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Changes in the amount of sulphated and unsulphated glycosaminoglycans in the spleen and the bone marrow of lethally irradiated mice were followed up to 11 days after irradiation. One day after irradiation, a sharp decrease in the amount of glycosaminoglycans was observed. In the absence of hemopoietic cells, the remaining stromal elements of spleen and bone marrow underwent subsequently marked changes in the amount of the sulphated and unsulphated components of the glycosaminoglycans in both organs. Reconstitution of irradiated mice with bone marrow cells affected the pattern of changes in the amount of glycosaminoglycans. Although no linear correlation could be observed between the amount of glycosaminoglycans and the number of hemopoietic cells present in the hemopoietic organs, these results suggest an interaction between hemopoietic cells and stromal cells in the hemopoietic organs with regard to the glycosaminoglycan metabolism.

The possible relation between haemopoietic activity and glycosaminoglycan levels in the haemopoietic microenvironment was examined in ectopic erythropoiesis, induced by phenylhydrazine treatment, in the liver of adult mice. The hepatic glycosaminoglycan content in the liver was determined biochemically over a period of 9 d following induction of haemolytic anaemia. After induction sulphated glycosaminoglycan levels increased up to d 4, then decreased to subnormal levels on d 5 and returned to normal values on the following days. The pattern of changes in GAG content coincided with changes in the number of CFU-S and with the number of erythroblasts in the liver. This observation fits in with the hypothesis of McCuskey et al (1972) stating that high sulphated glycosaminoglycan levels are favourable for stem cell proliferation, whereas low sulphated glycosaminoglycan levels favour erythropoiesis.

We have compared four assays to detect hemopoietic stromal damage induced by various cytostatic agents in young (4-week old) and adult (12-week old) mice. These assays included: (a) quantitation of the hemopoietic stem cell content of subcutaneously implanted spleens and femurs, (b) quantitation of fibroblastic colony-forming units per femur and spleen, (c) quantitation of the growth of normal hemopoietic progenitor cells in irradiated cytostatic drug-treated mice, and (d) measurement of splenic hemopoietic stem cell accumulation in response to bacterial lipopolysaccharide-induced hemopoietic stress. Busulfan caused a short- and long-term hemopoietic stromal defect. However, the four assays used showed different kinetics and severity of the stromal damage. Cyclophosphamide treatment resulted in a short-term stromal damage which was repaired within one week to three months, depending on the assay used. Methotrexate and vincristine did not cause long-term stromal damage as measured by the four assays used, whereas a short-term splenic stromal damage was detected using the subcutaneous implantation technique. No significant differences in stromal sensitivity to drug treatment were observed between young and adult mice. The presented data suggest that the four assays used to study stromal integrity measure different components of the hemopoietic microenvironment, and indicate that the use of a single assay may well lead to erroneous interpretations.

Serum of lipopolysaccharide(LPS)-treated LPS-high-responder C3H/He mice was shown to increase survival of low-responder C3H/HeJ CFUs in an otherwise serum-free suspension culture by initiating cell cycling. Post-LPS serum of low-responder mice and serum of phosphate-buffered-saline-injected high-responder mice was significantly less effective in this respect. Since prolonged maintenance of CFUs was also found when cell suspensions highly enriched for stem cells were used, it seems unlikely that assessory cells mediated the effect of the post-LPS serum activity on CFUs maintenance. The serum activity did not enhance the stimulatory effect of saturating levels of highly purified stem-cell-activating factor (SAF) on CFUs maintenance in vitro. Upon injection of post-LPS serum from C3H/He mice a relatively small splenic CFUs accumulation in C3H/HeJ mice was observed.

We have studied the in vivo proliferation and differential properties of murine bone marrow cells (BMC) differing in mitochondrial activity. Following centrifugal elutriation, the cells were sorted on the basis of rhodamine-123 (Rh) fluorescence intensity within a predetermined light scatter window. Unfractionated BMC colonized the spleen with a characteristic preponderance of erythrocytic nodules upon infusion into heavily irradiated mice. In contrast, Rh-dull BMC formed few macroscopic spleen colonies up to day 12, but relatively more microscopic colonies when spleens were sectioned, and the majority of the colonies showed megakaryocytic (Meg) and/or granulocytic (G) differentiation. On day 16 many megakaryocytes were found in these spleens, and very large erythroid colonies had developed since day 12. In contrast, Rh-bright BMC were highly enriched for cells forming erythrocytic spleen colonies that disintegrated after day 12, but the percentage of G and Meg colonies was low. Spleens obtained from recipients of Rh-bright cells contained a negligible number of megakaryocytes on day 16. Apparently, the mitochondrial content of cells that form hemopoietic colonies in the spleen related to the onset and duration of their lineage expression, and to the relative sizes of the lineage compartments. Thus, the colony founders of the majority of spleen surface nodules observed on days 8 and 12 are cells with high mitochondrial activity forming transient erythrocytic nodules. Such spleen colonies represent the bulk of the countable nodules that form the basis of the widely applied murine "stem cell" assay.

