to express CRP2 and CRP3. IIwill also demonstrate that ectopic expression of CRP2 and CRP3 in murine P388 B lymphoblasts confers the capacity for lipopolysaccharide (LPS)-inducible transcription of cytokine genes in vivol and acts as an inducer of certain aspects of ‘myelomonocytic differentiation. 12 will also propose the utility of this experimental system for further discrimination of the genetic mechanisms of nwelomonocytic differentiation. DEDICATION To Phyllis Jo Hallet Bretz and Janet Lynn Salzwedel for their unwavering support, which made this possible. iv ACKNOWLEDGMENTS I would like to thank my mentor, Richard C. Schwartz, whom I consider a friend as well as mentor. Peter F. Johnson and his lab at the National Cancer Institute for giving my research a much needed boost. Jerry Dodgson and his lab for their helpful discussions, and reagents and equipment provided. My fellow graduate students in Rich’s lab, Tim Weichert and Shu Chih Chen, for providing an educational, interesting and pdeasant work environment (and challenging euchre games). And especially, to all the people who gave me a second chance and allowed me to prove I was worthy of their confidence in me. Their are so many of you (I hope you know who you are, and realize what a big difference you have made in my life). PREFACE In this thesis, two chapters are based on collaborative efforts. Chapter 2 involves the derivation and characterization of lineage switch macrophages. The isolation of these cells and preliminary characterization was accomplished by Shu-Chih Chen. RT-PCR was performed by Hsun- Lang Chang. The cytokine assays were performed by Alfred Ayala. Chapter 3 involves manipulation and characterization of CRP transfected cell lines. Western analyses were performed and anti sera and oligonucleotides supplied by Peter F. Johnson, Mark Baer and Simon Williams at the National Cancer Institute. Hsien-Meng thI constructed Inna pBABE-CRPZ expression vector. vi TABLE OF CONTENTS List of Tables ............................................ 10 List of Figures............ ......................... ......11 Chapter 1: Literature Review.............. ................ 1 Hematopoiesis: an introduction. ........... ....1 Myelomonocytic differentiation ................ .3 Colony stimulating factors ..................... 6 B lymphocyte differentiation...................9 Long-term B lymphocyte cultures ....... . ....... 11 Inducible differentiation of cell lines ....... 12 Lineage switching ............................. 15 Inflammatory cytokines ....................... .16 Interleukin 6 ............................ 17 Interleukin 1 .......................... ..19 Tumor necrosis factor .................... 20 Monocyte chemoattractant protein-1 ....... 21 C/EBP-related transcription factors ........... 23 Acute-phase response .......................... 30 Chapter 2: Lineage Switch Macrophages Can Present Antigen .................................. 31 Abstract. ...... .......... ..... ................31 Introduction .................................. 31 Results ....................................... 32 Tumor 4 is derived from the R2 cell line.32 Tumor 4 cells possess macrophage characteristics ................... ..32 Tumor 4 cells also show differentiated lymphoid characteristics ....... .....34 The subclones of tumor 4 can function effectively in antigen presentation.36 Ia expression............................37 Discussion ..... ...... ........................ .37 Materials and methods. ..................... ...40 Cell 1ines.......... ....... ..............40 Nucleic acid analysis .................... 40 Reverse transcription-polymerase chain reaction (RT-PCR)00.0.0000000000000041 Cytological analysis ..................... 41 Flow cytometry........ ................. ..41 Acknowledgements........... ......... ..........42 References.. ................................. .42 vii Chapter 3: Chapter 4: viii C/EBP-related proteins confer LPS-inducible expression of IL-6 and MCP-l to a lymphoblastic cell line.. ........ . ..... . ................. ...44 Abstract..... ....... . ....................... ..44 Introduction .......... . ....................... 44 Materials and methods......... .......... ......46 Cells and cell culture .............. .....46 Transfections............ ..... ...........47 Expression vectors..... ........... .. ..... 47 Nucleic acid isolation and analysis ...... 48 Western analysis ......................... 49 Electrophoretic mobility shift assays (EMSA).......... .................. ..49 Metabolic labeling and immunoprecipitation ...................... 49 Antisera ................................. 50 Results ....................................... 50 Differential CRP2 and CRP3 expression between P388 lymphoblasts and their macrophage derivative P388Dl(IL1)...SO Ectopic expression of CRP2 and CRP3 in P388 B lymphoblasts ...................... 54 LPS-induced cytokine expression occurs in the transfectants that ectopically express CRP2 and/or CRP3 ............ 62 Inhibition of LPS-induced cytokine expression by CRP2 antisense RNA....67 Discussion .................................... 69 CRP2 and CRP3: Implications for differentiation and discussion ........................... 76 Abstract ...................................... 76 Introduction .................................. 76 Experimental procedures ....................... 77 Cell lines and cell culture .............. 77 RNA isolation and northern analysis......78 Immunoprecipitations and protein analysis ........................ ............78 Cell staining ............................ 78 Results ....................................... 79 Survey of hematopoietic cell lines for CRP expression...... ........... .........79 Immunoprecipitations were performed to show that the CRP2 protein was also expressed ....................... ....83 Morphological and cytochemical analysis of P388 8 lymphoblasts ectopically expressing CRP2 and/or CRP3 ......... 84 Discussion................... ..... .... ..... ...87 Elements of exclusivity in lineage commitment..........................91 Evidence for incomplete differentiation..91 ix Positive elements of myelomonocytic differentiation.............. ....... 93 Elements that block myelomonocytic differentiation.....................95 Combinatorial gene regulation in myelomonocytic cells by CRPs in cooperation with other transcription factors... ................... .......95 Conclusions .............................. 97 References......... ................... . ................... 99 LIST OF TABLES Chapter 2 Table 1 LPS-induced cytokine release by tumor-4 macrophage subclones...............34 Chapter 1 Figure Figure Figure Figure Figure Figure Figure Chapter 2 Figure Figure Figure Figure Figure Figure Figure Figure Chapter 3 Figure LIST OF FIGURES Hematopoiesis: All lineages derive from a totipotent stem cell ................ 2 Macrophage/monocyte differentiation......4 B lymphocyte differentiation .... ....... 10 Cis-acting elements of IL-6 and IL-IB regulationOOOOOOOOOO0.000000000000018 C/EBP-related protein family nomenclature. 0.0.0.0.... ......... 0.0.0.00000000024 Leucine zipper schematic................25 C/EBP-related protein recognition sequences (adapted from Akira et a1 1990).. ........ . .............. .....27 Viral integrations ............... .......32 Nonspecific phagocytosis of latex beads.33 RNA analyses of c-myc, c-myb, and c-fms.33 Kappa light chain rearrangements. ...... .35 Expression of mu heavy chain RNA ........ 36 RT-PCR analysis of CD45 ............... ..37 Antigen presentation .................. ..38 I-AF expression ......................... 39 Analysis of differential CRP2 and CRP3 xi Figure Figure Figure Figure Chapter 4 Figure Figure Figure xii expression between P388 and P388(IL- 1) 0.0.00.0...... 0000000000000 .051-53 Analyses of P388 cells transfected for CRP2 and CRP3 expression ........ 56-60 Northern analyses for cytokine expression in P388 cells transfected for CRP2 and CRP3 expression... ......... .64-66 Analysis of a second population of P388 cells transfected for CRP2 expression ................. .......68 Inhibition of IL-6 and IL-lfi expression by antisense CRP2 RNA in P388Dl(IL-l) cells ....................... ....70-71 Survey of hematopoietic lineages for CRP expression ...................... 80-82 Morphological analysis by Wright-Giemsa staining of CRP transfectants...85-86 Leukocyte alkaline phosphatase staining of P388-Nee and P388C3/C2..........88-89 Chapter 1 Literature Review Hematopoiesis: an introduction Hematopoiesis is the process of producing blood cells and related cellular components of the immune systemu The process consists of differentiation of a single totipotent stem cell into a variety of cellular lineages (Figure 1) (Ruby 1992, Roitt et al 1989). There are two major lineages: lymphoid and myeloid. Both derive from the totipotent stem cell. Pluripotent lymphoid stem cells give rise to the T and B lymphocyte populations. Pluripotent myeloid stem cells (CFU- GEMM) give rise to monocyte/macrophage, neutrophil, megakaryocyte, erythrocyte, basophil/mast cell, and eosinophil cell lineages, each with distinct and complex functions. These lineages. give. rise: to extremely specialized cells involved in immune system function and oxygen transport. The microenvironment, including cell-cell contact, of hematopoietic cells is important for differentiation (Mayani et al 1992, Kincade 1992). Hematopoeisis occurs in a number of specialized sites. The bone marrow is the source of hematopoeitic cells of the early lymphoid lineage as well as myeloid, and erythroid lineages in adult mammals (Metcalf 1988). Stromal cells of the bone marrow provide factors for proliferation and differentiation (Mayani et al 1992). Fetal liver is also a source of stem cells (Metcalf 1988). Resting 1 9mm 6) Plum Col '90 9 Mast Cell ' Figure 1: Hematopoieses: All lineages derive from a totipotent stem cell (from Nicola 1989). 3 B lymphoid cells transport to the adult spleen or lymph nodes. I'progenitor cells transport.to the thymus where they complete their differentiation (Metcalf 1988). A more detailed discussion of hematopoiesis relevant to this thesis will follow. Normally, early commitment to an individual lineage produces progenitor cells restricted to that lineage through terminal differentiation. Terminal differentiation occurs in the circulating blood, spleen, lymph nodes and, as is the case for macrophages, the resident peripheral tissue. Erythropoiesis continues in the spleen in adults (Metcalf 1988). Differentiation is generally considered antithetical to proliferaticwn As cells differentiate they become less proliferative. Consistent with this, inhibition of hematopoietic differentiation can lead to leukemogenic proliferation. .Alternatively, induction of hematopoiesis can inhibit leukemogenic proliferation. Myelomonocytic differentiation A bipotential stem cell (CFU-GM) gives rise to both the neutrophilic granulocyte and. monocyte/macrophage lineages (Figure 2) (Roitt et al 1989). A number of cell surface markers are specific for these lineages (CD 13, CDllb (Mac I), CDllc, CDw12, c017, CD31, and cow32) (Kuby 1992). CD34 is specific for the myeloid.progenitor stage (Roitt et al 1989). The currently defined stages of monocyte/macrophage Mycloid srcm cell Gmulocytemonocuc progcmtor cell i I ‘2 O MOHOQ'IC progenitor - o z o o /’ oo o l K # Promunocx'tc : 1 ti . J AK 1 i .-.- 2 E Vb .. \ T ! 5 .r- 2 :- Ostctx'last Macmphage Kuptt’cr cell Micmgliu cell Scnml mncmphagc Alveolar macrophage (bone i (lymphonl tissue l l liver ) (hmm i (pleural cant? ) (lung) Figure 2: Monocyte/macrophage differentiation (from Kuby 1992). _h ll. 5 differentiation from stem cell to terminal stage are: CFU- GEMM (bone marrow derived, GM-CSF responsive myeloid stem cell), CFU-GM (bone marrow derived, M-CSF responsive granulocyte/monocyte progenitor), monoblast (renewable, bone marrow derived, monocyte committed precursor) , promonocyte (no longer under goes cell division), monocyte (translocates to peripheral blood, CD14U,. macrophage (after’ migration. to peripheral tissue, CD23+) and activated macrophage (CDZ6+) (Roitt et a1 1989, Kurland 1984, Metcalf 1988). CD14, CD23 and C026 cell surface marker expression is maintained through terminal expression. The expression of proto-oncogene and transcription factor c-fos is upregulated with differentiation along this pathway (Gonda and Metcalf 1984, Lord et al 1993). Cells of this lineage can also be identified by gross morphology (comparitively large cells, large cytoplasm-to- nucleus ratio), cytochemical stains [non-specific esterase (Yam et al 1970)], and functional assays. The important functions of this lineage are not acquired until after activation which occurs in the peripheral tissues in response to signals such as endotoxin (LPS) or cytokines (Turpin and Lopez-Berestein 1993).. Such functions include antigen processing and presentation, phagocytosis of particulate matter including cells, mediation of the inflammatory response, secretion of factors controling hematopoiesis, keratinization, brain wound repair, iron storage and calcium metabolism (Turpin and Lopez-Berestein 1993, Adams 1982 and 1989). Depending on the extravascular tissue into which they 6 have infiltrated, macrophages may be called Kupfer cells (liver), Langerhans. cells (skin), microglia (brain), osteoclasts (bone) or tissue macrophages of the spleen, lymph node, etc. (Turpin and Lopez-Berestein 1993). Neutrophilic granulocyte differentiation can be characterized by function, gross morphology, cytochemical staining (chloroacetate esterase, leukocyte alkaline phosphatase [LAP] , myeloperoxidase [MPO]) (Yam et al 1970, Rambaldi et al 1990) , lactoferrin (LF) expression, and cell surface markers. The stages of this lineage are defined as the myeloblast (CD333 (Roitt.et al 1989), promyelocyte (MPO+, chloroacetate esterasefl CD33+) , myelocyte (MPO+, chloroacetate esterase“ CD33”) metamyelocyte (MPO*, LF“, chloroacetate esterase*, CD33*) and neutrophil (MPO+, LE”, chloroacetate esterasefi polymorphonuclear, CD33') (Valteiri et al 1987). Differentiation occurs entirely in the bone marrow (Metcalf 1988). The neutrophils will then translocate to the peripheral blood and eventually to the extravascular tissue. The lifespan of the neutrophil in peripheral tissue is short (~3 days) compared to macrophages which may live for years.The functions of neutrophilic granulocytes include