Biomedical subjects

R Gallily

Publications and source records attributed to R Gallily.

At least 37 records · Page 2Linked to original sources

PubMed1995

Identification and characterization of a novel protein associated with macrophage complement receptor 3.

CR3 is a member of the beta 2 integrin family which functions as a bidirectional signaling receptor. The CR3 is composed of the alpha (CD11b) and beta (CD18) subunits, which contain a short intracytoplasmic domain devoid of catalytic activity. It was therefore postulated that CR3 is associated with intracellular molecules that link it to the cytoplasmic signal transduction apparatus. However, no direct association between such molecules and CR3 have been identified so far. We searched for CR3 co-associated molecules that might regulate the function of this receptor. For this purpose CR3 was immunoprecipitated from radiolabeled bone marrow macrophages using a combination of anti-CD11b and anti-CD18 mAbs. Two-dimensional isoelectric focusing analysis of the immunoprecipitates revealed the two CR3 subunits and an additional 16-kDa protein with an apparent isoelectric point of 5.1. This protein, designated p16/5.1, was intracellular, monomeric, nonglycosylated and noncovalently associated with CR3 but not with CR4. CR3-associated p16/5.1 was also detected in four of six macrophage lines as well as in thymic large macrophages, all of which express cell-surface CR3. We suggest that p16/5.1 may be involved in CR3-mediated function.

Animals↗

PubMed1995

Mycoplasma fermentans activates the hypothalamo-pituitary adrenal axis in the rat.

Intracerebroventricular administration in rats of heat inactivated Mycoplasma fermentans caused a dose- and time-dependent increase in serum adrenocorticotrophin (ACTH) and corticosterone (CS). In rats with complete deafferentation of the mediobasal hypothalamus, which markedly depleted the median eminence CRF-41, the ACTH and CS responses to M. fermentans were completely inhibited. Pretreatment with dexamethasone abolished the adrenocortical response to M. fermentans. In lipopolysaccharide (LPS) unresponsive C3H/HeJ mice LPS failed to induce the adrenocortical response while administration of M. fermentans elicited a normal CS response. These results suggest that: M. fermentans can activate the hypothalamo-pituitary-adrenal axis via a central mechanism which involves hypothalamic ACTH secretagogue(s), and this effect is sensitive to the negative feedback of glucocorticoids. It is possible that the elevated glucocorticoid levels resulting from mycoplasma infection may be involved in the pathogenesis of mycoplasma-associated diseases.

Adrenocorticotropic Hormone↗

PubMed1995

Mycoplasma stimulates the production of oxidative radicals by murine peritoneal macrophages.

Mycoplasmas and mycoplasma membranes have been shown to induce the production of inflammatory cytokines, including tumor necrosis factor alpha and interleukin-6, as well as nitric oxide, by mouse macrophages and rat brain astrocytes. Luminol-enhanced chemiluminescence was used as a sensitive method to show that Mycoplasma capricolum membranes induce mouse peritoneal macrophages to produce reactive oxygen radicals. Coincubation of the mycoplasma with a secondary stimulus, namely macrophage-activating factor or interferon-gamma, increased the chemiluminescence. The augmentation was abolished by the nitric oxide synthase inhibitor NG-methyl-L-arginine, indicating the involvement of nitric oxide. The coproduction of superoxide and nitric oxide by the same cell allows the formation of the powerful oxidant peroxynitrite, which could be responsible for the increased chemiluminescence. Induction of oxidizing radicals by mycoplasmas may contribute to the clinical pathology seen in mycoplasma infections.

Animals↗

PubMed1995

Cytokines induced in vitro by Mycoplasma mycoides ssp. mycoides, large colony type.

