Colony-Stimulating Factors*

Transcription

1 ANNALS OF CLINICAL AND LABORATORY SCIENCE, Vol. 20, No. 1 Copyright 1990, Institute for Clinical Science, Inc. Colony-Stimulating Factors* ENRIQUE PIM ENTEL, M.D. Centro Nacional de Genetica, Instituto de Medicina Experimental, Universidad Central de Venezuela, Caracas 1050-A, Venezuela ABSTRACT The colony-stimulating factors (CSFs) are hematopoietic growth factors (HGFs) involved in regulating the formation of nonlymphoid blood cells. A brief review of the different types of CSFs and their biological properties is presented. The current nom enclature of CSFs and other HGFs is confused by the use of terms and abbreviations based on particular effects on target cells. However, a given cell is frequently recognized by more than one HGF/CSF, and a given HGF/CSF may recognize different types of target cells, including in some cases nonhematopoietic cells. A num berbased nomenclature is universally used for the interleukins. Since the macrophage CSF (M-CSF) and the granulocyte-macrophage CSF (GM- CSF) have been designated previously as CSF-1 and CSF-2, respectively, the author proposes the designation of the granulocyte CSF (G-CSF) as CSF-3. Introduction The colony-stimulating factors (CSFs) are hematopoietic growth factors (HGFs) involved in controlling the formation, from bone marrow progenitors, of blood cells which include neutrophils, eosinophils, basophils, and monocytes. The CSFs w ere discovered on the basis of th e ir specific biological activities as assessed in colony assays in vitro. The CSFs are glycoproteins and, in the last years, most of them have been purified to hom ogeneity and their amino acid sequences have been deduced from the * Send reprint requests to: Enrique Pimentel, MD, POBA International No 33, P.O. Box , Miami, FL respective cloned genes.14 An overview of the factors involved in the regulation of hematopoiesis was published recently in the Annals o f Clinical and Laboratory Science.74 Biological Activities of CSFs The biological activities of CSFs can be assessed through their ability to stim ulate cultured clonogenic hematopoietic progenitors (bone marrow stem cells) to form clusters or colonies of differentiated cells in semisolid medium. The colonies form ed by C SF-stim ulated precursors are re p re s e n te d by clones in w hich im m ature cells give rise to cells that exhibit m orphological and functional ch aracteristics of term in al d ifferentia /90/ $02.00 Institute for Clinical Science, Inc.

2 COLONY-STIMULATING FACTORS 3 7 t io n.66 T h e C S F s a re s p e c ific a lly involved in stimulating the formation of nonlym phoid cells from bone m arrow progenitor cells. However, CSFs exert im portant physiologic actions not only on the proliferation of marrow progenitors b u t also on the effector functions of m atu re blood cells including granulocytes and m acrophages. W hile CSFs secreted by the bone marrow m icroenvironm ent, including endothelial cells and fibroblasts, serve to stim ulate the proliferation and differentiation of m yeloid p ro g e n ito r cells, C S F s p r e s e n t in peripheral tissue sites such as areas of local infection w ould be m ore likely involved in the activation of m ature myeloid cell function.10 The CSFs are pro d u ced by m itogen-activated T lym phocytes as well as by a variety of other cell types including fibroblasts, m onocyte macrophages, and endothelial cells. The CSFs have been classified according to the types of m ature blood cells found in the resulting colonies. W hereas the m acrophage CSF (M-CSF) stim u lates the proliferation of progenitor cells com m itted to the macrophage lineage, the granulocyte CSF (G-CSF) stimulates the proliferation of progenitor cells comm itte d to th e g ran u lo c y te lin eag e. A nother factor, the granulocyte-macrophage CSF (GM-CSF), is not a lineagespecific b u t is a m ultilineage grow th factor, capable of stim ulating the proliferation of both th e m acrophage and granulocyte lineages. Factors M -CSF an d G M -C S F are fre q u e n tly called CSF-1 and CSF-2, respectively. In te r leukin 3 (IL-3) has CSF activity on multiple precursors and can be considered as a m ulti-csf. A nother C SF, the m egakaryocyte CSF, although little characterized as yet, is known to be involved in th e r e g u la tio n of m e g a k a ry o c y to - p o ie sis.65 The p artial purificatio n of megakaryocyte CSF from human urine has been reported recently.88 Several C S Fs ex ert effects on m ore than one blood cell type, and, in addition to their effects on the proliferation and differentiation of specific progenitors, CSFs may have important effects on the expression of differentiated functions by m ature blood cells.139 Moreover, CSFs may have important physiologic effects on nonhem atopoietic cells. The factors responsible for the regulation of CSF production and secretion in vivo are only partially known. Regulation of CSF biosynthesis takes place mainly at the level of transcription and different CSF genes can be selectively induced by specific stim uli in v itr o.133 In suspensions of freshly isolated hum an blood monocytes, IL-3 induces expression of the CSF-1 gene at the messenger RNA (mrna) level as w ell as secretion of C S F-1 b u t has m uch less effect on G-CSF gene expression. In contrast, lipopolysaccharide (LPS) induces expression of the G-CSF gene and secretion of G-CSF, b u t has a considerably lesser effect on expression of the CSF-1 gene. Bacterial endotoxin, IL-1, and tum our necrosis factor-alpha (TNF) stimulate the expression of mrnas for several types of CSFs in endothelial cells, acting through in d ep e n d e n t pathw ays.110 Both IL-1 alpha and TN F induce in vivo serum CSF activity in mice in a dose- and timedependent fashion.135 The T-cell product, interleukin 4 (IL-4), stim ulates in vitro the accum ulation of CSF-1 and G-CSF transcripts in human monocytes, and the stim ulated monocytes release the respective CSF proteins into the culture supernatants.141 The molecular events associated with the cellular actions of CSFs are little known.23 The CSFs seem to stimulate a num ber of consensus biochemical events regardless of the specificity detailed by unique ligand and receptor structures. Although initial m em brane signal transduction events may differ significantly for different types of CSFs, the distal events usually include phosphorylation