phagocytosis and destruction of antigen (Roitt et a1 1989, Kuby 1992). Colony stimulating factors Four colony stimulating factors, first identified by their ability to stimulate colony formation of bone marrow cultures, have been shown to promote survival, proliferation, 7 differentiation, and maturation of myeloid cells. IL-3 (multi-CSF), granulocyte-macrophage colony stimulating factor (GMCSF), macrophage colony stimulating factor (MCSF), and granulocyte colony stimulating factor (GCSF) are a family of glycoproteins that act through receptors on their target cells (Kuby 1992). Expression of CSFs is usually not constituitive but requires an inductive stimulus on the synthesizing cell. IL-3 is a 20-28kd glycoprotein synthesized by T lymphocytes (Metcalf 1988). IL-3 influences the differentiation.of all myeloid progenitors and.may even act on the totipotent stem cell. More specifically, IL-3 promotes survival and proliferation of these stem, cells. Other functions include proliferation and maturation of mast cells and proliferation of erythroid cells (Hapel et a1 1985). GMCSF is a 21-23 kd glycoprotein produced in vivo by a wide ‘variety' of cell types (T lymphocytes, macrophages, fibroblasts, bone marrow stromal cells, endothelial cells) (Metcalf 1988, Golde et al 1990). Similar to IL-3, it acts on all myeloid progenitors in survival and proliferation (Metcalf 1988). It induces differentiation and proliferation. of immature granulocytes and monocytes (Golde et al 1990). It also has a number of effects on the function of mature myeloid cell types. GMCSF enhances phagocytosis, intracellular killing, cytotoxicity, and antigen processing of macrophages, (Golde et a1 1990). Neutrophils have enhanced phagocytosis, intracellular killing, chemotaxis, antibody dependent cytotoxicity (ADCC), protein synthsis, and vascular adhesion, 8 cytotoxicity (ADCC) , protein synthsis, and vascular adhesion, due to GMCSF (Golde et al 1990). In addition GMCSF primes neutrophils for degranulation, release of arachidonic acid and leukotriene B“ and oxidative metabolism. (Golde et a1 1990) . Mature eosinophils also respond with enhanced functions (Golde et al 1990). MCSF is a glycoprotein homodimer comprised of 43kd subunits that can be synthesized by fibroblasts and bone marrow stromal cells in mouse and also macrophages and endothelial cells in humans (Metcalf 1988). MCSF induces differentiation and proliferation of progenitors of the monocyte lineage (Kuby 1992). It can also induce cytokine expression (GMCSF, GCSF, megakaryocyte potentiator [an enhancer of platelet production]), ADCC, tumor killing, and osteoclast production and activation (Metcalf 1988) . The MCSF receptor gene also known as c-fms, was originally discovered as the tranforming gene in an acute oncogenic retrovirus (Hampe et a1 1984, Sherr et al 1985). C-fms has been found expressed in some tumors, and with MCSF, induces growth through a paracrine or autocrine mechanism (Roussel and Sherr 1989). GCSF is a ~25kd glycoprotein synthesized by macrophages, fibroblasts, bone marrow stromal cells and, in humans, endothelial cells (Fukanaga et al 1990, Metcalf 1988). GCSF promotes differentiation and proliferation of neutrophil progenitors. For mature neutrophils, it enhances phagocytosis and ADCC (Kuby 1992). B lymphocyte differentiation Stages of B lymphocyte differentiation are generally defined by the state of immunoglobulin gene expression (see Figure 3). Cell surface markers are also used experimentally to determine the stage of development. Both T and B lymphocytes are derived from the same lymphoid stem cell characterized by its expression of the enzyme terminal deoxynucleotidyl transferase (TdT) , but lacking immunoglobulin (Ig) or T cell receptor gene rearrangements (Gregoire et a1 1979). TdT inserts nucleotides in the Ig heavy chain gene providing increased variability (Desiderio et a1 1984). The progenitor of the B lineage expresses the cell surface marker 8220 (CD45) which is diagnostic for the entire lineage (Kuby 1992). Commitment to the B lineage is first observable with )1. heavy chain Ig gene rearrangement (Roitt et a1 1989). Cytoplasmic expression of the u heavy chain Ig gene is diagnostic of a pre-B lymphocyte. CD20 is a cell surface marker, possibly part of a calcium channel (Tedder et al 1990), whose expression starts at this point and continues through terminal differentiation (Clarke and Lane 1991). This is followed by light chain gene (either kappa or lambda) rearrangement and expression (Tsubata and Nishikawa 1991). This allows for the heavy chain and light chain to combine and be expressed as IgM on the cytoplasmic membrane which is diagnostic of an immature or resting B lymphocyte. The immature B lymphocyte will then relocate to the spleen or lymph nodes (Ikuta et a1 1992). A mature B lymphocyte is 10 . + + 8.5. .5553 .5 2:5 E5. 3:5 85.. .26. 35. 1:.“ .5553. 589:2: 8.5. 0:89:98 .29; .22.» €88 33> 3. .5 a .« .5 2 x 5 2 1 + .5 9:0... » .5 w w .5 955.71 856+1 .d.w+1 8:3 «Ema... m..o.u m 12:25.. =3 8 as 3. .03 3.83m :3 m 1.589.). 38.: 5.4.55975354 x. mm. .5 .3: .01. .21. m0 1:.“ 1O .5 5.5.. ...cc 1U .c 8.5.. 0:85:98 .53; 5828.3: ”2.4; d .5 k & .5 2 “=52 w + 1 1 0.6.3583. 1 £3 a 83:2 3.5 m 9.38:8. 3.3 a 8.. .. 02:... Euu:u.n.u.5.-:uw=:< 0:52 0:02 ”1:22.?” 2.36.... “15.93.25... <2: 2.83 2m: .2129 >25: Hawaii» a. £3 a 51:05:...— 52.8: 53.5... ammo B lymphocyte differentiation (from Kuby 1992). Figure 3 11 generated after IgD is coexpressed with IgM on the cell surface. After initial antigen stimulation, the cell becomes an activated B lymphocyte secreting low levels of Ig after isotype switching but maintaining surface Ig. The expression of C039, possibly involved in B lymphocyte-dependent T lymphocyte activation (Harriman et al 1993), is specific for mature B lymphocytes (Clark and Lane 1991). Isotype switching may continue after further Ag stimulation and a changeover to exclusively the secreted form of Ig. This is the terminal plasma cell stage of differentiation which is marked by the expression of a number of specific surface markers including CD27, C038, and plasma cell associated-1 (PCA-l) (Clark and Lane 1991, Kuby 1992). The B lymphocytes used in this thesis work were characterized by southern analysis of Ig gene rearrangements and immunocytostaining for 8220 expression. Long-term B lymphocyte cultures In the studies reported in this thesis, the v-Ha-ras transformed pre-B lymphocytes that underwent B lymphocyte to macrophage conversion were derived from the “fitte-Whitlock long-term culture system (see chapter 2) (Bretz et al 1992). This system allows for continuous culture of primary cells of the B lineage for extended periods (>1 year) (Whitlock and Witte 1982 and 1987). Committed B lymphocyte precursors can be followed through differentiation to mature B lymphocytes. The culture is derived from bone marrow cells obtained 12 from the femurs of three to four week old mice. These cells are grown in culture medium supplemented with 5% fetal calf serum (lot tested; serum lot varies and is critical for successful cultures), and 10'5M 2-mercaptoethanol. A high density layer of adherent cells (feeder layer) derived from the bone marrow provides factors required to maintain and grow the non- adherent B lymphocytes. The bone marrow also provides B lymphocyte progenitors. Typically the predominant cell type early after establishment in culture (3-8 weeks) is pre-B. This is observed by the appearance of foci of small round cells growing on the feeder layer. After 3-6 months, mature B lymphocytes become predominant. Cells can. be maintained for extended periods after transfer to low density feeder layers (Whitlock and Witte 1987). High density feeder layers have a tendency to overgrow and after some time they will die. The cells maintained in this culture system can be grown in numbers suitable for biochemical analysis and can be cloned, infected with retroviral vectors (Whitlock et a1 1983), analyzed.by FACS, or'used.in reconstitution experiments of immune-deficient mice. Inducible differentiation of cell lines There are problems with using primary cell cultures for the study of hematopoiesis. The inability to maintain a homogenous population of cells for extended periods of time in culture can be overcome by the use of spontaneous or induced 13 (chemically or by viral infection) leukemic cell lines. But these cell lines are usually locked into a stage of differentiation. The isolation of cell lines capable of differentiating in response to chemical or biological agents has overcome this problem. These cells can be induced and studied along their differentiative path to determine the mechanisms involved. Three myeloid cell lines have relevance to my research. M1 is a murine monoblastic leukemia. Various subclones have been isolated that can differentiate in response to one or more of the following factors: IL-6, leukemia inhibitory factor (LIF), and IL-1 (Lotem and Sachs 1992). Induction may be indirect by some of these factors; one factor inducing expression of another factor(s). LPS causes an abortive differentiative effect where some immediate response genes are expressed but the cells fail to reach the terminal stage of differentiation. (Lord et al 1990). Cytokine induction leads to terminal differentiation into a functional macrophage. The end stage is non-tumorigenic. This system was used to determine the immediate response genes for’ myeloid differentiation (My0”) (Lord et al 1990a and 1990b). MyD genes include the transcription factors c-jun, jun B, jun 0, but curiously not c-fos Which is upregulated during normal and leukemic differentiation (Lord et al 1993, Gonda and Metcalf 1984) . Expression of transcription factors c-myb (Selvkumaran et al 1992) and c-myc (Hoffman-Leibermann et al 1991) block induced differentiation. Expression of these genes is 14 normally suppressed in the course of differentiation. 320C13 is an IL-3 dependent. myeloblastic cell line derived from a murine long term bone marrow culture (Greenberger et al 1993). If IL-3 is removed and GCSF is added to their growth medium, 320C13 cells differentiate into mature neutrophilic granulocytes (Valtieri et al 1987). This method has led to the discovery that expression of evi-l (a zinc finger transcription factor gene) (Moroshita et al 1992) or id (an inhibitor of certain bHLH transcription factors) (Benezra et al 1990, Kreider et a1 1992) can inhibit GCSF induced differentiation. .As with M1, 2: number of transcription factors are immediate early response genes to GCSF induction (c-fos, c-jun, jun B, egr-l), but c-myc expression remains unaltered (Krieder and Rovera 1992). In Chapter'4 I‘will show that transcription factors CRP2 and CRP3 may be involved in the differentiation of myeloid cells. The human cell line HL-60 was isolated from a patient with acute promyelocytic leukemia (Rovera et al 1979). This line has been very useful due to its ability to differentiate along either the neutrophilic granulocyte or the monocyte/macrophage pathway. Dimethyl sulfoxide and retinoic acid are the most commonly used compounds for induction of granulocyte differentiation (Breitman et al 1980, Collins et al 1978) . 1,25 dihydroxyvitamin 03 and phorbol-lZ-myristate- 13-acetate are used for induction of macrophage differentiation (Murao et al 1983). The induced cells become growth arrested and functional at the terminal stage. 15 Expression of the zinc finger transcription factor egr-1 is essential for and restricts differentiation to the macrophage lineage in this system (Nguyen et a1 1993). Lineage switching According to historical hematopoietic dogma, commitment to one lineage starts an irreversible path of differentiation along that lineage. This dogma has recently been challenged by the discovery of lineage switched cells. A lineage switch is defined as the changing of a cell line (or leukemia) of one lineage to another lineage. The first examples were discovered by clinical observation in the course of human leukemia. In the early 19805 it was shown that some patients with a leukemia of one lineage (usually lymphoid) would relapse after remission with a leukemia of a different lineage (usually myeloid) (Stass et al 1984, Stass et al 1986). It was not determined if these cases were a true lineage switch (perhaps caused by the chemical agents of therapy) or merely one leukemia being eradicated by chemotherapy allowing expansion of a secondary leukemia of a different phenotype. In the latter case both leukemias would have been derived from the same pluripotent stem cell. This has yet to be completely resolved. Occurences of a lineage switch in culture have also been observed. More recently, lineage switch has been observed 'through the expression of certain oncogenes. Davidson et al. (1988) used LPS to stimulate v-Ha-ras-transformed lymphoid 16 cell lines to differentiate into macrophage-like cells after long term culture selection. Klinken et a1. (1988) induced B lymphoid cells derived from a transgenic mouse expressing c- myc from the immunoglobulin mu enhancer to convert to macrophages by expressing the v-raf oncogene. Borzillo et al (1990) have expressed human c-fms (MCSF receptor) in murine pre-B lymphocytes in culture and induced lineage switch by stimulating with human MCSF. The above lineage switch systems evaluated the macrophage nature of the resultant cells by morphology, adherence, surface markers (including CDllb [Mac I] expression), phagocytosis, esterase activity, and lysozyme activity. All of these studies were lacking in assays to determine complete macrophage functionality of the macrophage- like cells derived from pre-B lymphocytes. In chapter two, we address this question. Our studies show that ras-transformed pre-B lymphocytes can undergo lineage switch on passage in animals and differentiate into functional macrophages with Ag presentation and cytokine production capabilities (Bretz et al 1992). This validates the lineage switch model as a system in which to study the regulation of myeloid differentiation (Chapters 3 and 4). Inflammatory cytokines Inflammation is the body’s response to injury or infection. It allows the body to direct the immune system to the site of injury. Several cytokines are involved in signaling the inflammatory response. Inflammatory cytokines 17 such as IL-1, IL-6, TNF, mop-1, IFNy, MIP-l, NIP-2, IL-8, and others are released by cells at the site of injury. Inflammatory response activities such as chemotaxis of immune cells to the site of tissue damage, adhesion of these cells to vascular endothelial cells and permeabilization of vascular tissue to allow the migration of immune system cells to the site of injury, as well as activation of antigen clearance mechanisms (e.g. phagocytosis, degranulation) are all signaled by these cytokines (Kuby 1992, Roitt et al 1989). Inflammatory cytokines are also involved in stimulating growth and differentiation of immune cells. The inflammatory cytokines interleukin (5 (IL-6), interleukin 1 (IL-1), tumor necrosis factor (TNF), and monocyte chemoattractant protein (MCP-l) are germaine to my research. These cytokines have a wide variety of activities in inflammation as well as parallel activities that enhance the immune response such as the fever and acute-phase responses. In the following summary, I will limit descriptions to these cytokines’ major activities in the inflammatory response. Interleukin 6 IL-6 is a 20-30kd glycoprotein that has a wide variety of activities that play an important role in the immune response. These activities include terminal differentiation and proliferation of B lymphocytes, stem cell survival, elicitation of acute-phase response, differentiation and 18 c-fo RCE (:qu Gill-3 AP-l 038 NW“ homsology NF-kB 'a 9%: =4.