Haemopoietic modulation in tumour-bearing animals: enhanced progenitor-cell production in femoral marrow

Altered haematopoiesis in the femoral marrow was observed in mice bearing the Lewis lung carcinoma (LLca). During tumour growth, a marked reduction was observed in the myeloperoxidase-positive cells (granulocytes) of the marrow 7 days after inoculation of the LLca tumour reaching a nadir (17% of control) by day 28. Accompanying this suppression of mature white cells was a gradual expansion of the CFUc-GM compartment followed by an increase in the number of femoral CFUs. Humoral-stimulating activity (HSA) increased through day 14 in the serum of these animals; then returned to control levels by day 28. During this same interval, the more primitive erythroid progenitor (BFUe) compartment expanded to 168% of control, while the more differentiated (CFUe) compartment was reduced (45% of control at day 28). Reductions in both 59Fe-incorporation and erythroblasts/femur confirmed the suppression of erythroid differentiation in marrow during tumour growth. Similar results were observed following the daily injection (188 mg equivalent dose; q 24 hr X 10) of the supernatant prepared from LLca tissue. Marked differences were observed between the response of the spleen and the marrow to the supernatant. The data suggest that the growth of the LLca tumour results in a dissociation of the normal continuity of haematopoietic steady-state differentiation in the marrow of tumour-bearing animals.     

THE EFFECT OF E TYPE PROSTAGLANDINS ON THE PROLIFERATION OF HAEMOPOIETIC STEM CELLS IN VIVO

The ability of protaglandins E₁ and E₂ to stimulate the proliferation of haemopoietic stem cells (CFU_s) was studied in vivo. PGE₂, in a dose range of 10^-4 to 10^-1μg/g body weight and PGE₁ in a dose range of 10^-5 to 10^-1μg/g body weight, produced a rapid cycling wave of CFU_s. The increase in the number of CFU_s in S phase was not followed by a rise in the femoral CFU_s content, and except for a transient increase in femoral CFU_c level, no increase in differentiation was found either. Therefore, it is proposed that haemopoiesis after PG-induced CFU stimulation is ineffective. PGE₂ did not stimulate regeneration of CFU_s in a perturbed state (after sublethal irradiation). All these findings support the idea that PGEs might represent potent stimulators of the haemopoietic stem cells acting in physiological doses. However, if acting concurrently with physiological control systems PGs lead to ineffective haemopoiesis (under normal conditions) or do not exert any measurable effect (after sublethal irradiation).     

PROLIFERATION OF ERYTHROID AND GRANULOCYTE PROGENITORS IN THE SPLEEN AS A FUNCTION OF STEM CELL DOSE

A study of the kinetics of cellular proliferation, in the morphologically unrecognizable haemopoietic progenitor cell compartment, as a function of injected CFU-S dose has been carried out in the spleens of lethally X-irradiated mice using ³H-TdR labelling. Amplification in this proliferating cell compartment was observed to decline as CFU-S dose increased. The number of divisions in the differentiated line arising from CFU-S up to the first appearance of recognizable erythroid precursors were calculated to be 9.2, 12.5, 15 and 17 for the 2, 0.35, 0.05 and 0.007 femur equivalent doses respectively. The growth of cell populations arising from CFU-S was biphasic, with a rapid initial phase having a doubling time of about 6.3 hr, and a slow phase of doubling time around 1 day. Analysis of the rapid phase by the FLM method gave a cycle time of 5.6 hr. Recognizable labelled erythroid precursors were detected at the same time as, or just after, the change in slope of the growth curve. Significant numbers of proliferating (labelled) granulocytes only appeared in the spleens of animals receiving the higher marrow doses (2 and 0.35 femur). The erythroid to granulocyte ratio was also a decreasing function of marrow dose.     

