Twenty-Fifth Annual National Meeting Reticuloendothelial Society GABRIEL Deportment
and Clinical Cnrolincr.
AND H. HUGH
trnd Microbiology. South Corolinn
The 25th annual national meeting of the Reticuloendothelial Society was held in Charleston, South Carolina, December 6-9, 1978. The program consisted of three of the Reticuloendothelial symposia (“Macrophage Enzymes, ” “Pharmacology System,” and “The Role of Macrophages in Tumor Immunology”), the address of by Dr. Elliot F. Osserman), and 12 general the keynote speaker (“Lysozyme” sessions for the presentation of 136 solicited free communications. The keynote lecture by Dr. Osserman summarized the current state of knowledge concerning the structure and biological function of lysozyme. In addition, the competition for graduate student presentations attracted a large number of entries and was won by P. A. Marino, with a presentation entitled “Specificity and Kinetics of Tumor Cell Binding to Activated Macrophages.” The competition for postdoctoral fellows was won by Dr. E. W. Ades, with a communication entitled “Surface Receptor Expression on Human Null Cells (E-Rosette Negative, sIg Negative).” The first symposium, entitled “Secretory and Regulatory Products of Macrophages,” chaired by Dr. N. R. Di Luzio, Tulane University School of Medicine, was designed to provide a basis for the sessions which followed. Dr. Philip Davies, Merck Institute for Therapeutic Research, opened the session with an overview on secretory products and biochemical alterations of activated macrophages. Dr. Davies defined the functional characteristics of macrophages and their responses to various stimulating agents. He described in detail the striking responses of macrophages to changes in environment and various stimuli. Examples of substances released on stimulation of mononuclear phagocytes, such as lysozyme, complement, pyrogen, prostaglandins, chemotactic substances, stimulants for T and B cells, colony-stimulating factor, and cytotoxic factors for tumor cells, were among those discussed. Additionally, such secretory products as plasminogen activator, collagenase, elastase, and interferon, were considered in detail. The role of each product in such diverse macrophage-mediated events as modification of infectious diseases and the role of macrophages in inflammation and control of tumor growth were discussed. An additional consideration in this presentation was the mechanism(s) whereby macrophages may be modulated in their activity by factors or products from tumor cells, bacteria, and fungi. 137 0090-1229/79/090137-06$01.00/O Copyright ci‘m1979 by Academic Press. Inc. All rights of reproduction in any form reserved
This overview was followed by a detailed discussion of “The Mechanism of Endo- and Exocytosis of Macrophages” by Dr. Samuel S. Spicer, Medical University of South Carolina, who reviewed the nature of the mononuclear cell system and its contributions to homeostasis and discussed in detail the intracellular localization of various monocyte-macrophage enzymes in a variety of tissues. The use of immunocytochemical procedures to define precise cytoplasmic or granular localization of many of the important enzymes discussed by Dr. Davies was described by Dr. Spicer. Additionally, the specific cell types producing various secretory and regulatory products and their significance in maintaining surveillance mechanisms of the host were discussed. Dr. Di Luzio reported on the potential use of lysozyme as an index of macrophage function. Previous studies in his laboratory demonstrated that animals receiving glucan, a potent macrophage-activating agent, showed increased resistance to a variety of bacterial, viral, fungal, and parasitic infections. These studies led to the observation that animals with activated macrophage activity possessed significant elevations in serum lysozyme. Studies were therefore undertaken by Dr. Di Luzio’s group to evaluate the possibility that serum lysozyme levels may provide an index of the functional status of the macrophage population, since one of the present limitations in clinical and experimental research is the absence of any con\‘enielzf index of macrophage function. The studies presented by Dr. Di Luzio demonstrated that serum lysozyme was significantly elevated in animals following administration of macrophage-activating agents; conversely, serum lysozyme was significantly depressed by the administration of reticuloendothelial depressants. Dose-response relationships were established for macrophage stimulators, and a correlation was shown between the level of serum lysozyme and resistance to tumor growth and development. Doses of glucan effective in promoting tumor regression were associated with macrophage activation and elevated serum lysozyme activities: while conversely, the administration of methyl palmitate, which suppresses macrophage activity, depressed serum lysozyme levels and enhanced tumor growth. The possibility that the secretion of lysozyme by activated macrophages may play a significant role not only in the antibacterial activity of glucan but also in tumoricidal mechanisms was discussed. The presentation on lysozyme was followed by a