Increased uptake and desulphation of heparin by mouse macrophages in the presence of polycations.

Heparin uptake and desulphation by cultured macrophages were investigated. Histones, polyamino-acids, protamine and eosinophil-basic protein stimulated both heparin uptake and desulphation, processes found to be non-related. Poly-L-ornithine and poly-DL-lysine increased the heparin uptake by about 33-fold, and histone produced up to 7.5-fold increase in the desulphation. The same polycations inhibited heparin desulphation by macrophage extracts.

Mode of binding and internalization into mouse macrophages of heparin complexed with polycations.

Heparin uptake by cultured macrophages was investigated from the standpoint of: (1) whether the increased uptake in the presence of polycations is due to charge neutralization, and (2) whether the heparin becomes internalized. Regarding the first point, our results are compatible with the notion that charge neutralization is mainly responsible for the enhanced uptake of heparin in the presence of protamine, histone, poly(DL-lysine) and poly(L-ornithine). As for the second point, chasing experiments at low and high temperatures strongly suggest that while heparin binds onto the cell membrane at both 4 degrees C and 37 degrees C, it undergoes internalization only at 37 degrees C.

Desulphation of heparin by mice and guinea pig leukocytes.

Leukocytes from mice and guinea pigs were tested for their sulphate-splitting activity on heparin. Mouse macrophages showed the highest degrading activity while mouse neutrophils and lymphocytes showed only a week degrading activity. Mouse macrophages maintained in tissue culture were also found to degrade heparin, the amount of sulphate released increasing with time up to 96 h. Spleen extracts were found to neutralize the anticoagulatory activity of heparin.

Sensitivity of hematopoietic progenitors of acute myeloblastic leukemia to new compounds derived from marine organisms.

Results of chemotherapy in acute myeloid leukemia (AML) have improved slowly or not at all in the last decade. We evaluated the effect of Eilatin and Norsegoline, two new aromatic alkaloids derived from the Red Sea purple tunicate Eudistoma sp., on in vitro proliferation and differentiation of leukemic cell lines and blast cells of three AML patients. These biological properties were studied in two complementary culture methods. The first is a clonogenic assay that supports colony formation in agar and reflects terminal divisions. The second is a suspension assay where clonogenic cells increase exponentially and reflects self-renewal. Eilatin and Norsegoline, at micromolar concentrations, suppressed, in a dose-dependent manner, both primary colony formation in agar and the recovery of clonogenic cells from suspension culture in the investigated cell lines and in fresh blasts. Furthermore, both alkaloids were more effective in inhibiting clonogenic cells grown in suspension than primary colonies grown in agar. In addition, these agents were able to induce immunophenotypic maturation of leukemic cell lines (upregulation of CD14 and CD11 and down-regulation of CD34 antigens). Our results indicate that Eilatin and Norsegoline significantly inhibit self-renewal capacity of leukemic progenitors and may provide a useful new tool for the treatment of AML patients.

Induction of murine macrophage fungal killing by interleukin 3.

The effect of recombinant interleukin 3 (IL-3) on the function of murine resident peritoneal macrophages was investigated. IL-3 enhanced the phagocytosis of Candida pseudotropicalis and Candida albicans and enhanced killing of the former. The enhanced killing is inhibited by scavengers of oxygen radicals, suggesting that IL-3 primes macrophages for enhanced oxidative metabolism in response to Candida.

Enhanced reconstitution of hematopoietic organs in irradiated mice, following their transplantation with bone marrow cells pretreated with recombinant interleukin 3.

Lethally irradiated C3H/eb mice were injected with syngeneic bone marrow cells that had been exposed for 4 h in vitro to purified bacterially synthesized interleukin 3 (rIL-3). Control mice were injected with cells exposed to incubation medium only. Mice injected with rIL-3-treated cells exhibited, on day 10 after transplantation an 8.2-fold and 2.7-fold increase in number of myeloid progenitors in their spleen and bone marrow, respectively, but the in vitro differentiation pattern of the myeloid progenitors was not affected. There was, however, an increase in the number of cells per individual in vitro myeloid colony (CFU-C) of the rIL-3-treated mice. The latter mice also showed a 1.6-fold increase in the number of splenic colony-forming units (CFU-S), a higher self-renewal capacity of hematopoietic progenitors, and a higher number of leukocytes in the peripheral blood. These results indicate that the injection into lethally irradiated recipients of bone marrow cells briefly pretreated in vitro with rIL-3 significantly enhances the reconstitution of their hematopoietic organs, and suggest that the in vitro pretreatment of bone marrow cells with appropriate stimulating factors could be useful in bone marrow transplantation.