We studied the ability of the hemopoietic organ stroma to recover from damage inflicted by 5 or 7 Gy gamma radiation administered during a period of stromal growth in 4-week-old mice. Irradiation resulted in an immediate depletion of femoral colony-forming fibroblastic progenitors (CFU-F) down to 10-20% of age-matched control values. A full recovery to normal numbers occurred between 120 and 240 days after irradiation and was followed by a secondary decrease 1 year after irradiation. This secondary decrease was accompanied by a decrease in the femoral CFU-S and CFU-C content. Femoral CFU-F attained normal numbers and it was demonstrated to occur from surviving CFU-F and could not be enhanced or prolonged following infusion of unirradiated bone marrow cells after irradiation. During the transient CFU-F recovery the hemopoietic stroma remained severely damaged as judged by the regenerative capacity of spleen and femur stroma after subcutaneous implantation, and the ability of the spleen to accumulate CFU-S in response to lipopolysaccharide injection. We have reported earlier that in similarly irradiated adult mice, no restoration of femoral CFU-F was observed. This difference between 4-week-old and adult mice could not be explained by a difference in in vitro radiosensitivity of CFU-F or in their in vivo regeneration kinetics following irradiation and subsequent lipopolysaccharide injection. We conclude from these observations that the recovery kinetics of the CFU-F population is different in young and adult irradiated mice, infused CFU-F do not contribute to CFU-F regeneration in an irradiated femur, CFU-F are not the sole determinants of stromal regeneration in femur and spleen following irradiation.

Mouse bone marrow cells (BMC) were subjected to countercurrent centrifugal elutriation and subsequently separated on the basis of light scatter and fluorescence intensity after being labeled with the supravital dye Rhodamine 123 (Rh-123). The sorted cells were then assayed for their in vivo spleen colony-forming ability (day -8, -12, and -16 CFU-S) and their ability to repopulate the bone marrow or spleen over a 13-day period with CFU-S-12, CFU-GM, or nucleated cells. Cells with marrow repopulating ability (MRA), as measured by the ability of the sorted cells to repopulate the marrow with secondary CFU-S-12 or CFU-GM, had low affinity for Rh-123. These cells showed minimal spleen colony-forming ability, and the ratio of MRA to CFU-S-12 in this preparation was 309. Cells with spleen repopulating ability (SRA), CFU-S-16, CFU-S-12, and CFU-S-8 retained increasing amounts of Rh-123, respectively, and CFU-S-8 were almost exclusively found among cells with high Rh-123 affinity. These cells also included about half of all day-12 CFU-S, and the ratio of MRA to day-12 CFU-S was 0. The results show that MRA cells, SRA cells, CFU-S-16, CFU-S-12, and CFU-S-8 can be sequentially ordered on the basis of increasing mitochondrial activity. The data also demonstrate for the first time, and without the application of negative selection by the use of cytostatic agents, that MRA cells are a separate class of primitive hemopoietic stem cells that fully meet the criteria of pre-CFU-S.

A significant volume of reviewed literature in combination with observations presented here suggest that fibroblastic reticulum cells of the hematopoietic stroma may play a decisive role in the regulation of granulopoiesis. The histology of both bone marrow and spleen demonstrates an association between granulopoiesis and fibroblastic reticulum cells. Using in vitro cultured primary fibroblastic cells from murine bone marrow (BM) as antigenic source, we have prepared a hybridoma cell line secreting a monoclonal antibody (mAb) to fibroblastic reticular cells of the nonlymphoid domains in the spleen and BM. The mAb (ER-HR1) was demonstrated to exclusively detect primary fibroblastic cells cultured from spleen and bone marrow and in situ reticular cells with characteristic dendritic morphology and a dispersed distribution pattern. These reticular cells had extensive ramifications in the hemopoietic parenchyma. With the exception of some sparse fibroblastic cells in the dermis of the skin, the mAb did not react with any other cell type or extracellular substance elsewhere in the mouse, nor did it cross-react with human hemopoietic tissues. Support for a role of fibroblastic reticular cells in the regulation of granulopoiesis was obtained from in vitro studies. Primary BM fibroblasts produced granulocyte-macrophage colony stimulating activity (CSA). In contrast, we have been unable to detect CSA production by BM macrophages. Rather, we have found that BM macrophages deplete exogenous CSA from their culture medium. It is concluded that CSA may be produced by fibroblastic cells in adherent BM cultures and that the low CSA levels detected in their medium is due to depletion of fibroblastic cell-derived CSA by macrophages. We conclude that fibroblastic reticulum cells of the hemopoietic stroma carry unique antigenic determinants and together with macrophages, endothelial cells, and lymphocytes contribute to a complex network of cellular interactions providing an integrated microenvironmental control of granulopoiesis.