3 3 8 PIMENTEL of specific cellular protein substrates on tyrosine and serine-threonine residues, and some of these substrates are shared by more than one CSF. Most or all CSFs stim ulate the expression of specific cellular genes w hich include proto-oncogenes, the ornithine decarboxylase gene, and m em bers of the ancient family of stress response genes. Com plex interactions betw een different CSFs, and between CSFs and other growth factors, are of great importance for the normal control of hematopoietic processes in the intact animal. The stimulatory action of CSFs on hematopoietic cell proliferation may be counterbalanced by factors with growth inhibitory properties. Transforming growth factor beta (TGF-beta) is a potent inhibitor of hem atopoietic cell growth in vitro and may have an important role in modulating the developm ent of the hematopoietic system in vivo.118 The role of C SFs in leukem ogenic processes is a very complex one. The blasts of the majority of acute myelogenous leukem ia (AML) patients do not express CSF-2 and G-CSF transcripts but in some cases an altered pattern of CSF expression is detected, including abnormally large CSF mrnas.12 Most leukem ic cells req u ire an exogenous supply of particular CSFs but in certain cases the leukemic cells may be able to supply partially th eir own grow th facto rs.134 In g en eral, C SFs are active growth factors for human AML cells and their effects are frequently additive in p ro m o tin g m axim um colony size in vitro. M alignant hem atopoietic cells may retain their ability to proliferate and differentiate in vitro in response to one or m ore C SFs in a high p ro p o rtio n of cases.17,71 The CSFs can regulate the growth and differentiation of AML cells even in vivo.60 However, the presence of the receptor for an H G F in a leukemic cell clone does not ensure a biological response. Combined treatm ent of HGFs with other differentiation inducers may be m ore effective than treatm ent with single H G Fs. Biological response m odifiers used for the treatm ent of leukemias may act, at least in part, through the production of CSFs which are capable of in d u cin g term in al d iffe re n tia tio n of m alignant hem atopoietic cells.109 The C SFs m ay have p o te n tia l beneficial properties for the therapy of selected hematologic diseases as well as in AIDS patients and cancer patients after intensive chemotherapy.32 Purified CSFs have considerable promise for therapy of bone marrow failure states. Nomenclature o f CSFs The litera tu re on C SFs and other HGFs is often confused by the use of different names and symbols to designate a single factor. Moreover, the same name has been used for different CSFs. For example, the term granulocyte-macrophage colony-stimulating factor has been used not only to designate G M -CSF itself but also when reference is made to the three factors, G-CSF, M-CSF, and GM-CSF.66 The name of each CSF was selected according to its most peculiar activity, as detected on the occasion of its discovery. However, owing to the diversity of biological actions attrib u ted to CSFs and other HGFs, a number-based nomenclature may be more conveniently applied to HGFs/CSFs than one based on particular physiologic effects. Both M -C SF and G M -C SF are frequently designated as CSF-1 and CSF-2, respectively. H ere I w ould like to propose G-CSF to be designated CSF-3. Colony-Stimulating Factor 1 The CSF-1 (M-CSF) is a lineage-specific H G F which stimulates primarily the survival, proliferation and differentiation of m ononuclear phagocytes and th e ir

4 COLONY-STIMULATING FACTORS 3 9 precursors. The mononuclear phagocyte system, consisting of monocytes and tissue m acrophages, is the major cellular com ponent of the reticuloendothelial system which participates in a variety of physiological and pathological events.128 Phagocytosis by macrophages is essential to m etazoan life, and macrophages are involved in the secretion of a num ber of im portant biological products.81 M acrophages are highly active cells that readily respond to hormonal and cellular signals, exhibiting noteworthy interactions with lymphocytes and participating in im m u nological responses. B io l o g ic a l E f f e c t s o f CSF-1 Both CSF-1 and IL-3 can support macrophage and neutrophil-macrophage colony formation from bone marrow cells of normal mice, but the macrophage colony-forming cells that respond to IL-3 are more primitive than those that are sensitive to CSF-1.50 In addition to its action on progenitor cells in the marrow, CSF-1 has effects on m ature blood cells. The CSF-1 has a direct effect on mature hum an m onocytes, increasing the pro d u c tio n of in te rfe ro n (IF N ), tu m o r necrosis factor (TNF), and myeloid colony stim ulating activity.136 The CSF-1 stim ulates glucose uptake and induces thromboplastin (tissue factor) activity in m u rin e and hum an m acrophages in v itro.37'61 Throm boplastin is the m ost p o te n t physiological trigger of blood coagulation presently known and consists of an integral m em brane glycoprotein, apoprotein III. Macrophages stim ulated by CSF-1 have inhibitory activity for proliferating lym phocytes, w hich suggests that CSF-1 may play a role in im m unoregulatory m echanism s.145 It is known that CSF-1 may have an im portant physiologic role in the pregnant uterus.2,94 In contrast to a two-fold elevation of CSF-1 concentrations in seru m and m ost tissues, preg n an cy results in a 1,000-fold increase in the concentration of uterine CSF-1, which is regulated by chorionic gonadotropin. Studies with in situ hybridization techniques dem onstrated the expression of b oth the CSF-1 and CSF-1 recep to r genes in the m aternal endom etrium of pregnant m ice.100 Expression of CSF-1 receptor mrna is confined to trophoblast cells. T he o n set tim e of CSF-1 receptor expression in the trophoblast and CSF-1 expression in the uterus are very similar, suggesting a functional role for interactions betw een CSF-1 and its receptor in trophoblast developm ent. S t r u c t u r e a n d S y n t h e s is o f CSF-1 The CSF-1 gene is located on human chromosome 5, at region 5q The general structure and com plete nucleotide sequences of the human CSF-1 gene have been d eterm in ed.53 The hum an CSF-1 gene contains 10 exons and 9 introns, which span 20 kilobases (kb) and encode a mature CSF-1 polypeptide of 224 amino acid residues. Recombinant human CSF-1 has been purified in milligram quantities from culture supernatants of SV 40-infected CV-1 m onkey cells that w ere transfected with a plasm id containing the hum an com plem entary DNA (cdna) sequence.36 T he purif ie d p r o d u c t o b ta in e d w ith th is procedure is a dimeric, disulfide-linked glycoprotein of 65 kilodalton (kda). Differential splicing of a large primary transcript of the CSF-1 gene may explain the presence of CSF-1 mrnas of different sizes found in cells producing the factor. A 4-kb mrna transcribed from the hum an CSF-1 gene encodes a 61- kda precursor protein which is very similar in its properties to those of CSF-1 isolated from the hum an u rin e.147 The high molecular heterogeneity observed in the CSF-1 protein results from both pre- and post-translational modifications, including mrna splicing, polypeptide