paper on another secretory product of macrophages, interferon. Dr. Richard M. Schultz, National Cancer Institute, presented a report on “The Role of E Type F’rostaglandins in Regulation of Interferon-Treated Macrophage Cytotoxic Activity.” Drs. Schultz and Chirigos found that normal mouse macrophages could be activated and rendered cytotoxic by treatment with highly purified virus-induced libroblast interferon, and that the treated mouse macrophages had specific cytotoxic activity toward a variety of tumor cells. Prostaglandins of the E series, administered either in Eitro or in \riw, suppressed the cytotoxic activity of interferon-treated macrophages. Dr. Schultz suggested that prostaglandin synthesis by macrophages following certain activation stimuli could produce negative feedback inhibition to limit macrophage activity, and/or that the ability of tumors to produce E-type prostaglandins might be a possible mechanism by which tumor cells escape surveillance by activated macrophages. Dr. Michael Chirigos, National Cancer Institute, presented the final paper, entitled “Factors Affecting Macrophage Cytotoxic Activity,” with particular em-
phasis on corticosteroids and acute stress. He reported that the tumoricidal activity of interferon-activated macrophages could be modulated in vitro by physiological concentrations of various pharmacologically active agents. Glucocorticoid hormones and their synthetic derivatives, as well as E-type prostaglandins, corticosteroids, and dibutyryl cyclic AMP, markedly inhibit the cytotoxic activity of interferon-treated macrophages. Dr. Chirigos suggested that the response of macrophages to tumor cells can be influenced by a variety of factors present in the environment of the host. Finally, Dr. Osserman commented on the findings presented during the symposium. He noted that the area of secretory and regulatory products of macrophages is a frontier of immunology and one that will, when defined, significantly contribute to the health and welfare of man. The second symposium, entitled “The Pharmacology of the Reticuloendothelial System,” was chaired by Dr. David R. Webb, Roche Institute of Molecular Biology. In his introductory statement, Dr. Webb noted that immunologists have only recently begun to investigate the details of the complex cell interactions and metabolic events which govern the function of immunocompetent cells. Specific drugs or naturally occurring mediators have been used increasingly to probe the immune system, and this has lead to the establishment of a new subdiscipline of immunology, immunopharmacology, which deals with the pharmacologic manipulation of the immune system. The first presentation of this symposium was on “Immunologic Effects of Corticosteroids, Personality, and Stress” by Dr. J. John Cohen, University of Colorado. Corticosteroids are known to have potent effects on the immune and reticuloendothelial systems. For example, after high doses of steroids, lymphocytes, especially T cells, change their recirculation patterns, and T cells are found sequestered in the bone marrow, apparently due to changes in the bone marrow rather than in the lymphocyte. Dr. Cohen reported that most of the effects of exogenous steroids could be mimicked by stress, and that large numbers of T cells were sequestered in the bone marrow of stressed mice. The number of T cells in the marrow of “normal” mice correlated with measures of “emotionality,” which may reflect the way in which mice cope with everyday stress. These data were discussed in light of reports that stress is immunosuppressive, and that personality can be used as a predictor of immunologically related disease, including cancer. Dr. Frank H. Valone, Harvard Medical School, next discussed “Regulation of the Polymorphonuclear Leukocyte Chemotactic Response,” beginning with a brief review of PMN activity in inflammation. The initial event in the stimulation of PMN leukocyte accumulation at sites of tissue injury involves the activation of humoral and cellular pathways for the generation and release of diverse chemotactic factors. The preferential accumulation of one cell type is then dependent on the potency and specificity of the chemotactic factors and on factordirected and leukocyte-directed modulatory principles. CSa attracts eosinophils, neutrophils, basophils, and monocytes with comparable activity; kallikrein preferentially attracts neutrophils; and ECF-A and HETE are most chemotactic for eosinophils. Chemotactic factors are reversibly inactivated by a,-macroglobulin and Cl inhibitor, whereas lysosomal proteases, chemotactic factor inactivator. and the anaphylatoxin inactivator irreversibly degrade chemotactic factors. Leukocyte-directed inhibition is achieved by the chemotactic factors which in-