A tentative characterization of haemopoietic stem cells with respect to their organ distribution, seeding fraction and colony formation in the spleen, radiosensitivity and humoral regulation was attempted in mice heterozygous for the mutant allele Slj and in their normal littermates. Slj/+ mice were characterized by a deficient CFU-s content of the blood and spleen and had slightly lower femoral CFU-s numbers. This CFU-s distribution could not be explained by differences in seeding efficiency 'f' between CFU-s of Slj/+ and +/+ origin in lethally irradiated recipients used in the CFU-s assay. The seeding fraction of CFU-s of +/+ origin did not differ in +/+ and Slj/+ recipients. However, in irradiated Slj/+ recipient mice a 30% decrease was observed in the number of the colonies derived from splenic and femoral CFU-s of both +/+ and Slj/+ origin. The serum level of SHSF (splenic haemopoiesis stimulating factor) was decreased in Slj/+ mice, but significantly increased in Sl/Sld mice, as compared to their respective normal +/+ littermates. Endogenous colony formation in Slj/+ spleens was deficient in comparison to that observed in +/+ spleens, and distinct sex differences were observed. However, mutant and normal CFU-s from spleen and bone marrow had a similar survival following in-vitro gamma irradiation. Femurs and spleens of both Slj/+ and +/+ origin were implanted into both Slj/+ and +/+ hosts. Six weeks later the Slj/+ grafts contained less CFU-s than the +/+ grafts. These data show that the splenic stroma of Slj/+ mice is not defective in its capacity to lodge injected CFU-s but is deficient in its ability to maintain CFU-s under 'steady-state' conditions and stimulate their colony formation in a 'perturbed state'. Some of the characteristics of Slj/+ mice segregate them from Sl/Sld mice, i.e. a deficient splenic CFU-s content, normal seeding fractions 'f' of CFU-s from spleen and bone marrow in the presence of an almost compensated anemia, and decreased serum levels of SHSF. The study of the Slj trait may be a useful extension of the current Sl/Sld model for exploration of hereditary defects in haematopoietic stroma.

Counterflow centrifugal elutriation (CCE) in combination with density flotation centrifugation and fluorescence-activated cell sorting on wheat-germ agglutinin-FITC(WGA)-binding cells within the light-scatter "blast window" were used consecutively to enrich pluripotent hemopoietic stem cells (HSC) in bulk from lipopolysaccharide-stimulated mouse spleen. The medium-to-strong WGA + ve fraction contained 3.10(6) cells isolated from 3-4 X 10(9) spleen cells, with an average of 126% day-12 CFU-S and 65% day-8 CFU-S as calculated on the basis of their seeding fraction, suggesting that virtually all cells represented in vivo macroscopic colony formers. In view of the large differences reported elsewhere between stem cell subsets differing in reconstitutive capacity and secondary stem cell generation ability, we also studied various isolated cell fractions with respect to spleen colony formation, radioprotective ability, and spleen- and marrow- repopulating ability. Day-8 and day-12 CFU-S copurified when isolated by CCE. Cells from a fraction with high affinity for WGA were most highly enriched for their radioprotective ability (RPA) and their ability to repopulate the cellularity of the spleen and femur of irradiated recipients. This fraction contained virtually pure day-12 CFU-S. However, the ability to generate secondary day-12 CFU-S and CFU-GM in irradiated organs was enriched most in the medium WGA + ve cell fraction. MRA and SRA, according to the latter criteria, could therefore be partly separated from day-12 CFU-S and RPA on the basis of affinity for WGA. The data strongly suggest that at least part of all day-12 CFU-S have a high potential to proliferate and differentiate into mature progeny, but a relatively low self-renewal ability, and may therefore not be representative of the genuine stem cell.

Incubation of bone marrow cells (BMC) with neuraminidase (NA) reduces their ability to form colonies in the spleen of lethally irradiated mice. In vivo the largest reduction was observed in the erythrocytic colonies, whereas in vitro an inhibiting effect of NA incubation was observed on the plating efficiency of erythrocyte precursors (CFUE and day 7 BFUE). Masking of the receptors for neuraminidase-treated cell surface determinants in the recipient's body by infection of desialated erythrocytes (NA-Ery) or erythrocyte membrane fragments (NA-Efr) did largely restore the reduced colony formation of NA-BMC with respect to both the total number of spleen colonies and their type. Pretreatment of irradiated host with NA-Ery, but with NA-Efr, led to a slight polycythemia as judged by the number of erythrocytic and undifferentiated colonies as well as by the surface colony diameter. The reduced erythrocytic colony formation of NA-BMC was considerably enhanced in anemic irradiated recipients (from 25-70% of respective controls). Under all experimental circumstances the erythrocytic colony formation of NA-BMC never exceeded that of normal BMC (N-BMC). Further, in normal or anemic recipients whether or not treated with NA-Efr, the diameters of the spleen surface colonies in NA-BMC injected animals were smaller than in recipients of control-incubated BMC. In vitro experimentation indicated that the reduced plating efficiency of CFUE and BFUE following incubation could not be attributed to a decreased sensitivity to erythropoietin. However, day 7 BFUE with deficient cell surface sialic acid residues had a decreased sensitivity to burst promoting activity. Among several other explanations, our data support the possibility that the desialated colony forming cells that give rise to erythrocytic colonies in vivo have higher requirements for early regulatory factors than granulocytic precursor cells. In contrast to the normal postirradiation situation the level of these factors could be increased in recipients, which are bled subsequently to irradiation. Evidence is presented in support of our concept that neuraminidase-treated CFU are fully capable to exhibit normal, although delayed, colony formation in vivo in animals with covered receptors for galactosyl residues.

