Stephen W. Russell

Regulations of macrophage-mediated tumor cell killing by prostaglandins: Comparison of the effects of PGE2 and PGI2

Mouse resident peritoneal macrophages stimulated in vitro by purified bacterial lipopolysaccharide (LPS) produced both prostaglandin E2 (PGE2) and prostaglandin I2 (PGI2), the latter detected as its stable metabolite, 6-keto PGF1 alpha. Maximum production, induced in each case by 1 ng/ml purified LPS, was in the range of 10(-7)M for PGI2 and 3 x 10(-8)M for PGE2. A quantitatively similar increase in intracellular levels of macrophage cyclic AMP (measured on a whole cell basis), with a similar duration of effect, was stimulated by PGE2 and PGI2; however, only PGE2 had a negative regulatory effect on macrophage activation for tumor cell killing. These data confirm that more than a whole cell increase in the concentration of cyclic AMP is needed to shut off nonspecific tumor cell killing mediated by LPS-activated resident peritoneal macrophages.

The strain of mouse and assay conditions influence whether MuIFN-gamma primes or activates macrophages for tumor cell killing.

Investigators from two different laboratories have compared several variables in the short‐term macrophage‐mediated cytotoxicity assays used by each group to study the role of MulFN‐ γin macrophage activation. The findings suggest that the capacity of MulFN‐ γto activate macrophages without the need for a second triggering stimulus is related to assay conditions and, most especially, the strain of mouse used to provide the macrophages.

Both the Kind and Magnitude of Stimulus Are Important in Overcoming the Negative Regulation of Macrophage Activation by PGE2

Macrophages activated for tumor cell killing by bacterial lipopolysaccharide (LPS) were shown to lose their cytolytic activity if exposed to physiological levels of prostagalandin E2 (PGE2). Increasing the LPS stimulus more than 100‐fold over the amount needed to activate the cells did not substantially increase their resistance to the negative regulatory effect of PGE2. By contrast, killing mediated by macrophages activated by a mixture of LPS and gamma interferon was maintained. The degree of resistance conferred was directly related to the magnitude of the stimulus employed, reaching the point where not even 10–5 M PGE2 would diminish killing. Killing by both activated resident and inflammatory peritoneal macrophages could be maintained, but it was easier to do so if the cells had been elicited by an inflammatory stimulus. A preparation of type I interferons produced by cells of the macrophage cell line J774A.1 behaved similarly, but was over 500 times less efficient at helping to maintain killing than gamma (type II) interferon was. Alpha interferon alone, i.e., without LPS, was capable both of activating macrophages and of maintaining the activated state in the presence of PGE2. The capacity for both activation and maintenance could be strikingly enhanced, however, by mixing alpha and gamma interferons together under conditions that were free of detectable LPS. The data reported here collectively suggest that induction and maintenance of macrophage activation may be separable mechanistically, and that the interferons are important to host defense not only because they participate in the induction of macrophage activation for tumor cell killing but also because they help to maintain the activated state once it has been induced.

IDENTIFICATION OF MONONUCLEAR PHAGOCYTES BY INGESTION OF PARTICULATE MATERIALS, SUCH AS ERYTHROCYTES, CARBON, ZYMOSAN, OR LATEX

Publisher Summary This chapter discusses the identification of mononuclear phagocytes by the ingestion of participate materials, such as erythrocytes, carbon, zymosan, or latex. Mononuclear phagocytes are generally less avidly phagocytic when in suspension, especially if particles do not bind strongly to their surfaces. In addition to increasing the efficiency with which uptake occurs, by forming a monolayer, one enriches for mononuclear phagocytes and other adherent cells, eliminates loss of adherent cells from suspensions during incubation steps, and facilitates the removal of uningested particles after incubation has been completed. The principal problem with carbon particles centers on their tendency to obscure the cells with which they are associated. Neutrophils engulf this material and, when engorged with it, can be indistinguishable from mononuclear phagocytes that are similarly filled. If neutrophils contaminate the monolayer, it is essential that the result with carbon be interpreted as indicative of the total number of phagocytic cells in the monolayer.

Gamma interferon interferes with the negative regulation of macrophage activation by prostaglandin E2

Activation of mouse macrophages for tumor cell killing is negatively regulated by prostaglandin E2 (PGE2). The effect of this hormone is to shut off cytolytic activity that is expressed as a consequence of activation. A lymphokine in the culture supernates of concanavalin A stimulated spleen cells has been shown to change the sensitivity of activated macrophages to the negative regulatory effects of PGE2, thereby maintaining activation, as manifested by the continued expression of tumor cell killing by these cells. Using a highly specific polyclonal antiserum and gamma interferon produced either by a T-cell hybridoma or by recombinant DNA technology we show here that one lymphokine responsible for mediating the maintenance effect is gamma interferon.

