British Journal of Haernatology, 1992, 82, 483-487

Annotation

IN V I T R O STUDIES OF NORMAL AND PATHOLOGICAL ERYTHROPOIESIS

The availability of recombinant haemopoietic growth factors for clinical use owes much to the in vitro bone marrow culture techniques which first helped to characterize their activities on early myeloid progenitors. This statement may appear to be less relevant in the case of erythropoietin whose central role in erythroid stimulation was defined long before the advent of clonal progenitor assays. Despite this it has been clear for many years that, in addition to erythropoietin, other microenvironmental factors operate in vivo to maintain physiological erythropoiesis. Perhaps the best and most widely quoted examples of this are the congenital macrocytic anaemias found in two strains of mutant mice, W/W” and ‘Steel’ (SI/Sld) mice, which have for so long fascinated geneticists and experimental haematologists (Russell, 19 79). These anaemias fail to respond to administered erythropoie- tin but stem cells from SI/Sld mice correct the anaemia in the W/w’ mutants. The reverse is not true but a graft of stroma from W/W” into Sl/SId mutants corrects the anaemia show- ing that in the ‘Steel’ mouse replacement of a missing microenvironmental factor is curative.

The earliest demonstration of an in vitro requirement for such putative additional growth factors was made soon after the first description of erythroid colonies in tissue culture. The two most commonly used techniques for erythroid colony culture involve immobilization of cells either in a plasma clot or in viscous methylcellulose. Although the clot technique results in permanent preparations, methylcellulose permits easier identification of colony types in situ and is the more commonly used method of culture (Ogawa & Leary, 1984). When human bone marrow is cultured for 2 weeks mature colonies appear sequentially. The earliest single clusters of 8- 50 cells are observed at about 7 d but during the ensuing week progressively larger colonies replace these and reach maximal size and haemoglobinization by 14-21 d. The 7 d colonies are derived from what are termed colony forming units-erythroid (CFU-e) and because the later appearing colonies are often, though not exclusively, multicentric they are termed ‘bursts’ and their progenitor a burst forming unit- erythroid (BFU-e). BFU-e but not CFU-e are also observed when peripheral blood mononuclear cells are cultured under the same conditions. Although the presence of erythropoietin was found to be an absolute requirement for all erythroid colony growth the less mature progenitors (BFU-e) were found to require an additional source of stimulation. The nature of this ‘burst promoting activity’ (BPA) has been the subject of intensive investigation and it is now clear that a number of polypeptide growth factors are capable of inducing proliferation and maturation within stem cell and committed progenitor cell compartments in the bone marrow.

Correspondence: Dr C. D. L. Reid, Department of Haematology, Northwick Park Hospital, Watford Road, Harrow HA1 3UJ.

The identification of burst promoting activity. This burst promoting activity (BPA), though first identified in fetal calf serum, was also observed in medium conditioned by a variety of cell types such as T lymphoblasts. lectin stimulated blood mononuclear cells or spleen cells. It was apparent that BPA and erythropoietin were acting sequentially since, provided that BPA was present at the start of culture, BFU-e would survive and proliferate optimally even if erythropoietin addition was delayed by up to 72 h (Iscove, 1978). Since these colony cultures comprise large numbers of different cell populations of which the progenitors form only a tiny fraction (less than 0.1%) BPA might be acting either directly on the progenitors themselves or else indirectly by stimulat- ing the production of growth factors by accessory cells.

Over the last few years two approaches have helped to identify the nature of BPA---on the one hand the preparation of relatively pure progenitors from bone marrow and on the other the elimination of contaminating cytokines in serum by using chemically defined culture conditions. Nathan et a1 (1 9 78a) first demonstrated that peripheral blood BFU-e depended on T lymphocytes for optimal growth in serum replete conditions and other work later extended these observations to show that monocytes and T cells interacted in synergistic manner to promote the growth of these primitive erythroid progenitors (Reid et al. 1981; Linch 81 Nathan, 1984). More rigorous exclusion of extraneous sources of BPA entailed the depletion of mature myeloid and lymphoid cells from bone marrow using panels of antibodies directed against these cells (Sieff et al, 1986; Emerson et al, 1989). These studies showed that only a small minority of BFU-e were capable of proliferation in vitro with erythropoietin alone and these disappeared if erythropoietin was absent during the first 72 h of culture. Optimal growth was restored either by a source of crude BPA or by purified growth factors, recombi- nant GM-CSF or IL-3. These two growth factors were identified as having the ability to maintain viability and to stimulate the proliferation of BFU-e into erythropoietin responsive cells over the first 72 h of culture. It was noted that fetal erythropoiesis differed from this pattern with many more BFU-e responsive to erythropoietin alone. Similar experi- ments in serum deprived conditions (Sieff et al, 1989) showed that the BPA activity of IL-3 was superior to that of GM-CSF. It was observed in this and in other studies (Migliaccio et al. 1988) that IL-3 together with GM-CSF had greater activity than either cytokine acting alone when serum was excluded from culture.

