Effects of recombinant human tumor necrosis factor (rhTNF) on normal human and mouse hemopoietic progenitor cells

We examined the effects of recombinant human tumor necrosis factor (rhTNF) on normal human and murine granulocyte-macrophage (CFU-gm) and erythroid (CFU-e, BFU-e) progenitor cells. We suppressed in vitro colony formation by human marrow CFU-gm, CFU-e and BFU-e or peripheral blood BFU-e by adding rhTNF to the culture in a dose-related manner. A half-maximal inhibition was observed with 1-10 ng/ml. Leukemic cell line K562 cells were found to be sensitive to rhTNF in the clonogenic colony assay. However, the clonal growth of murine marrow CFU-e and BFU-e colonies was less than 50% inhibited and CFU-gm growth was unaffected even at a concentration of 1,000 ng/ml. We observed slight to moderate inhibition after 24 h pulse exposure of both human and murine-committed progenitors to rhTNF prior to the culture. Intravenous injection of 1 mg/kg of rhTNF caused a marked decrease in marrow erythroid progenitors and consequently caused anemia in the mice. Our data indicate that rhTNF has a suppressive effect on normal human and murine hemopoietic colony formation in vitro and murine erythropoiesis in vivo.     

Effects of inhibitors and activators of protein kinase C on late erythroid progenitor (CFU-e) colony formation in vitro

Protein kinase C (PKC) plays a central role in external signal transduction for many cell types. To examine the involvement of PKC in the control of erythropoiesis, we tested the effects of PKC inhibitors on in vitro colony formation by late erythroid progenitors (CFU-e) from normal and Friend virus-infected mice. Inhibitors of PKC and other kinases (H-7 and H-8) inhibited CFU-e at concentrations which inhibit PKC. HA1004, an inhibitor of the cyclic nucleotide-dependent kinases and a weak inhibitor of PKC, had little effect on CFU-e. In the absence of erythropoietin, a combination of phorbol ester and Ca++ ionophore significantly increased normal CFU-e. These results suggest PKC plays a role in the transduction of regulatory signals for the growth of CFU-e.     

Anti-thoracic duct lymphocyte globulin stimulates human megakaryocytopoiesis in vitro

Anti-thoracic duct lymphocyte globulin (ALG) therapy is effective in patients with aplastic anemia. We examined the effect of ALG on human megakaryocyte progenitor cells (colony-forming unit-megakaryocyte, CFU-Meg) in vitro. Normal human bone marrow mononuclear cells (MNC) were cultured in plasma clots with varying concentrations of ALG or non-immunized horse IgG. After 12 days of culture, significant megakaryocyte colony formation was observed in cultures containing ALG but not in cultures containing non-immunized horse IgG. The peak stimulatory effect seemed to occur with 10-25 micrograms/ml of ALG. When marrow MNC, depleted of adherent and T cells, were cultured in plasma clots with ALG, its stimulatory effect on megakaryocytopoiesis decreased markedly. Finally, it was demonstrated that ALG stimulated marrow MNC to produce a factor stimulatory for CFU-Meg. The in vitro megakaryocytopoietic stimulatory effect of ALG may be related to its clinical efficacy in some patients with aplastic anemia.     

An assay for serum cytotoxicity against erythroid precursor cells in pure red cell aplasia

Several reports have indicated that a circulating serum inhibitor (antibody) is involved in the pathogenesis of acquired pure red cell aplasia (PRCA). In the present study, the pathophysiologic significance of this inhibitor was assessed according to the status of erythroid progenitor cells in the bone marrow. So far, direct proof for the antibody acting against erythroid stemcells was lacking. Employing an "in vitro" assay, erythroid colony forming cell (CFU-e) numbers in PRCA marrow were quantified and the cytotoxic effect of PRCA serum on CFU-e was investigated. It was revealed that the CFU-e population size in the marrow of PRCA patients was severely reduced; at the same time the relative number of myeloid colony forming cells was normal. The serum was demonstrated to contain a factor cell which was cytotoxic to CFU-e, in the presence of complement. The results indicate that inhibition of erythropoiesis in PRCA is achieved by a complement dependent plasma factor which eliminates or inactivates CFU-e and which constitutes an effective block at the precursor cell level in the differentiation pathway of the erythroid line. The data present a practical assay for measuring cytotoxic factors affecting erythroid stem cells.     