5 4 0 PIMENTEL glycosylation, and proteolytic processing.112 The several distinct but related species of CSF-1 could m ediate the different biological activities reported for C S F S tu d ies w ith a tru n c a te d cdna encoding a polypeptide lacking th e carboxyl-term inal m em brane-spanning segm ent of the CSF-1 m olecule indicate that CSF-1 amino acid residues are sufficient for biological activity.39 P r o d u c t io n o f CSF-1 The CSF-1 activity is produced by a wide variety of mammalian tissues. The CSF-1 transcripts are undetectable in unstim ulated fresh hum an monocytes, but activation of these cells with IFN - gamma or phorbol ester results in induction of CSF-1 gene expression.99 The CSF-1 transcripts are not detectable in hum an T lymphocytes, either unstim u lated or stim ulated. Many different factors may be involved in controlling the p ro d u ctio n of C SF-1. P ro d u ctio n of CSF-1 by hum an bone marrow stroma cell layers in long-term culture is stim u lated by IL-1.25 The CSF-2 induces transcription and translation of CSF-1 gene in human peripheral blood monocytes.41 This induction occurs during the continued expression of CSF-1 receptor transcripts. Bacterial endotoxin, IL-1, and TN F-alpha stim ulate the expression of the CSF-1 gene in cultures of hum an umbilical vein endothelial cells.110 CSF-1 is produced by some hum an cell lines. The pancreatic carcinoma lines MIA PaCa and PANC secrete high levels of CSF-1.97 These two cell lines stopped secreting CSF-1 when transferred to serum-free medium, but serum-free production of CSF-1 was rein itiate d by treatm ent with phorbol ester. T h e CSF-1 R e c e p t o r The CSF-1 binds specifically to a single class of high-affinity receptors which are present on mononuclear phagocytes and their precursors as well as on myelomonocytic cell lines. Studies with monoclonal an tib o d ies to e p ito p e s in th e extracellular domain of th e CSF-1 receptor protein, using flow cytom etric techniques, indicate that expression of the CSF-1 receptor on normal human cells is restricted to the mononuclear phagocyte lineage.1 The CSF-1 receptor protein was purified from the human BeWo chroiocarcinoma cell line and from normal hum an peripheral blood monocytes by means of a specific monoclonal antibody to phosp h o ty ro s in e.42 T he p u rifie d C SF-1 receptor protein possesses tyrosine-specific kinase activity.151 The CSF-1 receptor p ro tein is encoded by the c-fm s proto-oncogene.114 A hom ologous protein w ith transform ing activity is e n coded by the v-frns oncogene contained in the genome of the Susan McDonough strain of feline sarcoma virus (SM-FeSV). The v-fms and c-fms gene products differ by scattered point m utations in both their ligand-binding and kinase domains and by replacem ent of the 40 carboxylterm inal residues of the c-fms gene product by 11 unrelated amino acid residues. The product of the hum an c-fms/csf- 1 receptor gene is a 140-kDa protein whose expression is restricted m ainly to cells of the m onocyte-m acrophage lineage. H ow ever, th e gene is also expressed at high levels in the normal placenta as well as in hum an choriocarcinoma cell lines.104 The CSF-1 protein expressed by choriocarcinoma cell lines is biochemically indistinguishable from the protein expressed on the surface of norm al hum an peripheral blood mononuclear cells. P o s t r e c e p t o r M e c h a n is m s o f A c t io n o f CSF-1 The molecular events responsible for signal tran sd u c tio n afte r b in d in g of CSF-1 to its receptor on the cell surface

6 COLONY-STIMULATING FACTORS 4 1 are little known. The CSF-1 receptor is phosphorylated on tyrosine in vivo and rapidly degraded in response to CSF-1 binding.20 The activated CSF-1 receptor kinase induces phosphorylation of other cellular proteins on tyrosine, w hich w ould m ed iate th e biochem ical and functional changes that occur in target cells stim ulated by CSF-1. In m urine BAC1.2F5 macrophages, CSF-1 stim u lates the sequential phosphorylation on tyrosine of cellular proteins ranging in molecular weight from 41 to 260 kd a.111 A 56-kDa protein of unknown function is phosphorylated on tyrosine by the action of CSF-1 on the hum an Be Wo choriocarcinom a cell line as well as in m ononuclear leukocytes from human peripheral blood.12 Changes in ion fluxes may be involved in the transmission of the signal initiated by CSF-1 binding to its receptor. The CSF-1 rapidly stim ulates a N a+/k +- ATPase-mediated K + influx in quiescent m ouse bone m arrow -derived m acrophages.130 This effect is transient, and the degree of stimulation is dependent on the dose of CSF-1. The biological relevance of this change is unknown, but th e in tra c e llu la r K + levels m ay be involved in the regulation of the protein biosynthetic m achinery, which ev en tu ally exerts a control on the processes related to DNA synthesis and cell proliferation. The mitogenic actions of growth facto rs are fre q u e n tly asso ciated w ith changes in the expression of specific cellu la r g en es, in clu d in g p ro to -o n co - genes.92 Stimulation of C SF-l-deprived c u ltu r e d m a c ro p h ag e s w ith C S F -1 results in the induction of c-myc and c-fos transcription.76 Addition of CSF-1 to quiescent, synchronized cultures of bone marrow-derived mouse monocytic cells results in the induction of c-fos, c-myc, and c-fgr, but not c-src, c-yes, and c -fm s p ro to -o n c o g e n e s.144 T he kinetics of these inductions are different for each proto-oncogene. High levels of c-m yb proto-oncogene expression are observed during the normal processes of macrophage differentiation.21 The role of proto-oncogene expression in the m echanism of action of CSF-1 is not u n d erstood. CSF-1 induces c-fos expression in macrophages and this effect is associated with phospholipid breakdown, protein kinase C activation, and Ca2+ release.8 However, CSF-l-induced stimulation of proliferation in cells such as m urine bone m arrow-derived m acrophages is apparently independent of increased phosphoinositide turnover.143 R o l e o f CSF-1 in N e o p l a s t ic P r o c e s s e s The role of CSF-1 in neoplastic processes is little known, but activated macrophages may be able to inhibit tum or cell grow th. P re tre atm e n t of m urine p e rito n e a l m acrophages w ith CSF-1 results in a marked augmentation of their tum oricidal capacity.96 The tumoricidal activ ity of h u m an p e rip h e ra l blood m onocytes is enhanced by CSF-1 via mechanisms that include the induction oft N F.107 The effects of CSF-1 on AML blast cells are m ediated through binding to the specific CSF-1 receptor encoded by the c-fms gene. Constitutive activation of the c-/m s/csf-l receptor gene, either by m utation or gene rearrangem ent, could contribute to leukem ia.115 Coexpression of CSF-1 and its receptor would confer the phenotype of constitutively activated c-fms tyrosine protein kinase. Cotransfection of mouse NIH/3T3 cells with a constructed vector carrying the hum an c-fm s proto-oncogene and a cdn A clone encoding the CSF-1 p re cursor results in transformation by an autocrine mechanism.103 However, cells expressing the CSF-1 receptor in a constitutive fashion do not always exhibit a fully transform ed phenotype. Introduction of a cloned human CSF-1 gene into th e m o u se m a c ro p h a g e c e ll lin e