The sequential nature of the actions of IL-3 and erythro- poietin have recently been confirmed by a comparison between colony forming potential and erythropoietin recep- tor density on highly purified peripheral blood BFU-e (Sa- wada et ul, 1990). In order to obtain high purity CFU-e. immature ‘burst’ colonies were plucked after only 7 d of

483

484 Annotution

culture. Of these cells, 77% showed specific binding of isotopically labelled erythropoietin ( IJiI-rEP) compared to only 19% of the initially purified BFU-e. These latter cells remained viable for 4X h in liquid culture with IL-3 and in the absence of erythropoietin. If erythropoietin was also present with IL-3 there was a progressive acquisition of IJiI-rEP binding over the 3 d of culture and the colonies that grew from progenitors sub-cultured into clonal assays became progressively smaller and were identifiable as CFU-e by 72 h.

However. it should not be assumed that BFU-e are entirely unresponsive to erythropoietin. As noted above, a proportion of adult bone marrow BFI:-e are erythropoietin responsive and this has been confirmed by other studies (Dessypris Sr Krantz. 19 84). Umemura rt n l ( 1 989) separated primate bone marrow BFU-e and CFU-e and showed expansion of CFU-e numbers in vitro from BFU-e (but not from CFU-e themselves) following cultures with erythropoietin alone, IL-3 or a combination of the two growth factors. It is noteworthy that two recent studies of the in vivo effects of rHu-erythropoietin on human BFCe (Reid et nl . 1988b: Dessypris et al. 1988) have supported this conclusion and have shown an increase in cycling of these progenitors after patients had received large doses of erythropoietin for the correction of renal anaemia.

The recent cloning of the W and 'Steel' (SI) locus in mice, has shed new light on even earlier events in stem cell commitment (Zsebo et a/, 1990). Mutant alleles of these genes are responsible for the WiWv and SljSld mouse anaemias. The former allele is the proto-oncogene known as c-kit and encodes for a tyrosine kinase transmembrane receptor for the product of the latter gene-variously termed c-kit ligand. stem cell factor (SCF) or mast cell growth factor. Purified SCF corrects the anaemia in 'Steel' mice and is being found to have potent activity on stem cells and progenitors in vitro (Migliaccio et al. 1991: Dai et al, 1991).

Bone marrow cells expressing c-kit have been purified by immunoadherence (Papayannopoulou et 01. 1991) and are progenitors of the myeloid. erythroid and megakaryocytic lineages. A particularly high proportion of these cells appear to be committed to the erythroid lineage (BFU-e) or are multipotent erythroid-myeloid progenitors (CFU-mix). Their colony forming potential in vitro is sensitive to SCF which may also synergize with the other 'early acting' factors IL-3 and GM-CSF in the generation of erythropoietin responsive precursors. The c-kit+ colony forming cells were further found to be CD34'. DR-, CD38' cells expressing the erythroid Ep-1 marker. Other recent reports have confirmed the particular activity of SCF on RFU-e colony size as well as numbers and have also shown activity on a pre-colony forming cell population of CD34+ CD33 - cells which may be identical with the self replicating stem cell compartment responsible for repopulating long-term cultures and for long- term bone marrow engraftment (Bernstein et al. 1991: Broxmeyer et nl. 199 1 ), The cloning of these genes and the identification of their activity in haemopoietic regulation have thus come full circle from the earliest description of the mouse anaemias and promise a greater understanding of the relationships between the stem cell and its microenviron- ment. It is clear that stem cell factor, IL-3 and GM-CSF all

have activity in promoting erythropoiesis although their precise physiological significance still requires clarification.

Haernoglobin production in vitro. There is a switch from fetal to adult haemoglobin production around birth but a small number of F cells persist into adult life and their numbers may increase under certain conditions. Although the evidence suggests that a 'developmental clock' intrinsic to stem cells controls the switch in late fetal life (Stamatoyannopoulos & Nienhuis. 1987) studies in vitro indicate that the levels of gamma globin chain production postnatally are influenced by external factors. Thus adult BFU-e derived colonies express increased levels of HbF though the levels in CFU-e are close to those in the donor (Papayannopoulou et al, 1977). This implies that the potential for HbF production is preserved in early adult erythroid progenitors, is progressively lost during bone marrow maturation in vivo but that in vitro conditions of maturation somehow permit increased expression of gamma globin chain production.