Interaction between corticosteroid binding globulin and activated leukocytes in vitro

The interaction between human corticosteroid binding globulin and activated leukocytes is restricted to the granulocyte population, and is characterized by specific proteolytic cleavage of corticosteroid binding globulin which markedly reduces its steroid binding activity. A direct interaction between corticosteroid binding globulin and the activated cells appears to enhance this event, and does not involve cellular internalization of corticosteroid binding globulin or its proteolytic degradation products, which resemble those obtained after incubation of corticosteroid binding globulin with neutrophil elastase. These data suggest that corticosteroid binding globulin interacts with elastase on the surface of activated neutrophils, and may promote glucocorticoid delivery to these cells during inflammation.     

Characteristics of murine yolk sac erythroid progenitors and their population expansion in liquid culture

Remarkable differences were found between late erythroid progenitors (CFU-e) in cultures of murine yolk sac cells and those of fetal liver cells with respect to frequency, erythropoietin responsiveness and colony size. Cultures of yolk sac on day 11 of gestation showed a CFU-e population of lower frequency, less sensitivity to erythropoietin and smaller colony size than those from cultures of day 14 fetal liver cells. As the proportion of CFU-e to BFU-e was much lower in yolk sac than that in fetal liver, 48-96 h liquid culture experiments were done with these cells to examine the capacity of their precursors to generate a certain amount of CFU-e subpopulations. The cultures of yolk sac cells produced large numbers of CFU-e which formed some large-sized colonies but those of fetal liver cells generated only a small amount of CFU-e.     

Effect of recombinant human alpha interferon (INF) and antilymphocyte globulin (ALG) on normal and aplastic anaemia haematopoietic progenitor cell growth.

The effect of recombinant alpha interferon (INF) and of antilymphocyte globulin (ALG) to the colony stimulating factor (CSF) production was examined with in vitro culture of the bone marrow of healthy and of aplastic anaemia (AA) persons. In healthy persons the supernatant of lymphocytes preincubated with PHA and ALG was found to show a stimulating effect to clonogenic properties of marrow progenitors, the mentioned effect being not in proportion to the concentration value. Similar properties are shown by interferon in these persons. In patients with aplastic anaemia, a considerable stimulating ALG effect to the granulocytic formation of colonies and a lesser stimulating effect of interferon were shown.     

In vitro growth of erythropoietic progenitor cells in long-term remission of acute leukemia

In vitro growth of CFU-e and BFU-e in bone marrow and of circulating BFU-e in a group of adult long-term survivors of acute leukemia has been evaluated. Six patients with acute nonlymphoblastic and three patients with acute lymphoblastic leukemia in first continuous remission for more than four years (range 4-12 years) and without maintenance therapy for at least one year were studied. BFU-e and CFU-e growth in patients' bone marrow was not statistically different from a control group of 12 healthy adult volunteers. However, proliferation of BFU-e in peripheral blood of patients was significantly reduced (p less than 0.001). This growth pattern was found in both lymphoblastic and myeloblastic leukemia.     

A new candidate for the regulation of erythropoiesis. Insulin-like growth factor I

The effect of pure human insulin-like growth factor I (IGF I) on the colony formation of late stage erythroid precursor cells (CFU-e) from fetal mouse liver and adult bone marrow was studied in a serum-free culture system. We found that IGF I in physiological concentrations stimulated erythroid colony formation. The combined effect of IGF I and erythropoietin was smaller than the sum of their single effects. The number of colonies induced by IGF I was linearly dependent on the number of plated cells. Our results indicate that IGF I is the first clearly defined mitogen that stimulates the late stages of erythroid differentiation independently of erythropoietin.     

