Biomedical subjects

R Gallily

Publications and source records attributed to R Gallily.

At least 55 records · Page 3Linked to original sources

PubMed1990

Partial biochemical characterization of spiroplasma membrane component inducing tumor necrosis factor alpha.

We have recently found that membranes of Spiroplasma spp. strain MQ-1 (hereafter referred to as MQ-1) induce both tumor necrosis factor alpha (TNF alpha) secretion by bone marrow macrophages and blast transformation of lymphocytes via a mechanism different from that operated by bacterial lipopolysaccharide (LPS). This report presents evidence indicating that the MQ-1-derived membrane component(s) which activates bone marrow macrophages to secrete TNF alpha is, at least in part, protein. This conclusion is supported by our findings that TNF alpha secretion was reduced following exposure of MQ-1 membranes to elevated temperatures, extreme acidic pH treatment and incubation with protease K or pronase. Furthermore, following lipid extraction of MQ-1 membranes, most of both induction of TNF alpha secretion and blast transformation activities appeared in the 'protein' fraction. When membranes were chromatographed on a phenyl-Sepharose column, two major peaks were obtained, one containing most of the TNF alpha induction activity and the other the mitogenic activity. Neither peak coeluted with the peak of bulk membrane lipids. The possibility that the spiroplasma membrane component inducing TNF alpha secretion is acylated protein is discussed.

Animals↗

PubMed1990

Effect of strain and age on in vitro proliferation of murine thymus-derived macrophages.

In our laboratory we developed a technique which enabled us to carry out long term cultures of pure murine thymus-derived macrophage (TDM phi) populations having morphology, phagocytic capacity and surface markers characteristic of mononuclear phagocytic cells. We cultured thymic cells derived from various mouse strains on extracellular matrix coated tissue culture dishes, in the presence of conditioned medium. These cells developed into discrete TDM phi colonies and demonstrated a different proliferation rate. The TDM phi bearing haplotype H-2k (C3H/Crgl, CBA/LAC) proliferated more rapidly than TDM phi derived from mouse strains bearing the haplotypes of H-2b (C57BL/6), H-2d (BALB/c) and H-2k/d (A/J). To evaluate more precisely the number of precursor cells which generated TDM phi colonies and to study their growth kinetics, thymic cells were cultured in soft agar. A relatively large number of TDM phi precursor cells was derived from thymuses of H-2k haplotype origin. These precursor cells manifested a high rate of proliferation as compared with precursor cells of other murine haplotypes studied (H-2b, H-2d). These observations suggest that macrophage precursor cells are also located in the thymus and their numbers are controlled by genetic factors. We also observed a correlation between the number of precursor cells and the age of the mice. The percentage of TDM phi in new born and young mice was approximately four times higher than that found in older mice. It, therefore, seems that the degree of thymic involution affects the number of thymic macrophage precursor cells, which might indirectly affect thymocyte maturation and/or differentiation.

Age Factors↗

PubMed1990

Characterization and localization of human placental mononuclear phagocytes by monoclonal antibodies and other cell markers.

Human first-trimester placental macrophages were identified in frozen tissue sections and following cultivation as single-cell suspensions. In placental sections, the placental macrophages were shown to be the main cell type of the stromal zone. They clearly expressed macrophage-specific antigen, identified by EBM 11 monoclonal antibody; all were positive for nonspecific esterase (NSE), and 40-60% of them expressed the HLA-DR antigen. These cells were negative to anti-alpha-human chorionic gonadotropin (HCG) monoclonal antibody. After preparation of the placental single-cell suspension, the macrophages comprised about 15% of the total cell population. This fact was established by the following findings: 15% of the placental cell suspension expressed the antigen recognized by the OKM5 antibody, 16% expressed the EBM 11 antigen, 13% expressed the HLA-DR antigen, 5.5% were positive for NSE, and 24% phagocytosed Staphylococcus albus. Thirty percent of the cells were positive to anti-alpha-HCG antibody. After 48 h of cultivation in defined tissue culture conditions, the macrophages comprised more than 95% of the culture cell population. In the 48-h cultures, 98% of the cells expressed the macrophage antigens identified by OKM5 and EBM 11 monoclonal antibodies, and 75% expressed the HLA-DR antigen; 95% were positive for NSE, 88% of the cells had Fc receptors, and 95% of them were able to phagocytose S. albus. Only 0.1% of the culture cell population were positive to anti-alpha-HCG monoclonal antibody. These assays showed that the placental macrophages are the main cells in the placental stroma. In our tissue culture conditions, incubation time was shown to be the dominant factor in selecting macrophages over other cell types, most probably because the macrophages are the only placental cells that have the ability to adhere to plastic dishes. This observation might also explain the contradictory results in various studies over the last decade, regarding the incidence of the placental mononuclear macrophages.