Stimulation of differentiation and proliferation of haemopoietic cells in vitro

A liquid culture system, for haemopoietic cells, has been developed using bone marrow cells alone, or co-cultures of thymus and bone marrow cells, inoculated into four ounce medical bottles. After several days growth, such cultures consisted of an attaching population of cells, forming discrete colonies, and a non-attaching population. In the (co-cultures) there was a 2 X enhancement of monolayer colony development compared with the combined total present in the (marrow alone) plus (thymus alone) cultures. Also, better maintenance of non-attaching cells was seen in the (co-cultures). Normal CFUS and CFUC were present in both the (marrow alone) and the (co-cultures) for at least 14 days. In the (marrow alone) cultures, granulocytes in all stages of development were present for the first week, but by 12 days the culture consisted mainly of mono-nuclear cells. In the (co-cultures), however, at 12 days more than 60% of the cells were granulocytes, in all stages of differentiation. (Co-cultures) established using lethally irradiated thymus cells were not able to support this prolonged myeloid differentiation. By feeding the (co-cultures) it was possible to maintain production of (granulocytic) cells for at least ten weeks, although no fully mature granulocytes were observed. After the second feeding, no CFU_S were detectable, but variable numbers of agar colony forming cells (not classical CFU_C) were present at least for ten weeks.     

Tumour-induced altered gastrointestinal steady-states: absence of MHC restriction in the paraneoplastic gastrointestine

Abstract The growth of a number of experimental rodent tumours including the Lewis lung tumour (LLca) progressively compromises the integrity of the host's gastrointestine by inducing cytokinetic alterations in the small bowel resembling those generally defining the intestinal phase of a graft-versus-host reaction (GVHR). To determine whether the induction of this paraneoplastic gastrointestine (PGI) involves, similar to a GVHR, a disparity between the MHC of the donor (LLca tumour) and the recipient (host), PGI development was evaluated in various LLca tumour-bearing murine strains that were either ‘syngeneic’[C57BL/6 and BL/10 (H-2^b)], ‘semisyngeneic’[B6D2F₁ (H-2^bd) and B6C3F₁ (H-2^bk)] or ‘allogeneic’[C3H/HeJ (H-2^k) and DBA/2 (H-2^d)] to the H-2^b LLca tumour. The temporal appearance and magnitude of a PGI developing in either LLca-syngeneic or semi-syngeneic hosts, but not the allogeneic strains, suggested that the mechanism(s) involved in PGI development, like the GVHR, was restricted by the MHC. Subsequent studies using congenic strains [B10.A (H-2^k) and B10.D2/nSn (H-2^d)], however, demonstrated that the mechanism(s) responsible for the PGI was restricted by the non-MHC loci of the C57BL mouse. These observations were supported by the appearance of a LLca-induced PGI in various B10.A congenic strains carrying mutations at the I-A or I-E/I-J loci of the MHC. Not unlike the intestinal phase of a GVHR, development of the PGI required the participation of enhanced mucosal mast cells which were limited in the WCB6F₁ (S1/S1^d) but not the (+/+) murine strains. These observations are discussed in light of the postulated premature migration of immature thymocytes that accompany tumour growth and their ability to non-specifically enhance (or suppress) cell mediated immune reactions in the host.     

Survival of human bone marrow progenitor cells after freezing: Improved detection in the colony-formation assay

If cryopreserved suspensions of human bone marrow were stimulated by human placental conditioned medium in the same way as fresh unseparated marrows, less than 40% of granulopoietic progenitor cells (CFU_c) was identified. By adding α-thioglycerol (0.6 mM) to the culture medium, the concentration of detectable CFU_c in cryopreserved bone marrow was increased by a factor of 3.4, and the recovery of CFU_c after cryopreservation rose to 90%. The low recovery of CFU_c after freezing in the absence of α-thioglycerol is due to the destruction of accompanying cells. Noncolony-forming cells normally present in the fresh human marrow promote colony formation in cultures stimulated by placental conditioned medium. Their effect can be replaced by α-thioglycerol. It is concluded that, in order to detect all CFU_c independent of the cellular composition of the marrow suspension, this supplement is essential for CFU_c cultures stimulated by conditioned medium.     