New bone formation and bone marrow differentiation induced in rats by extracellular bone matrix implantation: effect of local preirradiation on the process.

Subcutaneous implantation of demineralized diaphyseal rat bone matrix in ACI rats initiates a developmental cascade that results in the formation of new endochondral bone and an associated hematopoietic bone marrow differentiation. Irradiation (1500 rad, 60Co) of the implantation site 24 h prior to implantation suppresses the formation of endochondral bone and bone marrow. All phases of the developmental cascade, including chemotaxis, proliferation, and differentiation, are arrested by the irradiation. Simultaneous implantation with the extracellular matrix of bone marrow, bone, pieces of a four-day-old implant or of thoracic muscle--but not of brain, liver, or spleen tissue--results in the development of endochondral bone and bone marrow at the irradiated site. Concurrent implantation with the extracellular matrix of in vitro growing fibroblasts of marrow or ossicle origin does not restore the developmental cascade.

Origin of stromal cells associated with osteoclast recruitment in s.c. implants of bone particles in chimeric mice.

Subcutaneous implantation of devitalized bone particles (BPs) in mice elicits a fibrovascular response with subsequent differentiation of multinucleated osteoclast-like cells. In bone marrow, stromal cells are known to play important roles in controlling hematopoiesis. Similarly, the stromal cells in the initial reaction to BPs may take part in supporting subsequent osteoclast recruitment and differentiation within the implants. Cross-gender chimeric mice were used to allow determination of whether these stromal cells were derived from local tissue or from hematopoietic stem cells. In radiation-chimeric mice, there was a 7-day delay in stromal recruitment and osteoclastic differentiation. Therefore cultures were established from the stromal tissue elicited 11 days after implantation, prior to osteoclastogenesis. Analysis of Y-chromatin DNA from these lines demonstrated that the majority (97%) of the lines were of recipient origin. It is possible that these fibroblast-like cells migrate to the site of BP implantation and play a role in the initiation of osteoclast development. This model can be used to define cellular interactions in osteoclastogenesis.

Effect of exogenous recombinant human granulocyte and granulocyte-macrophage colony-stimulating factor on neutrophil function following allogeneic bone marrow transplantation.

Functional activity of peripheral blood granulocytes was assessed in seven patients and in their normal donors following allogeneic bone marrow transplantation (BMT). Functions studied included superoxide generation (O2-), intracellular killing of Staphylococcus aureus, phagocytosis, and killing of Candida albicans. Neutrophils were tested following preincubation with 300 pM granulocyte-macrophage colony-stimulating factor (GM-CSF), 1.2 nM granulocyte colony-stimulating factor (G-CSF), or buffered solution (diluent) as control. Our data indicate that following BMT, both recipients and their normal donors show GM-CSF- and G-CSF-induced increases in: 1) O2- production in response to fMet-Leu-Phe (fMLP), 2) killing of S. aureus, and 3) phagocytosis of C. albicans. In two patients that showed low candidacidal activity, GM-CSF and G-CSF markedly enhanced the cytotoxic activity of the cells. Our studies indicate that GM-CSF and G-CSF increase "oxygen-dependent" oxidative activities in neutrophils from BMT recipients and their normal donors and enhance the antimicrobial activity of the cells.

Growth modulation and differentiation of acute myeloid leukemia cells by jaspamide.

