Search

Interleukin II, also called T-cell growth factor, induces proliferation of immature peanut agglutinin positive murine thymocytes (PNA+) and renders them responsive to concanavalin A. It also, as shown by others, provides a second signal to induce phytohemagglutinin-primed mature human T-lymphocytes to enter DNA synthesis. Both actions are associated with early increases (2-60 min) in cellular levels of cyclic 3'5' guanosine monophosphate (cyclic GMP) without change of cyclic 3'5' adenosine monophosphate (cyclic AMP) levels. Cyclic GMP is postulated to represent part of the mechanism by which IL-2 acts.

The action of the purified thymic factor, thymopoietin, on populations of post-thymic lymphocytes has been studied. Thymopoietin, at concentrations as low as 1.5 ng/ml, uniquely enhanced the proliferative response of peripheral T cells from lymph node and spleen to allogeneic stimulation. Enhancement of the allogeneic response (MLR) was not produced by several polypeptide hormones, including insulin, ACTH, HCG, or Ubiquitin. Treatment of spleen cells with anti-Thy-1 antiserum almost completely abolished the MLR. Thymopoietin's stimulatory effects could not reverse this. Thymopoietin treatment of Thy-1+-enriched spleen cell populations enhanced the MLR even when thymopoietin was removed as early as 2 min after incubation with responding cells. The interaction of thymopoietin with peripheral Thy-1+ cell populations produced a rapid and transient rise in cyclic GMP levels and slightly decreased cyclic AMP levels. These results suggest that thymopoietin interacts with one or more Thy-1+ subpopulations and that this interaction involves early changes in cyclic nucleotide metabolism.

Induction of thymus-dependent lymphocyte (T cell) differentiation was performed in vitro with thymic factors as inducers. T cell precursors from human bone marrow first expressed surface differentiation antigens and then acquired the capacity to form rosettes with sheep erythrocytes. The latter marker could not be induced when cells with differentiation antigens had been eliminated. The proliferative responses to phytomitogens or to allogeneic stimuli appeared to be characteristics of later stages in differentiation that also can be induced or amplified by in vitro incubation of marrow cells or thymocytes with thymic factors. When phytomitogen-responsive cells from peripheral blood were inactivated in vitro , the allogeneic response was enhanced. Although these responses are acquired almost concomitantly, they are therefore envisioned to be characteristics of separate T cell subsets. After immunological reconstitution of patients, the T cell development in vivo involves a succession of differentiation events similar to that described above. Our experiments with mice, using similar methods, have also shown that graft-versus-host inducing capacity is a function of a cell population distinct from that which yields a proliferative response to in vitro stimulation by phytohemagglutinin. These results support our model of sequential differentiation of human prothymocytes into various subsets of mature T cells.

The ability of glucocorticoids to modify lymphokine-induced macrophage proliferation, an in vitro correlate of cellular immunity in the guinea pig, was investigated. Lymphocyte production of macrophage mitogenic factor (MMF) was decreased in the presence of physiological concentrations of glucocorticoids. Inhibition was concentration dependent (IC 50 of triamcinolone acetonide (TA): 2 × 10 −9 M ), glucocorticoid specific, and reversed by cortexolone. In contrast, pharmacological concentrations of glucocorticoids were necessary to inhibit macrophage proliferation induced by suboptimal dilutions of MMF. This inhibition was concentration dependent (IC 50 of TA: 4 × 10 −7 M ), glucocorticoid specific, and reversed by cortexolone. At supraoptimal dilutions of MMF, glucocorticoids caused a twofold potentiation of MMF-induced macrophage proliferation. Potentiation was concentration dependent (EC 50 of TA: 3 × 10 −8 M ), glucocorticoid specific, reversed by glucocorticoid antagonists, and occurred in the presence of indomethacin. Thus, glucocorticoids regulate both the initiation and effector phases of this in vitro model of delayed hypersensitivity. However, the results indicate that the major mechanism of glucocorticoid-mediated anti-inflammatory action occurs at the level of the MMF-producing lymphocyte rather than at the effector macrophage, as MMF-induced proliferation is likely controlled by opposing glucocorticoid-sensitive mechanisms.