Comparison of Five Short‐Term Assays That Measure Nonspecific Cytotoxicity Mediated to Tumor Cells by Activated Macrophages

Five different short term assays (<48 h) used to measure macrophage‐mediated, nonspecific cytotoxicity were compared under similar conditions in the same laboratory using the same reagents. The purpose was to determine the extent to which results were comparable. Three of the assays were dependent on the release of a radioisotope to measure cytotoxicity, one was dependent on cell counting, and the last was dependent on flow cytometric quantification of remaining viable tumor target cells after they had been exposed to macrophages. The variables examined were the following: a) three different populations of macrophages; b) four different kinds of target cells; c) two types of radioisotopes; and d) two different agents that trigger the expression of cytolytic activity by primed macrophages. Recombinant gamma interferon was used as the priming agent in all the experiments. There was unexpectedly good agreement between the results of the various assays. No differences were found among the different macrophage populations, the isotopes or the triggering agents. Perhaps the most important finding was that differences in target cell susceptibility to killing by activated macrophages, which were ap‐

Heterogeneity among macrophages cultured from mouse bone marrow

SummaryThe development of macrophages in culture from mouse bone marrow was followed for 14 days by light and electron microscopy, ultrastructural cytochemistry, and flow cytometric analysis. By 10 days greater than 97% of the cells in culture were mononuclear phagocytes, and by 12 days greater than 99% were identifiable as macrophages. Ultrastructurally, three subpopulations of mononuclear phagocytes were distinguished based on the appearance of cytoplasmic structures. Early in culture, cells containing large, membrane-bounded vesicles predominated. With increasing time in culture these cells were replaced to varying degrees first by cells that contained vesicles filled with relatively dense, osmiophilic material and, finally, by macrophages that contained granules of various sizes, shapes and staining densities. Cytochemical (peroxidase and acid phosphatase) and colloidal gold uptake studies at the ultrastructural level suggested that many, if not all, of these cytoplasmic structures arose by pinocytosis and subsequent fusion of pinocytic vesicles with lysosomes. Analysis of DNA content of propidium iodide-stained nuclei by flow cytometry, coupled with the examination of cells treated with colchicine to arrest mitosis in metaphase, suggested that cell cycling was a negligible contributor to heterogeneity within cultured populations. Thus, by waiting until 12–14 days after bone marrow cultures were initiated, with partial replenishment of the culture medium at 7 days, heterogeneity could be greatly reduced in cultured macrophage populations. Taking this fact into consideration could help to reduce the variability seen in functional studies of macrophage populations that are less homogeneous.

Culture of macrophages from bovine bone marrow

Macrophages perform important immunoregulatory and host defense functions. Examination of this cell type in the bovine has been restricted because of lack of a means to obtain pure bovine macrophage populations reproducibly. We have developed a system for production of large numbers of macrophages from this species with greater than 99% purity. Stem cells were obtained from the bone marrow of neonatal calves and cultured in vitro in the presence of macrophage-colony-stimulating factor. Bovine bone marrow culture-derived macrophages were esterase-positive, expressed Fc receptors for aggregated IgG, and bovine macrophage differentiation markers. In addition, they displayed class I and class II major histocompatibility (MHC) antigens. The level of MHC antigen expressed could be further enhanced by treatment with recombinant bovine interferons. The macrophages exhibited expected functions, for example, Fc-mediated ingestion of opsonized sheep red blood cells. Augmentation of phagocytic capacity by either alpha or gamma interferon could also be demonstrated. The data reported here confirm that bone marrow culture is a convenient, reliable source of macrophages for investigations of this bovine cell type.

Enrichment and initial characterization of the solubilized receptor for mouse gamma interferon

The work reported here constitutes a first step in characterizing the receptor for mouse gamma interferon at the biochemical level. The myelomonocytic cell line, WEHI-3, was the source of starting material. Iodinated recombinant mouse gamma interferon incubated with WEHI-3 cells, as well as membranes prepared from them, bound specifically to a single class of sites with a Kd of 7 x 10(-9)M. Membranes were solubilized with the non-ionic detergent octyl-beta-D-glucopyranoside. As solubilization proceeded, binding activity could be assayed by precipitating the receptor with acetone in the presence of egg phosphatidylcholine liposomes. The Kd of the receptor in association with liposomes was 13 nM. Again here, only a single class of binding activity was found, and specificity for gamma, compared to other interferons, was maintained. This is the first time that the receptor for mouse gamma interferon has been solubilized and recovered in functional form. Further characterization included at least a 200-fold enrichment of binding activity by ligand affinity chromatography, resulting in the identification of a 95 kDa protein as the most likely candidate for either the receptor or a binding subunit thereof.

Autoregulation of mononuclear phagocyte function.

Among phagocytic cells the mononuclear phagocyte is particularly notable for the diversity of its functions. One explanation for such diversity is the fact that mononuclear phagocytes respond in a variety of ways to a number of different stimuli. Some of these stimuli can be produced by the mononuclear phagocytes themselves, raising the possibility that these cells are autoregulatory. It is my intention to consider this possibility here. Four classes of secretory products with autoregulatory potential will be considered, including proteinases, complement proteins, interferons, and prostaglandins.