Antibodies, Monoclonal↗

PubMed1989

Tumor necrosis factor as a mediator of Mycoplasma orale-induced tumor cell lysis by macrophages.

We and other investigators have previously demonstrated that mycoplasmas induce macrophage-mediated lysis of tumor cells, but the mechanism responsible for this process had, thus far, not been clarified. We now report that addition of either viable or heat-killed Mycoplasma orale to murine macrophages induces a cytolytic activity which, due to its neutralization by a specific antiserum against murine cloned recombinant tumor necrosis factor (rTNF), was identified as TNF-mediated. Both thioglycollate-elicited peritoneal macrophages and the normal macrophages cloned from our JBM phi 1.1 bone-marrow-derived cell line effectively produced TNF at levels similar to, or higher than, those obtained in the presence of high concentrations of lipopolysaccharide (LPS). Four other mycoplasma species demonstrated a varied capacity to induce TNF production by macrophages. Elevated TNF levels were also observed during macrophage-mediated cytolysis of murine A9 fibrosarcoma cells in the presence of either M. orale or LPS. Addition of the specific antiserum against rTNF at a concentration which neutralized all TNF activity in the co-cultures partially inhibited concomitant A9 cell killing. We can, therefore, conclude that M. orale induces TNF production which is, at least partially, responsible for subsequent tumor cell killing.

Animals↗

PubMed1989

Production of a factor (CIF) from normal fibroblast cells inhibiting tumor necrosis factor/cachectin production.

Murine embryonic fibroblast cells produce a factor designated cytotoxin-inhibiting factor (CIF) which inhibits tumor necrosis factor (TNF) and interleukin 1 production as well as tumoricidal activity by lipopolysaccharide-activated macrophages. This study determines the physiologic conditions of CIF production in serum-free medium. CIF production was largely dependent upon the presence of lipopolysaccharide. A quantitative correlation between fibroblast cell number, lipopolysaccharide concentration, and incubation time was established. Evidence is presented that CIF inhibited the production or release of TNF. CIF did not destroy TNF after production and release nor did it sequester secreted TNF. The supernatant fluids which inhibited TNF production did not suppress the capability of resting macrophages to phagocytize opsonized sheep erythrocytes, suggesting that only functions expressed in the activated state are inhibited.

Animals↗

PubMed1988

Modulation of lipoprotein lipase activity in mouse peritoneal macrophages by recombinant human tumor necrosis factor.

Thioglycollate-elicited mouse peritoneal macrophages spontaneously secrete lipoprotein lipase during culture. Exposure of the cultures to 50 ng/ml of recombinant human tumor necrosis factor (rTNF) for 48 h resulted in a 69% reduction in lipoprotein lipase activity in the culture medium with a concomitant decrease in cellular enzyme activity. The decrease in enzyme activity was not the result of rTNF-dependent reduction in the total protein synthesis, since the presence of rTNF did not affect [3H]leucine incorporation into cellular proteins. The effect of rTNF on lipoprotein lipase was reversible; upon TNF withdrawal, enzyme activity returned to basal levels after 60 h. The reduction of lipoprotein lipase in rTNF-treated cultures could be completely prevented by preincubation with a specific antiserum against recombinant human TNF. The late onset of decrease of lipoprotein lipase (LPL) activity suggests that rTNF might induce a mediator, which in turn suppresses LPL production. While rTNF was very effective in reducing lipoprotein lipase activity in mouse peritoneal macrophages, it did not affect lipoprotein lipase activity when added to the murine J774 cell line and to CT2 macrophage-like cells, a variant of the J774 cell line.