“— c-fos SRE homology ' TATA i NF-kB NF-iL6 l 09”! I +t-H—Ho—i— +15 IAP-t | SRE TATA -1 -H% q-—F Figure 4: Cis-acting elements of IL-6 (upper) and IL-13 (lower) regulation (from Akira and Kishimoto 1992) and (Zhang and Rom). 19 of T lymphocytes and macrophages (Akira and Kishimoto 1992). When stimulated appropriately, monocyte/macrophages, fibroblasts, keratinocytes, endothelial cells, mesangial cells, glial cells, chondrocytes, and T and B lymphocytes can produce IL-6. Depending on the producing cell, IL-6 can be induced by LPS, IL-l, TNF, IFNB and PDGF. Elevated levels of IL-6 have been found in several pathological conditions including certain lymphoid malignancies, rheumatoid arthritis,and HIV infection (Akira and Kishimoto 1992). Cis-acting elements of IL-6 gene transcription regulation are summarized in Figure 4. The NF-IL6 element confers LPS and IL-1 inducibility in a human glioblastoma cell line as detected by a chloramphenicol acetyl transferase (CAT) assay (Isshiki et al 1990). In chapter three, I will show that CRP2 (NF-1L6) and CRP3 can confer LPS inducibility of IL-6 when ectopically expressed in a lymphoblastic cell line. Interleukin 1 Interleukin 1 activity describes the effects of two structurally related 17k0 proteins that activate the same receptor (Scales 1992). Macrophages are the major in vivo source of both IL-la and IL-13 although many other cell types also Texpress 'them. including' lymphocytes and. neutrophils. Macrophages can be induced to produce IL-l by endotoxin (LPS) , as well as by cytokines IL-2, GMCSF, TGFB, TNFa, and all three interferons. Activities of IL-1 include T lymphocyte proliferation, B lymphocyte activation in response to antigen, 20 induction of acute phase proteins by the liver, induction of expression of adhesion molecules by vascular endothelial cells, and oxidative metabolism by neutrophils. Cis-acting elements involved in IL-lfi gene regulation and the factors that bind them are summarized in Figure 4 (Zhang and Rom 1993). The NF-IL6 enhancer elements of the IL-1 B promoter confer LPS inducibility in CAT assays using THP-1 cells (a human myelomonocytic leukemia line). In Chapter 3, I will show that anti-sense CRP2 and CRP3 gene transcripts can block LPS inducibility of IL-16 when expressed ectopically in a lineage switched macrophage cell line. Tumor necrosis factor Similar to IL-1 in that two distantly related 17k0 proteins bind to the same receptor, TNF(a and 8) also has an overlap of activities with IL-1 (Tsuji and Torti 1992). TNFa is produced by macrophages in response to LPS, gamma interferon, or IL-6. TNFB is produced by T lymphocytes. TNF induces lymphocyte proliferation, activation of neutrophils, induction of expression of adhesion molecules by vascular endothelial cells, tumor killing by macrophages and acute phase protein expression in liver, probably through IL-6 up regulation (Tsuji and Torti 1992, Dawson 1991). It is a mediator of septic shock (Buetler et al 1988). TNF inhibits adipocyte specific gene expression and may play a role in cachexia (wasting, wieght loss and muscle weakness) in immune disorders such as AIDS (Dawson 1991). 21 Regulation of TNFa gene transcription has not been well characterized. The TNFa core promoter can confer TPA inducibility when joined to a number of unrelated enhancer sequences (Leitman et a1 1992) . A TNF-responsive element contains AP-l and ATF/CREB binding sequences but these factors are not involved in its regulation (Leitman et a1 1991). Four LPS-responsive sites bind NFKB in primary macrophages (Drouet et a1 1991). Monocyte chemoattractant protein-1 Monocyte chemoattractant protein-1 (MCP-l) was first discovered in murine fibroblasts as a platelet-derived growth factor (POGF)-induced immediate early response gene and designated JE (Cochran et al 1983). It was later found by homology to be related to a number of cytokines and identical to the human monocyte chemoattractant protein-1 (MCP-l) (Rollins et al 1989). This 16-18k0 glycoprotein is specific for monocyte/macrophage chemotaxis with no activity on neutrophils or lymphocytes (Rollins et al 1989, Leonard and Yoshimura 1990). A wide variety of cells can be induced to express MCP-l. The literature is often contradictory as to that cell types respond to what stimuli. Peripheral blood :monocytes (PBMC) may (Yoshimura et a1 1989) or may not (Brach et al 1992) upregulate MCP-l in response to LPS. This may be «due to the need for given factors to act in synergy with other factors that may or may not be present from one assay system ‘to the next or, in the case of PBMCs, possible contaminating 22 MCP-l expressing cells. LPS, IL-1, IL-4, TNF, PDGF, and GMCSF have all been shown to induce MCP-l in at least one cell type [peritoneal macrophages (Introna et al 1987), pulmonary fibroblasts (Rolfe et al 1992), kidney tissue (Xia et a1), endothelial cells (Shyy et a1 1993) , alveolar epithelial cells (Paine et al 1993), or chondrocytes (Villiger et al 1992)]. In addition to chemotaxis, MCP-l also is involved in activation of macrophages (Rollins et a1 1991). Cis-acting elements involved in MCP-l gene regulation have not been mapped in detail nor have any specific transcription factors been implicated. A TPA-inducible element has been mapped to a region 88 to 141 base pairs upstream of the transcription start site without specific knowledge of the transcription factors involved (Timmers et al 1990). But a possible AP-l binding site (normally conferring TPA inducibility) in the -70 to -38 region does not confer TPA inducibility to a reporter gene construct (Timmers et a1 1990). A novel 7 base pair element 3’ of the MCP-l stop codon apparently is necessary for serum inducibility in conjunction with a 5' element that is also required (Freter et al 1992). No LPS inducible elements have as yet been identified. In chapter 3 I will show that LPS inducibility can be confered by expression of CRP2 or CRP3. I will also identify a putative promoter element. This is a dizzying array of cytokines and functions that make up the signaling required. to :maintain homeostasis. Hopefully the relevant facts have been communicated in a 23 Hopefully the relevant facts have been communicated in a meaningful way for proper understanding of the thesis to follow. C(EBE-related transcription factors C/EBP related proteins (CRPs) are a family of enhancer binding transcription factors (see Figure 5) (Williams et a1 1991, Cao et al 1991). They have been grouped together based on amino acid homology and their ability to dimerize. CRPs have extensive homology between their carboxy terminal regions (~100 a.a.). This region is the basic region-leucine zipper (bZIP) element which constitutes the DNA binding and dimerization domains. The leucine zipper domain is an alpha helix that has a leucine residue every seven amino acids (Landschulz et al 1988a). Computer modeling has predicted that this sequence places the hydrophobic leucines on the same side of the alpha helix in a linear fashion. X-ray crystallography has shown the alpha helices of leucine zippers are leii coiled-coil configuration (Ferre-D’ Amere et a1 1993, Ellenberger et al 1992) . This allows the hydrophobic leucines of one alpha helix to interact with those of a dimerization partner to form a dimer (see Figure 6). The dimerization allows juxtaposition of the basic regions which contact the DNA” The CRPs can both homodimerize as well as heterodimerize within the family (Williams et al 1991, Cao et al 1991). Heterodimerization outside of the family generally does not occur but an 24 NAflE SYNONYMS DERIVATIVE TISSUE PROTEINS EXPRESSION C/EBP C/EBPa, C/EBP-42 C/EBP-30 LIVER, MYELOID CELLS, ADIPOSE TISSUE CRPl ---- ---- UNKNOWN CRP2 NF-IL6, C/EBPfi, LAP, LIP, LIVER, MYELOID CELLS, IL-6DBP, AGP/EBP, LOP ADIPOSE TISSUE CRP3 C/EBPd, NF-IL63 ---- LIVER, MYELOID CELLS, ADIPOSE TISSUE IgEBP-l C/EBPY ---- UBIQUITOUS CHOP GADD153 ---- UBIQUITOUS Figure 5: C/EBP-related protein family nomenclature ital! C/EBP Figure 6: Leucine zipper schematic. An axial view (upper figure) of the a-helix of a leucine zipper (from Vinson et a1 1989) . The leucines are all on the same side. Leucine zipper (lower figure) allows for dimerization and DNA binding (from _ Akira et al 1993). 26 exception has recently been reported with IgEBP dimerizing with ATF4 (Vinson et al 1993). Heterodimerization with multiple partners may allow for exquisite fine tuning of DNA binding and transcriptional control. Interactions with other enhancer binding factors such as Spl, NFKB and the glucocorticoid receptor, which occur through non-bZIP dimerization, have also been investigated (P. Johnson personal communication, LeClair et al 1992, Stein et al 1993, Nishio et al 1993). The basic region.is coterminal to the leucine zipper and, as the name suggests, consists predominantly of basic amino acid residues. The basic amino acids interact with DNA bases and phosphate oxygens along the major groove of double stranded DNA (Vinson et al 1989). This allows the protein to bind in a sequence specific manner. Models of this structure have been described as a "scissors grip" or "forceps grip" (Vinson et al 1989, Ellenberger et al 1992). The CRPs bind DNA in a sequence specific manner. The consensus sequence T T/G N N G N A A T/G has been determined by DNA footprinting, electrophoretic 'mobility shift, and interference assays on a number of gene promoter sequences (Akira et a1 1990). This sequence is found in the promoter of a number of genes (Figure 7). These include genes that fall into five general categories: cytokines, liver specific genes (including many acute-phase proteins), immunoglobulin genes, adipocyte specific genes and viral enhancers (Akira et al .1992, Shirakawa et al 1993, Brooks et al 1992, Christy et a1 27 Cytokine genes AGATTGTGCAATGT IL-6 (human) GGATTTGGAAAGTT TNF—a (human) AACTTTCGCAAACA G-CSF (mouse) ATCAGTTGCAAATC IL-8 ACGTTGCACAACCT IL-lB Liver-specific genes TGATTTTGTAATGG albumin distal element 1 (0E1) AGATTGAGCAATCT albumin -3.5kb HS site GTCTTAAGCAAAGC cy-antitrypsin site C GTATTAGGACATGT transthyretin site 2 ATGTTGAGTAAGAT transthyretin site 3 ACCTTTTGCAATCC apolipoprotein B element IV ACAAGTTGCAACAT carbamyl phosphate synthetase 1 (acute-phase proteins) GTGTGAAGCAAGAG haptoglobin site A GAATTACGAAATGG haptoglobin site C AAGTTGTGCAATGG cn-acid glycoprotein TAGTGGCGCAAACT C-reactive protein CAGTGATGTAATCA hemopexin site A Immunoglobulin genes ATCTTAAGCAACTG ng enhancer GAATTGAGCAATAA IgH enhancer GCATTTTGTAATAA VH V1 promoter TCATGAGGCAAGGC VH 17.2.25 promoter Adipocyte-specific genes AAGTTGAGAAATTT 422(aP2) GGCTGAGGAAATAC stearolyl-CoA desaturase Virus enhancers GGGTGTGGAAAGTC simian virus 40 enhancer TGGTTTTGCAAGAG polyoma enhancer ATCTGTGGTAAGCA murine sarcoma virus enhancer Consensus TTNNGNAAT (G) (G) Figure 7: C/EBP-related protein recognition sequences (adapted from Akira et al 1990) 28 1989, Kardassis et a1 1992, Johnson et al 1987). The cytokine genes for IL-6, TNFa, GCSF, and IL-18 are of importance in our current research» I will propose the addition of the MCP-l to this list (see Chapter 3). The amino terminal section of CRPs contains the transcription activation domain. Mutations in this region inhibit the ability to trans-activate expression of target genes in cotransfection assays (Freidman et a1 1990, Pei and Shih 1991) without disrupting dimerization or DNA binding. Three CRP family members (CHOP, LIP and LOP) do not have an activation.domain (Ron.and.Habener 1992, Descombes et al 1991, P. Johnson personal communication). Due to their ability to heterodimerize and bind DNA with other family members they can act as repressors of transcription. 'This adds to the capacity for fine tuning of transcriptional control. IgEBP-1 and CHOP are ubiquitously expressed proteins (Roman et al 1990, Ron and Habener 1992). However, all other CRPs exhibit some tissue specificity (see Figure 7). This implies a possible involvement in control of tissue specific expression and differentiation. CRP2 and CRP3 are highly expressed in mature macrophages and neutrophils but have not been detected in abundance in other hematopoietic lineages (Natsuka et al 1992, Scott et al 1992, this thesis). They have also been shown to be upregulated in induced differentiation of M1 (Natsuka et al 1992) and 320c13 (Scott et a1 1992) myeloid cell lines. For M1, upregulation is observed as soon as three hours post 29 induction. CRP2 and, CRP3 are initially upregulated in. hormone induced differentiation of 3T3-L1 mouse fibroblasts to adipocytes (Cao et al 1991). CRP2 and CRP3 expression then declines as C/EBP expression increases. Anti-sense C/EBP expression can block adipocyte specific gene expression in the above system (Lin and Lane 1992). The CRP2 and CRP3 genes are LPS inducible (Akira et al 1990, Kinoshita et al 1992). This information along with the LPS-inducibility of many cytokines in macrophages and the presence of CRP binding sites in the promoter regions of these cytokine genes, suggests that CRP2 and CRP3 may be essential for inducibility of cytokines by LPS (see Chapter 3). The expression of CRPs in macrophages, coupled with induction of their expression at early times in M1 and 320c13 differentiation suggests that they play a role in myeloid differentiation (see Chapter 4). It has already been shown that CRPs play a role in another differentiative process, adipogenesis (Cao et a1 1991). C/EBP and CRP2 have also been shown to play a central role in the control of expression of liver genes both during normal liver metabolism as well as in response to a diseased state by expression of acute-phase proteins (Akira and Kishimoto 1992, Poli et a1 1989). 