Levamisole, an immunopotentiator, was compared to its parent molecule imidazole in its effects on lymphocyte proliferation and cyclic nucleotide levels. Both levamisole and imidazole augment phytohemagglutinin-induced proliferation of human peripheral blood lymphocytes, mouse thymocytes and T-cell enriched splenocytes. Both agents increase cyclic GMP levels of T-cell enriched mouse spleen cells with a dose—response curve which closely parallels their effects on proliferation. Both agents lower cyclic AMP levels in these cells. The data are consistent with the interpretation that the immunopotentiation induced by levamisole may result from imidazole-like effects on lymphocyte cyclic nucleotide levels, particularly cyclic GMP.

Muramyl dipeptide (MDP) and the butyl ester derivative, N- acetylmuramyl- l -alanyl- d -glutamine-alpha -n- butyl ester (MDP[Gln]OnBu), were shown to induce the in vitro proliferation of oil-induced guinea pig peritoneal exudate cells (PEC). Both agents induced 10–20 fold increases in tritiated thymidine incorporation in PEC cultures. The maximal effects occurred in 72 h cultures stimulated with either 0.1 μg MDP or 10 μg MDP[Gln]OnBu. The mitogenic effects of MDP appeared to be mediated by a macrophage product detected in the supernatants of MDP-stimulated cultures. Supernatants of MDP- or MDP[Gln]OnBu-stimulated PEC cultures were also inhibitory to normal fibroblast growth and cytotoxic to L929 tumor cells. These results indicated that these agents may stimulate macrophages by modulating secretory functions. In addition, either peptidoglycan was capable of activating bactericidal activity in in vitro macrophage cultures. Initial studies of possible mechanisms of action revealed an early increase in the level of cyclic GMP. The possible role of cyclic GMP in mediating the stimulation of macrophage secretory processes is discussed.

Adenosine 3′:5′-cyclic monophosphate (cyclic AMP) has been shown to have an antimitotic role in various cell types, and it has been hypothesized that a decrease of cyclic AMP concentration in the cell initiates or permits cell division. This hypothesis has been evaluated with respect to clonal proliferation of lymphocytes. Two potent mitogenic agents, phytohemagglutinin and concanavalin A, which induce thymic-dependent lymphocytes to undergo clonal proliferation, were examined for their ability to initiate proliferation and to alter the concentrations of cyclic AMP and guanosine 3′:5′-cyclic monophosphate (cyclic GMP) in purified human peripheral blood-lymphocytes. Optimal mitogenic concentrations of phytohemagglutinin and concanavalin A produced 10- to 50-fold increases in the concentration of lymphocyte cyclic GMP within the first 20 min of exposure to the mitogens. No changes were seen in the concentration of cyclic AMP after stimulation with either mitogen in purified form. Increases of less than 2-fold in the concentration of lymphocyte cyclic AMP observed with a less purified preparation of phytohemagglutinin could be attributed to the agglutinating rather than the mitogenic properties of the mitogen. A revised hypothesis is presented in which a temporally discrete rise in lymphocyte cyclic GMP concentration is viewed as an active signal to induce proliferation, while the elevation of cyclic AMP concentration in these cells is viewed as a regulatory influence that limits or inhibits mitogenic action.

Guanosine 3',5'-monophosphate (cyclic GMP) and cholinergic agents stimulate incorporation of phosphate into specific nuclear acidic proteins of horse peripheral blood lymphocytes. Agents that raise intracellular adenosine 3',5'-monophosphate (cyclic AMP) inhibit nuclear acidic protein phosphorylation. The opposing effects of cyclic GMP and cyclic AMP upon nuclear protein phosphorylation parallel the effects of the cyclic nucleotides upon induction of lymphocyte proliferation.