Measurement of human corticosteroid binding globulin by enzyme-linked immunoassay

Thirteen monoclonal antibodies have been raised against corticosteroid binding globulin (CBG). From four of those with highest affinity for the antigen, two were selected for development of a sandwich enzyme-linked immunoassay (ELISA). The sensitivity of the assay was such that 0.7 fmol CBG could be detected. Levels of the binding protein in men (740 +/- 67 nmol/l) and women (690 +/- 103 nmol/l) were not significantly different, while those found during the third trimester of pregnancy (1500 +/- 423 nmol/l) were approximately twice these levels. CBG denatured by heating to 60 degrees C could not be detected by the ELISA.     

Effects of dolichol and dolichyl phosphate on in vitro differentiation of hematopoietic progenitors

The exogenous addition of dolichyl phosphate (Dol-P), an active form of dolichol (Dol) that carries oligosaccharide chains for protein-N-glycosylation, significantly enhanced colony formation of mouse bone marrow hematopoietic progenitors (CFU-e, BFU-e, and CFU-gm) was stimulated by erythropoietin (Epo) and colony-stimulating factor (CSF), but Dol enhanced colony formation of CFU-e only. The effects of Dol or Dol-P on these hematopoietic progenitors were fully dependent on stimulation by Epo or CSF. Other mevalonate-metabolites, such as cholesterol, coenzyme Q10, and isopentenyladenine, had no effect on hematopoietic progenitors. These studies suggest that exogenous Dol-P enhances the frequency of differentiation of hematopoietic progenitors stimulated by Epo or CSF, and there may be a diversity in cellular response of these progenitors to Dol.     

Clinical course after corticosteroid therapy in IgG4-related aortitis/periaortitis and periarteritis: a retrospective multicenter study

Introduction

Immunoglobulin G4 (IgG4)–related aortitis/periaortitis and periarteritis are vascular manifestations of IgG4-related disease. In this disease, the affected aneurysmal lesion has been suspected to be at risk of rupture. In this study, we aimed to clarify the clinical course after corticosteroid therapy in IgG4-related aortitis/periaortitis and periarteritis.

Methods

We retrospectively evaluated clinical features, including laboratory data, imaging findings and the course after corticosteroid therapy, in 40 patients diagnosed with IgG4-related aortitis/periaortitis and periarteritis on the basis of periaortic/periarterial radiological findings, satisfaction of the comprehensive diagnostic criteria or each organ-specific diagnostic criteria, and exclusion of other diseases.

Results

The patients were mainly elderly, with an average age of 66.4 years and with a marked male predominance and extensive other organ involvement. Subjective symptoms were scanty, and only a small proportion had elevated serum C-reactive protein levels. The affected aorta/artery were the abdominal aortas or the iliac arteries in most cases. Thirty-six patients were treated with prednisolone, and the periaortic/periarterial lesions improved in most of them during the follow-up period. Two (50.0%) of four patients with luminal dilatation of the affected lesions before corticosteroid therapy had exacerbations of luminal dilatation after therapy, whereas none of the twenty-six patients without it had a new appearance of luminal dilatation after therapy.

Conclusions

The results of this retrospective multicenter study highlight three important points: (1) the possibility of latent existence and progression of periaortic/periarterial lesions, (2) the efficacy of corticosteroid therapy in preventing new aneurysm formation in patients without luminal dilatation of periaortic/periarterial lesions and (3) the possibility that a small proportion of patients may actually develop luminal dilatation of periaortic/periarterial lesions in IgG4-related aortitis/periaortitis and periarteritis. A larger-scale prospective study is required to confirm the efficacy and safety of corticosteroid therapy in patients with versus those without luminal dilatation and to devise a more useful and safe treatment strategy, including administration of other immunosuppressants.     

