Macrophages In Tissue Culture Research Articles

Haemocompatiblity of controlled release glass.

There are many medical applications which benefit from the use of soluble biomaterials, including the sustained release of drugs over a precise period of time, or temporary conduits for controlling nerve regrowth. We have manufactured a series of phosphate-based controlled release glasses (CRGs) in which the solubility could be controlled by varying the concentration of CaO and Na2O. Fibres of the CRG containing iron and cerium were placed into direct contact with human neutrophils and macrophages in tissue culture for 2.5 and 24 h respectively and the responses analysed by scanning electron microscopy (SEM) and confocal microscopy. The supernatants were analysed for the cytokine IL-1beta by enzyme-linked immunosorbent assay (ELISA). Disks of CRG of various compositions were placed in contact with whole blood for 30 min and platelet adhesion assessed by SEM. Activation of platelets, granulocytes and complement were quantified by ELISA for beta-thromboglobulin, elastase and iC3b. Intrinsic coagulation activation was measured by timing the clotting of recalcified plasma. Only the cerium fibre inhibited IL-1beta release from macrophages. No platelet adhesion was observed to any disk composition. Three compositions containing MgO inhibited plasma clotting and showed an insignificant level of complement activation. This study has demonstrated the development of a number of compositions of CRG, which have great potential in a wide variety of biomedical applications.

In vitro studies on the role of titanium in aseptic loosening.

This study was designed to define the role titanium debris plays in aseptic loosening. Macrophages exposed to commercially pure titanium (1-3 microns) exhibit the same mediator profile as those exposed to polymethylmethacrylate. This response consists of increased release of tumor necrosis factor but not prostaglandin E 2 or interleukin-1. Osteoblasts increase production of prostaglandin E 2 when exposed to media from titanium stimulated macrophages but not interleukin-6 or granulocyte macrophage colony stimulating factor. Media from macrophages exposed to titanium did not lead to bone resorption, as measured by calcium 45 release, in organ culture. The cellular response to titanium is characterized by release of tumor necrosis factor from macrophages and prostaglandin E 2 from osteoblasts exposed to the macrophage conditioned medium. A comparison of the results of this study with those of others involving exposure of macrophages in tissue culture suggests that titanium may not be as inflammatory as other particles in the aseptically loose joint.

Studies of the mechanism by which the mechanical failure of polymethylmethacrylate leads to bone resorption.

The purpose of this study was to examine the relationship between the mechanical failure of polymethylmethacrylate and bone resorption at the bone-cement interface of a prosthesis. Evaluation of tissue that had been retrieved from the cement-bone interface of eighteen femoral components of total hip prostheses that were loose without associated infection revealed that a critical factor associated with bone resorption was the presence of particles that were small enough (one to twelve micrometers) to be phagocytized by macrophages. To study this phenomenon in vitro, macrophages in tissue culture were exposed to three preparations of polymethylmethacrylate cement. A novel method of cement preparation was used with control for solid and soluble contaminants, which provided a sensitive and specific technique for the determination of which mediators were released from the macrophages. Electron microscopy demonstrated phagocytosis of particles of less than twelve micrometers in size, regardless of the type of cement preparation. Exposure to all three cement preparations resulted in toxicity, as reflected by inhibition of 3H-thymidine incorporation. Exposure also led to increased release of tumor necrosis factor, but none of the three preparations resulted in release of prostaglandin E2. Division of the cement preparations into two groups on the basis of the size of the particles demonstrated that exposure to particles that were small enough to be phagocytized led to inhibition of 3H-thymidine incorporation and release of tumor necrosis factor, while exposure to particles that were too large to be phagocytized did not. Neither exposure to small particles nor exposure to large particles of cement led to release of prostaglandin E2. Our results show that when the mechanical failure of cement produces particles that are small enough to be phagocytized, phagocytosis of the particles results in the increased production of tumor necrosis factor by the macrophages, which may in turn lead to bone resorption and prosthetic loosening. These small particles also decrease 3H-thymidine uptake by the macrophages.

Paraoxonase prevents accumulation of lipoperoxides in low-density lipoprotein

Oxidative modification of low-density lipoprotein (LDL) enhances its uptake by macrophages in tissue culture and in vivo may underly the formation of arterial fatty streaks, the progenitors of atheroma. We investigated the possible protection which high-density lipoprotein (HDL) affords against LDL oxidation. The formation of lipoperoxides and thiobarbituric acid reactive substances when LDL was incubated with copper ions was significantly decreased by HDL. The enzyme, paraoxonase (E.C. 3.1.8.1), purified from human HDL, had a similar effect and thus may be the component of HDL responsible for decreasing the accumulation of lipid peroxidation products.