Mice of the Sl/Sld genotype have an approximately 3-fold higher number of fibroblastoid progenitors (CFU-F) in their spleens than their normal +/+ littermates. Experiments were performed to determine whether the elevated Sl/Sld splenic CFU-F numbers were due to compensatory mechanisms acting in the presence of a functionally abnormal CFU-F population or to a nonspecific response to chronic anemia. Comparison of the functional ability of Sl/Sld splenic fibroblasts to produce granulocyte/macrophage colony-stimulating activity with +/+ splenic fibroblasts demonstrated that there was no difference. Similar results were obtained for Sl/Sld and +/+ femoral fibroblasts. Analysis of CFU-F in W/Wv mice revealed an approximately 3-fold higher number of splenic CFU-F than in either +/+ or heterozygous (W/+ and Wv/+) littermates. Since the anemia in W/Wv mice is attributed to a hemopoietic stem cell defect and that of the Sl/Sld mice is attributed to a microenvironmental defect, we suggest that the increased splenic CFU-F number in Sl/Sld mice is not specifically due to the microenvironmental defect, but is part of a general response to hemopoietic stress.

This paper describes the kinetics of fibroblastic colony forming units (CFU-f) in murine blood after phenylhydrazine-induced haemolytic anaemia and their subsequent migration into haemopoietic organs. Murine blood contained 5.3 +/- 0.8 CFU-f per 10(6) nucleated cells. Absence of particle ingestion and factor VIII-related antigen in addition to the enzyme pattern in CFU-f-derived cells confirmed that these cells did not have a macrophage-like or endothelial nature. Phenylhydrazine treatment of mice resulted in a 3-fold increase in blood CFU-f numbers which was accompanied by increases in blood cellularity and granulocyte-macrophage progenitor numbers. When both partners of CBA/N and CBA/T6T6 mice in parabiosis had been treated with phenylhydrazine, spleens and femoral bone marrow of both mice were shown to contain partner-derived CFU-f. These data suggest that circulating CFU-f represent a stromal cell population which can migrate into haemopoietic organs.

Mouse spleens were separated into white and red pulp fractions and into axial and marginal fractions for analysis of the relative concentration of hemopoietic stem cells (CFU-S) with respect to their spatial distribution in the spleen. Of the total splenic CFU-S population, 80% was in the red pulp fraction. The CFU-S concentration in this fraction was 11 times higher than in the white pulp fraction, which contained lymphoid follicles, periarteriolar sheaths, surrounding marginal zones, and occasional fragments of red pulp areas. Within the red pulp, CFU-S were spatially distributed with high concentrations in the subcapsular regions and decreasing frequency toward the median intersept of the spleen, where the CFU-S frequency was about one-fifth that found in the subcapsular area. The data suggest that marginal zones contain relatively more CFU-S than the follicles themselves.

A possible regulatory action of phagocytic cells on erythropoiesis was investigated by infusion of inert polystyrene latex particles (LAT). LAT appeared to induce changes in the femoral content of erythroid progenitor cells. These changes were most pronounced in primitive erythroid progenitor cells (BFUe) and appeared to be gradually damped in more differentiated populations (CFUe and erythroblasts). LAT did not influence granulocyte/macrophage progenitor cells (CFUc). The effects of LAT could not be attributed to changes in the systemic erythropoietin (EP) concentration. Administration of dexamethason nullified the effect of low doses of LAT, suggesting that phagocytosis of the particles is essential to the observed effects. Erythroid burst formation was previously found to be dependent on a bone marrow associated activity, termed BFA (burst feeder activity). BFA acts as an in vitro inducer of EP-responsiveness in BFUe. In this study it was found that LAT-induced changes in femoral erythroid progenitor cell content were characteristically preceded by corresponding changes in BFA. It was concluded that BFA-associated cells probably play a role in vivo in the early differentiation of erythroid progenitor cells. The present data are interpreted as direct in vivo evidence supporting a two-step regulatory model operating in erythropoiesis and provide evidence that phagocytic cells are a component of the erythroid haemopoietic inductive micro-environment.

The recovery of fibroblastic colony-forming units (CFU-F) in murine bone marrow hemopoietic stroma was studied during eighteen months after 9 Gy lethal total-body irradiation and reconstitution with syngeneic bone marrow cells. After an initial depletion, CFU-F numbers increased from 10% of normal values at three months to 40% at 18 months after treatment, irrespective of graft size and presence of CFU-F in the graft. Fourteen months after treatment 35% of all CFU-F present in the recipients' bone marrow was donor-derived independent of graft size. When mice were treated with high-dose lipopolysaccharide-W three months after irradiation and bone marrow transplantation, CFU-F numbers decreased to hardly detectable levels within one day, and then recovered to normal numbers four weeks later--whereas radiation control mice still had low CFU-F numbers. These data suggest that after lethal total-body irradiation the stroma still contained viable fibroblastic cells that had lost their in vitro colony-forming capacity as a result of radiation damage. In consequence there was no need for replacement of these fibroblastic cells by donor-derived or host-derived CFU-F. Only depletion of CFU-F from the bone marrow, as was induced with lipopolysaccharide, stimulated repopulation of the stroma with colony-forming fibroblastic cells.