ERYTHROID AND GRANULOCYTE-MACROPHAGE PROGENITOR CELLS IN PRE-NATAL 'STEEL' (SlJ/SlJ) ANAEMIC MICE

The erythropietin sensitivities of dissociated cell cultures and explanted fragments of fetal livers of congenitally anaemic Sl^J/Sl^J mice, and their normal littermates, have been compared. The erythropoietin responsiveness of Sl^J/Sl^J foetal liver cells is deficient in both types of culture. The maximum liver complement of erythroid colony forming cells (CFUe) occurs on the 16th day of development when ‘normal’ livers contain approximately 6 × 10⁵ erythroid colony forming cells/liver. In Sl^J/Sl^J fetuses the maximum reached is only 1 × 10⁵. Granulocyte-macrophage colony forming cells (CFU_C) in Sl^J/Sl^J fetal livers are also reduced to approximately 60% of normal numbers. Erythroid colony forming cells are also reduced in the spleen and femoral bone marrow of Sl^J/Sl^J mice in the 2–3 days preceding birth. Granulocyte-macrophage colony forming cells are rare in the femoral marrow of pre-natal Sl^J/Sl^J mice, but their production in the Sl^J/Sl^J pre-natal spleen appears unaffected.     

REGULATION OF HUMAN MYELOPOIESIS BY PROSTAGLANDIN E AND LACTOFERRIN

Studies on human myeloid stem cell proliferation indicate that progenitor cell populations committed to monocytoid differentiation are preferentially inhibited by prostaglandin E (PGE). the addition of PGE but not PGF to day 7 CFU_GM cultures upon initiation results in the dose-dependent inhibition of total colony and cluster formation. Morpholigical analysis of proliferating clones demonstrates that the effect of PGE on total colony and cluster formation results from the selective inhibition of monocyte-macrophage colony forming cells. Mixed monocytoid/neutrophil colony formation was markedly less sensitive and neutrophil and eosinophil colony formation essentially insensitive to the inhibitory effects of PGE. the inhibition of monocytoid colony formation by PGE₁ extends equally well to day 13 CFU_GM, but not to CFU_GM in suspension culture. the observed effects of PGE₁ on monocytoid committed pre-CFU_GM and day 7 and day 14 CFU_GM indicate that sensitivity to inhibition by PGE increases with progenitor cell maturity. Specificity analysis indicates that prostaglandins of the E series (PGE₁, PGE₂) are by far the most active naturally occurring prostanoate compounds inhibiting CFU_GM proliferation. The addition of iron saturated lactoferrin to liquid cultures of human peripheral blood monocytes, inclusion into mononuclear cell feeder layers or addition to agar cultures proliferating in response to endogenously produced CSFs, results in the equivalent inhibition of CSFs necessary for day 7 and day 14 monocytoid and/or neutrophil and eosinophil colony and cluster formation. These results indicate roles for PGE and lactoferrin in myeloid stem cell regulation in vitro and suggest that they may serve as physiological regulators of granulocyte and monocyte proliferation.     

Anethole prevents hydrogen peroxide-induced apoptosis and collagen metabolism alterations in human skin fibroblasts

Thyroid hormone stimulates erythropoietic differentiation. However, severe anemia is sometimes seen in patients with hyperthyroidism, and the mechanisms have not been fully elucidated. Bone marrow is comprised about 2–8 % oxygen, and the characteristics of hematopoietic stem cells have been shown to be influenced under hypoxia. Hypoxia-inducible factor-1 is a critical mediator of cellular responses to hypoxia and an important mediator in signal transduction of thyroid hormone [triiodothyronine (T3)]. The aim of this study was to investigate the effect of T3 on erythropoiesis under hypoxia mimicking physiological conditions in the bone marrow. We maintained human erythroleukemia K562 cells under hypoxic atmosphere (2 % O₂) and examined their cellular characteristics. Compared to that under normal atmospheric conditions, cells under hypoxia showed a reduction in the proliferation rate and increase in the hemoglobin content or benzidine-positive rate, indicating promotion of erythroid differentiation. T3 had no effect on hypoxia-induced erythroid differentiation, but significantly inhibited activin A/erythroid differentiation factor-induced erythroid differentiation. Moreover, GATA2 mRNA expression was suppressed in association with erythroid differentiation, while T3 significantly diminished that suppression. These results suggest that T3 has a direct suppressive effect on erythroid differentiation under hypoxic conditions.     