Cellular Membranes on Glass Lens Implants-Formed in Tissue Culture by Mouse Macrophages

Intraocular lens implants made of glass were exposed to a suspension of mouse macrophages in tissue culture for 48 and 96 hours. After 48 hours the macrophages had formed a dense cellular membrane on the glass surface and some of them had changed into fibroblast-like cells, epithelioid cells, and early stages of giant cells. After 96 hours a firm and continuous proteinaceous capsule had formed on the glass in the interspaces between the cells. The membranes on glass implants were more cellular and more firmly adherent than those seen on plastic implants under the same conditions.

Complement proteins C2, C4 and factor B. Effect of glycosylation on their secretion and catabolism.

Tunicamycin, an inhibitor of N-acetylglucosaminylpyrophosphopolyisoprenol-dependent glycosylation, was used to study the effect of glycosylation on the synthesis, post-translational modification, secretion and function of the complement proteins that are associated with the major histocompatibility complex in humans, mice and guinea pigs. Tunicamycin blocked glycosylation of pro-C4, C2 and factor B and inhibited secretion of the corresponding native complement proteins synthesized by guinea-pig peritoneal macrophages in tissue culture. In addition, underglycosylated pro-C4 was more rapidly catabolized intracellularly than the corresponding fully glycosylated pro-complement protein. C4 protein secreted by cells incubated with tunicamycin had approximately the same specific biological activity as the protein obtained from control culture media, suggesting that carbohydrate is not required for its activity in immune haemolysis. Direct studies of carbohydrate incorporation and the tunicamycin effect suggested an unequal distribution of sugar among the C4 subunits, with maximal incorporation of carbohydrate into alpha-, and less into the beta-chain of the native protein.

The use of a genetically incompatible combination of host and virus (MHV) for the study of mechanisms of host resistance.

In 1960 when we found that mouse hepatitis virus (MHV) could grow in, and would destroy, macrophages in tissue culture, and that macrophage behavior in vitro reflected the single gene for susceptibility in the mouse,1,2 we constructed a simple model of how genetic resistance and susceptibility to this virus functioned. Today, the basic experimental findings remain unchanged, but new findings have compelled a very different interpretation of the same data. We believe that this re-interpretation applies to many host-virus genetic systems. It may be useful to first follow each item that has been re-interpreted, and then illustrate how the more complex model can clarify one way in which the environment affects host-parasite interrelations. We believe that the effects of environmental pollutants could be tested in this system. The original view of the system and the change in viewpoint between 1960 and 1980 are shown in Table 1.

HEPES buffered media may induce prostaglandin release from macrophages in tissue culture.

On investigating the release of PGE2 from mouse peritoneal macrophages in tissue culture it was found that the spontaneous release of PGE2 was dependent on the culture conditions employed. The type of buffering of the medium was especially important. Using CO2- sodium bicarbonate buffers we observed little, if any, spontaneous release of prostaglandins (PGs) from macrophages, whereas in the presence of N-2-hydroxyethylpiperazine-N-2-ethane-sulfonic acid (HEPES) buffer in some cases a dramatic increase in PG release was observed.

Bromodeoxyuridine-induced relaxation of DNA-protein interactions in chromatin as revealed by nucleoprotein-celite chromotography

The effect of polysaccharides isolated from yeast-like fungi on diverse activities of murine peritoneal macrophages was studied in experiments in vitro. Yeast polysaccharides had a nonspecific activating effect on macrophages in tissue culture. They enhance the adhesion of macrophages to glass, stimulate phagocytosis and intracellular digestion, and change the chemotactic activity. The activity of polysaccharides directly correlates with their molecular weight. There is a relationship between the biological activity and chemical structure of polysaccharides. The beta-structural polysaccharides are capable of activating the functions of macrophages, while beta-structural dextrans do not possess such properties.

Effect of leukocytic pyrogen on the phagocytic properties of macrophages in tissue culture

The effect of rabbit leukocytic pyrogen (LP) on the phagocytic activity of the peritoneal macrophages of albino mice against shigellae was investigated. The dose-effect dependence was revealed: high LP doses depressed the phagocytosis, and low ones were insufficiently effective; addition of average LP doses against the kanamycin background stimulated both the absorption phase and that of shigella digestion. Phagocytosis stimulation with LP was also accompanied by an increase of the acid phosphatase lysosomal enzyme activity in macrophages; the RNA content was not changed.

The defectiveness of Mill Hill 2, a carcinoma-inducing avian oncovirus

The avian carcinoma virus Mill Hill 2 (MH2) transforms fibroblasts and macrophages in tissue culture. It is defective in replication and dependent on a helper virus, MH2 AV, for the production of infectious progeny. MH2 AV contains both subgroup C and A envelope determinants; its helper functions are required by MH2 in the env, pol, and probably also the gag genes. The defects of MH2 can be complemented only by helper viruses of the chicken leukosis group, further suggesting that MH2 is defective in gag or pol, or both. MH2 transformed nonproducer cells synthesize an aberrant viral polyprotein of 100,000 daltons. This MH2p100 carries antigenic determinants of the gag protein p27, but not of the env protein gp85. It is not cleaved into smaller, functional proteins and is not glycosylated.