In an agar-liquid double-layer colony assay in which myeloid leukemia colony-forming cells require the presence of both the lectin PHA and CSF for in vitro proliferation, colony formation of bone marrow cells derived from patients with a myelodysplastic syndrome (MDS) was studied. In five of 14 MDS and all five leukemic transformed MDS cases, colony formation was found to require both PHA and CSF. Three of these five PHA-dependent MDS cases progressed to overt leukemia within 1 year, one progressed from RA to RAEB, and one patient received AML chemotherapy. PHA-dependent colony formation was associated with higher bone marrow blast counts, but not directly to FAB type or cytogenetic abnormalities. In nine other MDS cases only CSF was required for colony formation. In these PHA-independent cases the course of the disease was stable during the observation time (5-17 months). Two types of colonies were observed in this in vitro system: colonies adherent and colonies nonadherent to the agar underlayer. The former consisted of terminally differentiated myeloid cells, and the latter comprised immature cells. This suggests that the percentage of adherent colonies formed in vitro may be used as a measure for the maturation defect in MDS. However, no correlation was found between the percentage of adherent colonies and progression to leukemia of the MDS cases. Our findings suggest that the dependency on PHA for in vitro colony formation of colony-forming cells in MDS is predictive for the progression to leukemia. However, the in vitro differentiation capacity has no apparent prognostic significance.

An adherent cell line (AP63) derived from murine spleen was characterized as fibroblastic, and several of its properties distinguished it from other adherent cells (i.e., macrophages and endothelial cells). The ability of the AP63 cells to regulate in vitro myelopoiesis was investigated. Medium conditioned by the cell line (CM) induced granulocyte-macrophage (GM) colonies, thus demonstrating the production of colony-stimulating activity by AP63 cells. A relatively large proportion of these colonies had a "tight" morphology and contained many early myeloid cells and cells capable of secondary cluster and colony formation. CM also contained a prostaglandinlike inhibitor of colony formation. Furthermore, AP63 cells inhibited GM colony formation by bone marrow cells in their immediate vicinity, whereas colony formation was stimulated at greater distances. These observations may reflect in vivo regulatory properties of hemopoietic stromal fibroblasts with respect to proliferation and differentiation of GM progenitor cells.

Treatment of mice with high doses of endotoxin (ET) affected the CFU(S) population in bone marrow, spleen and liver. By 7 days after injection the number of CFU(S) in bone marrow had decreased and the number in spleen and liver had increased. The experimental data show that if mice were irradiated and reconstituted with bone marrow 7 days after ET treatment, the lodging pattern of injected cells followed qualitatively the same changes as the changes in numbers of CFU(S) present before irradiation in these organs. However, elevated numbers of CFU(S) cannot be maintained in the liver and can only partly be maintained in the spleen. In retransplantation experiments, CFU(S) from bone marrow and from bone marrow or liver after treatment of mice with ET have the same seeding efficiency in the spleen, indicating that there is no preferential homing of subclasses of CFU(S) in bone marrow or liver. It is concluded that the presence of CFU(S) and hemopoietic tissue may be an expression of the capability of the tissues for the maintenance of lodging CFU, but the initial lodging in these organs is also affected by other trapping mechanisms.

We determined the number of fibroblastoid progenitors (fibroblastoid colony-forming units, CFU-F) in femurs and spleens derived from (CBA X C57BL)F1 mice of different ages. The femoral CFU-F population size increased from 350 at 1 week of age and plateaued at approximately 1900 CFU-F at 8 weeks of age. The mean incidence of CFU-F per 10(6) femoral marrow nucleated cells decreased from 82 at 8 weeks of age to 55 at 70 weeks of age; however, due to an increase in femur cellularity, there was no decrease in the CFU-F population size. The splenic CFU-F population decreased from 1700 at 1 week of age to 180 at 8 weeks of age; no further change was observed in mice up to 70 weeks' old. Analysis of colony-stimulating activity production by fibroblastoid colonies derived from young (6 weeks) and aged (70 weeks) mouse femoral marrow demonstrated no difference. These results indicate that there is no change in CFU-F numbers or fibroblastoid cell colony-stimulating activity production associated with the age-related increase in hemopoietic organ cellularity and hemopoietic progenitor content observed in this mouse strain. There were, however, major changes in the CFU-F population sizes during development of both femoral marrow and spleen in the first 2 months after birth.

Different glycosaminoglycans are described to be involved in processes of cell proliferation and differentiation. To investigate a possible direct involvement of glycosaminoglycans within the haemopoietic organs in erythropoiesis, biochemical separation and quantification of splenic and bone marrow glycosaminoglycans in anaemic and polycythaemic mice were performed. Hyaluronic acid, chondroitin sulphate A, B and C were present in both organs under both conditions. Both in spleen and bone marrow of polycythaemic mice very low amounts of chondroitin sulphate A, B and C, and a higher amount of hyaluronic acid were found in comparison to normal mice. In anaemic mice only the amount of splenic chondroitin sulphate C was found to be lower than in normal mice. It is demonstrated that considerable changes in sulphated and unsulphated glycosaminoglycans occur during erythropoietic stimulation and suppression. The present findings do not indicate a causal relationship between sulphated glycosaminoglycan levels in the haemopoietic organs and the extend of erythropoietic maturation.