Some studies have suggested that accessory cell derived BPA or recombinant growth factors act directly on progeni- tors to determine HbF synthesis in colony progeny. Thus it appears that HbF production in BFU-e is greater in unfractio- nated cells cultured in fetal calf serum than when monocyte depleted or enriched progenitor cell fractions are cultured under these conditions (Baptista & Reid, 1985: Gabianelli et al. 1990). Exclusion of serum from cultures was also found to reduce HbF synthesis but both IL-3 and GM-CSF stimulation of erythropoiesis in the absence of serum restored this to the original high levels by a direct action on progenitors (Gabianelli et al. 1989, 1990). Curiously this has not been confirmed by other reports that have attributed the regula- tion of HbF expression solely to a n as yet uncharacterized factor in fetal calf serum and have failed to show any effect of these growth factors in its absence (Fujimori et al, 1990: Migliaccio et al. 199 0). The dominant role of such a factor has been supported by evidence that sheep serum may inhibit HbF synthesis (Papayannopoulou et al, 1982).

Thus the issue remains unresolved. It is noteworthy that, i n viw, the erythroid expansion that follows the administra- tion of erythropoietin to baboons increases F cell production and this effect is enhanced by also giving GM-CSF and IL-3 (McDonagh et al, 1989). In man, one study failed to show such a n effect of erythropoietin treatment in patients with sickle cell anaemia (Goldberg et al. 1990). However, a more recent study has documented rising F levels a t high doses of this hormone (Rodgers et aI, 1990). It remains to be seen whether this will be a clinically useful adjunct to hyroxyurea therapy in this condition and whether, as is suggested by the experimental studies, the administration of other growth factors, together with erythropoietin, will result in greater HbF production.

Pathological erythropoiesis Antibody mediated aplasins-pure red cell aplasia (PRCA) and

transient erythroblastopenia in children ( T E C ) . In these dis- orders profound anaemia is usually associated with the absence of erythroid precursors in the marrow. Culture of bone marrow cells in PRCA shows normal CFU-e and BFU-e numbers in 60% of cases but in the remainder numbers are

Annotation 48 5

Virus mediated aplasia-B19parvovirus (HPV) injection. The B19 HPV is the virus responsible for the benign exanthem of childhood called “Fifth’s disease”. A temporal association between B19 HPV infection and aplastic crisis in sickle cell disease led to the first identification of a direct viral toxic effect on haemopoiesis and subsequently the virus has also been associated with transient aplasia in other types of chronic haemolysis (Young & Mortimer, 1984). Mortimer et ul (1983) observed a correlation between serum viral titres and the suppression of CFU-e and BFU-e growth in vifru and subsequent studies (Young et al, 1984) have shown not only that HPV inhibits the growth of CFU-e, obtained from 7 d BFU-e cultures, but they also identified virus in these progenitors by direct immunofluorescence after 24-48 h of culture. Parvovirus particles in crystalline arrays werk seen by electron microscopy within the nucleus of infected CFU-e. Deliberate infection of normal volunteers with B19 caused a significant fall in haemoglobin, disappearance of erythroid precursors in the marrow and of circulating BFU-e (Potter et al, 1987). Oddly the numbers of bone marrow progenitors in these individuals were very variable so that the exact mechanism of erythroid suppression remains uncertain. Although transient erythroid aplasia is the hallmark of B19 infection it seems that chronic aplasia may also occur. There is a reported association between B19 and chronic PKCA in immunologically compromised individuals with defective antibody responses and especially those infected with HIV (Frickhofen et al, 1990). Very recently it has been possible to detect B19 DNA in the sera of eight patients with PKCA and in bone marrow cells of some of these. Some of these patients had ‘idiopathic’ disease and not all had obviously impaired IgM responses (Frickhofen et ul, 199 1). It is possible that B19 may be responsible for a higher proportion of these cases than was previously thought.

Anaemia of chronic disorders. The anaemia in active rheu- matoid arthritis has often been taken as a paradigm of the anaemias that so often accompany chronic infection, inflam- mation and malignant disease. It is now thought to be mainly due to a failure of bone marrow response although erythro- poietin levels may also be sub-optimal in some cases. Inhibition of in vitro CFU-e and BFU-e growth has been demonstrated when progenitors are cultured in serum from patients with anaemia and active disease. It seems that interleukin 1 (IL-1 alpha) and tumour necrosis factor (TNF) may mediate this suppression and both of these cytokines are products of activated macrophages that are found in this disease. The respective roles of inappropriate erythropoietin secretion, activated bone marrow macrophages and humoral inhibitors are still disputed and the topic has been extensively reviewed recently by Baer et a1 (1990).