We have examined the effect of Jaspamide, a peptide isolated from the marine sponge Hemiastrella minor, on in vitro proliferation and differentiation of leukemic cell lines and blast cells of three AML patients and compared it to that of cytosine arabinoside (ARA-C). The biological properties were studied in two complementary culture methods. The first is a clonogenic assay that supports colony formation in agar and reflects terminal divisions. The second is a suspension assay in which clonogenic cells increase exponentially and which reflects self-renewal. Jaspamide, at micromolar concentrations and in a dose-dependent manner, suppressed both primary colony formation in agar and the recovery of clonogenic cells from suspension culture in the investigated cell lines and in fresh blasts. Furthermore, Jaspamide was more effective in inhibiting clonogenic cells grown in suspension than primary colonies grown in agar. In addition, Jaspamide, similarly to ARA-C, was able to induce immunophenotypic maturation of leukemic cell lines (upregulation of CD14 and CD11 and downregulation of CD34 antigens). Our results indicate that Jaspamide significantly inhibits the self-renewal capacity of leukemic progenitors and may provide a new useful tool for the treatment of acute myeloid leukemia (AML) patients.

The hormonal milieu in early stages of bone cell differentiation modifies the subsequent sex-specific responsiveness of the developing bone to gonadal steroids.

We have established previously that rat bone tissue, as well as rat and human-derived bone cells in culture, show a sex-specific response to gonadal steroids in stimulation of the specific activity of the BB isozyme of creatine kinase (CK) and DNA synthesis. This response could be modified by manipulation of the endocrine environment during early stages in rat development. To further examine the influence of changing hormonal steroid milieu and vitamin D status on the action of gonadal steroids in developing bone tissue, we used two models of ectopic bone formation: demineralized tooth matrix (DTM) implanted under the skin, and femoral bone marrow (BM) transplanted under the kidney capsule of a syngeneic recipient mouse. The response to gonadal steroids in ossicles developed from implanted DTM depended on the recipient's gender; injection of estradiol 17beta (E2; 5 microg) into young female mice 21 days after DTM implantation increased, 24 h later, CK activity in the newly formed ossicles by approximately 60%, whereas injection of dihydrotestosterone (DHT; 50 microg) had no effect on CK activity. In contrast, in male mice, DHT but not E2 increased CK activity in the ossicles by approximately 50%. This sex-specific response was abolished in gonadectomized mice resulting in a similar response of the ossicles to both E2 and DHT. When DTM was implanted into vitamin D- deficient female mice, there was a lower basal CK activity and a significantly diminished response to E2 in the newly formed bone tissues. When BM, which contains mesenchymal and stromal cells and committed osteoprogenitor cells, was transplanted into 6-week-old intact or gonadectomized female or male mice, the response of the newly formed bone ossicles, 21 days after transplantation, to E2 or to DHT was according to the gender of the donor. Bone formed from BM obtained from female mice responded to E2 only and those formed from male BM responded to DHT only. Ossicles developed from BM obtained from gonadectomized mice showed lack of response to either gonadal steroid. Furthermore, only approximately 25% of the BM transplants obtained from castrated (CAST) male donors developed into ossicles. Ossicles formed from BM obtained from vitamin D-deficient female donors showed lack of response to gonadal steroids. These findings suggest that the manipulation of the hormonal milieu in early stages of the differentiation sequence of bone cells modifies the subsequent selective responsiveness of the developing bone tissue to gonadal steroids.

Stimulation of myeloid colony development elaborated by colony forming cells--fibroblasts from rat developing ossicles.

Subcutaneous implantation of demineralized bone matrix in rats results in endochondral bone formation and bone-marrow development. The cascade of events leading to this process occurs following the accumulation of mesenchymal cells with colony forming cell fibroblasts (CFC-F) potential in the implanted area, a process which commences already 72 h post implantation. It is demonstrated herein that CFC-F from various stages of ossicle development (days, 3, 7, 10, 14 or 18) stimulate hemopoiesis to the same extent as judged by the number of granuloid-macrophage-progenitors (CFU-GM), developed as hemopoietic colonies, and by the ratio of granuloid to macrophagic colonies. High concentrations of CFC-F, however, tend to diminish the stimulatory capacity. Prostaglandin E, CFU-GM, CFC-F, ossicle, growth factors, microenvironment, hemopoiesis, development.

The effect of 13-cis retinoic acid on hematopoiesis in human long-term bone marrow culture.