Radiation damage to femoral hemopoietic stroma in mice was measured 6 weeks after irradiation and reconstitution with syngeneic bone marrow by quantitation of fibroblastic progenitor cells (CFUF), and by assaying hemopoietic progenitors and CFUF numbers in regenerated subcutaneous femur implants. A dose-dependent effect of radiation on both preimplantation CFUF numbers and implant regeneration was observed. Changes in bone marrow cell (BMC) graft size did not alter these stromal parameters. Therefore, transplanted CFUF, which were present in the BMC graft, did not alter either the CFUF content of femurs or their regenerative capacity. The strong correlation between CFUF numbers and implant regenerative capacity after irradiation, leads us to suggest a function for CFUF in femoral implant stromal regeneration.

Injection of bacterial lipopolysaccharides (LPS) in LPS-responsive mice produces a transient increase of CFUs in spleen and blood but not in bone marrow. The cellular aspects of the mechanism underlying this response of the hemopoietic system to LPS were investigated. Bone marrow cells from LPS-high responder mice (BMC-H) of the C3Heb/FeJ and C57BL/ScSn strains were transferred to lethally irradiated histocompatible LPS-low responder C3H/HeJ and C57BL/10/ScCr mice and vice versa. Six to ten weeks after reconstitution recipient mice were tested with LPS. Six days after injection of LPS, CFUs numbers in blood and spleen of low-responders reconstituted with BMC-H showed a 10-17 fold increase compared with PBS-injected controls. Lethally irradiated LPS high-responders reconstituted with low-responder bone marrow cells (BMC-L) still produced a small but significant increase of splenic and blood CFUs numbers. These results suggest that relatively radioresistant stem cell-derived cells play an important role in the generation of a stimulus inducing the splenic CFUs accumulation following LPS injection. The decrease of femoral CFUs numbers was less prominent in mice reconstituted with BMC-L than in those reconstituted with BMC-H. Thus expression of the LPS locus is evident in both medullary and extra-medullary sites.

A variety of substances can mobilize haemopoietic stem cells (CFUs) into the peripheral blood. In this study the involvement of the complement system in the mobilization process was investigated. Pretreatment of mice with the complement-activating factor of cobra venom (CoF), which lowered the serum C3 levels to 10-25% of the normal value, could completely prevent CFUs mobilization induced by high doses of CoF, endotoxin (ET) from Salmonella typhosa, inulin, zymosan and the proteolytic enzymes proteinase and trypsin. On the other hand, mobilization induced by the polyanions dextran sulphate and the copolymer of polymethacrylic acid and styrene could not be prevented, or at least affected only slightly. There appears to be a relationship between the extent of decomplementation by CoF and the extent of CFUs mobilization induced by ET. The results indicate that certain agents mobilize CFUs via the complement system, whereas other agents induce CFUs mobilization independent of the availability of complement components.

In a patient who developed Richter's syndrome, complex cytogenetic abnormalities of the centroblastic non-Hodgkin lymphoma (NHL) was associated with chemotherapy resistance. The clonal origin of the preexisting chronic lymphocytic leukemia (CLL) and the subsequent NHL cells was investigated. Both cell populations were present in the peripheral blood and could be separated efficiently by counterflow centrifugal elutriation. In the lymph node biopsy mainly NHL centroblasts were found and only a minor population of small lymphocytes. The separated CLL and NHL cells from blood as well as the lymph node cells were found to express mu and kappa Ig chains. Since expression of identical light chains is not synonymous with common clonal origin, Southern blot analysis of the Ig heavy chain genes was also performed, which showed that the two cell populations had identical Ig heavy chain gene rearrangements. Therefore, it was concluded that the CLL cells and the NHL cells in the present case originate from the same precursor cell and that the NHL has to be regarded as a malignant progression of the CLL. These findings are different from our previous report on another patient with Richter's syndrome, in whom the CLL and the NHL represented two unrelated malignancies. Therefore, the occurrence of NHL in Richter's syndrome apparently may represent either a clonal progression of the CLL or a second lymphoid malignancy.