3O Acuge-phase response The acute-phase response accompanies the inflammatory response by altering the level of several plasma proteins known as acute-phase proteins (APPs) (Akira and Kishimoto 1992). The APPs are thought to act to protect against general tissue destruction by inflammation (Kuby 1992). Several cytokines as well as LPS can induce a full acute-phase response when injected into animals. But in cultured hepatocytes, IL-l, TNF, IFNy, and TGFB induce only a subset of APPs. IL-6, which can be induced by both IL-1 and TNF, has been shown to be a principal mediatior of APP induction along with oncostatin M, and leukemia inhibitory factor (LIF) (Akira and Kishimoto 1992). Several APP genes possess CRP binding sites in their promoters (Table 2). The sites from the C-reactive protein, hemopexin, and haptoglobin genes have been shown to confer IL- 6 inducibility when co-transfected with either CRP2 or CRP3 expression vectors (Ramji et a1 1993). CRP2 has also been shown to transactivate the al-acid glycoprotein gene and can synergize with the glucocorticoid receptor (Nishio et al 1993). Jun B expression can down-regulate this gene (Baumann et al 1991). Developmental Immunology. 1992. Vol. 2, pp. 249—261 Reprints available directly from the publisher Photocopying permitted by license only © 1992 Harwood Academic Publishers CmbH Printed in the United Kingdom Lineage Switch Macrophages Can Present Antigen JAMES D. BRETZ, SHU-CHIH CHEN, DIANE REDENIUS, HSUN-LANG CHANG, WALTER]. ESSELMAN, and RICHARD C. SCHWARTZ‘ Department of Microbiology, Michigan State University, East Lansing, Michigan 48824-1101 Recent reports of "lineage switching" from a lymphoid to macrophage phenotype have left unresolved the question of whether such cells are functional macrophages or nonfunctional products of differentiation gone awry. This study demonstrates that several "macrophage-like" cell lines derived from v-Ha-ras-transformed pre-B cells have gained the capacity to effectively present antigen in MHC-restricted fashion. Using an assay involving the cocultivation of putative antigen-presenting cells with chicken ovalbumin (COVA) and a cOVA-specific T-cell hybridoma, ”lineage switch” cell lines were found to present antigen as effectively as macrophage-containing peritoneal exudates. Neither the original pre—B-cell precursors nor B-cell lymphomas derived from them present antigen. Thus, we have demonstrated that these "lineage switch" macrophages are capable of antigen presentation, a mature differentiated function. While gaining macrophage characteristics, these cells have also rearranged their kappa light-chain immunoglobulin locus, suggesting that macrophage differentiation and immunoglobulin rearrangement are not mutually exclusive processes. The existence of both lymphoid and myeloid characteristics in a cell fully capable of antigen presentation suggests greater plasticity in hematopoietic lineage commitment than conventionally thought to be the case. KEYWORDS: Lineage switch, macrophage, antigen presentation. INTRODUCTION The concept that hematopoietic differentiation involves an early and irreversible lineage com- mitment is brought into question by numerous observations of leukemias and lymphomas that express myeloid or lymphoid markers outside their respective lineages. The coexpression of dif- ferentiation markers has been interpreted as being either an aberrant phenomenon caused by leukemogenesis (McCulloch, 1983) or a reflection of the normal but transient existence of bipoten- tial progenitors in hematopoiesis (Greaves et al., 1986). In particular, the existence of a number of transformed cell lines with both lymphoid and macrophage characteristics has suggested a close relationship between these lineages. Murine mac- rophage cell lines have been derived from lymphoid tumors and from in vitro trans- formants induced either by murine leukemia viruses or chemical carcinogens (Boyd and 'Corresponding author. Schrader, 1982; Holmes et al., 1986; Hanecak et al., 1989). Three groups have studied systems in which a transition from a lymphoid to a mac- rophage phenotype could be induced. Klinken et al. (1988) demonstrated that B lymphoid cells from transgenic mice that express c-myc using the immunoglobulin mu enhancer could be induced to take on macrophage-like characteristics when infected with a retrovirus expressing v-raf. Davidson et al. (1988) showed that a v-Ha—ras- transformed lymphoid cell line could be stimu- lated by lipopolysaccharides (LPS) to differen- tiate along either the lymphoid pathway into pre—B-like cells or along the myeloid pathway into macrophage-like cells. Recently, Borzillo et al. (1990) reported the CSF-l-dependent mac- rophage lineage transition of a pre-B-cell line expressing the human CSF-l receptor. The macrophage-like cell lines that have been derived from B lymphoid cells have been classi- fied as macrophage on the basis of their mor- phology, expression of MAC-l, MAC-2, a-naph- thyl acetate esterase and lysozyme, and their 249 31 150 ability to phagocytose latex beads. More func- tional assays for antigen presentation and tumor- icidal activity that would establish whether these cells could act in vivo similarly to authentic mac- rophages have not been presented. In this paper, we demonstrate the ability of several macro- phage cell lines derived from v-Ha-ms-transfor- med pre-B cells to present antigen to a T-helper cell hybridoma. RESULTS A tumor consisting of adherent cells with a mac— rophage morphology was identified during our studies on the tumor progression of a pre- B lymphoid cell line expressing v- --H -rus (Chen et al., 1991). This tumor, designated tumor 4, was derived from a clonal cell line, designated R2, that was generated by infection of fresh murine bone marrow with a mixture of a v-Ha-ras- expressing retrovirus and Moloney murine leu- kemia virus (MoMuLV) (Schwartz et al., 1986b). The R2 cell line was classified as being a pre-B cell on the basis of several criteria. It possessed a blast—cell morphology with a large nucleus and scant cytoplasm. It expressed the B lineage- specific marker, 8220 (Coffman and Weissman, 1981). Though not expressing a detectable Immu- noglobulin mu chain, R2 showed a rearrange- ment In the DNA of that locus. The Immunoglob- ulin kappa-chain locus was in a germline configuration. Tumor 4 is Derived from the R2 Cell Line In order to ascertain whether we had identified a probable instance of lineage switching, It was necessary to demonstrate that tumor 4 was derived from R2. To that end, the sites of Inte- gration of the v-Ha-ms-expressing retrovirus and MoMuLV were compared between the tumor and the cell line. Southern hybridization analysis of EcoRI-digested DNA with a v-Ha-ms probe showed that tumor 4 contained the same 5.3-kb proviral integration fragment as R2 (Fig. 1A). This proviral integration fragment is defined by a 3‘ EcoRI site internal to the viral genome and a 5' EcoRI site peculiar to the site of integration. In addition to the 5.3-kb fragment, there is a 23-kb fragment representing the endogenous c-Ha-rus In all the DNAs. Southern hybridization analysis 32 JD. BREI'Z el .ll. of BglII-digested DNA, using a probe for the eco- tropic MuLV env gene, revealed similar MoMuLV integration fragments In R2 and tumor 4 (Fig. lB). The MoMuLV genome possesses a BglII site within env. such that the foregoing hybridization would detect a fragment extending from that BgIII site to a BglII site in the host-cell genome flanking the 3' terminus of the provirus. These data demonstrate that the putative macrophage tumor was derived from the pre-B-cell line. Tumor 4 Cells Possess Macrophage Characteristics Tumor 4 was initially suspected to be a macro- phage because of the large size of its cells and its adherent growth in cell culture. Microscopic examination of Wright-CIemsa-stained cells con- firmed their large size and revealed the cells of tumor 4 (Fig. 28) to have a much more extensive and granular cytoplasm than R2 (Fig. 2A). An Immunoperoxidase detection procedure found tumor-4 cells to have retained some expression of 3220, and to have gained expression of high levels of MAC-1 (data not shown), MAC-l is gen- erally considered to be a marker for cells of the myeloid lineage (Springer et al., 1979). Histo- B: ENV A: RAS L H: Ti ~—-—-—-— w 9.4» 3‘ 6.6» 2'. 4.0 FIGURE 1. Viral Intergrah‘ons. Southern blot analysis of DNAs from liver (L) R2, and tumor 4 (T4). (A) DNA was digested with EcoRI and l0 ug of each 0sawmpe was electrophoresed through 0.8%.] a.mse The srop obed or v- Ha- ms. (8) DNA was digested with Bgllll0 The blot was rmurine ectotropic (nz' seque esSIze markers are the posItIons of an ethidium bromIde—stanmee‘sd Hindlll digest of bacterIophage 4‘. and are denoted in klIOJ uses. 33 A\TI(IL\ PRESENTATION AFTER LIINEACIF. SWITCH ZS] chemical procedures revealed a high level of a- naphthvl acetate esterase actintv In tumor—4 cells. which is not found In R2 cells (data not shown). This Is an enzyme activity generally associated with cells of the monocyte-macro- phage lIneage (Rogers et al.. 1980) Tumor- 4 cells (Fig. 28) were posItI\e for the nonspecific phago— cytosis of latex beads, whereas R2 cells (Fig. 2A) were not. Nonspecific phagocytesis Is another marker of the monocyte-macrophage lineage (Raschke et tal. 1978). These data strongly sug- gest a macrophage phenotype for tumor- 4 cells. At late stages of myeloid differentiation the levels of c—mtu .Ind C-Hll/fI mRNA decrease, whereas the level of c-IIIIs mRNA Increases (Gonda and Metcalf, 1°84; SlIeng-Ong et al., 1987). The levels ()I mRNA from these proto- oncogenes detected In tumor-l cells were consist- . ' ‘5‘? FIGURE- 3 Nunspmific phaencx tnsis (It l.)IL'\ heads. The cells were Wrightt .lt ms.) sl II I -II Ind photochliplu‘t M 2X00 magnihcalmu I \I R“ .. and III) “In“ r 4 FIGURE 3 RNA analises of Northern blot annlvses were performed on pIIlvA' RNA from ent with tumor 4 having advanced to a late stage of myeloid differentiation C\toplasm1c poly A’ RNAs of the parental R2 cell line, tumor 4, and six other tumors derived from R2 that had lymphoid characteristics were examined by C-IVC IIT'ITITS‘I’ITSTIT'I “m In.‘ Iu'nnnrirs'nn "flh..0u c- IIIIII‘, c- mill) and c—fms. and sewn tumors (T14.- VA traction selected from lat) III; III total cvtoplasmic RNA. ) FILII sample Hf RNA was the poly Snnde blot (upper pan cl) was pmluti ‘U\(L‘\.\|\CIV tor both c- m vc dB-micruglobulm. and the (Ithtr blot (lower panel) was probed successIIelI Iurc- ‘Hl/I‘t -'I-I~ .Ind rt.. 34 252 JD. BRETZ ct al. Northern hybridization analysis (Fig. 3). One blot was hybridized successively with c-myc and [32- microglobulin probes. Another blot was hybridized successively with c-myb, c—fms, and rat glyceraldehyde-3-phosphate dehydrogenase (GAPDH) probes. Hybridization to the [lg-micro- globulin and CAPDH probes provided a control for gel loading. Tumor-4 cells clearly show reduced levels of c-myc and c-myb expression in comparison to R2 cells and lymphoid tumors. In contrast, c—fms expression is elevated in tumor-4 cells. A cell line with macrophage characteristics has also been isolated from tumor-S cells, which show elevated c-fms expression (Fig. 3). Another aspect of macrophage function is the ability to release cytokines in response to LPS stimulation. We examined the presence of lL-l, IL-6, and TNF in the media of cells cultured in the presence or absence of 10 ,ug/ ml of LPS for 24 hr. Cellular proliferation assays for lL-l and IL-6, and a cytotoxicity assay for TNF revealed varying levels of cytokine release for six sub- clones of tumor 4, whereas the parental R2 pre-B- cell line did not elaborate any of these cytokines except low levels of lL-l (Table 1). The LPS- inducible release of cytokines was again consist- ent with a macrophage phenotype for tumor-4 cells. Tumor 4 Cells Also Show Differentiated Lymphoid Characteristics Davidson et al. (1988) found that a v-Ha-ras- transformed lymphoid cell line could be stimu- lated to differentiate along either the myeloid or lymphoid pathways. Because tumor-4 cells showed a variety of DNA rearrangements in the kappa light-chain locus (data not shown), it was of interest to determine whether the cells that had gone on to rearrange the kappa locus were the same cells that had progressed toward a mac- rophage phenotype or whether the tumor-4 cells were a mixed population of B cells and macro- phages. To that end, tumor-4 cells were plated in soft agar medium and six subclones were recov- ered. Southern hybridization analysis of EcoRl- digested DNA isolated from the subclones showed that they all contained the same 5.3-kb v- l-la-ras proviral integration fragments as R2 and tumor~4 cells (data not shown; see Fig. 1A). The six subclones of tumor-4 possessed the same myeloid characteristics described before for the uncloned tumor, but varied in their pattern of kappa light-chain gene rearrangement. Southern hybridization analysis of Baml—Il-digested DNAs with a kappa probe revealed that subclones 3 and 5 possessed one germline and one rearranged kappa allele, and subclones 1, 2, 4, and 6 pos- sessed rearrangements in both alleles (Fig. 4). All the subclones possessed a rearranged BamHI fragment of approximately 7kb. Tumor 4 was apparently derived from an outgrowth of R2 that had undergone this rearrangement. Some sub- clones then proceeded to rearrange their other kappa allele. Clearly, tumor 4 contained cells that individually had differentiated along both the lymphoid and myeloid pathways. Having observed kappa light-chain rearrange- ments in macrophage subclones of tumor 4, we next examined the status of immunoglobulin expression by Northern blot analysis. Kappa light-chain transcript could not be detected (data TABLE I LPS-Induced Cytokine Release by Tumor-4 Macrophage Subclones‘ lL-l (U/ml) lL-tHU/ml) TNF(U/’ml) -Ll’$ +LPS ~Ll’S +Ll’S -Ll’S +LT‘S R2 0 2 0 0 0 0 T4.l 0 2 0 l 0 0 T42 0 6 0 100 O 40 T43 0 6 0 l500 0 85 T44 0 l9 0 39 0 34 T45 l 4 0 0 0 0 T46 0 4 0 35 0 0 The capactv of cell lines to release the tvtnlunes IL-L lL-h, and tumor na-(rivsis tactur (TVH was dctvrmmnl bv ~|.