Besides being activated through the T-cell receptor in response to antigen presented in the context of self MHC molecules, T lymphocytes can also be activated by mitogens such as plant lectins. In this case two separate signals — one from the mitogen the other from interleukin 2 — are required to complete the cell cycle. Here, John Hadden details the biochemical events associated with mitogenic activation and emphasizes the importance of calcium and arachidonic acid metabolism in these processes.

In cancer, there appears to be a direct relationship between immunosuppression and the ability of neoplastic cells to flourish. Because the immune system is compromised, tumor growth is unchecked by the normal immune regulatory mechanisms. For this reason it has been suggested that immunomodulators, agents that directly affect immune function, might be used therapeutically in the treatment of cancer. Such compounds could restore the body’s capacity to mount an immunologic response leading to the eventual destruction of the neoplasm. Perhaps better supported by clinical evidence, is the possible use of immunomodulators as adjuvants to standard cancer therapies, such as chemotherapy or radiotherapy. The generalized immunosuppression resulting from these procedures often leads to severe problems with opportunistic infections. Used in combination with standard chemotherapy or radiotherapy, immunomodulators might speed the recovery of normal immune function thereby not only reducing the incidence of recurrence but also reducing the likelihood of infection. Immunomodulators, therefore, might be used either as a primary cancer therapy or as an adjuvant to established therapies. In this paper we review recent investigations which support the potential application of two immunomodulators, Isoprinosine and NPT 15392, in cancer therapy.

The mononuclear phagocyte system (MPS) is centrally important in both specific and nonspecific immune responses. Once regarded primarily as scavenger cells with remarkable phagocytic capacity, macrophages are now recognized as effector cells that function in the induction, amplification, expression, and regulation of humoral and cellular immunity. Through numerous, diverse secretory products, mononuclear phagocytes contribute to inflammatory reactions and delayed-type hypersensitivity, participate in tissue repair and wound healing, and regulate proliferation and differentiation of various tissues. Cells of this system are responsible for processing antigen and presenting immunogen to lymphocytes. Activated macrophages are capable of intracellular destruction of microbial pathogens and extracellular lysis of tumor cells. Thus, mononuclear phagocytes are considered vital to host defenses against a wide variety of pathogens and contribute to immunosurveillance against neoplasia.

Two major questions concerning the role of cyclic adenosine 3′,5′-monophosphate (cyclic AMP) in lymphocyte metabolism and function have been focused upon in recent years: (1) What role does cyclic AMP play in mediating hormonal influences to modify lymphocyte proliferation and function? (2) Do mitogens initiate proliferation by using a second-messenger system like cyclic AMP?

The role of thymic epithelium in T cell development has given rise to a number of studies, but less information is available concerning the factors regulating thymic epithelial cells (TEC) themselves. Several cytokines, natural or recombinant, were investigated for their effects on human TEC proliferation. This study presents evidence for the first time that human recombinant interleukin 1 (IL1) and IL1-containing mixed cytokine preparations induced DNA synthesis of TEC as measured in a 48-hr stimulation assay. The effects of IL1 were dose dependent and sustained in time. The following recombinant cytokines, IL2, IL3, IL4, interferon-gamma (IFN-gamma), IFN-alpha, tumor necrosis factor-alpha (TNF alpha), and TNF beta, as well as thymosin fraction 5 and Escherichia coli lipopolysaccharide (LPS), were not found to modify TEC proliferation but IFN-gamma and TNF alpha enhanced the effects of IL1. We also report that IL1 induced a profound change in the morphology of TEC. Our observations suggest that TEC are targets for the action of cytokines and emphasize the important role played by IL1 within the thymus.