7 4 2 PIMENTEL B A C 1-2F5, w hich ex p re sse s C SF-1 re c e p to rs, re s u lts in a b ro g atio n of growth factor dependence but does not re n d th e cells tu m o rig en ic in n u d e m ice.106 Thus, growth factor independence is not necessarily associated with a tum origenic phenotype. In a study of fresh human AML cells using N o rth ern blot analysis, CSF-1 transcripts w ere detected in 10 of 17 cases, CSF-1 recep to r transcrip ts in seven of 15 cases, and coexpression of CSF-1 and CSF-1 receptor transcripts was observed in five cases; in five additio n al cases, n e ith e r g en e was expressed.98 Coexpression of CSF-1 and its receptor could result in stim ulation of cellular proliferation through an autocrine mechanism. In a study of human myeloid leukem ia using monoclonal antibodies to epitopes in the extracellular domain of the CSF-1 receptor protein and flow cytometric techniques, CSF-1 receptors were detected on the leukemic blasts from 15 of 30 children with AML, compared with four of 26 adults with the disease.1 By contrast, detectable CSF-1 re c e p to rs w ere uniform ly ab sen t on blasts from 19 children with acute lymphoblastic leukemia (ALL). Exposure of hum an leukem ic blast progenitors to CSF-1 in vitro may result in weak stimulation of terminal cell divisions but not in stim ulation of self-renewal of leukemic blast progenitors.123 Thus, CSF-1 is not able to induce the differentiation of leukemic blast progenitors into normal macrophage-monocy tes. Colony-Stimulating F acto r 2 The CSF-2 (GM-CSF) was first identified in crude preparations of conditioned m edia that stim ulated im m ature hem a topoietic cells to proliferate and differentiate in appropriate systems in vitro into m ature granulocytes and macrophages. However, there are several factors capable of controlling the form ation and function of granulocytes and m acrophages. Moreover, the factor originally defined as CSF/-2 may have important effects on a diversity of hem atopoietic and nonhem atopoietic cells. B io l o g ic a l E f f e c t s o f CSF-2 The CSF-2 is required for the formation of colonies of neu tro p h il granulocyte/macrophage progenitor cells in soft agar cultures. Removal of CSF-2 from the m edium results in accumulation of the precursor cells in the Gx phase of the cell cycle whereas readdition of CSF-2 to such quiescent cells is followed by progression of the cells from Gx to S phase with a lag period of 10 hours.93 Both CSF-2 and IL-3 have overlapping but distinct hematopoietic activities on hum an progenitor cells.23 The IL-3 supports the differentiation of a larger num ber of erythroid and megakaryocytic progenitor cells than does CSF-2, while CSF-2 supports m ore m yeloid progenitors. The hem atopoietic effects of a continuous i.v. infusion of IL-3 into p r i m ates are greatly p o ten tia te d by the subsequent administration of a low dose of CSF-2 to the animals.19 These results suggest that IL-3, acting as a multi-csf, expands an early cell population in vivo that subsequently requires the action of a later factor such as CSF-2 to complete its developm ent. The stim ulating activity of CSF-2 on granulocyte/macrophage progenitor cells is counterbalanced by th e action of an inhibitor which is re leased by m ature granulocytes.5 In addition to its effect on progenitor cells, CSF-2 exerts a diversity of effects on m atu re blood cells. B iosynthetic CSF-2 is a powerful stimulator of mature hum an eosinophils and n e u tro p h ils, enhancing the oxidative metabolism and phagocytosis of b a c te ria by n e u tro phils.27,59,140 Purified hum an recom binant CSF-2 stimulates hum an peripheral blood monocytes to becom e tumoricidal