Complement-dependent cytotoxic factor to megakaryocyte progenitors in sera from patients with idiopathic thrombocytopenic purpura

The influence of sera from patients with idiopathic thrombocytopenic purpura (ITP) was examined on colony formation from megakaryocyte (M) progenitors. Though incubation of marrow cells in Iscove's modified Dulbecco's medium (IMDM) containing 50% sera from several ITP patients stimulated M-colony formation in 8 of 13 cases, incubation in the sera from the patients and in baby rabbit serum as a source of complement significantly suppressed the colony formation. Experiments showed that sera of immunoglobulin G from ITP patients had significant complement-dependent cytotoxicity to M-progenitors in normal marrow cells or in the marrow cells from corresponding patients, but not to CFU-e, BFU-e or CFU-gm. Cytospin preparations of individually collected M-colonies from marrow cells treated with ITP patients' sera and complement revealed a reduction of megakaryocyte colonies containing cells of multilineages. These results indicate that autoantibodies detected in ITP patients can bind not only to platelets and megakaryocytes, but may also bind to M-progenitors.     

Effects of recombinant interferons on the clonogenic growth of leukemic cells and normal hemopoietic progenitors

We have examined the effects of recombinant immune and leukocyte interferons (rIFN-gamma and rIFN-alpha) on the clonogenic growth of leukemic cells and normal hemopoietic progenitors using in vitro colony assays. Both interferons suppressed the colony formation by granulocyte-macrophage progenitors (CFU-gm) and erythroid progenitors (CFU-e and BFU-e) in a dose-dependent manner. Six myeloid leukemic cell lines were less sensitive to rIFN-gamma than CFU-gm. The colony formation of some myeloid leukemic cell lines was suppressed more potently by rIFN-alpha than by CFU-gm. Four lymphoid leukemic cell lines of the T-cell type were very resistant to both recombinant interferons. Reduced sensitivity of leukemic cells to rIFN-gamma, a possible hemopoietic regulator, may explain partially the unregulated proliferation of leukemic cells in vivo.     

Effect of recombinant human interleukin 3, granulocyte-macrophage colony-stimulating factor and granulocyte colony-stimulating factor on human BFU-e in serum-free cultures

The effects of recombinant human hemopoietic growth factors on early and late human erythroid progenitors (BFU-e and CFU-e) were investigated in serum-free cultures. Recombinant human erythropoietin (rhEpo) induced the formation of not only human CFU-e-derived colonies but also human BFU-e-derived bursts. Recombinant human interleukin 3 (rhIL-3) alone did not induce the formation of human BFU-e-derived bursts and human CFU-e-derived colonies. In the presence of rhEpo, rhIL-3 dose dependently increased the number of bursts stimulated by rhEpo, although rhIL-3 did not have the augmentative effect on human CFU-e growth. On the other hand, rhIL-3 did not stimulate the formation of murine BFU-e-derived bursts, and murine IL-3 did not stimulate the formation of human BFU-e-derived bursts. The results indicated that the burst-promoting activity of IL-3 was species-specific between human and murine cells. Recombinant human GM-CSF (rhGM-CSF) or recombinant human G-CSF (rhG-CSF) failed to induce human burst formation and did not augment the effect of rhEpo on human burst formation. The results of the present study suggest that in vitro, IL-3 can stimulate BFU-e in collaboration with Epo, but GM-CSF and G-CSF do not stimulate BFU-e growth in the presence or absence of Epo.     

Improvement of culture conditions for human megakaryocytic and pluripotent progenitor cells by low oxygen tension

The effect of low oxygen tension on the growth of human hemopoietic progenitor cells in bone marrow was investigated using the semisolid methylcellulose colony assay. The clonal growth of granulocyte-macrophage progenitors (CFU-gm), early (BFU-e) and late (CFU-e) erythroid progenitors, megakaryocyte progenitors (CFU-meg) and pluripotent progenitors (CFU-mix) improved more markedly incubation at the low oxygen tension (5%) than in conventional air (20%). The thiol compound 2-mercaptoethanol had a strong additive effect on colony growth in conventional air, but little or no effect in the low oxygen tension. These results suggest that enhancement of colony growth in the low oxygen tension may be due to a decrease in the production of oxygen intermediates.     

A Leu----His substitution at residue 93 in human corticosteroid binding globulin results in reduced affinity for cortisol.