The modulatory effect of 13-cis retinoic acid (RA) on the growth, differentiation and function of hematopoietic cells in human long-term cultures was studied. RA (5 X 10(-8) M) induced enhancement of myeloid progenitor cell growth in the non-adherent layer throughout 6 weeks of incubation while it did not affect the number of myeloid progenitors in the adherent layer. The vitamin did not alter the differentiation pattern of colony forming unit-culture (CFU-C). The addition of RA to cultures for 5 weeks did not alter the cellular composition of the adherent layer. Prolonged exposure of hematopoietic cells to RA did not affect the functional activity of neutrophils and macrophages, i.e. the cells were active in phagocytosing Candida albicans (CA).

Heparin receptors on mouse macrophages.

This study deals with the nature of the attachment between the macrophage cell membrane and heparin molecules. Treatments intended to remove or internalise macrophage receptors (trypsinisation and stimulation of phagocytosis respectively) were shown to considerably modulate the attachment of heparin. An excess of heparin fractions ranging in mean molecular weight from 8100 to 25700 all inhibited attachment of 35S heparin as did a mixed isomer chondroitin sulphate preparation. Our study provides evidence for the presence of receptors for sulphated glycosamino-glycans on the mouse macrophage cell membrane.

Differential effect of polycations on uptake and desulphation of heparin.

Previous work has established that macrophages in culture release sulphate from heparin. We now report that increased uptake and desulphation of heparin occurred in the presence of polycations (poly-L-ornithine and poly-DL-lysine) and that the increase in heparin uptake was by about 30-fold. The desulphation was less related to uptake than to the nature of the bound polycation. Serum was found to have an inhibitory effect on heparin uptake while polycations inhibited heparin desulphation by macrophage extracts.

The role of non-calcemic analogs of vitamin D in differentiation of cultured rat bone marrow into osteoblast-like cells: age and sex differences.

We have previously demonstrated that rat bone in vivo and rat bone cells in vitro, responded sex-specifically to gonadal steroids in stimulation of the specific activity of the BB isozyme of creatine kinase (CK), a marker for hormonal responsiveness. Pre-treatment with vitamin D analogs up-regulated the sex-specific responsiveness and sensitivity to gonadal steroids. We also found that mice cultured femoral bone marrow (BM) in the presence of dexamethasone (DEX) and 1,25(OH)2D3 (1,25D) or both differentiated into osteoblast-like cells (Obs), which acquired sex-specific responsiveness to gonadal steroids. This response was significantly augmented in the presence of both agents. In the present study, we examined the effect of age, sex and vitamin D non-hypercalcemic analogs on the differentiation of rat derived femoral BM into Obs. In female or male derived BM from intact but not gonadectomized rats DEX and DEX+1,25D increased the constitutive levels of CK. BM derived from old females showed lower stimulation of CK than BM originated from young females by estradiol (E2) or raloxifene (Ral) in the presence of both DEX and 1,25D. The non-hypercalcemic analogs of vitamin D: CB 1093 (CB), EB 1089 (EB) and MC 1288 (MC) were more effective than 1,25D in both age groups in stimulating CK in the absence of DEX. In the presence of DEX, there was a further increase in CK with the same differential effectiveness. BM from gonadectomized male or female rats lost the sex-specific response, responding to both E2 and dihydrotestosterone (DHT). BM derived from both intact and gonadectomized males and females, growing with DEX or DEX+1,25D showed increased specific activity of constitutive levels of alkaline phodphatase (AP). No significant stimulation of AP was seen in any BM by gonadal steroids. These findings suggest that manipulation of the hormonal milieu in early stages of differentiation sequence of Obs determines the subsequent selective responsiveness of the developing bone tissue to sex steroids. Also non-calcemic vitamin D analogs were more effective in this process than 1,25D and showed activity even in the absence of DEX and may be applied to the differentiation process for bone tissue engineering.

Colony forming cell-fibroblast development in extracellular matrix-induced bone and bone marrow formation in rat.

The development of murine endochondral bone and bone marrow as a result of demineralized bone matrix implantation is preceded by the accumulation and proliferation of colony forming cell fibroblasts. These cells appear first at 24 hours post-implantation, after which they increase in 2 swells, achieving peak number between days 10-14. The observed differences in developmental kinetics of colony forming cell fibroblasts in culture were not found to be related to qualitative differences in the synthesis of collagens, fibronectin, laminin or proteoglycans. The colony forming cell fibroblasts were shown to be radiosensitive, with the Do = 339 +/- 63.