In adult mice suffering from a phenylhydrazine (PHZ)-induced hemolytic anemia, erythropoietic islands were observed in the liver. These islands were studied with the light and electron microscope. Within two days after the beginning of four daily injections of PHZ, erythoid elements appeared in the sinusoids and central veins. A maximum number of erythroblasts was found on day 7. Light and electron microscopic observations revealed that the erythropoietic islands consisted of centrally located macrophages(CM) with a Kupffer cell-like morphology, surrounded by erythroblasts, which were often of the same maturation stage. CM in central veins (CM-V) and in sinusoids (CM-S) were found to have a different morphology. The CM-V phagocytized less circulating red blood cells and were in contact with a smaller number of erythroblasts. Furthermore, the contact areas between erythroblasts and CM-S extended for a much longer distance than those between erythroblasts and CM-V. The progenitor cell for the CM-V is most likely a monocyte, since cells which were morphologically determined as monocytes were found to appear on the first day of the PHZ treatment and differentiated into macrophages within about 2 days. The origin of the CM-S population was less clear, but could be monocytic as well. These data are tentatively explained as a migration of a progenitor of a cellular component of the erythroid micro-environment into the liver after appropriate stimuli. In contrast to fetal liver erythropoiesis, erythroblasts in the adult liver occurred only incidentally extrasinusoidally. Furthermore, specialized membrane contacts between erythroblasts and CM or hepatocytes could not be observed in adult liver. Ferritin could not be detected on the erythroid cell membrane or located in coated vesicles. Also, no ferritin could be observed within or attached to the finger-like processes of CM. The observations suggest that the coated vesicles in erythoid elements are partly exocytotic vesicles and are not specific for ferritin transport. The morphological aspects of PHZ-induced extramedullary erythropoiesis is discussed in relation to the hemopoietic microenvironment.

We investigated the in vitro effects of ASTA-Z-7595, ASTA-Z-7557, ASTA-Z-7654, and 4-hydroperoxycyclophosphamide (4HC) on murine stromal fibroblastoid colony-forming units, committed hemopoietic progenitors (erythroid burst-forming units and granulocyte/macrophage colony-forming units), and pluripotent hemopoietic stem cells assayed by the spleen colony-forming unit (CFU-s) assay. In general, the drugs showed a time-and dose-dependent effect on colony-forming unit survival, and the relative toxicities were in the order in which the drugs are listed above. We found a relative sparing of day 12 CFU-s compared with day 7 CFU-s and committed hemopoietic and stromal progenitors, although colony size of day 12 CFU-s was reduced. Our results support two possible mechanisms for delayed or inadequate hemopoietic reconstitution in clinical studies using bone marrow purged with 4-hydroperoxycyclophosphamide or ASTA-Z-7557, i.e., damage to (a) transplantable stromal cells or (b) the hemopoietic stem cells.

We studied the short- and long-term effects of a fractionated injection of cis-diamminedichloroplatinum (II) (CDDP) upon the haemopoietic stroma and the haemopoietic precursor cell compartment of young and adult mice. The integrity of the stromal microenvironment was investigated using three different assays including quantification of (a) the fibroblastoid progenitor cell compartment, (b) the regenerative capacity after subcutaneous implantation of spleen and femur, and (c) the growth of normal bone marrow progenitors in lethally irradiated CDDP-treated mice. CDDP treatment induced a slight anaemia which lasted for the observation period of 1 year, and could not be restored by infusion of normal bone marrow cells. The population size of haemopoietic progenitors was severely decreased immediately after CDDP treatment and the CFU-S recovery in the bone marrow was slow and temporary. Stromal function was significantly decreased and normalization occurred within approximately 40 d, depending on the stromal parameter measured. Subsequently, the regenerative capacity of the stroma showed a second decrease which was still detected at 1 year. This pattern of stromal damage has not been reported for any other cystostatic agent. Since the other two assays did not detect a second decrement in stromal integrity it is implied that the three stromal assays used detect different stromal functions. We conclude that CDDP treatment of both young and adult mice results in severe short-term damage and a late occurring secondary regenerative defect of the haemopoietic organ stroma.

Phenylhydrazine treatment of normal mice elicited a rise in the numbers of CFU-S in blood, spleen and liver. High numbers of CFU-S were found in blood and liver 4 d after the first phenylhydrazine injection. CFU-S in the liver decreased slowly and were absent after 2 weeks. Blood CFU-S returned to normal levels by day 6, whereas spleen CFU-S numbers remained high upto day 12 with a 20-fold increase being apparent between days 5 and 8. Bone marrow CFU-S numbers were relatively unaffected except for a dip between days 4 and 7 with a nadir at day 5 where numbers decreased to 50% of the control levels. Approximately 40% of liver, spleen and blood CFU-S present on the 4th d after initiation of phenylhydrazine treatment, were killed with a single dose of hydroxyurea whereas bone marrow CFU-S numbers were not significantly reduced by the drug. Splenectomy performed before (21 d) or during phenylhydrazine treatment did not diminish the number of CFU-S found in theliver on day 4. A 3 d interval was observed between peak numbers of CFS-U and erythroblasts in the liver which suggests that hepatic CFU-S are able to undergo differentiation along the erythroid pathway. The presence of maceophages was correlated with that of erythroblasts in the hepatic central veins. These macrophages may be essential to the liver environment for induction of erythropoiesis.