Septicemic disease in goats and sheep caused by the large colony type of Mycoplasma mycoides ssp. mycoides has clinical and pathological features in common with septic endotoxemia. We studied the ability of the mycoplasma to induce mediators of biological responses to endotoxin, such as TNF alpha, IL-1 alpha, IL-6 and nitric oxide. Heat-killed suspensions of mycoplasmas in a concentration of 25 micrograms protein ml-1 induced all mediators in macrophages from peritoneal cavity and bone marrow of both C3H/HeN (LPS responsive) and C3H/HeJ (LPS low-responsive) mice. Lipopolysaccharide (LPS) in a concentration of 100 ng ml-1 induced the mediators in C3H/HeN derived macrophages, only. Simultaneous stimulation of macrophages with interferon-gamma enhanced the secretion of nitric oxide (measured as nitrite) but not the cytokines. We conclude that heat-killed Mycoplasma mycoides ssp. mycoides, large colony type, has cytokine inductive activity similar to bacterial endotoxin, but with an induction mechanism different from LPS.

Animals↗

PubMed1995

Pasteurella multocida toxin induces IL-6, but not IL-1 alpha or TNF alpha in fibroblasts.

Pasteurella multocida toxin (PMT) is the major virulence factor in Progressive Atrophic Rhinitis of swine. Other workers' previous findings that PMT was mitogenic for 3T3 fibroblasts, were confirmed in the present study. In addition, PMT stimulated 3T3 cells to release IL-6, but IL-1 alpha or TNF alpha were not detected in fibroblast supernatants sampled 24, 48, or 72 h after stimulation. In view of the role of IL-6 in osteoclastic bone resorption, these findings provide a new working hypothesis for investigations into the molecular pathogenesis of this important disease.

3T3 Cells↗

PubMed1995

Differential H-2 antigen expression in teratocarcinoma-fibroblast hybrids.

Embryo-derived teratocarcinoma cells, like early embryonic cells, do not express the classical MHC class I antigens. The mRNAs for both the H-2 alpha chain and beta2-microglobulin are also undetectable in these cells. We observed that upon fusion of H-2-negative mouse P19 teratocarcinoma cells (H-2k allotype) with H-2-positive embryonic fibroblasts of C57BL/6 origin (H-2b allotype), teratocarcinoma-like cell hybrids were obtained which express the H-2Kb antigen derived from the embryonic fibroblasts, but not the H-2KkDk antigens of the teratocarcinoma. This finding demonstrates that the teratocarcinoma H-2 genes do not respond to the positive regulatory factors present in the hybrids. The H-2k allele was not lost during fusion, as shown by its expression in retinoic-acid-differentiated hybrids treated with interferon-gamma (10 U/ml, 4 days). H-2KkDk antigen expression could also be induced in the undifferentiated hybrids by treating the cells with the protein synthesis inhibitor cycloheximide (1-10 mu g/ml, 18 h), but not with the demethylating agent 5-azacytidine (5 mu M, 2-4 days). These data suggest the presence of a labile, negative regulating protein factor which selectively prevents the expression of the teratocarcinoma-derived H-2 antigens. When the level of this factor(s) is reduced, the teratocarcinoma H-2 genes are capable of responding to the positive regulatory factors.

Animals↗

PubMed1994

Mycoplasma triggering of nitric oxide production by central nervous system glial cells and its inhibition by glucocorticoids.

The same cytokines that have been implicated in the pathology of central nervous system (CNS) inflammatory diseases and demyelinating diseases are also associated with the induction of nitric oxide (NO) production by macrophages and other somatic cells. Recently we have showed that mycoplasma can trigger the production of tumor necrosis factor (TNF)alpha and eicosanoids in rat astrocytes. In the present study, the effect of mycoplasma on NO production in rat glial cells was assessed. The addition of 10 micrograms/ml of membranes derived from M. capricolum (sheep isolate), M. fermentans (human isolate), or lipopolysaccharide (LPS) led to a 15- to 20-fold increase in NO production. The glucocorticoids dexamethasone and corticosterone, but not progesterone, markedly inhibited NO production. The addition of glucocorticoid prior or conjointly with the activator prevented large amounts of NO from being formed. Even when glucocorticoids were added 5 or 24 h after activation, effective inhibition of NO production was obtained. Thus, it is likely that glucocorticoids exert some of their ameliorating effects in neurological diseases by reducing the production of NO, cytokines and prostaglandins in the CNS.

Animals↗

PubMed1994

Characterization and purification of a mycoplasma membrane-derived macrophage-activating factor.