8 COLONY-STIMULATING FACTORS 4 3 against malignant cells such as the A375 hum an malignant melanoma cell line.34 The C SF-2 p roduced by T cells may have a direct action on m ature neutrophils as w ell as lym phokine activity, enhancing the production of antibody by B cells.30,33 Subcutaneous administration of recom binant human CSF-2 to rhesus monkeys results not only in a prom pt (within 24 hours) rise in circulating white blood cells b u t also in enhanced oxidative m etabolism and activation of the phagocytic function of m ature granulocytes.64 N eutrophils from patients w ith the acquired im m une deficiency syndrome (AIDS) can be activated in vitro by incubation with CSF-2.3 Adm inistration of recom binant hum an CSF-2 to cancer patients results in enhancem ent of the cytotoxicity and secretion of TN F and IFN by peripheral blood m onocytes.146 The CSF-2 has been recognized recently as a multilineage growth factor, capable of stimulating the formation of colonies d e riv e d from m ixed p ro g e n ito rs.116 Recombinant m urine CSF-2 can support the form ation of m ultipotential hem atopoietic progenitors that are also supported by IL-3.49 The CSF-2 is a regulator of megakaryocytopoiesis, augmenting the effect of IL-3 in the form ation of m urine megakaryocyte colonies.105 R e c o m b in a n t h u m a n C S F -2 can induce in vitro the formation of colonies from e ry th ro id b u rst-fo rm in g u n its (B FU -E ), eosinophil colony-form ing units (CFU-Eo), and m ultipotential colony forming units (CFU-GEMM). However, CSF-2 has little, if any, capacity for stim ulating the growth of granulocyte colony-form ing units (CFU-G), m acrophage colony-forming units (CFU-M), and granulocyte-m acrophage colonyforming units (CFU-GM), when tested on highly enriched hum an hem atopoietic progenitor cells derived from the bone m arrow of norm al adults.6 The effect of CSF-2 on the developm ent of hum an granulocyte-macrophage colonies in vitro may be indirect and is probably m ediated by accessory cells in the culture. Thus, the designation of this factor as GM-CSF may be, at least for human cells, a m isnom er. M oreover, C SF-2 may exert physiological effects not only on hem atopoietic cells b u t also in a diversity of normal and transformed cells of non-hem atopoietic origin.16 S t r u c t u r e a n d S y n t h e s is o f CSF-2 T he hum an C S F-2 gene has b e e n cloned, sequenced, and expressed.11,148 The predicted hum an CSF-2 polypeptide contains 144 amino acids and shows 54 percent homology with mouse CSF-2. Native human CSF-2 is a glycoprotein of 22 kd a that may contain up to 34 p e r cent carbohydrate by weight. Recom binant hum an CSF-2 produced in Escherichia coli is deglycosylated and displays higher immunoreactivity and bioactivity than native human CSF-2.72 Removal of N-linked oligosaccharides from recom binant human CSF-2 proteins produced in yeast or animal cells results in significant increases in th eir im m unological and biological properties. Expression of the human CSF-2 gene is cis-regulated by 5 '-flanking DNA sequences and depends on the interaction of cell-specific and inducible pro teins with a specific region of the CSF-2 gene prom oter.113 A complex pattern of regulation of CSF-2 expression exists in T cells, and both positive and negative regulatory DNA sequences m ay play critical roles in controlling the expression of the CSF-2 gene in the bone marrow microenvironment and in localized inflam m atory resp o n ses.85 The gene encoding CSF-2 has been localized to hum an chrom osom e region 5q23-31, which is involved in interstitial deletions that occur in the 5q-syndrom e.57 This syndrome is associated with refractory anem ia, low leukocyte count, and ele

9 4 4 PIMENTEL vated platelet counts. Total or partial monosomy of human chromosome 5 has been described in acute nonlymphocytic leukemia. P r o d u c t io n o f CSF-2 The CSF-2 is produced and secreted by various types of cells under the regulation of a diversity of stimuli. Phagocytosis and inflammatory stimuli induce C SF-2 mrna expression in m acrophages through regulatory mechanisms depending on post-transcriptional p ro cesses.125 Cytokines may be involved in the regulation of CSF-2 synthesis. In addition, IL-1 induces the release of CSF-2 from hum an m ononuclear phagocytes.26 Purified T lym phocytes stim u lated by IL-1 secrete CSF-2, and this effect of IL-1 takes place at the level of gene transcription.40 The prostaglandin PG E2 may function in synergy with IL-2 to ilicit CSF-2 production by T cells.95 Substantial amounts of CSF-2 are produced by unstim ulated rat microvascular endothelial cells.62 Recom binant IL-1 and TN F-alpha stim ulate hum an endothelial cells to produce the CSF-2.110,117 Normal and transformed m urine keratin - ocytes produce C SF-2.13 Keratinocytederived CSF-2 may play an im portant role in regulating cutaneous macrophage responses occurring in infections and other cutaneous diseases. In addition, IL-2 stimulates the production of CSF-2 by stroma cells in vitro and probably also in vivo.150 Tumor cells may be able to produce CSF-2. A factor secreted by the human bladder carcinoma cell line HTB9 and capable of contributing to the proliferation of clonogenic blast cells was identified as C S F The ROS clonal rat osteosarcom a cell line secrete CSF-2 u nder the stim ulus of bone-resorbing agents such as parathyroid hormone and LPS.138 The CSF-2-like factors produced constitutively by tumor cells may play a role in the process of metastasization, m ediated through functional suppression of lymphoid tissues of the host.126 T h e CSF-2 R e c e p t o r High-affinity CSF-2 receptors of 130 kda are present in normal and leukemic hum an m yeloid cells.29,89 Fresh blast cells of AML possess CSF-2 receptors, b u t th e s e re c e p to rs are sm a lle r in num b er and affinity than th e CSF-2 receptors found on normal neutrophils.46 The tum or cells from some patients with chronic myelogenous leukem ia (CML) may also express CSF-2 receptors. P o s t r e c e p t o r M e c h a n is m o f A c t io n o f CSF-2 Little is known about the post-receptor m echanism of action of CSF-2. Addition of recom binant hum an CSF-2 to isolated hum an granulocytes in concentrations sufficient to induce maximal stimulation of the proliferation and differentiation of granulocyte-macrophage progenitor cells does not result in alteration of th e restin g tran sm em b ran e potential, the intracellular ph, or the c o n c e n tra tio n of fre e cytosolic c alcium.122 However, the intracellular conc e n tra tio n of C a2+ is in c re a se d by CSF-2, as well as by erythropoietin, in norm al im m ature precursors derived from cultured hum an cord blood erythroid progenitors whose proliferation and differentiation is regulated, at least in part, by CSF-2.70 The CSF-2 induces release of arachidonic acid from the plasm a m em brane w ithin m inutes of addition to culture, which suggests that direct activation of phospholipase C and/ or protein kinase C is part of the cellular response to CSF-2. Stimulation of m urine hematopoietic cell lines w ith C SF-2 results in rep ro ducible increases in the phosphorylation of distinct cellular proteins on tyrosine