Animals↗

PubMed1988

Lipoprotein lipase in heart cell cultures is suppressed by bacterial lipopolysaccharide: an effect mediated by production of tumor necrosis factor.

Exposure of rat heart cell cultures, consisting mainly of nonbeating mesenchymal cells, to 50 ng/ml of bacterial lipopolysaccharide (LPS) for 24 h resulted in a more than 80% reduction in lipoprotein lipase activity. The loss of enzymic activity was accompanied by a concomitant reduction in enzyme protein, as shown by immunoblotting. Addition of LPS to the culture medium resulted also in the production of tumor necrosis factor (TNF), and the fall in lipoprotein lipase in LPS-treated cultures could be prevented by an antibody to TNF. Addition of recombinant human TNF to the heart cell cultures also depressed lipoprotein lipase activity. LPS treatment of preadipocytes in culture resulted in a fall in lipoprotein lipase activity and TNF production. Since TNF is known as a macrophage product, the cultures were tested for phagocytic capacity, and only 0.2-1.3% of the cells were shown to engulf Staphylococcus albus. Immunofluorescent staining with monoclonal antibodies OX-1, which identify leukocyte common antigen, was negative, and only 0.1 +/- 0.07% of the cells were positive after staining with OX-42 antibody to iC3b receptor. Both antibodies stained more than 98% of rat peritoneal macrophages used as controls. Since LPS treatment of macrophages at numbers comparable to or exceeding the number of phagocytic cells present in the heart cell cultures did not induce measurable amounts of TNF, it is suggested that in the heart cell cultures, TNF may be produced by cells other than macrophages.

Animals↗

PubMed1988

Interaction between thymocytes and thymus-derived macrophages. I. Surface components participating in mutual recognition.

Incubation of C57BL/6 thymus-derived macrophages (TDM phi) with syngeneic thymocytes resulted in binding of thymocytes to macrophages and rosette formation. Up to 60% of the TDM phi formed rosettes with thymocytes after 6 hr of interaction at 4 degrees C. Rosette formation of the immature PNA+ thymocyte fraction was up to fivefold higher than that of PNA- and cortisone-resistant thymocytes. Pretreatment of PNA- thymocytes with neuraminidase enhanced thymocyte binding to macrophages up to sevenfold, whereas a marked reduction of rosette formation was seen following (1) incubation of thymocytes with tunicamycin; (2) incubation of macrophages with 20 mM D-galactose, GLCNaC, or GalNaC; (3) treatment of macrophages or thymocytes with trypsin; (4) treatment of macrophages with anti-1-Ab mAb and its F(ab')2 fragment; (5) treatment of thymocytes with anti-Lyt-2.2 mAb; and (6) addition of EDTA and EGTA to the interacted two cell populations.

Animals↗

PubMed1988

Interaction between thymocytes and thymus-derived macrophages. II. Engulfment of thymocytes by macrophages.

A high percentage (80-90%) of immature thymocytes were engulfed by syngeneic thymus-derived macrophages (TDM phi) following cocultivation for 3 days. Elimination occurred via internalization of thymocytes by the macrophages. We unequivocally demonstrated the presence of many live thymocytes inside the TDM phi by means of specific staining. Mature PNA- thymocytes were phagocytized to a lower degree than immature thymocytes, and T splenocytes were not eliminated at all. Bone marrow-derived macrophages internalized immature thymocytes to a degree similar to TDM phi. Since thymocyte survival was not at all affected by M phi culture supernatants alone, we conclude that cell to cell contact is necessary for thymocyte elimination. To identify the surface molecules which participate in internalization of thymocytes by the macrophages, both cell types were pretreated with a variety of agents. Treatment of thymocytes with tunicamycin (N-glycosylation inhibitor) and anti-Lyt-2 mAb decreased their elimination by M phi. Similarly, treatment of M phi with neuraminidase, trypsin, and anti-Ia mAb markedly suppressed their capacity to engulf thymocytes. On the other hand, thymocyte elimination was unaffected by (1) cell cultivation in syngeneic serum rather than heterologous serum; (2) use of allogeneic rather than syngeneic thymocytes; and (3) use of X-irradiated M phi and LPS-activated M phi rather than nontreated M phi.