Lysis of antibody-treated human erythrocytes by human leukocytes and macrophages in tissue culture.

The <sup>51</sup>Cr-chromate method was used for quantitation of lysis of antibody-treated human RBC which had been labilized by pretreatment with 1% trypsin. RBC treated with heat-inactivated isoantiserum to A, B or Rh antigens were lysed within 3–6 h by blood leukocytes or peritoneal macrophages. Macrophages or suspensions containing mainly granulocytes and monocytes were cytotoxic at the leukocyte-RBC ratio 1:5 while an excess of purified lymphocytes ( > 99%) had no hemolytic effect. Erythrophagocytosis by monocytes and some granulocytes was frequently seen and is assumed to be the main mechanism of hemolysis. There was no evidence of hemolytic factors released by leukocyte suspensions. This method may be useful for studies of cell mediated hemolysis in human immune hemolytic diseases.

Demonstration in Tissue Culture of Lymphocyte-Mediated Immunity to Tuberculosis

We have shown that splenic lymphocytes from mice immunized with viable attenuated mycobacterial cells (H37Ra) or ribonucleic acid prepared from these cells will bring about inhibition of multiplication of virulent tubercle bacilli (H37Rv) within normal mouse peritoneal macrophages in tissue culture. Apparently these lymphocytes, when stimulated with viable virulent tubercle bacilli, elaborate a filterable substance(s) which is responsible for this intracellular growth inhibitory effect.

Multiplication of Mycobacterium tuberculosis Within Normal and “Immune” Mouse Macrophages Cultivated With and Without Streptomycin

Acquired cellular immunity to infection with Mycobacterium tuberculosis is believed to reside in the capacity of mononuclear phagocytes of immunized animals to inhibit intracellular multiplication of the parasite. However, in macrophage tissue culture systems, it has been customary to employ streptomycin in the medium for the purpose of restricting extracellular, but not intracellular, growth of M. tuberculosis. In contrast, our data show that small amounts of streptomycin markedly inhibit intracellular as well as extracellular growth of M. tuberculosis in normal mouse peritoneal macrophages, and that the degree of this inhibition is directly proportional to the concentration of streptomycin used. In the absence of streptomycin, virulent tubercle bacilli grew as rapidly in "immune" macrophages as in normal macrophages. "Immune" macrophages, however, were slightly more resistant to destruction by the intracellularly multiplying mycobacteria. In the presence of streptomycin, however, intracellular mycobacterial growth was inhibited more in "immune" macrophages than in normal macrophages, and this effect also was directly proportional to the concentration of streptomycin used. Virulent mycobacteria grew somewhat more slowly within mouse peritoneal macrophages obtained after induction of a peritoneal exudate with glycogen than in noninduced cells. The rate of multiplication, though, was the same within normal and "immune" induced peritoneal cells except in the presence of streptomycin. As with noninduced macrophages, this drug inhibited the intracellular multiplication of virulent tubercle bacilli more effectively within "immune" induced than within normal induced cells. It would appear, therefore, that the greater inhibition of intracellular multiplication of virulent tubercle bacilli in "immune" macrophages in tissue culture noted by a number of investigators in the past may have been an artifact created by the use of streptomycin in the tissue culture medium.

Host-parasite relationships in brucellosis. I. Infection of normal guinea pig macrophages in tissue culture.

Host-parasite relationships in brucellosis. I. Infection of normal guinea pig macrophages in tissue culture.

Opsonic Factors in Normal and Immune Sera in the Differential Phagocytosis of Normal, Trypsinized and Virus-Treated Human and Rabbit Erythrocytes by Macrophages in Tissue Culture

Summary Maximum erythrophagocytosis with normal serum required heat-stable, specific, absorbable factors and complement. Quantitative and qualitative aspects of complement were demonstrated. The opsonic action of specific antibody was increased in the presence of complement. Complement appeared to have no effect on the specificity or absolute degree of the opsonic reaction in immune serum, but only on the rate of phagocytosis.

Differential Phagocytosis of Normal, Trypsinized and Virus-Treated Human and Rabbit Erythrocytes by Macrophages in Tissue Culture

Summary Human and rabbit erythrocytes modified by either enzymes or viruses have been shown to be more susceptible to phagocytosis by macrophages from rabbit spleen grown in tissue culture compared to normal red cells. The factors responsible for this differential phagocytosis were opsonic properties of serum and the capacity of macrophages to ingest modified red cells maximally in a certain time. The roles of other factors such as age of macrophages, evaluation of phagocytic index and the per cent of susceptible erythrocytes available were also examined.

Effect of variations in osmotic pressure on macrophages in tissue culture.

Summary1. The optimal growth in tissue culture of macrophages and the exoerythrocytic forms of Plasmodium gallinaceum occurred between 7 and 11 atmospheres of pressure. Growth was reduced to 50% at 5 and 13 atmospheres.2. High concentrations of serum (over 50%) were inhibitory to both the malarial parasites and the host cell, the macrophage.