\\1‘\ln|: culturi- supt'rnatants for this purpuse, cell lines Wm Imuhutcd for 24 h at 25- IU' u‘lh ml with Ill-urn ml Ll‘S In KI'MI lbw supplemented mth Ill“. tt-tal (alt scrum and "- l” \1 1mm thtk‘lhanul. Culture suru-matants were collected, passed through a 0 Shannon ”lift, and stun-d .it -70 ‘C until assauxl lL-l .u'm ”V was .t\\l\\\l bx its JblllfV to unduu- “ft‘lllt’fJIIt‘n ut DHHZJ I cells in the presence of Concanavalln ~\ as dearth-d bv .-\\.I|.I rt .II (lWlbI ll_-0 .icttwtv was detrrrmnvd bi. Its .ihilits to induu- the prulitrutmn ut the WDI Bat-ll hvbndoma as prrwuuslv described by llultncr 1" al IIU‘NI T\F activity was assessed bv its (ytutnuuti tn WHll-lh-l tlum- l} u-ils .is pvt-\it-Usls derlh" by -\\.i|.l t-t .Il il‘NIl.” The NLITIVK‘ units of cytokine.- JC"\'I(\ vu-n- dz-termnns-d by comparison at the adults 0t dilution st-ncs nt npvrimrntal supernatants tn the arm (tn-s ut dilution series 0! purities! human lL-l (Cenlvmet, ru‘umhmant human ll.-o(:\mgr:n (mp nvr murme TNF-alpha (Amgt-n L'urp » standards. ANTlG EN PRESENTATION not shown). A mu heavy-chain probe revealed a diverse range of RNAs in the macrophage sub- clones (Fig. 5) that correspond in size to 1.9-, 2.1-, 2.3-, and 2.9-kb transcripts reported to be initiated in the mu—switch region of myeloid cell lines (Kemp et al., 1980). The R2 pre—B-cell line possesses predominantly larger RNA species that include those that correspond in size to mature mu mRNAs of 2.4 and 2.7 kb. These species are diminished upon lineage switch. Comparison to a hybridization of the same blot with a probe for GAPDH (Fig. 5) shows the R2 RNA to be underloaded and thus the diminution of mu tran- scription in the macrophage is even more dra- matic than apparent from casual inspection of the data. Apparently, the macrophage subclones of tumor 4 lose the capacity to transcribe functional mu mRNA, even though the rearrangement of the kappa locus suggests progress in lymphoid differentition. Since CD45 isoforms have been reported to be lineage-specific (Ralph et al., 1987; Saga et al., 1987; Streuli et al., 1987), the expression of this surface marker was examined among the sub- clones of tumor 4. An immunoperoxidase—detec- tion procedure detected 8220, the B lymphoid 35 AFTER LINEAGE SWITCH :53 isoform of CD45, in tumor-4 cells. The expression of CD45 was further examined among the tumor- 4 subclones in order to determine the relative expression of the 8220 isoform in comparison to the isoform that predominates in myeloid cells. Recently, Chang et al. (1989) described the use of a reverse transcription-polymerase chain reaction (RT-PCR) technique to detemiine the pattern of alternate exon use in CD45 expression of hemato- poietic cells. They found that B lymphoid cell lines uniquely expressed a form of CD45 mRNA possessing three optional exons, whereas two myeloid cell lines (a macrophage and a mast cell) predominantly expressed a form lacking these exons. We utilized RT-PCR to examine CD45 expression among R2 and the subclones of tumor 4 (Fig. 6). All of the cell lines expressed multiple species of CD45 mRNA. Subclones 2, 3, 4, and 6 expressed a CD45 mRNA containing three optional exons, typical of B lymphoid cells, whereas subclones 1 and predominantly expressed mRNA lacking these exons, typical of myeloid cells. R2 expressed the expected three exon B lymphoid isoform. Thus, the pattern of CD45 expression is heterogeneous among mac- rophage subclones of the same tumor. in- w .y 7. 'l 9". . 'f ,. s" '2 FlCURE 4. Kappa light-chain rearrangements. Southern blot analysis of DNAs from liver (L). R2. and six subclones of tumor 4 (1-6). DNA was digested with BamHl and 10 pg of each . kappa light-chain constant region ' sequences. Size markers are the positions of an ethidium bromide- stained Hindlll digest of bacteriophage A and are denoted in ses. 36 254 ].D. BREI'Z et al. T4 subclones ‘32»1 23 4 56 FIGURE 5. Expression of mu heavy-chain Northern blot analysis was performed by poly A' A from R2 and six subclones of tumor 4 (l-6). Each sample of RNA was the poly A' fraction selected from 100 ug of total cytoplasmic RNA. The blot was probed for mu heavy chain. The positions of ethidium bromide-stained rRNAs are noted on the right. The positions or mu RNA 5 res are marked on the left and denoted in kilobases. The lower panel shows the same blot probed for GAPDH as a control for loading. The Subclones of Tumor 4 Can Function Effectively in Antigen Presentation in order to test the ability of tumor-4 subclones to present antigen, an assay system required an antigen-specific T-helper cell line that could be stimulated to produce interleukin-2 (IL-2) upon presentation. For these experiments, the putative antigen-presenting cells were cocultivated with a T-cell hybridoma specific for chicken ovalbumin (COVA) and restricted for l-A" (the haplotype for BALB/c), DO.11.10/54.4. In the presence of COVA, authentic macrophages such as those in a peritoneal exudate stimulate the hybridoma to produce lL-2 (Fig. 7A). lL-2 production was assayed by the application of media supernatants trom cocultivations to an lL-Z-dependent cell line, CTLL-Z. All of the macrophagelike tumor-4 subclones displayed antigen-presentation capacities comparable to peritoneal exudates (Fig. 7A). Furthermore, all of the tumor-4 sub- clones showed antigen-presentation capacities dramatically greater than either the parental R2 pre-B-cell line or tumor 1, a B-cell tumor derived from R2 (Fig. 7A). lL-Z production was depen- dent on the presence of cOVA during coculti- vation of presenting cells with cells of the helper T-cell hybridoma. Supernatants produced in the 37 ANTIGEN PRESFJ‘JTATION AFIER LINEAGE SWITCH 255 T4 subclones C65432IM No. of Exact: FIGURE 6 RT— PCR analysis of CD45. RT-PCR was performed on the polyA' RNAs of R2. the six subclones of tumor P3880] (a myeloid control). The and rodu cts were electro ohop oresed through 2% agarose and stained with ethidium bromide (C) 3- exon plasmid control; (1-6) T4 subclones; (M) 123-b p ladder: (Mo) P388 D. PCR rod ucts smaller than the 0 exon product may P represent an RNA species lacking an additional exon (Chang and Esselman, unpublished results). absence of cOVA were analyzed for all the cell lines and the values for IL-2 production were found to be near zero (data not shown). These control values were subtracted from those deter- mined for supernatants produced in the presence of cOVA to generate the data presented in Figs. 7A, 7B, and 7C. IL-2 production was also depen- dent on the presence of T-cell hybridoma cells. Supematants produced by incubations of puta— tive presenting cells with cOVA in the absence of T-cell hybridoma cells had no detectable lL-2 (data not shown). The ability to present antigen was not stimulated by exposure to LPS for any of these cell lines (data not shown). A macrophages like outgrowth from tumor 5 (also derived from R2) and the cells of a macrophagelike tumor derived from the pre—B-cell line RI (9) showed levels of antigen presentation similar to those observed for the tumor-4 subclones (Fig. 7B). The ability to present antigen, therefore, may be a common phenomenon among v—Ha-ras—transfor- med B lymphoid cells that acquire macrophage- like characteristics. Authentic antigenic presentation should be MHC-restricted, so all of the putative antigen- presenting cells were also cocultivated with a T- cell hybridoma specific for cOVA and restricted for l-A“, 3Q023-24.4. As exemplified by subclone 4 of tumor 4 (T44) and the macrophage tumor derived from R1 (RIT), the antigen presentation observed is MHC-restricted (Fig. 7C). I-A Expression Antigen presentation to T cells requires la expression and the observation of l-Ad-restricted presentation (Fig. 7C) indicates that these "lin- eage switch" macrophages express I-A“. In order to assess whether the acquisition of presentation capacity correlated with acquisition of la expression, in particular I-A“, we performed flow cytometry with FITC-conjugated antimouse l-A‘ on the macrophage cell lines and their pre-B-cell precursors. Although both R1 and R2 (pre-B cells) displayed no detectable l-A“, the macro- phage cell lines represented by RIT and TIA showed a low expression of l-Ad (Fig. 8). DISCUSSION This study demonstrates the capacity of several macrophage-like tumor cell lines derived from v- Ha-ras-transformed pre-B—cell lines to present antigen with MHC—restriction. This finding estab- lishes that cells having undergone "lineage switching" can perform a function normally associated with a fully differentiated macrophage 38 256 1.1). BRETZ ct al. 12 7 A 1‘ ” ( l 0 R2 (C) ' - 1° . '2 :2“ .- E 9 P 0 "Mi :7 \ , 5 _ 3 8 _ -/I V T42 E '\.V 'E v T43 g 3 7 n T4.4 g ‘ ” w d '8 6 I T45 v R1T(I—A) E A T46 3 3 r V R17 (Ht‘1 3 5 3 o T4.4 (l-Aq) a. ‘ E 2 r o T44 (I-A) 1’ .. t d 3 N 1 / I 2 : o 1 0 O 3 o lVIVT'l'lfl'l "l'—I'I'r'1 I'fi'r'i 0 2 4 6 810121416182022 No. of presenting cells/culture (x 10‘) 14 13 - (B) / 0 R2 12 0 RI 1‘ 0 R11” 10 0 T5 to IL-2 produced (units/ml) .._A O‘NUJDUICIVU ' I I l f I ' l ' 0 2 4 6 8101214161820 4 No. of presenting cells/culture (x 10 ) FIGURE 7 or B cell. Although numerous examples exist of B lymphomas with the capacity to present antigen (Chesnut et al., 1982; Walker et al., 1982), neither the pre-B-cell precursors of the macrophage-like cell lines nor B-cell lymphoma cell lines derived from those precursors could present antigen. Thus, the capacity to present antigen appears to correlate with the differentiation of these cells along the macrophage lineage. Indeed, two cell O 1 2 3 4 5 6 7 8 9‘ 10 No. of presenting cells/culture (x 10 ) FIGURE 7. Antigen presentation. (A) Antigen-presentation assays were performed on R2. the tumor-4 subclones (4.1-4.6), tumor 1 (a B-cell tumor derived from R2), and peritoneal exudates (pMo). (B) Antigen-presentation assays were performed on R2, a macrophage outgrowth of tumor 5 (TS; derived from R2 in a different animal), RI (another preB—cell line transformed by v-Ha-ras), and RIT (a macrophage tumor derived from RI). (C) Antigen-presentation assays were performed on a tumor-4 subclone (T 4.4) and RIT with DO- 11.10/ 54.4 (LIV-restricted) or 3Q023-24.4 (LN-restricted). Each point represents an average value obtained from a dilution series for each presentation supernatant. testing the response of CILL-Z cells to IL-2 in those supernatants. The results shown are representative of at least two experiments with each cell line. lines with the most dramatic level of antigen presentation had lost expression of the B-cell iso- form of CD45 and displayed a pattern of CD45 more typical of a myeloid cell (subclones 1 and S, Fig. 6; T4.1 and T45, Fig. 7A). Perhaps, loss of the B-cell isoform of CD45 is indicative of further maturation along the myeloid lineage. It may be worthwhile to investigate the role of CD45 in macrophage function. The fact that similar antigen-presentation abilities were found in mac- rophage derivatives of two completely indepen- dent cell lines (R1 and R2) suggests the generality of this phenomenon. Because it is well established that IL-1 along with antigen presentation is an important coacti- vator of T cells, it is surprising that the induc- ibility of cytokine release by LPS (Table I) does not correlate with the effectiveness of antigen presentation by the T4 subclones (Fig. 7A). 39 ANTIGEN PRESENTATION AFTER LINEAGE SWITCH 257 Apparently the low levels of lL-l that some of these macrophages are capable of elaborating is sufficient for T-cell activation. The observation that two of the best lines for antigen presentation (T4.1 and T45) have a weak response to LPS sug- gests that LPS-induced cytokine release may not be an adequate measure in itself for evaluating macrophage function. The "lineage switch” macrophages reported here express a low level of la (Fig. 8). This is con- sistent with the previous report of Davidson et al. (1988). The precursor pre-B cells lack detectable Ia..Perhaps la expression is the critical property determining the capacity to present antigen among these cells. Certainly, la expression is necessary for antigen presentation, but its suf- ficiency for antigen presentation among the cell lines we have studied will require further experi- mentation. The six macrophagelike subclones of tumor 4, while possessing a common rearranged kappa allele, displayed a variety of kappa light-chain gene rearrangements at their other kappa allele. Compared to their parental cell line, these cells have progressed along the B as well as the monocyte/ macrophage lineage. The varying rearrangements of one kappa allele suggest rearrangement subsequent to macrophage con- version and that at least certain elements of lymphoid and macrophage differentiation pro- grams are not mutually exclusive. The fact that the R2 cell line can also generate a lymphoma (T1, Fig. 7A) that expresses both mu and kappa chains (data not shown) demonstrates the poten- tial of this cell line to differentiate quite far along either the lymphoid or macrophage pathways. The relationship between lymphoid and mac- rophage differentiation revealed in these cells differs somewhat from that seen in cases of ”lin- eage switch" previously reported. Klinken et al. (1988) found ”lineage switch” macrophages at both the pre-B- and B-cell stages of immunoglob- ulin rearrangement. However, they did not find macrophages that had progressed in their immu- noglobulin rearrangement compared to their lymphoid cell precursors, as we have. Davidson et al. (1988), examining v-Ha-ras-transformants similar to those reported here, could induce those cells to differentiate into either lymphoid or mac- rophage cells upon exposure to LPS. The lymph- oid derivatives they reported did not progress beyond the pre-B-cell stage, whereas we have identified an immunoglobulin-producing tumor derived from a pre-B-cell line that also gave rise to a macrophage tumor. Perhaps the more com- plex environment provided during tumor chal- T41 L L FLUORESCENCE FIGURE 8. I-Ad expression. Flow cytometry was performed on R1, R2, T4.,4 and RIT after reactions with FITC-conjugated anti-IA“ (solid line) or, as a control. FITC- coniugated mouse IgGhK (dashed line). For R1 and R2, the plots of experimental and control are Virtually coincidental. 