Efforts to establish an immunopharmacology for cancer immunotherapy have been frustrated by the lack of animal tumor models which directly relate to human cancer. Initially, emphasis was placed on transplantable tumors which were often inadvertently immunogenic by nature of infection with murine viruses or were of nonsyngeneic origins. The easily demonstrated effectiveness of BCG, C. parvum and high molecular weight polysaccharide preparations led to the erroneous conclusion that such therapy in man would be equally effective. Considerable clinical trials since have not generally supported this conclusion. The relative failure of these agents in the human can be attributed to a number of factors including the inadequacy of the models. Complexity and ambivalence of their actions in humans have played a major role. The evolution of immunotherapy in human cancer has increasingly moved away from the complex agents towards the use of biologicals and chemically defined agents. It is clear that seldom are the agents effective in cancer therapy when used as a monotherapy. The apparent rule, both in murine tumor systems and in human cancer trials, has emerged that these agents act to increase the number of individuals remaining in remission following effective cytoreductive therapy with chemotherapy, surgery or irradiation. They have not been particularly active in treating progressive disease. Levamisole offers a case in point. In 34 animal tumor models in which it was used as a monotherapy no effect was observed; however, following cytoreductive therapy it was active in 13 of 21 systems. The experience in human cancer generally parallels these results. If one accepts the contention that levamisole is the only agent whose animal experience correlates with the human then logically the models in which it has been effective are the most appropriate for considering related agents. One of th difficulties in this consideration is that very little is known about the detailed antitumor mechanisms of the action of levamisole in either the animal models or in human cancer.

The importance of macrophages as effectors of cell-mediated immunity to a variety of microorganisms, particularly to facultative intracellular parasites, is well established. Recent evidence indicates that macrophages are important participants in the host’s reaction to engraftment and to spontaneous or induced neoplasia. The effectiveness of macrophages in these aspects of host resistance appears to depend on a process of activation by which monocyte-derived macrophages acquire a nonspecific enhancement of microbicidal and tumoricidal capacity. While this process of activation has been extensively analyzed in vivo, it is only recently that it has been examined in vitro and its mechanisms have been probed. This chapter, in attempting to develop what is known about this process, is admittedly premature in its presentation, since the specific mechanisms of macrophage activation have yet to be clarified.

In the future, the development of an effective immunopharmacology will service clinical immunotherapy by removing the current empiricism in which the latter is steeped. By dissection of critical features of the cell targets of action and intracellular mechanisms of action and relating these to primary effects to modify host immune responses, in a predictable, consistent manner, the primary goal will be achieved. Once achieved and appropriate nonimmunologic aspects of host resistance and status are taken into account, the second goal of effective delivery of a restored or enhanced immune system to combat the invading tumor or pathogen can be made. Finally, by overcoming the resistance and suppressor mechanisms derived from the pathogen or tumor, immunotherapy will achieve an efficacy and safety which will ultimately justify a more general use. To date, cyclic nucleotide pharmacology and in vitro analysis of cellular targets and function have provided important first steps towards achieving these goals. Once achieved, the clinical potential for therapeutic benefit in cancer, immunodeficiency, aging, chronic infections, and autoimmune disorders seems great.

Summary Proteins extracted from calf thymus nuclei with 2 M NaCl and separated by Bio-Rex 70 column chromatography were analyzed for their ability to bind to cyclic nucleotides and to calf thymus DNA. Several nuclear protein fractions contain both cyclic GMP-binding and DNA-binding activities. One protein fraction binds cyclic GMP with an apparent high affinity, as determined by equilibrium dialysis. At low concentrations (10 −6 M – 10 −9 M) cyclic GMP slightly enhances the ability of proteins in this fraction to bind 125 I-labelled DNA, while at concentrations >10 −5 M the cyclic nucleotide is strongly inhibitory to DNA binding.

The thymus-derived system is under the regulation of a variety of exogenous and endogenous mediators. Mediators of the autonomic nervous system, the “classic” endocrine system, and the thymic endocrine system act in concert with endogenous lymphokines, prostaglandins, eicosanoids, and inflammatory molecules to regulate the system. The former can be envisioned to provide the molecular “background” for development of the system and the maintenance of the functional integrity of the system. The latter signals derive from its activation in response to antigenic stimuli and, therefore, more likely qualify as “acute phase” regulators signaling abrupt changes in direction. Recent advances in understanding the mechanisms of transmembrane signal transmission including phospholipid methylation, PI turnover, AA release, Ca2+ influx, protein kinases (c, cAMP, and cGMP) cyclic nucleotide cyclases, ATPases, etc. offer a wealth of opportunity to study the mechanisms involved in neuroimmunomodulation.