10 COLONY-STIMULATING FACTORS 4 5 resid u es.73 Since the CSF-2 receptor does not possess tyrosine protein kinase activity, the CSF-2-stim ulated phosphorylation of cellular proteins on tyrosine residues m ust depend on the action of intracellular tyrosine-specific kinases which could include, at least in part, products of the src proto-oncogene family. The physiological actions of CSF-2 are exerted, at least in part, at the level of gene expression. CSF-2 induces, for e x a m p le, th e e x p re ssio n o f C S F -1 mrna and protein by human peripheral blood m onocytes.41 Expression of protooncogenes may be altered by the action of C SF-2. The factor induces m yelomonocytic leukem ia W EHI-3B cells to differentiate into both monocytes-macrophages and granulocytes in the presence of actinomycin D. This differentiation is accompanied by a decreased expression of the proto-oncogenes c-myc and c-myb and a markedly increased expression of c-fos, w hereas the expression of other proto-oncogenes remains unaltered or is not detectable.31 Expression of c-fos may be tightly correlated with monocyte differentiation in both normal and leukemic cells and may also be required for the expression of some macrophage-specific functions. T he p ro to-oncogene c-fes may be involved in regulatory phenom ena associated with the action of HGFs, in particular with the action of CSF-2.24 The KG-1 hum an m yeloblastoid cell line expresses the c-fes protein and can be induced to differentiate in response to CSF-2, while the K G -la variant of the same line does not express the c-fes protein and, concom itantly, has lost th e capacity to differentiate and respond to CSF-2 and/or pluripotent CSFs. Two other hem atopoietic cell lines, LTBM and IO-3, have an absolute dependence on CSF-2, IL-3, or both for their growth and c-fes protein are expressed by both cell lines. These correlations suggest that the c-fes protein may be involved in the cellular response of myeloid cells to specific HGFs. R o l e o f CSF-2 in N e o p l a s t ic P r o c e s s e s The CSF-2 is frequently required for the establishm ent and continuous proliferation of human AML cells in vitro.55,75 T he C S F-2, and to a slightly lesser extent CSF-3, is capable of supporting the growth of AML cell colonies.132 For m any cases of AM L, C SF-2 can com p letely replace standard conditioned m edia; in o th e r cases, grow th w ith CSF-2 alone is suboptimal, and, in some cases, no growth of AML colony-forming cells is observed at all. Furtherm ore, in some cases the effects of C SF-2 and CSF-3 may be additive in prom oting maximum colony size. C o n stitu tiv e expression of C SF-2 mrna and protein occurs in certain tumors. Using a full-length CSF-2 cdna as a probe, expression of the CSF-2 gene was detected in three of 24 human solid tumors and tum or cell lines and in one of 12 human leukemia cell lines.63 No rearrangem ent of the tumor cell DNA was detected by the CSF-2 cdna probe. C onstitutive expression of transcripts from the CSF-2 gene was detected in the leukemic cells of 11 of 22 cases of AML, whereas no expression of the gene was found in 11 cases of common (pre-b) ALL and four cases of CM L.152 Biologically active CSF-2 was detected in six of 11 cases of AML which were positive for the presence of CSF-2 transcripts. The results of these studies suggest that expression of the CSF-2 gene may contrib u te to the growth of some hum an tumors. Expression of a retroviral vector carrying a cdna coding for CSF-2 in the growth factor-dependent m urine cell line FDC-P1 resulted in proliferation of th e cells in th e absence of exogenous

11 4 6 PIMENTEL CSF-2 and the transfected cells, unlike the parental cells, exhibited tumorigenic properties and produced transplanted leukemias in recipient syngeneic m ice.54 A lth o u g h th e c lo n e d C S F -2 g e n e behaved in these experiments in a way w hich is sim ilar to th at o f active oncogenes, the recipient cells are not normal cells but can be considered as cells that have already undergone at least one of th e changes associated w ith transform a tion. C S F -2 in t h e T r e a t m e n t o f C a n c er an d AIDS Treatm ent of mice with purified recombinant CSF-2 resulted in increased num bers of circulating granulocytes and m acrophages as w ell as in enhanced phagocytic activity of the macrophages.67 A d m in istra tio n o f h u m an C S F -2 to rhesus m onkeys resu lted in th e induction of leukocytosis and the stimulation of phagocytic function of m ature neutrophils.64 Administration of recombinant human CSF-2 to cancer patients treated with high-dose chemotherapy and autologous bone marrow transplantation resulted in accelerated myeloid recovery.7 Administration of CSF-2 to patients with sarcoma during the leukocyte recovering period following chem otherapy resulted in a marked increase in the num ber of granulocyte-macrophage colony-forming units (CFU-GM) and erythroid-burst-forming units (BFU-E) in the peripheral blood.119 Both CFU-GM and BFU-E correspond to hematopoietic stem cells and are normally found in small amounts in human peripheral blood. Treatment of patients w ith refractory carcinoma with recom binant hum an CSF-2 resulted in enhancem ent of the oxidative metabolism (superoxide release) from p o ly m o rp h o n u clear leukocytes while chemotaxis was not changed.45 Infusion of CSF-2 to cancer patients increased the num ber of monocytes and neutrophils in peripheral blood and activated the antitum or potential of peripheral blood monocytes, with secretio n of T N F -alp h a and IF N by these cells.114 These results suggest that CSF-2 may be a useful adjunct in the therapy of patients w ith cancer. N eutrophils from AIDS patients re spond normally to CSF-2 in vitro, and in vivo administration of CSF-2 to AIDS patients is well tolerated and may correct neutrophil functional defects in phagocytosis and intracellular killing of germs such as Staphylococcus aureus,4,35 The num ber of circulating neutrophils increases in AIDS patients treated with CSF-2 in a dose-dependent manner. Colony-Stimulating Factor 3 The CSF-3, previously called G-CSF, was first detected in the mouse through its capacity to stimulate the proliferation of committed precursors in the marrow to form differentiated granulocytes.84 The human analogue of m urine CSF-3 is also able to stimulate granulocytic colony formation in vitro.82 The structure of the hum an and m urine CSF-3 genes and proteins, as well as the functions of these proteins, have been characterized re cently.77 B io l o g ic a l E f f e c t s o f CSF-3 The CSF-3 stimulates the proliferation and differentiation of com m itted precursors in the bone marrow to form granulocyte colonies. W hereas IL-3 supports th e proliferation of the hem atopoietic stem cell population, CSF-3 induces the terminal differentiation of the cells into neutrophilic granulocytes.131 In addition to its gran u lo p o ietic effects, CSF-3 exerts a strong stim ulus for th e functional activity of granulocytes, including th e p ro d u ctio n of alkaline p h o sp h a tase.47,58,108 It has been suggested that CSF-3 would also be capable of stimulât-