duce chemotactic deactivation, leukocyte aggregation, and, after systemic infusion, leukocyte depletion. Neutrophil immobilizing factor (NIF), a neutrophil product, suppresses migration without influencing cellular metabolism. Leukocyte inhibitory factor, a lymphokine, suppresses migration both directly and through the release of NIF. Chemotaxis is suppressed by nonchemotactic agents such as colchicine, cytochalasin B, and amphotericin B, as well as agents which elevate intracellular cyclic AMP such as isoproterenol, PGE2, and histamine. Although the role of cyclic GMP is unclear, chemotaxis is enhanced by ascorbic acid, serotonin, levamisole, and PGF,,. Thus, the influx of leukocytes at sites of tissue injury is regulated at multiple functional levels. Dr. James S. Goodwin, University of New Mexico School of Medicine, next discussed “Prostaglandin-Producing Suppressor Cells in Humans.” It is known that prostaglandins (PGs) of the E series inhibit many assays of T and B cell function in vitro. PGE, is produced by glass-adherent peripheral blood mononuclear cells (PBMC) and inhibits the T-cell response to mitogens or antigens. Addition of indomethacin or other prostaglandin synthetase inhibitors to mitogen- or antigen-stimulated cultures of human PBMC blocks the production of PGE, and enhances the T-cell response, as measured by [“Hlthymidine incorporation. Prostaglandin-producing suppressor cells appear to be partly responsible for the depressed cellular immunity associated with several human conditions. In Hodgkin’s disease and sarcoidosis there is increased activity of the prostaglandin-producing suppressor cell; i.e.. increased PGE, production in culture. Elimination of PGE, production, either by addition of indomethacin or by removal of the glass-adherent cells on glass-wool columns, eliminates the depressed mitogen responses of PBMC from patients with these disorders. lndomethacin also restores the depressed mitogen responses of PBMC from healthy old people to the normal range. In this case the PG-producing suppressor cell is not more active, but the T cells of old people are more sensitive to inhibition by PGEz. Further studies have shown that indomethacin in ri~o can partially restore the depressed cellular immunity of patients with common variable immunodeficiency. Two patients who were anergic to skin testing became reactive while on indomethacin and returned to anergy after indomethacin was stopped. Indomethacin administration also enhances the antibody response to influenza vattine in normal humans. The third symposium, entitled “The Role of Macrophages in Tumor ImmunoiDuke University. The relative ogy,” was chaired by Dr. Ralph Snyderman, biological importance of immune function in providing resistance to tumor development and spread is not yet fully understood; however. there is substantial evidence that macrophages may be instrumental in mediating this resistance. Moreover, there is substantial evidence that macrophages are necessary for the mediation of tumor destruction in many forms of immunotherapy. Therefore. this symposium was organized in an attempt to bring together leading investigators in the field of macrophage immunobiology and its role in tumor immunity. The speakers discussed various aspects of macrophage biology and the role of this cell type in tumor surveillance and tumor destruction. Dr. Monte S. Meltzer, National Cancer Institute, spoke on “Macrophage Activation for Tumor Cell Cytotoxicity-Analysis of Intermediary Reactions.” He defined the mechanisms whereby macrophages undergo activation and acquire tumoricidal capacity. He
demonstrated that for a macrophage to be activated (i.e., to have the potential to kill tumor cells) requires the presence of effective activation signals and a normal response to such signals. He described the existence of a macrophage activation sequence, based on an analysis of lymphokine-induced activation of macrophages in vitro. He reported the use of resident peritoneal macrophages from normal mice to show that tumoricidal activity in vitro could be induced in these cells after treatment with supernatants of antigen-stimulated lymphocyte cultures. He described the time course for lymphocyte-induced macrophage activation and showed that such activation was short-lived when the lymphocyte supematants were removed. The loss of lymphokine responsiveness by macrophages in tissue culture was irreversible but not due to cell death. He provided data which suggested that only a subpopulation of resident macrophages could respond to lymphokines under tissue culture conditions. He also described some in \givo studies which suggested that BCG activation of tumoricidal potential in macrophages involves the production of lymphokines which activate immature, peroxidase-positive, blood-derived mononuclear phagocytes which are newly arrived at inflammatory sites. He concluded that macrophage activation for tumoricidal cytotoxicity is the final result of a cascade of short-lived intermediary reactions. The tumoricidal activity of fully activated cells and the responsiveness of noncytotoxic intermediates to activation stimuli are all short-lived macrophage functions and can be modified by many factors, at the local reaction site, from serum, or from tumors themselves. Dr. J. Stephen Haskill, University of North Carolina School of Medicine, described the correlation of in vitro and in tvivo anti-tumor defense mechanisms. In the studies reported, tumors were removed from animals with progressing or regressing neoplasms. By the use of enzymatic digestion the tumors were broken down into