Lipopolysaccharides (LPS) are responsible for the characteristic immunopharmacologic activities of the endotoxins of gram-negative bacteria. This chapter will discuss their immunopharmacologic actions including some new observations and the evidence that their actions are mediated in part by cyclic nucleotides. The lipopolysaccharides are to be distinguished, for the purposes of this discussion, from bacterially derived protein toxins, like choleratoxin and E. coli enterotoxins, which have also been implicated as immunomodulators acting via cyclic nucleotide pathways.

Mitogen activation of human peripheral blood lymphocytes (HPBL) occurs as a consequence of several early membrane events. One of the earliest is the Ca2+ -dependent stimulation of guanylate cyclase (Coffey et al., 1981). Experiments with the T-cell mitogens phytohemagglutinin (PHA), concanavalin A (Con A), and phorbol myristate acetate (PMA) suggested that phospholipase and lipoxygenase activation are important in the sequence of early membrane events leading to guanylate cyclase stimulation, cGMP accumulation, and DNA synthesis (Coffey and Hadden,1981, 1983a,b). Cyclooxygenase (Coffey et al., 1981) and thromboxane synthetase activities (Gordon et al., 1981) were not found to be essential for mitogenesis.

This book and numerous others with similar titles emphasize the fact that cyclic 3′,5′-adenosine monophosphate (cyclic AMP) has been accorded center stage in the field of biological regulation during the past 10 years. Almost a decade ago, a second cyclic nucleotide, cyclic 3′,5′-guanosine monophosphate (cyclic GMP), was found to occur in mammalian urine (Ashman et al., 1963) and subsequently to be a naturally occurring component in mammalian tissues (Goldberg et al., 1969; Ishikawa et al., 1969) and in lower phylogenetic forms including bacteria (Goldberg et al., 1973). The biological importance of cyclic GMP has remained relatively obscure since its discovery primarily because of the technical difficulties associated with its detection and what appear now to be a number of subtle properties that may distinguish its biological characteristics from those of cyclic AMP.

Immunopharmacology, as defined by the contents of this volume, represents a broad endeavor to elucidate the basic molecular aspects, both intercellular and intracellular, of the regulation of immune response. While this scientific enterprise is in its infancy, perhaps the most critical evaluation of its progress to date lies in the appreciation of the extent to which principles have emerged that will be therapeutically useful. The application of therapeutic agents that suppress the immune response and its associated inflammation has developed empirically, as a result of the need for symptomatic relief, effective organ transplantation, and amelioration of immunopathologic processes such as allergy and autoimmunity. In contrast, the development of agents that augment immune responses has been more recent, as a result of the need to combat cancer, to reverse iatrogenic immunosuppression, and to treat immunodeficiency and chronic infection. As is always the case with therapeutic endeavors, it turns out to be far easier to generate agents that inhibit than agents that augment physiologic functions. This chapter will attempt to summarize the progress that has been made in the development of approaches to the potentiation of immune responses, with the ultimate goal of effective therapy.

Cyclic GMP (guanosine 3′:5′-cyclic monophosphate) accumulation and Ca2+ influx have been correlated with early nuclear events associated with increases in RNA polymerase I activity and decreases in RNA polymerase II activity in phytohemagglutinin-stimulated lymphocytes. In the present study we demonstrate that cyclic GMP in the presence of Ca2+ stimulates RNA polymerase I activity in lymphocyte nuclei isolated from both non-stimulated and phytohemagglutinin-stimulated lymphocytes. In addition, cyclic GMP in the presence of Ca2+ decreases RNA polymerase II activity in both non-stimulated and phytohemagglutinin-stimulated lymphocyte nuclei. These observations suggest that cyclic GMP and Ca2+ may represent components of a plasma membrane-to-nucleus “mitogen signal sequence”.