Animals↗

PubMed1988

Prostaglandin E2-mediated suppression of human maternal lymphocyte alloreactivity by first-trimester fetal macrophages.

We examined the immunosuppressor role of the first-trimester human placental macrophages on maternal lymphocyte alloreactivity in vitro to determine whether these macrophages and their secreted mediator(s) participate in the local immunoregulation at the fetomaternal interface. These cells were tested for their effects on maternal lymphocyte proliferation when added alone (as stimulator cells) or as regulator cells in 1) a one-way mixed lymphocyte reaction for six days and 2) lymphocyte cultures in the presence of phytohemagglutinin for three days. We detected a dose-dependent inhibition of phytohemagglutinin-induced proliferation with 48-hour macrophage culture supernatants. Placental macrophages, when used alone as stimulator cells in the mixed lymphocyte reaction, were stimulatory only at a concentration of 1-2%; the stimulation was abolished at a concentration of 10%. When used as regulator cells, they showed a strong inhibition of mixed lymphocyte reaction and phytohemagglutinin-induced proliferation at a concentration of 10%; no effect was observed at a concentration of 1-2%. The major class of the molecules mediating the suppression was identified as prostaglandins, primarily prostaglandin E2 (PGE2), based on the findings that the presence of indomethacin (10(-5) M) or various dilutions of an anti-PGE2 antibody abrogated suppression substantially or completely. Prostaglandin E2 levels measured in the mixed leukocyte culture wells containing placental macrophages as regulator cells correlated positively with the macrophage dose and its suppressive effect.

Dinoprostone↗

PubMed1987

Establishment and characterization of murine bone marrow-derived spontaneously immortalized cell lines and clones expressing properties of normal macrophages.

Murine bone marrow (BM) cells were cultivated on bacteriological grade culture dishes (BCD) in liquid medium containing L-cell-conditioned medium (LCM). The first month of rapid exponential multiplication was always followed by an interim phase of slow growth, and then by continuous proliferation. These established lines were called Jerusalem bone marrow macrophages (JBM phi). One of these, which had been derived from a C3H/Crg1 female mouse and was designated JBM phi 1.1, was studied in more detail. Its cloning efficiency when grown in LCM-containing soft agar was 65%. Of several clones isolated, one, C1.26, was selected for further cultivation and propagated for about 600 days. Cells from all cultures were surface adherent with limited proliferative capacity on tissue culture plastic. The properties displayed by all cells in a culture or clone include a typical macrophage (M phi)-like morphology, effective ingestion of killed bacteria and zymosan, staining for nonspecific esterase, and expression of Fc receptors and of F4/80 surface antigen. Addition of lymphokine (LK) induced Ia antigen expression on a high percentage of the cells. The JBM phi 1.1 cells also secreted high levels of lysozyme, produced a zymosan-induced respiratory burst, and, upon addition of lipopolysaccharide (LPS), released interleukin-1 and tumor necrosis factor. Efficient tumoricidal activity could be induced by LK and LPS. No evidence for the production of colony-stimulating factors, even in the presence of LPS, could be found. The JBM phi 1.1 or C1.26 cells did not develop into tumors following subcutaneous injection in x-irradiated syngeneic or in nu/nu mice and were also incapable of growing in soft agar without LCM. All the properties studied were expressed at similar levels by the "young" BM-derived M phi during their first exponential growth phase, as well as by other JBM phi lines and clones. It is concluded that the established JBM phi lines consist of homogeneous cell populations which, according to all markers and functions studied, could be classified as non-activated, functional, and mature M phi, resembling in all aspects BM-derived M phi during their first few weeks of cultivation. This shows that cell lines expressing properties of normal M phi may develop spontaneously by continuous cultivation of BM cells in growth factor-containing liquid medium on BCD.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1986

Correlation of macrophage-mediated tumor-cell lysis with the production of macrophage cytolytic factor (CF). Preliminary characterization of a factor inhibiting CF production.