A steroid binding capacity assay and a radioimmunoassay were both used to measure corticosteroid binding globulin (CBG) in serum samples from 22 patients with sepsis. An approximately 50% discordancy between the two values in one patient suggested the presence of a CBG variant with reduced affinity for cortisol, and this was confirmed by Scatchard analysis. We therefore used the polymerase chain reaction to amplify exons that encode for human CBG from the genomic DNA of this patient. This revealed two mutations within the coding sequences: one of which results in a Leu----His substitution at residue 93 and another which encodes a Ser----Ala substitution at residue 224 of the human CBG polypeptide. To assess the impact of each substitution on the steroid binding affinity of CBG, each mutation was introduced separately into a normal human CBG cDNA, and the normal and mutated cDNAs were expressed in Chinese hamster ovary cells. Scatchard analysis of the CBG produced in culture indicated that the His93 mutation (Kd = 2.24 +/- 1.75 nM) reduced the cortisol binding affinity of CBG (mean +/- SD) significantly (P less than 0.024) when compared to normal CBG (Kd = 0.64 +/- 0.31 nM), while the Ala224 mutation (Kd = 0.63 +/- 0.33 nM) did not influence cortisol binding affinity. We therefore conclude that residue 93 may play an important role in determining the structure of the CBG steroid binding site.     

In vitro culture studies of blood and marrow cells in chronic myeloid leukemia at different phases of the disease

The in vitro culture growth of peripheral blood (PB) and bone marrow (BM) cells were studied simultaneously from 100 adult patients with chronic myeloid leukemia at different phases. Sixty-five patients were investigated at initial diagnosis, 30 patients in control phase, and 41 patients in blast phase. In untreated chronic phase, the relative concentrations of granulocyte-macrophage progenitor cells (CFU-GM) in BM were not significantly different from those of normal controls, but there was generally a marked increase in circulating CFU-GM. The 6 Ph1-negative patients did not show different growth characteristics. We were unable to correlate the CFU-GM number to any of the hematologic parameters as well as to the response to busulfan therapy. Pretreated patients with excessive cluster formation did not necessarily indicate impending blast crisis. In hematologic remission, the numbers of CFU-GM in both BM and PB were well within the ranges of normal controls. Culture results in blast phase revealed a spectrum of abnormal growth. In myeloid crisis, 14/29 BM and 12/29 PB samples showed increased colony and cluster formations which were composed predominantly of immature cells with variable degeneration. Marrow cells in lymphoid crisis produced low numbers of both colonies and clusters in 5 out of 8 patients, while blood cells from 8 out of 10 patients formed large amount of colonies of normal morphology. This study indicates that the in vitro CFU-GM assay may have diagnostic utility in differentiating lymphoid crisis from myeloid crisis.     

Effect of lithium on granulopoiesis in culture.

Lithium carbonate therapy is associated with polymorphonuclear leukocytosis. In vitro studies have shown that lithium ions stimulate formation of granulocytic colonies. In a study undertaken to determine how lithium acts, colony-forming cells uncontaminated by monocytes (which elaborate colony-stimulating factor [CSF] in vitro) were obtained by means of a two-step cell separation procedure. The effects of lithium on colony formation were then studied in (a) cultures stimulated by humoral CSF, (b) cultures in which monocytes were relied upon to synthesize CSF de novo and (c) unstimulated cultures. Lithium enhanced the action of CSF but did not stimulate colony formation in the absence of CSF. In monocyte-stimulated cultures, colony formation increased with lithium concentrations up to 1 mmol/L but this increase paralleled that in CSF-stimulated cultures and therefore was not due to increased CSF production by monocytes. At higher concentrations of lithium, colony formation decreased in the monocyte-stimulated cultures but increased in the CSF-stimulated cultures. A lithium concentration of 4 mmol/L gave the greatest enhancing effect on colony formation in CSF-stimulated cultures and a concentration greater than 1 mmol/L inhibited de novo synthesis of CSF by monocytes.