40 258 JD. BREI'Z ct al. lenge allowed the cells described here to more fully develop along the lymphoid lineage when that pathway was selected. At any rate, the v-Ha- res-transformed pre-B cells described here seem truly bipotential. The ability of these cells that undergo an apparent "lineage switch” to perform a fully dif- ferentiated function presents the possibility that they may represent an unusual but normal subset of hematopoetic cells rather than an oddity induced by transformation. The existence of both lymphoid and macrophage characteristics in a cell fully capable of antigen presentation sug- gests greater plasticity in hematopoietic lineage commitment than conventionally thought to be the case. MATERIALS AND METHODS Cell Lines R1 and R2 are v-Ha-ras-transformed murine pre- B-cell lines described in Schwartz et al. (1986b). Tumors derived from R1 and R2 were generated as described in Schwartz et al. (1986a, 1986b) in syngeneic BALB/c mice and in BALB/c athymic nude mice. Briefly, cells were washed twice in RPMI 1640 and were then resuspended in the same at 8x10° cells per ml. Five-week-old mice were injected intraperitoneally with 0.25 ml of the cellular suspension. Tumor 4, in particular, was isolated from an inguinal lymph node at 74 days postinjection. Tumor cell lines were readily produced from explanted tumors by dispersal and transfer to feeder cultures of adherent bone marrow cells (Whitlock et al.? 1983). All of these cell lines were cultured over feeder cells in RPMI 1640 supplemented with 5% fetal calf serum and leO‘5 M 2-mercaptoethanol. The subclones of tumor 4 were generated from single colonies grown in soft agar medium as described by Whitlock et al. (1983). The T-cell hybridoma, DO-11.10/ 54.4, was a generous gift of Drs. Philippa Marrack and John Kappler (University of Colorado, Denver) (White et al., 1983). This hybridoma is specific for chicken ovalbumin in the context of l-Ad and cross reacts weakly with chicken ovalbumin in the context of I-A". 3Q023-24.4, another T-cell hybridoma, was also a gift of Drs. Marrack and Kappler. This hybridoma is specific for chicken ovalbumin in the context of either I-Aq or I-E. CTLL-Z is a T-cell line responsive to IL-2 and was obtained from the ATCC. All of these cell line were cultured in RPMI 1640 supplemented with 10% fetal calf serum and 5x10'5M 2-mercaptoethanol in the absence of any feeder cells. Peritoneal exudates containing macrophages were produced from BALB/C mice treated 1 week previously with a 0.5-ml intraperitoneal injection of pristane. Nucleic Acid Analysis Cytoplasmic RNA was isolated from actively growing cells by a sodium dodecyl sulfate-urea procedure as described by Schwartz et al. (1981). Poly A‘ RNA was selected by oligo-dT cellulose chromatography (Aviv and Leder, 1975). RNA was denatured, electrophoresed in a formal- dehyde-1% agarose gel (Rave et al., 1979), and transferred to Nytran (Schleicher and Schuell) (Thomas, 1980). High molecular weight DNA was isolated from nuclei collected in the preceding RNA isolation procedure as described in Schwartz et al. (1986b). DNA was digested with restriction enzymes as noted in the figure legends, electrophoresed through 0.8% agarose, and transferred to Nytran (Southern, 1975). Hybridization probes were prepared by nick translation (Rigby et al., 1979) through the incor- poration of [tr-”Pl dATP (3000 Ci/mmol; ICN). The v-Ha-ras probe was the replicative form of phage Ml3mp10 containing a 0.46-kb EcoRl frag- ment corresponding to v-Ha-ras encoding sequences (Ellis et al., 1980). The arm probe was a 0.8-kb BamHI fragment from the env region of Friend murine leukemia virus and is specific for the env sequences of murine ecotropic retro- viruses (Silver and Kozak, 1986). The c-myc probe was the 4.7-kb genomic HindIII fragment of murine c-myc (Stanton et al., 1984). The murine c- myb probe was a cloned 2.4-kb cDNA (a generous gift of Dr. Timothy Bender, University of Vir- ginia, Charlottesville). The fms probe was a cloned 2.7-kb Clal-BamHl fragment of the McDonough strain of feline sarcoma virus (Donner et al., 1982). The murine fi1-microglobu- lin probe was a cloned 0.5-kb cDNA (Parnes et al., 1981). The rat glyceraldehyde-3-phosphate dehydrogenase (GAPDH) probe was a cloned 1.3-kb cDNA (Fort et al., 1985). The murine ANTIGEN PRESENTATION AFTER LINEAGE SWITCH kappa light-chain probe was the replicative form of phage M13mp10 containing a genomic 0.48 kb Hpal-Bglll fragment extending from a point about 50 base pairs within the 5' terminus of the kappa light-chain constant region gene to the poly A addition site (Seidman and Leder, 1978). The murine mu heavy-chain probe was a cloned cDNA (#12) that extends from CH2 to the 3'- untranslated region of the secreted form of mu mRNA (Rogers et al., 1980). All hybridizations were performed under aqueous conditions in 5x SSC at 65 °C and washed to a stringency of 0.1x SSC at 65 °C. Reverse Transcription-Polymerase Chain Reaction (RT-PCR) RT-PCR was performed according to the pro- cedure of Chang et al. (1989, 1991) using poly A’ RNA as substrate. The primers were a sense primer specific to exon 2 (GCCC'ITCTGGACACAGAAGT, base positions 167—186) and an antisense primer specific to exon 9 (AATTCACAGTAATGTTCCCAAACAT; base positions 764-740) of the cDNA of murine CD45 (Thomas et al., 1987). cDNA was prepared by incubating lpg of poly A’ RNA for 60 min at 37°C with 200 units of MoMuLV reverse tran- scriptase in a 20411 reaction volume containing 50 mM Tris-HCI (pH 8.3), 75 mM KCl, 3 mM MgClz, 5 mM DTT, 100 pg/ml BSA, 40 units RNa- sin, 500 pM dNTP, and 200 ng of antisense primer. A S-pl aliquot was used directly for PCR amplification in a 50-11] reaction volume contain- ing 50 mM KCl, 10 mM Tris-HCl (pH 9.3), 3 mM MgC12, 0.1% w/v gelatin, 500 11M dNTP, 400 ng of sense and antisense primers, and 2.5 units of Taq polymerase. PCR was performed in a DNA Thermal Cycler (Perkin-Elmer-Cetus, Inc.) for 24 cycles. Each cycle consisted of 405 at 94 °C for denaturation, 15 s at 55 °C for annealing, and 30 s at 72 °C for elongation. The first cycle was pre- ceded by a 5-min incubation at 94 °C and the last cycle followed by a 4-min incubation at 72 °C. Cytological Analyses Cells were cytocentrifuged onto a microscope slide and allowed to air dry overnight. The cells were then incubated with either rat anti-8220 (monoclonal 14.8) or rat anti-MAC-l (Boehringer Mannheim). Goat antirat immunoglobulin- 41 259 horseradish peroxidase (Boehringer Mannheim) was used in a secondary incubation for detection. The presence of a-naphthyl acetate esterase was determined by cytochemical staining (Yam et al., 1971) with a Sigma research kit. Nonspecific phagocytosis of latex beads was assayed by the method of Raschke et al. (1978). Antigen Presentation Assays for antigen presentation were performed in a manner similar to that described by Marrack et al. (1989). Briefly, the cell lines to be assayed for antigen presentation were titrated into 200-111 microcultures containing 105 cells of either the T- cell hybridomas DO-11.10/54.4 or 3Q023-24.4, both of which produce IL-2 in response to the presentation of chicken ovalbumin (cOVA) in the context of I-Ad or I-A“, respectively. These assays were carried out in RPMI 1640 supplemented with 10% fetal calf serum, 5x10" M 2-mercapto- ethanol and, where required, COVA at 1 mg/ml. After 24 hr, incubation supernatants from these cultures were assayed for IL-2 using CTLL-Z, an IL-2-dependent cytotoxic T-cell line. Twofold ser- ial dilutions of supernatants were added to 5x103 CTLL-Z cells in 100-111 microcultures and incu- bated for 48 hr at 37 °C. MTT (Sigma), a substrate for production of a colored product indicative of cell survival (Mosmann, 1983), was added at 0.5 mg/ml and the cultures incubated for an additional 4hr at 37°C. Acid-isopropanol (40- mM HCl) was then added to dissolve the M'IT formazan reaction product. The optical density of each well was quantitated by an ELISA reader at a wavelength of 540 nm. The specific activity of lL-Z in the supernatants was determined by com- parison to a standard curve produced through the use of purified recombinant IL-2 (Cetus Inc.). Flow Cytometry Cells were stained in PBS, 2% FCS with either FITC-conjugated monoclonal antibody AMS-32.1 (antimouse l-A") (Phar Mingen) or FITC-conju- gated mouse lgGZb, K (Phar Mingen) as an isot- ype-matched control. Cells were then fixed in PBS, 2% FCS, 0.5% formaldehyde, and stored at 4 °C until analysis. Flow cytometry was perfor- med using an Ortho Diagnostics Cytofluoro- graph SO-H. 42 260 JD. BRE'I'Z ct al. ACKNOWLEDGMENTS This work was supported by Public Health Service grants CA45360 (RC8) and GM35774 (WJE) and by a grant from the Elsa U. Pardee Foundation (RCS). We are indebted to Alfred Ayala for the cytokine assays. The authors thank Donna Paulnock for helpful dis- cussions, and Susan Conrad and Michele Fluck for thoughtful comments on this manuscript. (Received October 8, 1991) (Accepted November 22, 1991) REFERENCES Aviv H., and Leder P. (1975). Purification of biologically active globulin messenger RNA by chromatography on oli- gothymidylic acid-cellulose. Proc. Natl. Acad. Sci. USA 69: 1408-1412. Ayala A., Perrin M.M., Meldrum D.R., Eretel W., and Chau- dry I.H. (1990a). Hemorrhage induces an increase in serum TNF which is not associated with elevated levels of endo- toxin. Cytokin 3: 170-175. Ayala A., Perrin M.M., Wagner M.A., and Chaudry l.H. (1990b). Enhanced susceptibility to sepsis following simple hemorrhage: Depression of EC and C3b receptor-mediated phagocytosis. Arch. 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Immunol. 130: 1033—1037. Whitlock C.A., Ziegler S.F., Treiman L.J., Stafford J.l., and Witte ON. (1983). Differentiation of cloned populations of immature B cells after transformation with Abelson murine leukemia virus. Cell 32: 903—91 1. Yam L.T., Li C.Y., and Crosby W.H. (1971). Cytochemical identification of monocytes and granulocytes. Am. J. Clin. Path. 55: 283-290. Chapter 3 C/EBP-related proteins confer LPS-inducible expression of IL-6 and MCP-l to a lymphoblastic cell line1 Abstract C/EBP-related proteins 2 and 3 (CRP2 and CRP3) are differentially expressed by P388 lymphoblasts and their derivative P388D1(IL-1) macrophages. We have ectopically expressed CRP2 and CRP3, either singly or together, in P388 lymphoblasts. The expression of either CRP2 or CRP3 is sufficient to confer the LPS-inducible expresson of IL-6 and MCP-l (macrophage chemoattractant protein) to a cell type not typically displaying LPS induction of inflammatory cytokines. Consistent with these findings, the expression of CRP2 antisense RNA blocks the LPS-induction of IL-6 expression in P38801(IL—l) macrophages. This work clearly establishes the essential role of C/EBP-related proteins in the induction of cytokine genes by LPS. Additionally, these data add MCP-l to the list of cytokines showing an involvement of either CRP2 or CRP3 in their expression. Introduction C/EBP-related proteins (CRPs) are a family of basic region-leucine zipper (bZIP) transcription factors. These dimerize through a leucine zipper and bind to DNA through an 1 Adapted from JD Bretz, 8 Williams, PF Johnson, and RC Schwartz. Proc. Natl. Acad. Sci. (submitted). 44 45 adjacent basic region (Johnson et al 1987, Landschulz et a1 1988, and Vinson et a1 1989). Several lines of evidence implicate CRPs in the regulation of inflammatory cytokines. The promoter regions for the IL-6, IL-la, IL-lB, IL-8, TNFa and G-CSF genes contain sequences that bind CRPs (Akira et al 1990, Furatani et al 1986, Shirakawa et al 1993, and Zhang and Rom 1993). The best fit consensus CRP binding site is T(T/G)NNGNAA(T/G) (Akira et al 1990). Both CRP2 (Williams et al 1991) (also known as NF-IL6 [Akira et a1 1990] and C/EBPB [Cao et a1 1991]) and CRP3 (Williams et a1 1991) (also known aerF-ILGB [Kinoshita et a1 1992] and C/EBP6 [Cao et al 1991]) can transactivate a reporter gene driven by the IL-6 promoter in transient expression assays (Akira et al 1990 and Kinoshita et al 1992). Additionally, the LPS-induced expression of IL- 13 (Shirakawa et al 1993 and Zhang and Rom 1993) and G-CSF (Akira et al 1990, Nishizawa et a1 1990 and Nishizawa and Nagata 1990) requires one or more elements that bind a CRP- like activity. Among the hematopoietic lineages, mature macrophages and granulocytes are specific in their expression of high levels of CRP2 and CRP3 (Natsuka et al 1992 and Scott et a1 1992). Macrophages are also notable for their lipopolysaccharide (LPS)-induced trancription of the genes for various inflammatory cytokines, the same genes that posses CRP binding sites in their promoters. Indeed, CRP2 and CRP3 are LPS- inducible (Akira et al 1990 and Kinoshita et a1 1992) and the CRP binding sites of the IL-18 and G-CSF genes have been 46 characterized as LPS-responsive elements (Shirakawa et al 1993, Zhang and Rom 1993, Nishizwa et a1 1990 and Nishizawa and Nagata 1990). These data suggest that CRP2 and CRP3 may be necessary for the LPS-induced cytokine response in macrophages. The evidence for the function of CRP2 and CRP3 in the regulation of IL-6 and other cytokines has rested on the transient transactivation of reporter genes rather than the activation of endogenous cytokine genes with intact promoter regions. Additionally, the relative activities of these two co-expressed and structurally related transcription factors have not been tested in vivo. In this paper, we have directly assessed the capacities of CRP2 and CRP3 for conferring LPS- induced cytokine expression to a lymphoblastic cell line normally lacking these activities. We have found that either singly or together, the ectopic expression of CRP2 and CRP3 in the lymphoblastic P388 cell line (Bauer et a1 1986) confers LPS-inducible expression of the genes encoding IL-6 and.MCP-1 (macrophage chemoattractant protein). Consistent with this, the expression of CRP2 antisense RNA blocks the induction by LPS of IL-6 in P38801(IL-l) macrophages. The roles of CRP2 and CRP3 in the LPS-induction of IL-6 and MCP-l are clearly established. Materials and methods Cells and cell culture. P688 lymphoblasts are P38801 cells (ATCC CCL 46). We have denoted these cells as P388 to 6). by Hi we of re tr ar 9t Vi Tn th 47 void confusion with their macrophage derivative which is usually referred to as P388D1 (Bauer et al 1986). P388 cells (13388D1 ATCC CCL 46) are lymphoblastic in morphology and lack appreciable expression of Mac 1, Mac 