Macrophage-mediated cytolysis of thymidine-prelabelled murine A9 fibrosarcoma cells was compared to the level of cytolytic factor (CF) present in the cultures by assaying supernatant aliquots on actinomycin (AcD)-treated A9 fibrosarcoma cells. A good correlation between the level of A9 killing and CF titer was observed when different concentrations of lipopolysaccharide (LPS) were added to various macrophage populations: murine peritoneal cells, short-term bone-marrow (BM)-derived macrophages and JBM phi macrophage lines. Optimal A9 killing and CF secretion, equivalent to the killing of about 1000 AcD-pretreated A9 cells by a single macrophage, were obtained following activation of JBM phi by LPS. CF production by BM-derived macrophages was enhanced in serum-free medium when compared to its release in the presence of fetal calf serum. The LPS-activated macrophages could be restimulated by the activating agent to produce CF following one week of propagation in the absence of LPS. On the other hand, CF activity was absent from the supernatants of activated macrophages co-cultured with normal embryonic fibroblasts, which are resistant to macrophage-mediated killing. This effect could be attributed to a factor, secreted by normal fibroblasts but not by A9 cells, which suppressed CF release from the activated macrophages. Our data strongly support earlier observations, suggesting that CF [which appears to resemble the tumor necrosis factor (TNF)] is responsible for LPS-induced macrophage-mediated tumor cell lysis. It is suggested that suppression of the latter process by the fibroblast-derived factor proceeds via inhibition of CF/TNF production from the macrophage.

Animals↗

PubMed1986

Suppression of both macrophage-mediated tumor cell lysis and cytolytic factor production by a factor (CIF) derived from normal embryonic fibroblasts.

We had previously established a murine bone marrow-derived cell line, designated JBM phi 1.1, which displayed properties of normal macrophages, including the ability to perform macrophage-mediated cytolysis. It was also found that these cells could be induced by lipopolysaccharide (LPS) to produce reproducibly high levels of a cytolytic factor (CF) resembling tumor necrosis factor (TNF). This cell line was therefore selected for further studies on macrophage-mediated tumor cell lysis and CF production. Moreover, the CF production during incubation with LPS was higher in the absence of serum than in its presence, with a maximum at days 2-3 following the addition of LPS. A factor inhibitory to CF production (CIF) was detected in our laboratory in the supernatant of embryonic fibroblast cultures. We established the experimental conditions required for the optimal production and suppressive effect of CIF. High levels of CIF activity were obtained under conditions that promote fibroblast proliferation. Addition of embryonic fibroblast culture supernatant to the macrophages shortly before LPS suppressed both LPS-induced CF production and tumoricidal activity. CIF did not affect macrophage protein synthesis in the presence or absence of LPS. However, LPS-induced interleukin 1 release was partially (55%) suppressed by embryonic fibroblast culture supernatant. Our results show that CIF does not exert a general inactivating effect on the macrophages, although it may possibly affect other functions in addition to CF production and tumor cell lysis. The strong inhibition of both the latter properties further indicates that TNF-like CF is an important mediator in macrophage-mediated tumor cell lysis.

Animals↗

PubMed1986

Dual effect of lectins on macrophages: potentiation of bacterial uptake and suppression of bactericidal activity.