A highly hydrophobic component derived from the membrane of Mycoplasma capricolum has been characterized, purified and assessed for its ability to activate macrophages to tumor cytotoxicity. Initially, crude membranes were evaluated for their solubility in a wide range of solvents. Despite differential solubility in the various solvents, the mycoplasma membranes retained their ability to potentiate macrophage tumor cytotoxicity. Mycoplasma membranes were further characterized by appraising their macrophage-activating ability subsequent to various chemical treatments: cleavage of ester and thioester bonds, oxidation of vicinal hydroxyl groups, and exposure to a broad range of pH. Only strong alkaline treatment (pH > 12) caused a reduction in mycoplasma membrane activity; all other chemical treatments were inconsequential. With potential therapeutic applications in mind, mycoplasma membranes were subjected to various physical treatments including heating, freezing/thawing, sonication, lyophilization and storage. The ability of the membranes to induce macrophage activation was stably maintained following all these treatments. Purification of membranes was initiated by a chloroform/methanol lipid extraction. Macrophage-activating ability was found predominantly in the interphase. Proteolytic cleavage with trypsin increased specific activity at least sixfold. Trypsinized fractions were solubilized in 2-chloroethanol and gel filtration was performed on a hydroxylated Sephadex LH-60 column. The active fraction from this column had a further tenfold increase in specific activity. Subsequent rounds of reverse-phase HPLC on this fraction yielded three to four peaks absorbing at 280 nm, of which only one had macrophage-activating ability.

Animals↗

PubMed1994

Closed head injury triggers early production of TNF alpha and IL-6 by brain tissue.

In a model of closed head injury (CHI) in the rat we have shown the activation of phospholipase A2 and the production of eicosanoids after injury: at 15 min, mainly 5-hydroxyeicosatetraenoic acid (5-HETE), and at 24 h, mainly prostaglandin E2. The present study was designed to test whether CHI can also trigger the production of cytokines in the brain. CHI was induced in ether-anesthesized rats by a weight-drop device falling over the exposed skull covering the left hemisphere, 1-2 mm lateral to the midline in the midcoronal plane. In the posttraumatic period (1-24 h), the rats were decapitated, cortical tissue from the injured zone of the contused and contralateral hemispheres was removed and sonicated, and cytokine activity was assessed. Whereas no tumor necrosis factor alpha (TNF alpha) activity was found in normal brain tissue, it was detectable in the contused hemisphere (approximately of 72 +/- 50 pg/mg protein) as early as 1 h post-CHI. TNF alpha levels increased at 2 h, peaked at 4 h, (approximately of 609 +/- 540 pg/mg protein), and declined thereafter. At parallel intervals, only low levels of TNF alpha were detected in the contralateral hemisphere. In normal brain, interleukin-6 (IL-6) was nondetectable. Following CHI, high levels of IL-6 were present, although their accumulation lagged behind that of TNF alpha by 2-4 h, peaking at 8 h (62 +/- 31 ng/mg protein). We suggest that the rapid production of TNF alpha and IL-6 following CHI is a local inflammatory response of brain tissue to primary insult.

Animals↗

PubMed1993

Stimulation of tumor necrosis factor-alpha production by mycoplasmas and inhibition by dexamethasone in cultured astrocytes.

Elevated levels of tumor necrosis factor-alpha (TNF alpha) and other cytokines and eicosanoids in the central nervous system (CNS) have been noted in several human neurologic diseases, including multiple sclerosis and AIDS dementia. Recently it was shown that glial cells, especially astrocytes, are a major source of cytokines and eicosanoids. In the present study we have shown that astrocytes derived from fetal rat brain triggered by mycoplasmas produce TNF alpha and prostaglandin E2 (PGE2). Addition of mycoplasma (Mycoplasma capricolum isolated from sheep and M. fermentans KL-4 from human) at a concentration of 1-50 micrograms protein/ml (2 x 10(7)-10(9) colony forming units/ml), as well as lipopolysaccharide (5 micrograms/ml), led to a 200-500-fold increase in TNF alpha and a 2.5-4.5-fold increase in PGE2 production. Preincubation of the cells with the synthetic glucocorticoid, dexamethasone (2 x 10(-5)-2 x 10(-8) M), as well as with the natural hormone, corticosterone, markedly inhibited the secretion of both TNF alpha and PGE2. Thus, mycoplasmas can be added to the wide variety of agents that stimulate glial cells to produce cytokines and eicosanoids, and may contribute to various CNS pathological manifestations. In addition, the ability of glucocorticoids to inhibit particularly the stimulated productions of TNF alpha and PGE2 may explain at least in part the therapeutic benefit of these agents in CNS inflammation and demyelination.