Endogenous erythroid clones (EEC) and the myeloproliferative disorders (MPD). The response of erythroid progenitors to erythropoietin in culture is abnormal in polycythaemia Vera (Prchal & Axelrad, 1974). Although high concentrations of erythropoietin are generally required for normal in vitro colony growth, a variable proportion of both CFU-e and BFU-e proliferate in the absence of added hormone both in pplycythaemia Vera (PV) and in the other MPD (Reid, 1987). Colonies forming in this way are termed endogenous eryth-

reduced or even absent (Lacombe et al, 1984). Although usually a primary haematological disorder, PRCA frequently occurs in association with thymomas or secondary to other systemic disorders. Following the description of IgG anti- bodies inhibitory to in vitro erythropoiesis in PRCA by Krantz & Kao (1967) it has also been shown that such antibodies may inhibit the growth of CFU-e and BFU-e but not CFU-GM in clonal assays (Dessypris et al, 1984). Though antibody may persist even after successful therapeutic intervention, its removal by plasmapheresis has been associated with norma- lization of erythropoiesis (Messner et al, 198 l). TEC resembles adult PKCA in that 66% of children have IgG antibodies to progenitors that disappear spontaneously during recovery (Dessypris et ul, 1982). In some cases the antibody was shown to be active against CFU-e even after a short incubation in vitro although this activity was complement dependent in only a proportion of cases.

PRCA also occurs in chronic lymphoproliferative disorders (B-CLL and T-CLL). No humoral inhibitors have been demon- strated in these cases but T lymphocytes from both types of patient have been shown to inhibit erythropoiesis in vitro. This topic has recently been extensively reviewed (Dessypris, 1991).

Non-antibody mediated aplasia-Blackfan-Diamond anaemia (BDA). The mechanism of erythroid failure in DBA is complex and still poorly understood. The laboratory findings have been very variable and this has suggested that the disorder may be heterogeneous not just in its responsiveness to steroid therapy but also in its pathogenesis. The numbers of CFU-e and BFU-e are generally though not invariably reduced and early reports suggested inhibition of erythropoiesis by humoral factors, cell mediated suppression or a microenvir- onmentaldefect (Alter, 1987). Nathanetal( 1978b) observed erythropoietin insensitivity of erythroid progenitors and this intrinsic progenitor defect has been further defined recently. Tsai et a1 (1989) showed that there is an increase in the concentration of erythropoietin required to produce 50% of maximal BFU-e growth ([Epo]+max) in this condition and they also demonstrated a relative failure of response to BPA. There is some evidence that IL-3 may enhance growth in vitro (Halperin et al, 1989) though this has not been confirmed in all studies (Bagnara et al, 1991). It now seems that growth factor unresponsiveness in BDA may be due to either a lack of stem cell factor (SCF) or to anomalies of the c-kit receptor itself. Very recently Abkowitz et a1 (1991) have shown that SCF stimulates a three-fold increase in the BFU-e colony numbers generated by either erythropoietin alone or with IL- 3 or GM-CSF and this was accompanied by an increase in colony size. Differing patterns of response were apparent in different patients. In some a reduced sensitivity to SCF suggested an abnormality of c-kit receptor expression where- as in others the normal pattern of response was consistent with possibly reduced SCF production. No gross defect in either the c-kit or SI gene has so far been detected by Southern blot analysis (Olivieri et al. 1991). The nature of any molecular defect of either c-kit or its ligand remains specula- tive: however, these studies open the way not only for a better understanding of the pathology of BDA but also for a possible new approach to treatment of this disorder.

486 Annotatiotl

roid clones and it seems that the progenitors respond to very low levels of erythropoietin rather t han being truly indepen- dent of this hormone. There appear to be two populations of progenitor in PV. one with heightened erythropoietin sensiti- vity (FZC) and one with normal dose response characteristics. Contrary to expectation both populations belong to the same malignant clone (Prchal et al. 1978) suggesting tha t the level of erythropoietin responsilreness m a y be determined late in the erythroid commitment of affected stem cells. Numerous studies have investigated the application of in vitro culture to the diagnosis of polycythaemia. In one study of 3 7 patients with erythrocytosis virtually all those with clear clinical criteria of PV had EEC whereas none of 11 patients with ‘idiopathic erythrocytosis’ showed this feature (Reid et ul. 1988a) . Not only are secondary and idiopathic erythrocyto- sis so differentiated but a French study emphasized tha t t he appearance of EEC may facilitate diagnosis in those patients in w h o m red cell mass is only modestly increased to less t han 2 SD above the normal population mean (Lernoine et nl. 1986). These reports and others (Eridani e t a/, 1983) have confirmed tha t this is an almost constant clonal marker in PV and is therefore useful in differentiating this condition from other causes of polycythaemia. It should be emphasized tha t the demonstration of EEC is the single most useful clinical application of erythroid colony culture and deserves to find a wider application in the diagnosis of PV and primary thrombocythaernia.

Department of Hi~ematolog!{. Northwick Park Hospitul. Harrow

C. D. L. REID

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