Ilan Bleiberg

Increased uptake and desulphaton 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.

Antibody to Mol abrogates the increase in neutrophil phagocytosis and degranulation induced by granulocyte‐macrophage colony‐stimulating factor

We studied the ability of the human hemopoietic growth factors, granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) and granulocyte colony‐stimulating factor (G‐CSF) to activate polymorphonuclear neutrophils (PMN) for increased phagocytosis of opsonized Candida albicans and enhanced degranulation. Exposure of neutrophils to these two growth factors resulted in an increased number of Candida phagocytosed. Pretreatment of the neutrophils with the monoclonal antibody anti‐Mol abrogated the enhanced phagocytosis associated with GM‐CSF priming but not that of G‐CSF primed PMN. In examining the effect of these two colony‐stimulating factors (CSFs) on neutrophil degranulation we found that GM‐CSF induced enhanced release of lysozyme from cytochalasin‐treated PMN in the presence of Candida; however, G‐CSF did not. The effect of GM‐CSF on lysozyme release was abrogated by anti‐Mol antibody. These data suggest that GM‐CSF and G‐CSF prime PMN for certain enhanced functional activities by distinct mechanisms. The differential effect of the CSFs on neutrophil degranulation may relate to the more common inflammatory symptoms seen when GM‐CSF is used clinically as compared to the experience with G‐CSF.

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.

Biological properties in vitro of a combination of recombinant murine interleukin-3 and granulocyte-macrophage colony-stimulating factor

The effect of recombinant murine interleukin‐3 (rIL‐3) and recombinant murine granulocyte‐macrophage colony‐stimulating factor (rGM‐CSF) on in vitro murine myeloid progenitor cell (CFU‐C) growth and on the function of murine resident peritoneal macrophages was investigated. both rIL‐3 and rGM‐CSF are known to support the growth of CFU‐C and, when combined, were found to act synergistically to induce the development of an increased number of CFU‐C. The distribution pattern of myeloid colonies in the presence of these two growth factors was in general similar to that in the presence of rGM‐CSF alone. both rGM‐CSF and rIL‐3 enhanced the phagocytosis of Candida albicans (CA) by mature macrophages producing an increase in the percentage of phagocytosing cells as well as an increase in the number of yeast particles ingested per cell. No additive effect on the phagocytosis was observed when the two growth factors were added concurrently. rGM‐CSF, but not rIL‐3, enhanced the killing of CA by macrophages. This killing was inhibited by scavengers of oxygen radicals.

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).

Differentiation of cultured mice bone marrow into osteoblast-like cells results in acquisition of sex-specific responsiveness to gonadal steroids

We have previously demonstrated that mouse skeletal tissue, rat bone as well as rat or 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). This response could be modified by manipulation of the endocrine environment during early postnatal development. Moreover, pretreatment with vitamin D up-regulated the sex-specific responsiveness and sensitivity to gonadal steroids. In the present study we examine the differentiation pattern into osteoblast-like cells using dexamethasone (DEX) and 1,25 dihydroxy vitamin D3 (1,25D) and their effect on the acquisition of responsiveness to gonadal steroids by the differentiated cells. Cultured femoral bone marrow in the presence of DEX or 1,25D or both, were examined for their response to gonadal steroids by measuring the specific activities of alkaline phosphatase (AP) and CK BB. The constitutive level of CK in both male- and female-derived bone cells was decreased by DEX, by 1,25D or by both, whereas the constitutive level of AP was increased by DEX while decreased by 1,25D or by both. Following incubation of the bone marrow cultures with DEX, treatment with estradiol 17β (E2, 30 nM, 24 h) stimulated CK activity in female derived bone cells, with no effect of treatment with dihydrotestosterone (DHT, 300 nM). In contrast, in male derived bone cells, DHT but not E2 increased CK activity. This sex-specific response was also achieved upon culturing with 1,25D and was significantly augmented by culturing with both. No response to gonadal steroids was seen with undifferentiated bone marrow cells. All cultures responded to IGF-I when cultured with or without DEX and/or 1,25D but with no augmentation by 1,25D. Gonadal steroids increased AP to a much lesser extent; but enzyme activity decreased in the presence of 1,25D. IGF-I stimulated AP slightly with no effect of 1,25D. These findings suggest that manipulation of the hormonal milieu in early stages of differentiation sequence of osteoblast-like cells, determines the subsequent selective responsiveness of the developing bone tissue to gonadal steroids.

The Effect of 1,25-Dihydroxyvitamin D3 on Hematopoiesis in Long-Term Human Bone Marrow Cultures

Abstract The modulatory effect of 1,25-dihydroxyvitamin D3 (vit D) on the growth of myeloid progenitors and on the composition of the stromal layer in human bone marrow long-term cultures was studied. Vit D (2 × 10-8 M) caused an enhancement in myeloid progenitor cell (CFU-C) growth in the nonadherent and adherent layers during the entire 5-week incubation period. The vitamin did not alter the differentiation pattern of CFU-C (monocyte-macrophage progenitors CFU-M, granulocytic progenitors CFU-G, or monocyte-granulocyte progenitors CFU-GM). Vit D caused a marked increase in the percentage of lipid-containing cells in the adherent layer and an increase in the number of cells that specifically bound My4 monoclonal antibody (McAb), that reacted positively to fluoride-sensitive α-naphthyl acetate esterase, and that phagocytosed Candida albicans (CA). Concentrated supernatants harvested from control cultures showed significant levels of myeloid colony stimulating factor (CSF) activity. The addition of vit D to cultures for 5 weeks did not alter CSF levels. These results suggest that vit D may play a role in hematopoiesis by acting directly on the progenitor cells or via the stromal cell production of stimulatory factors).

The Effect of Heat Treatment on the Damage and Recovery of the Protein Synthesis Mechanism of Human Kidney Cell Line

SummaryThe effect of supraoptimal temperature on the suppression and recovery of protein synthesis activity in cultures of human kidney cell line was studied.It was shown that 44°C is a critical temperature to these cells, and after exposure to this temperature for 3 hours no recovery of protein synthesis activity could be detected. The cloning efficiency of cells exposed to 44°C for 2 hours was irreversibly reduced to zero. Protein synthesis at 43°C was much less affected, showing considerable recovery after eight hours exposure. Differences in heat susceptibility of individual cells in the culture were observed, but the reason for this heterogeneity is not yet known.

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.