Animals↗

PubMed1993

Trophoblasts protect the inner cell mass from macrophage destruction.

Several mechanisms have been suggested to account for the survival of the semiallogeneic fetus in the maternal uterus. However, no data are available to explain how the blastocyst resists the high number of macrophages in the uterus at the time of implantation. The present study examines the in vitro development of murine 3.5-day-old syngeneic or semiallogeneic blastocysts in the presence of nonactivated or lipopolysaccharide (LPS)-activated macrophages. It was found that the in vitro development of blastocysts was undisturbed by the presence of nonactivated or LPS-activated macrophages. The outgrowing trophoblasts were not only nonadhesive to the macrophages but also repelled them actively, thus preventing them from reaching the inner cell mass (ICM). Removing the zona pellucida by use of pronase or killing the ICM by irradiation did not alter the repulsion of macrophages by the trophoblasts. On the other hand, removal of the trophectoderm by antibody and complement treatment rendered the macrophages adhesive and destructive to the ICM. Four of 15 ICM (27%) were destroyed by nonactivated macrophages, and all of the ICM (15/15) were destroyed by LPS-activated macrophages. It is noteworthy that the addition of colchicine, cytochalasin B, proteinase inhibitors, anti-transforming growth factor-beta (TGF beta) antibodies, and indomethacin had no effect on the repulsion of macrophages by the trophoblasts. Therefore, it seems that microtubular proteins, microfilaments, extracellular matrix-degrading enzymes, TGF beta, and prostaglandins are not involved in the repulsion process. These results indicate that trophoblasts protect the ICM from the destructive action of macrophages by a repulsion mechanism of an as yet unknown nature.

Animals↗

PubMed1992

The unique killing of embryo-derived teratocarcinoma cells by nonactivated murine macrophages is not due to a lack of H-2 antigen expression.

It is well documented that activated macrophages, but not nonactivated ones, kill tumor cells in vitro without damaging normal cells. We, however, have previously shown that embryo-derived teratocarcinoma cells (F9, P19, PCC4) are efficiently killed by nonactivated macrophages as well as by activated ones. Whereas other tumor cells are killed extracellularly by macrophages, we found that F9 teratocarcinoma cells are phagocytosed alive by macrophages and subsequently killed intracellularly by a process dependent on intact lysosomal function. Neither the H-2 antigens nor the mRNAs for the alpha-chain and beta 2-microglobulin are detectable in embryo-derived teratocarcinoma cells. An obvious explanation for this unique killing is that the nonactivated macrophages recognize and kill these cells due to their lack of class I MHC antigen expression, assuming that class I MHC gene products on the target cells switch off the cytolytic machinery of nonactivated macrophages. Our present findings demonstrate that there is no correlation between H-2 antigen expression on tumor cells and their susceptibility to killing by macrophages. Retinoic acid-differentiated F9 cells and P19 cells expressing H-2 antigen after exposure to MAF (IFN-gamma) were sensitive to the killing by nonactivated macrophages. Hybrids that arose from fusion of P19 teratocarcinoma cells with embryonal normal fibroblasts (C57BL/6), which displayed the morphology of embryonal carcinoma stem cells and expressed H-2 antigens, were also sensitive to the killing by nonactivated macrophages. On the other hand, the H-2-negative testicular 402AX teratocarcinoma cells and K1735P melanoma cells were both resistant to the killing by nonactivated macrophages. We concluded that the unique killing of embryo-derived teratocarcinoma cells by nonactivated murine macrophages is not related to a lack of H-2 antigen expression.