12 COLONY-STIMULATING FACTORS 4 7 ing m ultipotential hem atopoietic precursors.68,120,121 However, CSF-3 is a lineage-specific H G F and the effects of CSF-3 on multipotential hematopoietic progenitors reported in some studies are probably due to its synergistic action with other factors such as IL-3.43 Combination of CSF-3 and interleukin 6 (IL-6) enhances the formation of granulocyte/ m acrophages colonies in m ouse bone m arrow cell cultures.101 The efficacy of CSF-3 has been verified in intact animals. Injection of native or recom binant human CSF-3 in mice, ham sters, or m onkeys results in the induction of m arked neutrophilic granulocytosis.15,28,124,142 Daily administration of hum an CSF-3 accelerates the recovery from granulocytopenia induced by total-body irradiation in m ice.48 The CSF-3 may act in concert w ith other monokines, such as IL-1 and TNF, to induce changes in the num ber of circulating leukocytes in endotoxemic rats.127 Studies on healthy volunteers and patients with various disorders using an enzyme immunoassay specific for human CSF-3 indicated that CSF-3 plays an im portant role as a circulating neutrophilopoietin.137 Administration of human CSF-3 to patients with myelodysplastic syndrom es resulted in repair of neutrophil anomalies with enhanced activity of neutrophil alkaline phosphatase.153 The production of superoxide anion (0 2~) by neutrophils is frequently decreased in patients with myelodysplastic syndromes and treatm ent with CSF-3 may induce a significant increase of this anion in most of the treated patients. T reatm ent of patients with small-cell lung cancer with recom binant human CSF-3 administered by continuous infusion resulted in an early fall in peripheral neutrophils, followed by stimulation of proliferation and differentiation of neutrophil precursors in the bone m arrow.9 Neutrophils re leased into the circulation were normal in tests of their mobility and phagocytic activity. The results of these and other studies suggest that CSF-3 may be clinically useful for the treatm ent of prem a- lignant and malignant diseases. S t r u c t u r e a n d S y n t h e s is o f CSF-3 H um an CSF-3 is a 19-kDa glycoprotein com posed of 174 am ino acids.87 Mutagenic analysis of the human CSF-3 p rotein has indicated that m ost m utations located in th e in te rn al and carboxyl-term inal regions of the m olecule abolish its biological activity, w hereas m utants lacking particular amino-terminal am ino acids retain the activity.52 Moreover, some substitutions of aminoterm inal amino acid sequences result in a significant increase in the specific biological activity of the CSF-3 molecule. The chromosomal gene structure and two mrnas coding for hum an CSF-3 were characterized.79 cdna coding for human CSF-3 was cloned from a human squamous carcinoma cell line (CHU-2) which constitutively produces CSF-3. Expression of this cdna in monkey cells gave rise to a protein showing authentic C SF-3 activ ity.78 The hum an C SF-3 gene was also cloned from the bladder c a rc in o m a c e ll lin e 5637 a n d w as expressed in Escherichia coli and the native hum an CSF-3 protein was p urified from this cell line.120 The purified protein is glycosylated and has a molecular weight of 19 kda. The gene coding for CSF-3 was also cloned, by using a synthetic oligonucleotide probe, from a cdna library of LPS-stimulated human peripheral blood m acrophages.51 M olecular hybridization analysis of DNA from norm al hum an leukocytes and C H U -2 cells in d ic a te d th a t th e human genome contains only one gene coding for CSF-3 and that some re a r rangem ent occurred within one of the alleles of th e CSF-3 gene in C H U -2 cells. No structural hom ology was d e tected betw een the hum an CSF-3 pro