Influence of Fructose and Fatty-Rich Diet Combined with Vanadium on Bone Marrow Cells

The aim of the study is to investigate the influence of diet treatment on bone marrow cells. Normal male Wistar rats were divided into six groups (n?=?6 per group): control with normal diet (C), increased fructose (31 % w/w in fodder) (Fr) and high fatty (30 % w/w of animal fat in fodder) diet (Fa), and the same diets with vanadium complex ([VO(4,4′ Me₂-2,2′ Bpy)₂]SO₄)?·?H₂O (CV, FrV and FaV). During 5 weeks, the animals had unlimited access to food and water. Immediately after anaesthetizing and sacrificing the animals, bone marrow smears were prepared from the femurs. Different types of cell lines in the animal smears were examined under the microscope: erythroid line, myeloid line, monocytic line, megakariocytic line and lymphoid line. Addition of fructose or animal fat had evident influence on the proportional composition of the bone marrow cells. In erythroid precursors, addition of both investigated products resulted in a statistically significant increase of percentage of this type of cells. A reverse effect was observed for the lymphoid cell line where addition of both tested diets decreased quantity of these cells in comparison to the control diet. In the same lines, addition of vanadium intensified the observed changes. In the case of other types of cell lines, statistically significant changes were not observed.     

Proliferation of erythroid and granulocyte progenitors in the spleen as a function of stem cell dose.

A study of the kinetics of cellular proliferation, in the morphologically unrecongizable haemopoietic progenitor cell compartment, as a function of injected CFU-S dose has been carried out in the spleens of lethally X-irradiated mice using 3H-TdR labelling. Amplification in this proliferating cell compartment was observed to decline as CFU-S dose increased. The number of divisions in the differentiated line arising from CFU-S up to the first appearance of recognizable erythroid precursors were calculated to be 9-2, 12-5, 15 and 17 for the 2, 0-35, 0-05 and 0-007 femur equivalent doses respectively. The growth of cell populations arising from CFU-S was biphasic, with a rapid initial phase having a doubling time of about 6-3 hr, and a slow phase of doubling time around 1 day. Analysis of the rapid phase by the FLM method gave a cycle time of 5-6 hr, Recognizable labelled erythroid precursors were detected at the same time as, or just after, the change in slope of the growth curve. Significant numbers of proliferating (labelled) granulocytes only appeared in the spleens of animals receiving the higher marrow doses (2 and 0-35 femur). The erythroid to granulocyte ratio was also a decreasing function of marrow dose.     

Myeloproliferative sarcoma virus: its effects on erythropoiesis in adult DBA/2J mice

Adult susceptible mice (DBA/2J) infected with MPSV (myeloproliferative sarcoma virus), a defective RNA tumour virus, develop splenomegaly and progressive disruption of the haematologic system culminating in death. The present study was specifically directed toward determining the effects of the virus on erythroid differentiation. Early and late precursor cells (erythroid burst-forming units; BFU-E and colony-forming units; CFU-E, respectively) were evaluated by the ability of bone marrow and spleen cells to form colonies of fully differentiated erythroid cells in vitro. MPSV caused substantial modification of both the BFU-E and CFU-E populations in the bone marrow and spleen of infected animals. Changes were detected in the CFU-E population preceding any significant increase in spleen weight. In the bone marrow, the proportion of CFU-E cells increased almost twofold by days 5-10 after virus infection but decreased by day 15. In the spleen, CFU-E frequency rose 40-fold by days 10-15 and then declined steadily prior to death. At the peak of CFU-E expansion, a small proportion of the population appeared to be erythropoietin (Ep) independent, although there was no evidence of a complete switch to Ep-independence which occurs in Friend virus-induced erythroleukemia. Dose-response curves showed that none of these data could be explained in terms of a changing responsiveness to Ep. However, evidence is presented that indicates that BFU-E from MPSV-infected animals lose or have a reduced requirement for burst-promoting activity (BPA) relative to normal cells although their progeny still need Ep for terminal erythroid differentiation.     