2, and Mac 3 as determined by flourescent activated cell sorting (FACS). P388D1(IL-1) macrophages are P38801 (IL-1) cells (ATTC T18 63). These cells are macrophage-like in morphology and express Mac 1, Mac 2, and Mac 3 at high levels as determined by FACS. Cells were cultured in RPMI 1640 medium supplemented with 5% fetal calf serum and 50pm 2-mercaptoethanol. Transfections. Transfections of G418-resistant vectors were carried out with 106 cells, Sug of plasmid DNA and 40ug of lipofectin Reagent (Gibco BRL) in 3 m1 of Opti-MEM I reduced-serum medium (Gibco BRL) . Cells were incubated in the transfection cocktail for 16 hours followed by the addition of RPMI 1640 supplemented with 20% fetal calf serum. After 72 hours the medium was replaced with standard growth medium supplemented with 0.67 mg/ml G418 (Gibco BRL) . Transfections of puromycin-resistant vectors were carried out similarly except that 10 pl of Transfectam Reagent (Promega) was used and selections performed with 3.75 ug/ml puromycin (Sigma). Expression vectors. pSV(X)Neo is pZIP—NEO SV(X)1 (Cepko et a1 1984). This vector contains Moloney murine leukemia virus (MoMLN) long terminal repeats (LTRs) and expresses the Tn5 neo gene through a subgenomic mRNA from the 5’ LTR. pSV(X)CRP2 and pSV(X)CRPZAS were constructed by insertion of the 1.55 kb NcoI/EcoRI genomic fragment encoding rat CRP2 48 (Williams et al 1991) into the BamHI site of pSV(X)Neo in both sense and antisense orientations. pSV(X)CRP3 was similarly constructed by insertion of the 0.8kb NcoI/HindIII genomic fragment encoding murine CRP3 (Williams et a1 1991) into the BamHI site of pSV(X)Neo. pBABE-Puro (Morgenstern and Land 1990) contains MoMLV LTRs and expresses the pa_c gene for puromycin resistance from the SV40 early promoter. pBABE-CRPZ was constructed by insertion of the 1.55 kb NcoI/EcoRI CRP2 fragment into the BamHI site of pBABE-Puro. Nucleic acid isolation and analysis. Cytoplasmic RNA was isolated by an SDS-urea procedure as described by Schwartz et al (1981). Genomic DNA was isolated from nuclei collected in the preceding procedure by a method described in Schwartz et al (1986). Restriction enzyme-digested DNAs were electrophoresed through 0.8% agarose. RNAs were electrophoresed through 1% agarose-formaldehyde gels. Transfers to membranes were hybridized and washed to a stringency of 0.1xSSPE in 0.1% SDS. Hybridization probes were prepared by random priming using a kit from United States Biochemical Corp. with the incorporation of 5’-[a-”P]dATP (3,000 Ci/mmol; Dupont/NEN). The probes for CRP2 and CRP3 were genomic fragments described above for expression vectors. The glyceraldehyde phosphate dehydrogenase (GAPDH) probe was a 1.3 kb rat cDNA (Fort et a1 1985). The IL-6 probe was a 0.65 kb murine cDNA (from Drs. N. Jenkins and N. Copeland, NCI-FCRDC). The MCP-l probe was a 0.58 kb murine cDNA (Rollins et a1 1988). The IL-la probe was 49 a 1.7 kb murine cDNA (Lomedico et al 1984). The IL-lfi probe was a 1.0 kb murine cDNA (Tannenbaum et a1 1988). Western analysis. Nuclear extracts were prepared as described below. These extracts (500g) were suspended in Laemmli buffer and electrophoresed through 12% SDS- polyacrylamide gels. The gels were electrophoresed onto Immobilon-P membranes (Millipore), and antibody-antigen complexes were visualized.with the Enhanced Chemiluminescence detection kit (Amersham) exactly as recommended by the supplier. Electrophoretic mobility shift assays (BMSAs). Nuclear extracts were prepared as described by Lee et a1 except that the samples were not dialyzed into buffer D. Extracts were incubated with a double-stranded oligonucleotide probe homologous to nucleotides -107 to 90 of the rat albumin promoter (the DEl site) or to a probe containing an optimal C/EBP binding site (Johnson et a1 1993). Reactions were carried out by the method of Fried and Crothers (1981), as modified by Nye and Graves (1990). Samples were electrophoresed through 6% polyacrylamide gels in 0.5 x TBE at 150 volts. For "supershifts", antisera were preincubated with nuclear extracts for 30 minutes at 49C prior to the binding reaction. Metabolic labeling and.immunoprecipitations. Cells were washed twice in methionine-deficient DMEM (Gibco BRL) and then labeled in the same medium containing 200 pCi of EXPRE”S”S protein labeling mix (Dupont/NEN) and 5% dialyzed fetal calf 50 serum. After 3 hours, cells were collected and lysed, and immunoprecipitation was performed and analyzed as described by Whitlock et a1 (1983). Antisera. Rabbit anti-CRP2 was generated by immunization with a peptide corresponding to amino acids 1-12 of CRP2 (Williams et al 1991). Rabbit anti-CRP3 antiserum ‘was generated by immunization with a peptide corresponding to amino acids 255-266 of the murine CRP3 protein. Results Differential CRP2 and CRP3 expression between P388 lymphoblasts and their macrophage derivitive P388D1(IL-1) . Upon screening' a jpanel of Zhematopoietic cell lines for differential CRP2 and CRP3 expression by Northern blot analysis, we found that the P388 B lymphoblastic cell line lacked CRP2 and CRP3 transcripts, while its macrophage derivative P388D1(IL-1) expressed these transcripts abundantly (Figure 1A). C/EBP transcripts were not detected in either cell line (data not shown). These findings suggested to us that these cell lines could provide a model system in which to test the capacity of CRP2 and CRP3 to confer the characteristics of macrophages to a lymphoid cell line not normally expressing CRP2 and CRP3. In particular, we sought to examine the ITS-induced transcription of genes encoding inflammatory cytokines. Having observed differential expression at the RNA level we sought to confirm that P388D1(IL-1) expressed active CRP2 51 Figure 1. Analysis of differential CRP2 and CRP3 expression Ibetween P388 and P388D1(IL—1). (A) Northern analysis of 200g of cytoplasmic RNA. The same blot was successively hybridized to probes for CRP2, CRP3, and GAPDH. Hybridization to GAPDH served as a control for loading. The positions of ethidium bromide-stained 288 and 18S rRNAs are marked. (B) Western analysis of proteins derived form nuclear extracts. CRP2- specific antiserum was used for detection. (C) EMSA analysis of nuclear extracts. Samples were treated in the absence of antiserum, and in the presence of normal rabbit serum (NRS), CRP2 antiserum (CRP2), and CRP3 antiserum (CRP3). IRecombinant bacterially-produced CRP2 (bact CRP2) and recombinant bacterially-produced CRP3 (bact CRP3) were analyzed as positive controls for DNA binding and "supershift". Species a represents CRP2 homodimer, while b and c probably contain heterodimeric forms of CRP2; all are "supershifted" with CRP2 antiserum. Species d is not "supershifted" and may represent ILIP (Descombes and Schibler 1991; see text). The positions of species supershifted by antisera are indicated by arrows. 52 P388 P38801(IL1) < 285 4183 CRP2 . < 288 < 188 4 288 < 18$ GAPDH Figure 1A Figure 1B 1 R ‘ “ rifiS-.. lAntiserum: ’11 p m a. a: O SdUO :oeq ZdHO 1er (L1Dl0888d ' 888d SdUO 199‘! ZdHO laeq (HDLOBBSd 888d ZdHO zoeq (L1I)L0888d 888d SdUO (seq ZdHO 139C} (L1|)L0888d 888d [ l { gure ' 10" 54 and CRP3 proteins while P388 did not" A western blot analysis of nuclear extracts for the two cell lines revealed CRP2 protein to be present in P38801(IL-l) cells, but not in P388 cells (Figure 18). An EMSA using an oligonucleotide probe containing an optimal CRP binding site found that the nuclear extract of P388D1(IL-1) cells formed several species (a, b and c) that could be "supershifted" by incubation with antiserum specific for CRP2 protein (Figure 1C). Species a, b, and c may represent various modified or heterodimeric CRP binding forms. Species d was not supershifted and it may represent the LIP product, an amino terminal-truncated form of CRP2 (Descombes and Schibler 1991) that would not be reactive with our amino terminal-specific antiserum. CRP3 protein was not observable (data not shown) but a "supershift" species was detectable with antiserum specific for CRP3 (Figure 1C). ‘This may be explained by the low abundance of CRP3 protein coupled with an antiserum of relatively low titer. Nonetheless, it is clear that P388D1(IL-1) macrophages display CRP binding site activity which is absent in P388 B lymphoblasts. Ectopic expression of CRP2 and CRP3 in P388 3 lymphoblasts. Three murine retrovirus vectors were utilized to ectopically express the CRP2 and CRP3 genes in P388 cells. pSV(X)CRP2 and pSV(X)CRP3 express CRP2 and CRP3, respectively, from the MoMLV LTR. Both vectors also express the gene for G418-resistance from a subgenomic mRNA. pBABE-CRP2 expresses CRP2 from the MoMLN LTR and the gene for puromycin-resistance from the SV40 early promoter. Populations of P388 cells were 55 transfected with pSV(X)CRP2, pSV(X)CRP3 or their parental vector lacking an expressed insert, pSV(X)Neo. Populations transfected with pSV(X)CRP3 were transfected,in turn, with pBABE-CRPZ or’ its parental vector lacking an expression insert, pBABE-Puro. Stably transfected populations were obtained after selection with the appropriate drug. CRP2 and CRP3 expression were initially analyzed in the transfected populations by Northern blot analysis (Figure 2A) . P388-C2 (transfected with pSV(X)CRP2) expressed a ~5.6 kb CRP2 RNA corresponding to the expected genome length retroviral transcript. P388-C3/C2 (transfected with pSV(X) CRP3and pBABE- CRP2) expressed a ~1.8 kb CRP2 RNA corresponding to a transcript extending from the 5’ LTR to the genomic CRP2 poly A. addition site. ‘P388-C3, P388—C3/Puro, and P388-C3/C2 (transfected with pSV(X)CRP3, pSV(X)CRP3 and pBABE—Puro, and pSV(X)CRP3 and pBABE-CRPZ, respectively) all expressed a ~4.8 kb CRP3 RNA corresponding to the expected genome length retroviral trancript. A Western blot analysis of nuclear extracts from the transfected populations confirmed the ectopic expression of the transfected CRP2 gene at the protein level (Figure 28). An EMSA detected protein-DNA complexes that could be "supershifted" by antiserum specific for CRP2 protein in nuclear extracts of the P388-CZ and P388-C3/C2 populations (Figure 2C). Neither CRP3 protein nor a "supershift" species was observable with antiserum specific for CRP3 protein, perhaps indicating a rather low level of CRP3 protein expression. \e‘ 3.1 56 Figure 2. Analyses of P388 cells transfected for CRP2 and CRP3 expression: P388 + pSV(X)Neo (P388-Neo): P388 + pSV(X)CRP2 (P388-C2); P388 + pSV(X)CRP3 (P388-C3); P388 + pSV(X)CRP3 + pBABE-Puro (P388-C3/Puro); P388 + pSV(X)CRP3 + pBABE-CRPZ (P388-C3/C2); P388D1(IL-l) + pSV(X)Neo (P388D1(IL- 1)-Neo). (A) Northern analysis of 20 pg of cytoplasmic RNA. Blots were successively hybridized to probes for CRP2 and GAPDH, or CRP3 and GAPDH. Hybridization to GAPDH served as a control for loading. The positions of ethidium bromide- stained 28S rRNAs are'markedn (B) Western analysis of protein derived from nuclear extracts. CRP2-specific antiserum was used for detection. Recombinant bacterially-produced CRP2 and CRP3 (bact CRP2; bact CRP3) are included as controls for specificity of the antiserum. The position of CRP2 protein is indicated. (C) EMSA analysis of nuclear extracts. Samples ‘were treated in the absence of antiserum, and in the presence of normal rabbit serum (NRS), CRP2 antiserum (CRP2), and CRP3 antiserum (CRP3) . Recombinant bacterially-produced CRP2 (bact CRP2) and CRP3 (bact CRP3) were analyzed as positive controls for DNA binding and "supershift." The positions of CRP2 EMSA species are indicated with asterisks and those species supershifted by antisera are indicated by arrows. (D) southern analysis of long of genomic DNAs. P388-C2-2 is an independent transfectant similar to P388-C2. DNAs were digested with BglII for CRP2 vector integraton sites and BamHI for CRP3 vector integration sites. DNAs were digested ‘with XbaI for internal vector fragments. Blots were hybridized to probes for either CRP2 or CRP3. Size markers are the positions of an ethidium bromide-stained HindIII digest of bacteriophage lambda DNA. 57 ooZIA—szoonna «OED-mama chafinoucanm oozuAde—bomnm nonownm «Gunman. ooznowna < 283 GAPDH.... .* GAPDH C .‘ Figure 2A 58 ammo aocn memo «can «030$an San—EOIQmmm acummmn «Gunman. oozéoma I ...————v4~ Figure ZB a mom 0 «can 2:0 88 ~ «28.82 .1 3 0528-82 1 8-82 3.82 82-82 NR fl 3:682 h 59 2a.... .28 25 88 «28-82 _ 2286-82 8-82 8.82 . 82-82 3:582 - CRP3 25 Ban 0.. . ammo .oam «GEO-.08.“. Sumac-cann— . nOunnnn. «Canaan ooznogn . f] 3.52381 . Nam—O «can 2280-82 a 8-82 . No-82 . 82-82 fi 3.5:;an «28-82 I . 8 2:0 88 ‘ Figure 2C 60 Xbal Bglll e NuNOnconn NOEOngm OBEMOIQRK "Gunman. «Olga 002:8»2. «INC-$3.“ NOEUIQQMA OSQBOnnonn nOnQonm «Uncann— ooz Iowan cann— b k 1. 3 2 4 < 4.4 Xbal I «IND-coma. «030:3 Sumac-own“. 