13 4 8 PIMENTEL tein and other proteins, including CSF-2 and IL-3.78 The gene encoding CSF-3 is located on hum an chrom osom e 17, at region 17q21-q22, which is involved in translocations frequently associated with a c u t e p r o m y e l o c y t i c l e u k e m i a (APML).44,56 The proto-oncogenes c-erb- A and c-neu/erb-b-2, as well as the NGF receptor gene, are located on hum an chrom osom e reg io n 17q21-q22, b u t these genes are apparently not involved in the developm ent of APML. P r o d u c t io n o f CSF-3 Mouse peritoneal cells can be used to obtain CSF-3.69 Cultured human umbilical vein endothelial cells express CSF-3 transcripts w hen they are stim ulated with LPS, IL-1, or TNF.9 Production of CSF-3 by human bone marrow stromal layers in long-term culture is stimulated by IL-1.25 This factor also stimulates the production of CSF-3 by the human stromal fibroblast cell line ST The CSF-3 is produced by a diversity of cell lines.18 Using an enzym e im m unoassay specific for CSF-3, the factor could not be detected in the serum of most healthy persons, but significantly elevated levels w ere present in patients w ith abnormal absolute neutrophil count.137 High serum levels of CSF-3 w ere found in patients with idiopathic aplastic anemia, Fanconi s anemia, myelodysplastic syndrome, chronic lymphoid leukemia, and chronic lymphoid leukemia as well as in cases of acute leukemia without any blast cells in the blood. In addition, some patients with lung cancer, cyclic neutropenia, malignant lymphoma, and acute infection had high levels of circulating CSF-3. Interestingly, a reverse correlation was found betw een blood neutrophil count and serum CSF-3 level in patients w ith aplastic anem ia. T hese findings support the concept that CSF-3 is one of th e circulating horm ones specific for neutrophilopoiesis. T h e CSF-3 R e c e p t o r The CSF-3 acts by binding to specific, high-affinity receptors expressed on the su rfa c e of C S F -3 -re s p o n s iv e cells p re s e n t in th e bone m arro w.83 The human CSF-3 receptor is a 150-kDa single subunit protein which is expressed on circulating n e u tro p h ils.129 In the m u rin e m y e lo m o n o c y tic c e ll lin e W EHI-3B D +, the CSF-3 receptor is also represented by a single subunit protein of approximate 150 kd a.86 No gross difference exists in CSF-3 binding capacity betw een norm al blast cells and the blast cells of myelomonocytic leukemia (cell line W EH I-3B D + ). M urine and hum an C SF-3 show alm ost com plete biological and receptor-binding crossreactiv ities to norm al and leukem ic murine or human cells.82 POSTRECEPTOR MECHANISM OF ACTION o f CSF-3 Little is known about the postreceptor mechanism of action of CSF-3. Addition of recombinant human CSF-3 to isolated hum an granulocytes in concentrations sufficient to induce maximal stimulation of the proliferation and differentiation of granulocyte-macrophage progenitor cells does not result in alteration of the resting m em brane electric potential, the intracellular ph, or the concentration of free cytosolic c alciu m.122 H ow ever, CSF-3 induces release of arachidonic acid from the plasma m em brane within m inutes of addition to the cell culture, which suggests that direct activation of phospholipase C and/or protein kinase C is part of the cellular response to CSF-3. R o l e o f CSF-3 in L e u k e m o g e n ic P r o c e s s e s L eu k em ic cells m ay have C S F-3 receptors and may respond to CSF-3, suggesting that CSF-3 may contribute to leukem ogenic processes. Both C SF-2

14 COLONY-STIMULATING FACTORS 4 9 and IL-3 act synergistically with CSF-3 in supporting the growth of human AML cells in vitro.90 The results of these studies indicate that AML cells respond to H G F in the same way as normal hematopoietic cells and that stimulation by a second, late-acting growth factor, such as CSF-3, may be required to yield optimal growth. Purified CSF-3 has a high capacity to suppress leukem ic stem cell self-regeneration and induces differentiation in m urine m yeloid leukem ia cell lines.84 Recombinant human CSF-3 can induce the term inal differentiation of cultured m urine and hum an leukem ia cells into m acrophages and granulocytes.120 Recombinant hum an CSF-3 can stimulate in vitro the self-renewal and term inal differentiation of blast progenitor cells from AML patients, although the effects of these progenitors are heterogeneous among different patients.80 Differentiation of hum an leukemic cells in vitro by CSF-3 and other granuloid inducers (retinoic acid or DMSO) is closely linked with the phosphorylation on serine of a 22-kDa cytosolic protein.149 However, the m olecular m echanism of the differentiation of leukem ic cells induced by CSF-3 and other HGFs is unknown. Summary and Conclusions The colony-stimulating factors (CSFs) are hematopoietic growth factors (HGFs) that stim ulate the proliferation and differentiation of nonlym phoid cells. In addition, C SFs may exert im portant effects on the expression of differentiated functions by the m ature blood cells and may have physiologic effects on nonhem atopoietic cells. The genes encoding hum an and m urine C SFs have been cloned and expressed in bacteria and m am m alian cells, and the amino acid sequences of th eir respective protein products have been deduced from the nucleotide sequences of the com plem entary DNA (cdna) copies. The nom enclature of CSFs and other H G Fs is often confusing by the use of different names for the designation of a single factor. The names selected on the occasion of the discovery of CSFs were b a sed on th e m o st p e c u lia r e ffect detected in the assay but are not appropriate for describing the m ultiple physiologic effects of CSFs on hem atopoietic and nonhematopoietic cells. A num berbased nom enclature, as that used u n i versally for interleukins, can be conveniently applied to CSFs. Since the terms CSF-1 and CSF-2 are widely used at p resen t for the m acrophage CSF (M- CSF) and the granulocyte-macrophage CSF (GM-CSF), the author proposes the designation of the granulocyte CSF (G- CSF) as CSF-3. References 1. A s h m u n, R. A., L o o k, A. T., R o b e r t s, W. M., R o u s s e l, M. F., etal: Monoclonal antibodies to the human C S F -1 receptor (c-fms proto-oncogene product) detect epitopes on normal mononuclear phagocytes and on human myeloid leukemic blast cells. Blood 73: , Ba r to c c i, A., P o ll a r d, J. W., and Stanley, E. R.: Regulation of colony-stimulating factor 1 during pregnancy. J. Exp. Med. 164: , Ba l d w in, G. C., G a sso n, J. C., Q u a n, S. G., F leisch m an n, J., et al: Granulocyte-macrophage colony-stimulating factor enhances neutrophil function in acquired immunodeficiency syndrome patients. Proc. Natl. Acad. Sci. U.S.A. 85: , Ba l d w in, G. C., G a sso n, J. C., Q u a n, S. G., F l e is c h m a n n, J., et al: Granulocyte-macrophage colony-stimulating factor enhances neutrophil function in acquired immunodeficiency syndrom e patients. Proc. Natl. Acad. Sci. U.S.A. 85: , B0YUM, A., L0VHAUG, D., KOLST0, A. B., H e l g e st a d, J., and M elby, T. : Colony inhibiting factor in mature granulocytes from normal individuals and patients with chronic myeloid leukemia. Eur. J. Haematol. 38: , Bo t, F. J., van E ijk, L., Sc h ip p e r, P., and L ô w e n b e r g, B.: Human granulocyte-macrophage colony-stim ulating factor (GM-CSF) stimulates im mature marrow precursors but not C FU -G M, C FU -G, or C FU -M. Exp. Hematol. 17: , Br a n d t, S. J., P et e r, W. P., Atw a ter, S. K., K u r t z b e r g, J., et al: Effect of recombinant human granulocyte-macrophage colony-stimulating factor on hematopoietic reconstitution

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