Animals↗

PubMed1992

Mycoplasma fermentans (incognitus strain) induces TNF alpha and IL-1 production by human monocytes and murine macrophages.

We have demonstrated that Mycoplasma fermentans (incognitus strain), as well as M. fermentans KL4, PG 18 and IM 1 strains have the ability to activate human peripheral blood monocytes and murine macrophages of two inbred strains to secrete a high level of tumor necrosis factor alpha (TNF alpha) in a dose-dependent manner. Secretion of interleukin-1 (IL-1) was also stimulated following the incubation of human monocytes with the organism. We suggest that cytokine secretion following infection with M. fermentans (incognitus strain) that was detected in AIDS patients may contribute to the pathological manifestations, including cachexia, in this disease.

Animals↗

PubMed1991

Radiation leukemia virus (RadLV)-induced leukemogenesis is associated with an increased number and activity of thymic macrophages.

The radiation leukemia virus (RadLV) is a chronic leukemia retrovirus that induces thymic lymphomas in C57BL/6 mice after a latency of 3 to 6 months. During the pre-leukemic (PL) period, the number of thymic macrophages gradually increased up to 100 fold. Of the cells in a RadLV-induced lymphoma, 0.3% were large macrophages packed with infected lymphoma cells. These thymic lymphoma macrophages (TLM) also ingested RadLV-induced lymphoma cells in vitro. Cultured RadLV-induced lymphoma lines could activate and fix C3 fragments through the alternative complement pathway (ACP). C3-bound lymphoma cells elicited an oxidative burst (OB) response in TLM but not in bone-marrow macrophages (BMM). However, IL4 treatment of BMM rendered them capable of responding with an OB following triggering by C3-opsonized cells. Thymic macrophages (TM) responded moderately with OB to C3-opsonized cells and this response was elevated if the TMs were treated by rIL4. The OB reaction of the TLMs could be partially inhibited by anti-LFA-I or anti-MALA-2 antibodies, and was completely inhibited by anti-CR3 antibodies. These results suggest that IL4 can prime macrophages for triggering an OB reaction and that the interaction between C3-opsonized cells and IL4-primed macrophages is mediated primarily through CR3.

Animals↗

PubMed1991

Thymocyte maturation following interaction with thymus-derived macrophages.

Murine C57BL/6 thymocytes were cultivated together with syngeneic thymus-derived macrophages (TDM phi) for up to 96 hr to determine whether TDM phi participate in thymocyte maturation. The expression level of H-2b and Thy-1.2 antigens served as thymocyte differentiation surface markers as analyzed by flow cytometry. Indirect immunofluorescent staining profiles of the thymocytes demonstrate a dramatic increase in H-2b expression and a profound decrease in Thy-1.2 expression during cultivation with TDM phi. A similar phenomenon was observed when enriched populations of immature thymocytes were cocultivated with TDM phi. These changes were not observed when thymocytes were cultivated alone or with trypsin-treated TDM phi; neither were they observed when cortisone-resistant thymocytes manifesting mature characteristics were cultivated together with TDM phi. These findings suggest that interaction of thymocytes with TDM phi, involving binding and engulfment, results in the appearance of mature thymocyte subsets.

Animals↗

PubMed1990

In vitro induction of tumor necrosis factor alpha, tumor cytolysis, and blast transformation by Spiroplasma membranes.

Membranes of Spiroplasma sp. strain MQ-1 (hereafter referred to as MQ-1) were potent inducers of tumor necrosis factor alpha (TNF alpha) secretion and of blast transformation. Specific anti-recombinant murine TNF alpha antibodies markedly inhibited macrophage-mediated tumor cytolysis of A9 fibrosarcoma target cells following activation by MQ-1 membranes. Thus, TNF alpha plays a major role in mediation of tumor cytolysis induced by MQ-1 membranes, which is similar to its role in lipopolysaccharide (LPS)-induced tumor cytolysis. Two findings, however, suggested that the mechanism of macrophage activation by MQ-1 membranes differs from that by LPS: (a) macrophages, taken from C3H/HeJ mice showing a low responsiveness to LPS, were activated by MQ-1 membranes to enhanced TNF alpha secretion, resulting in a high-level tumor cytolysis compared with the negligible tumor cytolysis induced by LPS; and (b) MQ-1 membranes and LPS synergized to highly augment TNF alpha secretion by macrophages of C57BL/6 mice. MQ-1 membranes were capable of inducing blast transformation of murine lymphocytes as well. In addition, they activated human monocytes to secrete high levels of TNF alpha. Further studies need to be carried out using in vivo models to evaluate the therapeutic potential of MQ-1 membranes in the treatment of malignant diseases.