The immunodeficiencies of trauma and burns have various levels of complexity and it is difficult to treat them as a single common entity (Davis and Shires 1986). They seem to have in common an acute phase reaction which, to a significant degree, is glucocorticoid mediated. This acute phase reaction is associated with lymphopenia and leukocytosis. Depending on the circumstances, a number of mediators are released including cycloxygenase products (thromboxanes and prostaglandins) lipoxygenase products (eicosanoids and leukotrienes), histamine and other mast cell, granulocyte and platelet products, and complement derived products. As the acute phase resolves, the situation may become further complicated by blood and serum losses, fluid and electrolyte imbalances, protein/calorie and nutrient deficiencies, and the many consequences of sepsis. All in all, not a simple metabolic or immunologic circumstance.

The last fifteen years have seen a marked increase in the experimental evidence supporting the central roles played by cyclic nucleotides in the regulation of diverse processes in cells and tissues of organisms throughout the plant and animal kingdom. Beginning with the observations of Sutherland and Rall (1960) that indicated that cyclic 3′,5′-adenosine monophosphate (cAMP) mediates the intracellular action of epinephrine and glucagon to induce glycogenolysis in liver, the concept of the cAMP “second messenger” system has been generalized to virtually every cell of the mammalian organism, and the system has been linked to the induction and regulation of central cellular processes in these cells. In general, cAMP participates in those processes that involve the promotion of preprogrammed events consistent with the differentiated phenotype, i.e., the dominant functions for which that cell type is developed—for the liver, glucose production from glycogen stores; for the adrenal gland, steroid production; for fat tissue, lipolysis; and so on.

That host-defense mechanisms control viral infection and eventually limit the spread of disease has been recognized for many years. Preceding modern medicine, Chinese physicians in the eleventh century observed that the inhalation of smallpox crusts prevented the subsequent occurrence of disease. Later, in the eighteenth century in England, variolation was practiced by Lady Montagu as a primitive form of immunization to protect against smallpox.

Two purine immunoenhancing drugs, isoprinosine and NPT 15392, were evaluated as inducers of IL-2 or enhancers of IL-2 induction by phytohemagglutinin (PHA). Both drugs were found to have significant enhancing activity on PHA induction of IL-2 by human lymphocytes over a wide range of concentrations. Alone they were unable to consistently induce IL-2 activity. NPT 15392 was able to enhance PHA-induced IL-2 production at 10–100 times lower concentrations than isoprinosine. The actions of these compounds to enhance IL-2 production may explain their effects on PHA-induced lymphocyte proliferation.

Four immunopotentiators, levamisole, isoprinosine, muramyl dipeptide (MDP) and SM1213 were analysed in vitro on phytohemagglutinin-induced lymphocyte proliferation and lymphokine-induced macrophage proliferation and activation to kill Listeria monocytogenes . Levamisole and isoprinosine augmented all three functions. MDP and SM1213 induced macrophage activation directly. The effects of these agents on these cells at concentrations relevant to in vivo therapy supports their immunopharmacologic capacity to modify cellular immunity.

Purified podophyllotoxin (CPH-86) is an inhibitor of microtubular aggregation used in the treatment of cancer, psoriasis and rheumatoid arthritis. To better understand its immunopharmacology we examined its effects on human lymphocytes and monocytes and guinea pig macrophages. CPH-86 inhibits mitogen-induced human lymphocyte proliferation and macrophage growth factor-stimulated macrophage proliferation with ID50s of approximately 10 −7 M. The effect of CPH-86 on lymphocytes in conjunction with mitogen is nonlethal, evident during the early but not the late phases of proliferation, and associated with early increases in cyclic AMP levels. In contrast to these obviously inhibitory effects, CPH-86 (10 −7 M) alone induces IL-1 by human monocytes and, with mitogen, it induces IL-2 production by human lymphocytes. It directly stimulates macrophage pproliferation and potentiates the effects of low doses of macrophage growth factor to do so. The latter effects may be mediated by colony stimulating factor production. The effects of CPH-86 are not mediated by inhibition of prostaglandin synthesis. The stimulation of monokine and lymphokine production by CPH-86 may represent positive features of its action and may be immunotherapeutic.