Humoral regulation of CFUs proliferation was investigated in S1/S1d mice characterized by a stromal defect, which severely limits in situ proliferation of in vivo colony-forming cells (CFUs). Injection of LPS-W evoked a large enhancement of CFUs numbers in the spleen of normal +/+ mice. S1/S1d mice were found to be refractory to low doses of LPS-W (up to 15 micrograms/mouse) and to have a diminished response to high doses (up to 150 micrograms). Serum transfer experiments showed that S1/S1d mice are not defective in the early elaboration (6 h) of a humoral factor (SHSF), which mediates the LPS-induced splenic stem-cell accumulation. In a serum-free in vitro system post-LPS S1/S1d and +/+ sera induced a similar degree of CFUs proliferation, indicating the ability of S1/S1d mice to produce normal levels of stem-cell-activating factor (SAF). Transfer of potent post-LPS serum from normal mice evoked a poorer splenic CFUs accumulation in S1/S1d mice as compared to normal +/+ littermates. The population size of splenic stem cells in S1/S1d mice parabiosed with normal +/+ mice also showed a limited increase in response to LPS-W injection. This diminished in vivo response of S1/S1d mice was not due to a decreased sensitivity of their CFUs for SAF, since S1/S1d and +/+ CFUs showed similar survival rates in vitro in the presence of SAF. We propose that the defective response of S1/S1d mice to LPS-induced humoral regulators is due to a nonmigratory component of the S1/S1d splenic stroma, which limits splenic CFUs proliferation either by a short-range inhibitory activity or by a deficiency of a local stimulatory activity or nutrient unlike SAF or SHSF, which might act in synergy with SAF.

We investigated a haemopoietic stromal defect, in mice heterozygous for the Slj allele, during haemopoietic stress induced by treatment with bacterial lipopolysaccharides (LPS) or lethal total body irradiation (TBI) and bone-marrow cell (BMC) reconstitution. Both treatments resulted in a comparable haemopoietic stem cell (CFU-s) proliferation in Slj/+ and +/+ haemopoietic organs. There was no difference in committed haemopoietic progenitor cell (BFU-e and CFU-G/M) kinetics after TBI and +/+ bone-marrow transplantation in Slj/+ and +/+ mice. The Slj/+ mice were deficient in their ability to support macroscopic spleen colony formation (65% of +/+ controls) as measured at 7 and 10 days after BMC transplantation. However, the Slj/+ spleen colonies contained the same number of BFU-E and CFU-G/M as colonies from +/+ spleens, while their CFU-s content was increased. On day 10 post-transplantation, the macroscopic 'missing' colonies could be detected at the microscopic level. These small colonies contained far fewer CFU-s than the macroscopic detectable colonies. Analysis of CFU-s proliferation-inducing activities in control and post-LPS sera revealed that Slj/+ mice are normal in their ability to produce and to respond to humoral stem-cell regulators. We postulate that Slj/+ mice have a normal number of splenic stromal 'niches' for colony formation. However, 35% of these niches is defective in its proliferative support.

The effects of acid mucopolysaccharides (AMPS) on in vitro erythrocytic and granulocytic colony formation of murine bone marrow cells have been studied. High concentrations of chondroitin sulphate A, B and C and heparitin sulphate partly or completely inhibited the response of CFU-E to erythropoietin stimulation whereas addition of heparin, hyalyronic acid and keratan sulphate II in concentrations up to 100 microgram/ml did not elicit an inhibition of erythrocytic colony formation. The granulocytic colony formation was not significantly affected by AMPS-addition under these circumstances. Low concentrations of chondroitin sulphate A and B evoked a stimulatory effect on the CFU-E number. The synthetic polyanion dextran sulphate did not affect the erythrocytic and granulocytic colony formation. It is concluded that AMPS can affect the in vitro erythrocytic proliferation and differentiation in concentrations which do not affect the granulocytic maturation. Since stromal cells, i.e. macrophages and reticular cells, in bone marrow in vivo have the ability to produce and remove AMPS in the extravascular matrix we postulate that stomal cells may be involved in the regulation of erythroid progenitor cell maturation.

We have studied the in vivo spleen colony-forming ability and marrow repopulating ability of murine bone marrow cells differing in mitochondrial activity. Following centrifugal elutriation the cells were sorted on the basis of rhodamine-123 fluorescence intensity within a predetermined light scatter window. It is shown that a class of hemopoietic stem cells exists that differs from the majority of day-12 spleen colony-forming units (CFU-S) in that it has low mitochondrial activity and a high capacity to generate in time new day-12 CFU-S and cells that rescue recipients from radiation-inflicted death. These data add direct evidence for the identity of pre-CFU-S and CFU-S by physical separation.

Several properties of fibroblastic colony-forming units (CFU-F) from murine bone marrow and their in vitro progeny were evaluated. CFU-F had a high buoyant density relative to total bone marrow cells; they were noncycling in situ and adhered to nylon wool. The fibroblastic cells stained positively for fibronectin, lipid, alkaline phosphatase, and nonspecific esterase, while phagocytosis assays were negative, and ultrastructural analysis failed to reveal desmosomes. These properties contrasted bone-marrow-derived fibroblastic cells to both endothelial cells and macrophages. Fibroblastic cells derived from several hemopoietic organs and skin were screened for antigenic determinants present on hemopoietic cells using monoclonal antibodies. Mac-1 and B220 were absent from all fibroblastic cells studied, whereas the Forsmann and Pgp-1 antigens were always present. Thy-1 was not detected on bone-marrow-derived fibroblasts, but was present on fibroblastic cells derived from other sources. T200 was found on all hemopoietic organ-derived fibroblastic cells, but not on those derived from blood and skin. Thus, analysis of antigenic determinants allowed distinction between fibroblastic cells from different organs.