HAEMOPOIETIC STEM CELL (CFU) KINETICS IN THE CULTURE

Haemopoiesis continued for over 2 months in organ culture of embryonal mouse liver, and haemopoietic stem cells (CFU_s) capable of DNA-synthesis were found in it all that time. Between the 10th and 40th day the number of stem cells in the culture was sustained in a steady state. Both in normal and in regenerating adult bone marrow haemopoiesis ceased within a short time in the culture. Induction of proliferation in haemopoietic stem cells combined with undamaged or improved micro-environment resulted in a little better maintenance of CFU_s in the adult bone marrow culture, The results are discussed in the light of current concepts of haemopoietic stem cell regulation.     

Iron kinetics effects of 88 millirads

Rats were irradiated with one tibia shielded (95% marrow exposure), total body exposed (TBI, 100%), and only one tibia exposed (5%), or they were sham irradiated (SI, 0%). Plasma Fe-59 clearance time (T _1/2) and Fe-59 content ratio in the right and left tibia (RT/LT) were assayed to determine the erythroid activity of the overall marrow of the animals and the relative marrow activity in the exposed and shielded tibias, respectively. When a major fraction of the overall marrow was shielded or irradiated, the overall erythroid activity levels were identical to those of the SI and TBI animals, respectively. Interestingly, enhanced normoblastosis was observed in the marrow of the exposed tibia of individual animals exhibiting normal erythroid activity in 95% of the marrow. Conversely, localized marrow with normal erythroid activity was found in a shielded tibia of individual rats, demonstrating an enhanced erythroid activity in a major fraction of the total body. It was concluded that 88 mrad can alter marrow functions in a small isolated skeletal region as effectively as in the whole body, and tandem assays of the Fe-59T _1/2 and Fe-59 RT/LT can facilitate ultra-low-dose X-ray studies involved with partial body exposures.     

Hematopoietic differentiation cell surface antigen switching in the bone marrow of different aged chickens

Abstract. Immune cytolysis and immunofluorescence were used to examine chicken fetal antigen CFA) and chicken adult antigen (CAA) expression on the differentiation/maturation series of definitive erythroid cells obtained from the bone marrow of different aged chickens. We found that erythroid cells undergo changes in CFA/CAA antigenic expression dependent on their differentiation/maturation stage as well as the developmental age of the chicken. All differentiation/maturation stages of erythroid cells in the bone marrow of 12 and 18-day-old embryos express CFA only. Erythroblasts obtained from 7-day post-hatched chickens express either CFA or CAA. All three CFA/CAA phenotypes (i.e., CFA, CAA, and CFA + CAA) are observed in subsequent maturation stages, but only the CFA + CAA phenotype is observed in mature erythroid cells in the bone marrow of 7day post-hatched chickens. Erythroblasts from 62 day post-hatched chickens exhibit all three CFA/CAA phenotypes. Cells in the subsequent maturation stages express various CFA, CAA, or CFA + CAA phenotypes resulting in a majority of the mature erythrocytes expressing both CFA and CAA, and a small population of mature erythrocytes expressing CAA only. Erythroblasts from adult chickens express both CFA and CAA; however, CFA is lost during erythroid maturation resulting in mature erythrocytes which express CAA only. These studies indicate that both the erythroid differentiation/maturation stage and the developmental age of the chicken influence CFA and CAA antigenic expression on erythroid cells undergoing cellular differentiation/maturation in the bone marrow.     

Modulations in mouse hemopoiesis after engraftment with Lewis lung (3LL) carcinoma cells

Hemopoietic changes in male C57BL/6Cum BR mice engrafted with Lewis lung carcinoma (3LL) were evaluated between day 7, when palpable tumors were present, to day 30 postengraftment. All experimental animals demonstrated decreasing hematocrits (down 40% by day 30) with concurrent leukocytosis which by day 30 postengraftment had reached levels 13.4 times normal. The myelocytic/erythrocytic ratio for normal animals was 1:3 (bone marrow: spleen). The ratio for engrafted animals ranged between 10:1 and 40:1. This apparent shift in production priorities is even more significant in light of the fact that femoral bone marrow cellularity had decreased by 33% on day 17. Splenomegaly, evident by day 7, was seven times control by day 17. Clonogenic analysis of erythroprogenitor cell concentrations revealed an inverse relationship between bone marrow and spleen. 27 days after engraftment, splenic populations demonstrated significant increases in colony forming unit-erythroid (115-fold), burst forming unit-erythroid (7.4-fold), whereas bone marrow concentrations had decreased (6-fold). This report suggests that initiation of 3LL tumor in mice results in a change in the degree of hematopoietic priorities and participation of erythroid organs.     