The first part of this paper focuses on the secondary immunodeficiencies caused by immunosuppressive drugs and environmental chemicals. The mechanism of action and the therapeutic effects of azathioprine, glucocorticosteroids, cyclophosphamide, cyclosporin A, and anticancer drugs will be discussed. The immunotoxic actions of benzene, polychlorinated and polybrominated biphenyls, tetrachlorodibenzo-para-dioxin, polycyclic aromatic hydrocarbons, various insecticides, metals and abused drugs or drugs with side effects are also described. Immunorestorative approaches are discussed in the second part, based on our current knowledge of the immune system and the lymphokines and hormones involved in its function. The usefulness of thymic hormones, interleukin 2, macrophage growth factor/colony stimulating factor, and interferons as immunotherapeutic agents is considered. In addition, we address chemically defined drugs, such as levamisole, diethyldithiocarbamate, isoprinosine, muramyl dipeptides, azimexon, ciamexon, bestatin, tuftsin, and pyrimidinoles, and finally conclude that combined use of different immunostimulants may be the most effective way to treat the above mentioned immunodeficiencies.

Antimicrobial agents which act on nucleic acids (e.g., rifampin, sulfonamides, trimethoprim, metronidazole), on ribosomes (e.g., tetracyclines, chloramphenicol, aminoglycosides) or on cytoplasmic membranes (e.g., amphotericin B, griseofulvin, polymyxin, imidazoles) positively influence the outcome of infections by acting against the offending pathogens but they may also have negative effects on the host’s immunocompetence (22). Although the immunosuppressive properties of most of the antimicrobials are not sufficient to warrant restricting their use, under certain circumstances a “pro-host” immunopharmacological approach seems warranted. In this regard it is important to remember that the immune system “cures” an infection, not the antimicrobial agent, and under circumstances where antimicrobial therapy is lifesaving, the host’s immune defenses are almost invariably compromised. To expedite the development of immunorestorative therapy it is important first of all to characterize the immunological deficiency which precedes, accompanies or follows a disease in order to select the appropriate immunorestorative agent. Secondly, new therapeutic strategies, where the antimicrobials are associated with prohost immunomodulating drugs, need to be studied in the appropriate animal systems.

NPT 15392 [9-erythro-(2-hydroxy,3-nonyl)-hypoxanthine] was administered in a single intraperitoneal injection to Balb/c mice at a dose of 0.1 mg/kg. Modifications of immune parameters were evaluated 1–14 days after the treatment. NPT 15392 potentiated antibody responses to both T-dependent (SRBC, TNP-KLH) and T-independent (TNP-LPS) antigens and delayed-type hypersensitivity to oxazolone. The proliferative response of spleen cells from NPT-treated mice to stimulation with PHA was depressed, but that to dextran sulphate was augmented. The responses to Con A or LPS were inconsistently modified. NPT 15392 augmented killer cell functions, including both T cell-mediated cytotoxicity against allogeneic tumor cells and NK cell activity against YAC-1 tumor cells. It slightly augmented or depressed ADCC activity against antibody-coated chicken erythrocytes (CRBC) depending on the time of its administration. Concerning the stimulation of NK cell activity, the effect was more marked on spleen effector cells when NPT 15392 was given i.v. and on peritoneal effector cells when it was given i.p. From these results, T helper cells, B cells, and NK cells appeared to be target cells of NPT 15392 action. The various stimulatory effects peaked at different times according to the immune function tested. In addition, the prolonged, sometimes double-peaked action (antibody response to T-dependent antigens, NK activity) indicates complex mechanisms of action which may involve indirect interactions mediated by lymphokines or monokines.

Mature peritoneal and alveolar macrophages of the guinea pig as well as non adherent bone marrow cells can be induced to undergo mitosis when cultured in the presence of lymphokine containing supernatants. The factor responsible for induction of proliferation is macrophage growth factor/colony stimulating factor (MGF/CSF). This report details the isolation, purification and characterization of the antigen-induced lymphokine MGF/CSF. Its properties are similar to the macrophage specific CSF-1 found in human urine and produced by mouse L cells. It is proposed that the lymphokine MGF/CSF is responsible for the macrophage proliferation observed during inflammation and infection.