00:89“. «Olga ooZusnm nonn— «IND-wann— NOBOIQona San—30-39“. ”Dianna «Uncann— ooz-uann— gnu <23.1kb <94 46.6 <44 “0 CC' C... a u .0 o u o . >>>> .0464 k96.4 1 2 3 P R C Figure 2D 61 In order to assess whether the transfected cells were comprised of single clonal outgrowths or complex populations of many transfectants, a Southern blot analysis was performed (Figure 2D). For the detection of CRP2 vector integration sites, genomic DNAs from the transfected populations were digested with BglII, which cleaves only once‘within.the pSV(X) vectors and not at all within the pBABE vectors. For the detection of CRP3 vector integration sites, genomic DNAs from the transfected populations were digested with BamHI, which does not cleave at all within the pSV(X)CRP3 vector. For evaluation of the integrity and abundance of the transfected genomes, the DNAs were digested with XbaI, which cuts once in each vector LTR to produce restriction fragments of a predicted size diagnostic of the transfected vectors. The predicted 5.6 kb CRP2-specific XbaI fragment was detected with at least haploid abundance in DNAs from P388-C2 and P388-C2-2, an independent population of cells produced in a manner identical to that of P388-C2. The DNA of P388-C3/C2 displayed the predicted 4.0 kb CRP2-specific XbaI fragment as well as several larger fragments, all at subhaploid abundance. Perhaps this indicates instability of the transfected pBABE- CRP2 vector. Nonetheless, P388-C3/C2 shows ectopic expression of CRP2 RNA, protein and EMSA activity. All of the DNAs possess identical endogenous CRP2-specific fragments of about 9 and 4 kb. The predicted 4.9 kb CRP3-specific XbaI fragment was detected with at least haploid abundance in DNAs from P388-C3, P388-C3/Puro, and P388-C3/C2. .All of the DNA possess 62 an.identical endogenous CRP3-specific fragment.at about.20 kb. Digestion of the transfected populations with BglII revealed two unique CRP2-specific restriction fragments for P388-C2 and one unique CRP2-specific restriction fragment for P388-C2-2. All of the DNAs possessed an identical endogenous CRP2-specific fragment of about 20 kb. P388-C2 and P388-C2-2 are clearly clonal or biclonal. Unique CRP2-specific fragments were not observed for P388-C3/C2, but this may be attributable to the instability of the transfected vector rather than the presence of a :multitude of independent transfectants. A unique CRP3—specific restriction fragment of about 30 kb was observed for P388-C3, P388-C3/Puro and P388- C3/C2 upon digestion with BamHI. ‘These populations are clonal for ectopic CRP3 integration. All of the DNAs possessed an identical endogenous CRP3-specific fragment of about 20 kb. LPS-induced cytokine expression occurs in the transfectants that ectopically express CRP2 and/or CRP3. Cultures ofI . P388-C3 . P388-03/Puro . e be ’t k . s . P388-CW2 Figure 2 87 for LAP (Figure 3). It is important to note the location of the staining; tGranulocytes often stain in areas surrounded by the nucleus or within an indentation of the nucleus while lymphocytes may show weak staining in the cytoplasm. Discussion The P388 B lymphoblast cell line was chosen for these transfection experiments for a number of reasons. Its previously mentioned capacity to switch lineages was important. The R2 cell line developed in our lab was also capable of a lineage switch (see chapter 2). The phenomena of differential expression of CRP2 and CRP3 during a lineage switch was first shown in the R2/T4 lineage switch pair; R2’s dependence on a feeder layer of stromal cells and its possession of a drug resistance marker (neo) made it difficult to use for multiple transfections. Attempts to transfect R2 with CRP2 using a selection other than neo resistance were unsuccessful. M1 and 32DC13, cell lines capable of induced myeloid differentiation, also proved difficult to transfect by several different methods. This suggested to us that CRP2 expression was deleterious when ectopically expressed. CRP2 lethality was further confirmed by the low co-transfection frequency of the fibroblast NIH3T3 cell line with a CRP2 expression vector and neo resistance on a seperate vector. Fewer resistant clones were obtained in the cotransfection than with the neo resistant vector alone. CRP3 transfections were more efficient than CRP2. But our desire to transfect 88 Figure 3: Leukocyte alkaline phosphatase staining of P388-Neo and P388C3/C2 (400x magnification). 89 i P388-Neo . 8’ . E P " Ie __ nip-calm. I v I C Figure 3 1.3 90 both CRP2 and CRP3 narrowed our choice of a recipient to P388. P388 did not require special handling nor did it possess any drug resistance markers. The above mentioned CRP2 lethality may be due to the abnormally high expression from the retroviral promoter or from expression in a genetic background incapable of tolerating CRP2 protein. Alternatively, the expression of CRP2 may induce immediate terminal differentiation which leads to a loss of immortalizing elements that allow the cell line to be cultured indefinitely. This problem would be especially acute for granulocytic differentiation due to the short lifespan of normal terminally differentiated neutrophils (~3 days). This cell death by terminal differentiation would be undetectable in the stable transfections that we performed, but might be observed by expressing CRP2 from an inducible promoter. It is apparent that expression of CRP2 and CRP3 have a dramatic effect on the morphology of P388 B lymphoblasts. Several lines of evidence suggested that a macrophage morphology would be the expected result of expression.of these genes in this particular background. The lymphoid P388 cell line was chosen.due to its previously described lineage switch capability to a macrophage morphology with expression of markers such as F} receptor, Mac 1, Mac 2 and Mac 3, and the ability for phagocytosis (Bauer et a1 1986). Also, in chapter 3 of this thesis we showed that CRP2 and CRP3 transfectants of P388 expressed IL-6 and MCP-l in response to LPS stimulation. 91 LPS induction of cytokines IL-6 and.MCP-1 is a common function in macrophages but not granulocytes. It was entirely unexpected that the CRP transfectants of P388 would acquire a morphology and LAP staining more similar to that of neutrophilic granulocytes. Elements of exclusivity in lineage commitment. The monocyte/macrophage and neutrophilic granulocyte lineages are closely related and derive from the same myeloid precursor which is incapablezof lymphoid differentiation (see chapter 1, figure 1). It is possible that CRP2 and CRP3 are involved in differentiation of both lineages (refer to Scott et a1 1992). CRP2 and CRP3 may have different functions depending on which lineage background they are expressed within. {Hue P388 B lymphoblast background may be deficient of elements required for exclusive commitment to either of the two lineages. This may account for the acquisition of both macrophage and granulocyte characteristics by P388. Candidates for such an element might be expression of the id (Benezra et al 1990) or evi-l (Moroshita et al 1992) genes which can inhibit GCSF induced granulocytic differentiation of 32DCl3 cells, or the egr-l gene (Nguyen et al 1993) which inhibits HL-60 differentiation along the granulocyte but not the macrophage lineage. Both evi-l and egr-l are transcription factors and id inhibits certain.bHLH transcription factors. 'The status of the expression of these genes in the P388 transfectants is unknown. Evidence for incomplete differentiation. A number of 92 experiments ‘were performed on. the P388 transfectants to further substantiate their' putative lineage switch to a myelomonocytic cell type and to determine at what point along that lineage they had proceeded. These included cytochemical assays for both specific and non-specific esterases, and myeloperoxidase (data not shown). As a functional assay, the ability to phagocytose latex beads was tested (data not shown). Molecular markers such as GCSF receptor, lactoferrin, and myeloperoxidase were assayed by northern analysis (data not shown). All of the above experiments were negative in showing myeloid markers for the P388 transfectants. Many markers during differentiation, such as lactoferrin and MPO, are expressed only transiently and this may account for the inability to detect these markers. Also, the P388 transfectants may still be relatively immature, not having yet acquired these markers through terminal differentiation. However, the polymorphonuclear appearance and leukocyte alkaline phosphatase expression are both late markers occurring at or near terminal granulocytic differentiation. This data suggests that P388 differentiation in response to CRP2 and CRP3 expression may be partial or incomplete. While probing northern blots for transcripts of myeloperoxidase, GCSF receptor and lactoferrin, we noticed that although a discrete band could not be seen for P388-C2, P388-C3, or P388C3/C2, a smear was evident. This only occurred with genes specific for the granulocytic lineage (data not shown). Other probes used on the'same blot showed 93 discrete bands demonstrating the intact quality of the RNA. This may have been due to the possible accumulation of degradative enzymes in terminally differentiated.granulocytes of the population. These cells might degrade mRNA locally (within one cell) during RNA isolationc 'This may indicate the occurence of differentiating granulocytes among the CRP expressing lymphoblasts. 'These cells may be the ones that are more granulocytic in appearance and stain more intensely with LAP. Positive elements of myelomonocytic differentiation. In addition to the possible lack of lineage suppressive regulatory elements mentioned above, the P388 B lymphoblasts may also lack certain positive elements required for full myelomonocytic differentiation. The expression of CRP2 and/or CRP3 in P388 B lymphoblasts in combination other factors may promote complete myelomonocytic differentiation in these cellsu The genes mentioned in the following discussion may be involved in terminal myelomonocytic differentiation. Expressing them.in«our P388 B lymphoblasts may provide insight into the mechanisms of myelomonocytic differentiation. The transcription factors c-fos, c-jun, jun B, and jun D are all upregulated during M1 myeloblast differentiation (Abdollahi et al 1991). C-fos is induced during normal and leukemic myelomonocytic differentiation (Gonda and Metcalf 1984, Liebermann and Hoffman-Liebermann 1989). Forced expression of c-jun and c-fos enhanced inducible differentiation of M1 myeloblasts. C-fos is slightly 94 upregulated in P388 lymphblasts expressing CRP2 (data not shown): the expression status of c-jun is unknown. The tyrosine kinase genes c-fgr and hck are expressed predominantly in myelomonocytic cells and are associated with early commitment and differentiation (Ziegler et a1 1991, Willman et al 1991). Expression of hck was undetectable by northern analysis in P388 lymphoblasts expressing CRP2 and/or CRP3. The status of c-fgr expression in these cells is unknown. V-raf expression has been shown to be involved in lymphoid to myeloid lineage switch (Klinken et al 1988). The expression of v-Ha-ras can activate c-raf. This may explain the involvement of v-Ha-ras expression in our lineage switch model (see chapter 2). Perhaps activation of c-raf may be required for myelomonocytic differentiation. The status of c- raf and c-ras activation is unknown in P388 lymphoblasts expressing CRP2 and/or CRP3. Of course, temporal gene regulation may be important during myelomonocytic differentiation. Any of the above mentioned genes that influence differentiation may only need to be expressed transiently to contribute to differentiation. Two genes are known to be expressed early in myelomonocytic differentiation, but are not expressed at terminal differentiation. C/EBP (this chapter, Scott et al 1992) and c-myb (Gonda and Metcalf 1984) are such genes. C/EBP is not expressed in P388 lymphoblasts (Figure 1). C-myb expression is detectable in P388 lymphoblasts and is not influenced by 95 ectopic CRP expression. Elements that block myelomonocytic differentiation. Alternatively, the P388 lymphoblasts may express genes that block differentiation. CRP2 and/or CRP3 may only be able to overcome part of this block. Removal of these blocking elements may lead to full differentiation. Candidates for such blocking elements are the c-myb and c-myc protooncogenes. Suppression of these transcription factors has been shown to be linked to terminal differentiaton of M1 myeloblasts (Hoffman-Liebermann and Liebermann 1991a). C-myb and c-myc inhibit induced differentiation when ectopically expressed in M1 myeloblasts (Hoffman-Liebermann and Liebermann 1991b, Selvakumaran et al 1992). C-myb mRNA expression remains unchanged while c-myc mRNA expression is slightly reduced in P388 lymphoblasts when CRP2 and/or CRP3 is expressed (data not shown). Inhibiting the expression of these genes with antisense constructs may possibly remove the block.on complete differentiation. The SCL transcription factor (expressed in M1 myeloblasts, but down-regulated during differentiation) and the Hox-2.4 gene (not expressed in M1 myeloblasts) can both inhibit differentiation of M1 when ectopically expressed (Tanigawa et a1 1993, Blatt et al 1992). The status of these genes is unknown in P388 B lymphoblasts. SCL and Hox-2.4 could possibly be involved in blocking differentiation in P388 B lymphoblasts. Combinatorial gene regulation.in.myelomonocytic cells by CRPs in cooperation with other transcription factors. Until 96 this point in the discussion, I have referred to CRP induced myelomonocytic differentiation in a general way. But as transcription factors, the CRPs act to promote transcription of specific genes. In chapter 3 we demonstrated CRP involvement in expression of genes normally associated with myelomonocytic cell types. Cooperative interactions with other transcription factors may provide an additional level of transcriptional control, including lineage specificity and temporal specificity, of gene expression during differentiation. Direct proteinzprotein interactions of CRPs with other transcription factors have been demonstrated with the NFKB family (Stein et al 1993), glucocorticoid receptor (a transcription factor) (Nishio et al 1993), as well as Spl (P. Johnson personal communication). The Rel homology domain, common to NFKB p65, p50, and C-rel, physically interacts with C/EBP, CRP2, and CRP3. These transcription factors also synergistically stimulate promoters with C/EBP binding sites, while inhibiting promoters with KB binding sites. CRP and NFKB binding sites have been found closely juxtaposed in several promoters including those of the IL-6, IL-8, angiotensinogen, and GCSF (Himes et al 1993) genes and expression of CRP2 and NFKB synergizes to activate transcription of the IL-6 and IL-8 genes (Stein et al 1993). The glucocorticoid response element (GRE) and the CRP binding site are found adjacent in the promoter of the al- acid-glycoprotein gene. Physical interaction of the glucocorticoid receptor' with CRP2 has been shown to ‘be 97 responsible for transcriptional synergy of these two factors (Nishio et al 1993). Several studies focusing on the chicken homologue:of CRP2 (NF-M) (Katz et a1 1993) have shown it to cooperate with other factors involved in transcription of myelomonocytic lineage specific genes. Chicken myelomonocytic growth factor (related to mammalian GCSF and IL-6) promoter activity requires both NF-M and AP-l binding to adjacent regions (Sterneck et al 1992). The myeloid specific mim-l gene promoter contains binding sites for both c-myb and NF-M that are required for full activity (Burk et al 1993). Expression of NF-M and c-myb in heterologous cell types was able to confer mim-l expression (Ness et al 1993). This also induced expression of the lysozyme gene that does not contain a known c-myb binding site but does have a NF-M site. ‘This implies that this combination of transcription factors may also induce a more generalized switch to the myelomonocytic differentiation pathway than either factor alone. 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