single cell suspensions, and by density gradient sedimentation the various cell types were removed. With this methodology, Dr. Haskill has been able to isolate inflammatory cells which migrate into neoplasms. Lymphocytes and macrophages can be isolated from both progressor and regressor neoplasms. At least two different types of macrophage-like cells were isolated from tumors: one a very large, phagocytically active cell and the second a smaller, less phagocytically active cell. Cells with capacity to participate in antibody-directed cellular cytotoxicity were present in both the blood and tissues of tumor-bearing animals. Large macrophages isolated from tumor sites were capable of actually phagocytizing rapidly growing tumor cells and were found in greater numbers in regressor than in progressor neoplasms. These studies provide new insight into the immunological events occurring within tumors themselves, and should permit the dissection of immune responses within tumors of animals undergoing neoplastic regression or progression, as well as definition of the factors which correlate with successful or unsuccessful host responses to neoplastic growth. Dr. Robert Evans, Jackson Laboratories, discussed host cells in murine tumors and their possible relevance to tumor growth. He concluded that the composition of progressive tumors represents a complex interaction between cells and humoral factors, the net result being a solid tumor mass that overwhelms the host. He suggested various ways in which host cells may influence tumor growth directly or indirectly and concluded that the greatest problem facing tumor immunobiologists is that of equating in vitro observations with events seen to occur in tivo. He
described situations in which tumor-associated host cells could either destroy or stimulate neoplastic cells and showed that progressor tumors contained increased numbers of host inflammatory cells. He also described the seemingly paradoxical finding of enhanced lymphoma cell growth in the presence of supematants from cytotoxic macrophages. Despite the fact that under many irz vitro conditions it can be shown that lymphoid cells have the capacity to destroy neoplastic cells, Dr. Evans presented data which indicated that host cells alone or with humoral factors could perhaps inadvertently encourage tumors to grow. The next speaker, Dr. Michael G. Hanna, Jr.) Frederick Cancer Research Center, discussed the cells of the macrophage-histiocyte series in nonspecific and specific immunotherapy. Dr. Hanna described a method whereby guinea pigs could be immunized actively and specifically with line 10 tumor cells so that they could resist intravenous inoculations of viable tumor cells. This method involved immunizing animals with dispersed irradiated solid tumor cells, accompanied by BCG. He showed that with proper immunization protocols he could protect animals from mortality with injections of doses of viable tumor cells ranging from ten thousand to one million. BCG was absolutely necessary for the immunization of animals with the irradiated tumor cells; if BCG was not given with the tumor cells. the animals died upon subsequent rechallenge with nonirradiated tumor cells. Dr. Snyderman next described “The Effects of Neoplasms on Macrophage Migration-A Mechanism for Subversion of Surveillance.” His data indicated that neoplastic cells produce and release a low-molecular-weight factor capable of depressing macrophage accumulation in response to delayed hypersensitivity reactions in biro. This factor is extremely potent in \?\YI; material derived from as few as 200 tumor cells can significantly depress macrophage accumulation in I~\Y). It can be found in transplantable tumors and can also be isolated from spontaneous mammary carcinomas in C3H/Hen mice. Dr. Snyderman proposed that tumors may release low-molecular-weight factors which depress macrophage migratory ability, thereby protecting themselves from immune surveillance during critical phases of their establishment as progressor neoplasms. The final speaker, Dr. Anne Weeks, University of South Carolina School of Medicine, described the effects of age and nutrition on macrophage function. She demonstrated that malnourished guinea pigs had significantly depressed cellmediated immune reactions in l,i\~> and in \aitro. She showed that, in addition to delayed hypersensitivity reactions in rvi\~>. chemotactic activity and phagocytic activity in rlitro were depressed in malnourished animals. Aging similarly had depressive effects on macrophage function. Dr. Weeks proposed that the depression of macrophage function in malnourished animals and in extremely young or old animals may be responsible for the increased incidence of neoplasia in such animals. In summary, the speakers discussed the requisites for the acquisition of tumoricidal capacity by macrophages, methods for immunotherapy which successfully prevent the growth of tumors injected in \Ylv. the biological characteristics of immune cells derived from tumors, and mechanisms whereby tumors may themselves abrogate immune surveillance. ACKNOWLEDGMENTS This meeting was supported in part by the Office assistance in preparing the report.