Age-related changes in hematopoietic stem cell populations of a long-lived hybrid mouse

Age-related changes in the number and concentration of pluripotential and unipotential hematopoietic stem cells in the femoral bone marrow and spleen of BC3F₁ mice were investigated. Pluripotential stem cells were assayed by the spleen colony technique, and unipotential stem cells were determined by an agar cloning method and by erythropoietin responsiveness in polycythemic mice. Changes with senescence were observed in the concentration of both uni- and pluripotential stem cells in the bone marrow; the size of the stem cell compartment in the marrow did not change significantly with age. Also, a reduction in the seeding of transplanted spleen colony-forming units into the spleens of aged recipients was demonstrated. The implications of these findings for the kinetics of hematopoietic stem cell proliferation in aged animals are discussed.     

Multiple members of the TNF superfamily contribute to IFN-gamma-mediated inhibition of erythropoiesis

IFN-gamma inhibits the growth and differentiation of erythroid precursor cells and mediates hemopoietic suppression through mechanisms that are not completely understood. We found that treatment of human erythroid precursor cells with IFN-gamma up-regulates the expression of multiple members of the TNF family, including TRAIL and the recently characterized protein TWEAK. TWEAK and its receptor fibroblast growth factor-inducible 14 (Fn14) were expressed by purified erythroblasts at all the stages of maturation. Exposure to recombinant TWEAK or agonist anti-Fn14 Abs was able to inhibit erythroid cell growth and differentiation through caspase activation. Because other members of the TNF family such as TRAIL and CD95 ligand (CD95L) are known to interfere with erythroblast growth and differentiation, we investigated the role of different TNF/TNFR family proteins as potential effectors of IFN-gamma in the immature hemopoietic compartment. Treatment of erythroid precursor cells with agents that blocked either TRAIL, CD95L, or TWEAK activity was partially able to revert the effect of IFN-gamma on erythroid proliferation and differentiation. However, the simultaneous inhibition of TRAIL, TWEAK, and CD95L resulted in a complete abrogation of IFN-gamma inhibitory effects, indicating the requirement of different receptor-mediated signals in IFN-gamma-mediated hemopoietic suppression. These results establish a new role for TWEAK and its receptor in normal and IFN-gamma-mediated regulation of hematopoiesis and show that the effects of IFN-gamma on immature erythroid cells depend on multiple interactions between TNF family members and their receptors.     

Altered colony-forming activities of bone marrow hematopoietic stem cells in mice following short-term in vivo exposure to parathion

This paper describes a study of hematopoiesis in parathion-treated mice. Adult mice (48 C57B1/6) were given a daily dose of parathion (4 mg/kg p.o.) or corn oil vehicle (5 ml/kg p.o.) for 14 days. During the pesticide and the examination period, treated animals showed no signs of poisoning and had normal body weights. On days 2, 5, 7, 9, 12 and 14 following parathion or corn oil, femoral marrow cells were assayed in vitro for granulocyte/monocyte (CFU-gm), erythroid (CFU-e and BFU-e), megakaryocyte (CFU-meg), stromal (CFU-str) and multipotential (CFU-mix) hematopoietic stem cells. Leukocyte counts were elevated on days 2 and 5, while platelet counts were not increased until day 12. No change was observed in either hematocrits or numbers of marrow cells. BFU-e were reduced (23% of control) by day 7, then increased to 137% of control by day 14. CFU-e were reduced (41% of control) on day 9, then increased to 71% of control by day 14. CFU-mix were 130% of control (day 2), then declined to control values by day 5. On days 12 and 14, CFU-mix colonies decreased to 40% of control. CFU-str were reduced at all time points examined. CFU-gm were 123%, 136% and 130% of control on days 7, 12 and 14, respectively, while CFU-meg were increased (145% of control) on day 7. The data suggest that parathion alters the cloning potential of bone marrow precursor stem cells.