Pretreatment of murine bone marrow-derived macrophages with wheat germ agglutinin (WGA) and concanavalin A (Con A) markedly enhanced the uptake of Staphylococcus aureus H and Escherichia coli 08, respectively. Pretreatment of the respective bacteria with these lectins also greatly increased their uptake by the macrophages. An increase of up to 40-fold in uptake of E. coli 08 by the macrophages was observed following their pretreatment with Con A in the absence of serum and at a low temperature. On the other hand, pretreatment of macrophages with either WGA or Con A suppressed very markedly or abolished completely their bactericidal activity against S. aureus H and E. coli 08, respectively. As macrophage chemiluminescence reactions to zymosan were also very greatly suppressed following pretreatment with WGA and Con A, we suggest that these lectins suppressed macrophage bactericidal activity by inhibiting reactions involving chemiluminescence.

Animals↗

PubMed1985

Non-immune non-activated chicken macrophages destroy murine fibroblasts.

Non-immune non-activated chicken bone marrow-derived macrophages (BM M phi) killed murine embryonic fibroblasts in vitro. Following precultivation for 10-35 days chicken BM M phi had the capacity to destroy normal murine embryonic fibroblasts at effector: target ratios of 10:1 to 1:1. Optimal killing was observed following cocultivation of M phi and fibroblasts for 48-72 hrs. Addition of LPS neither initiated nor potentiated M phi-mediated killing. This study demonstrates that chicken M phi have the capacity to destroy, in vitro, cells of phylogenetically distant species, similarly to the ability of murine M phi to kill chicken fibroblasts. It is suggested that vertebrate M phi xenolytic potential is analogous to the capacity of invertebrate phagocytes to destroy xenografts.

Animals↗

PubMed1985

Antigen presentation by proliferating thymic macrophages to A (T,G)-A-L specific T cell line in an H-2 restricted manner.

Long-term cultures of murine homogeneous populations of Ia-bearing thymus-derived murine macrophages were tested for their ability to present antigen to a (T,G)-A-L specific IL-2-dependent continuous T cell line. Thymus-derived macrophages, with and without pretreatment for Ia induction, triggered efficiently antigen-specific T cell proliferation in an MHC restricted way. This experimental system, consisting of two normal proliferating homogeneous populations of macrophages and antigen specific T cells, provides an ideal tool for studying the mechanism of antigen presentation to T cells and for elucidation of the role of macrophages in T-B cell collaboration for antibody production.

Animals↗

PubMed1985

Lysis of primitive teratocarcinoma cells by non-activated macrophages.

The capacity of non-activated murine thioglycollate-elicited macrophages and bone marrow-derived macrophages to lyse primitive F9 teratocarcinoma cells lacking H-2 antigens was investigated. Both populations of non-activated macrophages killed F9 cells efficiently whereas they were not cytolytic against murine fibrosarcoma targets. In vitro activation by lipopolysaccharide induced the macrophages to lyse fibrosarcoma cells but did not significantly increase the level of cytolysis against F9. These results are consistent with the hypothesis that the absence of H-2 expression on target cells may serve as a signal for macrophage "foreign" recognition and cytolysis.

Animals↗

PubMed1985

Thymus-derived macrophages in long-term culture: release of IL-1, stimulation of MLR and expression of tumoricidal activity.

The present report examines the behaviour of a slowly proliferating pure population of thymus-derived macrophages in long-term culture, regarding their ability to secrete interleukin-1 (IL-1) and prostaglandin E2 (PGE2), to stimulate a mixed leucocyte reaction (MLR) and to lyse tumour cells in vitro. Following stimulation with LPS, high levels of IL-1 were released to the medium. IL-1 release was significantly augmented by the addition of indomethacin during stimulation. Thymus-derived macrophages constitutively secreted significant levels of PGE2. These cells served as excellent stimulators in a one-way MLR, substantiating the claim that pure populations of macrophages effectively stimulate an allogeneic response in vitro. Thymus-derived macrophages showed tumoricidal activity following activation with either high concentrations of LPS or suboptimal concentrations of LPS and T-cell lymphokine. These findings portray a close interrelationship and reciprocal regulation between thymus-derived macrophages and T lymphocytes.

Animals↗