Animals↗

PubMed1990

Mycoplasma capricolum membranes induce tumor necrosis factor alpha by a mechanism different from that of lipopolysaccharide.

Heat-inactivated (60 degrees C, 45 min) Mycoplasma capricolum strain JR cells activate murine macrophages to secrete high levels of tumor necrosis factor alpha (TNF alpha) and to lyse tumor target cells efficiently. Fractionation of the intact M. capricolum cells, obtained from cells harvested at the exponential phase of growth, shows that their capacity to induce TNF alpha secretion by macrophage resides exclusively in the membrane fraction. The macrophage-mediated cytolysis following activation by M. capricolum membranes was significantly inhibited by specific anti-recombinant murine TNF alpha antibodies. M. capricolum membranes are a potent inducer of TNF alpha as the commonly used bacterial lipopolysaccharide, indicated by their dose-response curve for macrophage activation. Our study further showed that M. capricolum membranes and lipopolysaccharide synergize to augment TNF alpha secretion by C57BL/6-derived macrophages markedly. Moreover, lipopolysaccharide-unresponsive C3H/HeJ-derived macrophages, were pronouncedly activated by M. capricolum membranes, which do not contain lipopolysaccharide. These findings suggest that the mechanism by which M. capricolum membranes activate macrophages differs from that of lipopolysaccharide. Results of preliminary experiments show that human monocytes as well secrete TNF alpha following activation by M. capricolum membranes. Thus, in contrast with the prohibitive toxicity of lipopolysaccharide to animals and humans, M. capricolum membranes, which contain no lipopolysaccharide and are nontoxic in nature, may be of therapeutic value in the treatment of cancer.

Animals↗

PubMed1990

Engulfment and intracellular killing of F9 teratocarcinoma cells by non-activated murine macrophages.

Activated macrophages kill several types of tumor cells in vitro, whereas non-activated macrophages lack this capacity. We, however, observed that non-activated macrophages efficiently kill F9 teratocarcinoma as well as other teratocarcinoma cell lines. Dexamethasone, a glucocorticoid known to prevent macrophage activation, did not perturb the killing of F9 teratocarcinoma cells. Neither tumor necrosis factor alpha, nor the reactive oxygen intermediates, i.e. hydrogen peroxide, superoxide anion, and hydroxyl radical, nor serine proteases participated in this killing, shown by employing various agents which interfere with their production, secretion, or function. Using acridine orange/ethidium bromide vitality staining, the F9 teratocarcinoma cells were shown to be phagocytized alive by macrophages and subsequently killed intracellularly. Intact lysosomal function is required for the killing of F9 cells, as the lysosomotropic drugs chloroquine and ammonium chloride markedly inhibited this killing without perturbing their engulfment. The signal transduction pathway induced in the macrophages upon interaction with F9 teratocarcinoma cells seems to differ from that induced by macrophage activation. Neither the protein kinase C inhibitors polymyxin B and H-7 [1-(5-isoquinolinylsulfonyl)-2-methyl piperazine] nor the protein kinase C activator phorbol 12-myristate-13-acetate affected the killing of F9 cells. However, chlorpromazine (a powerful inhibitor of calmodulin), dibutyryl cAMP (a cAMP analog), and prostaglandin E2 inhibited the macrophage-mediated killing of F9 cells. In vivo studies indicate that an increased number of macrophages at the F9 tumor inoculation site (the peritoneal cavity) as a result of elicitation by thioglycollate prevents F9 tumor development. Our findings indicate that non-activated macrophages kill teratocarcinoma cells using a mechanism which differs from that employed by activated macrophages in the killing of other tumor cells.

Animals↗