Macrophages are critical in the expressions of immune response, especially in the defense against facultative intracellular bacteria and viruses and in the defense against the development of cancer. Historically, studies of the macrophage since the days of Metchnikoff focused on the important role played by this cell in the resistance to infection. With increasing understanding of the immune response, the macrophage was considered the principal cell in the expression of cell-mediated immunity. With the discovery of the role of the thymus and the functions of the T lymphocyte, the macrophage was placed in a role secondary to that of the lymphocyte. Currently, important interrelationships of the two cell populations are being elucidated, and a central role for the macrophage in tumor immunity is emerging.

Much of the study of immune regulation over the last twenty years has focused on the dissection of cellular function and interactions “in vitro”. While clearly “in vivo” studies have supported the models of regulation which have derived, this type of extrapolative approach tells only part of the story of immune regulation. The “in vitro” studies generally employ artificial media containing a drab of serum often from different species; they do not pretend to offer a dynamic fluid environment akin to that which exists “in vivo”. It is becoming abundantly clear that the immune system operates “in vivo” within a homeostatic milieu replete with neurologic, neuroendocrine, endocrine and microenvi-ronmental regulatory influences clearly as complex as those regulating the internal network of intercommunication among the cells of the system.

The recent advances in the immunopharmacologic development of immunotherapeutic drugs and biologicals have been significant. The recent discoveries of cloned anticancer biologicals like α and γ interferons, tumor necrosis factor, and lymphotoxin and of monoclonal antibodies to several human tumor antigens add important anticancer potential to the therapeutic repertoire. Based upon many examples of synergistic interactions of the various immunotherapeutic agents it seems warranted to use, in conjunction with cytoreductive therapy, new strategies involving the combined use of immunorestorative agents, anticancer biologicals and monoclonals, and antisuppression therapy in the experimental treatment of cancer.

The induction of T lymphocyte proliferation is thought to involve cell-cell interaction and molecular communications (1). The nature of the intercellular communication on a cell-cell contact basis is not understood; what evidence is available points to important molecular communications. It is generally accepted that T cells are triggered to proliferate by two signals (Fig. 1). The mitogen in the relative but not complete absence of accessory cells can induce T cells to enter a G1 phase of the cell cycle from a resting G0 or “restricted” G1 phase of the cell cycle. This first stage of activation is associated with cellular enlargement or blastogenesis and both protein and RNA synthesis. The lymphocyte does not enter DNA synthesis (S phase) unless a second signal is presented. This second process is thought to be initiated by mitogen interaction directly or indirectly with adherent accessory cells, (i.e., monocytes/macrophages) which results in the production of interleukin 1 (IL-1). The IL-1 thus acts on T lymphocytes to induce the production on interleukin 2 (IL-2), and the appearance of cell surface receptors for IL-2. Little is known of the metabolic events associated with IL-1 and IL-2 action. T cells triggered by mitogen to enter the cell cycle transit aG1-S boundary as a result of the second signal and complete the cycle. The subsequent divisions do not require re-exposure to the mitogen but do require the presence of IL-2. The majority of the studies to date have concentrated on early events following mitogen addition to peripheral blood lymphocytes with the assumptions that the events occur in T cells and are contributory to the replication process.

MACROPHAGE proliferation occurs in vivo as a component of the inflammatory response1 and particularly intense proliferation is characteristic of the delayed hypersensitivity reaction to infection with facultative intracellular pathogens2–4. The thymus-derived (T) lymphocyte is considered responsible for the expansion of the reticuloendothelial system in this type of infection5. Although the mechanisms by which lymphocytes induce macrophage proliferation and activation remain unclear, a number of lymphocyte-produced soluble mediators which influence the functions of macrophages have been described, including a biochemically distinct migration inhibitory factor (MIF) and chemotactic factor (MCF)6. The speculation that a lymphocyte-produced mitogenic factor may be responsible for the induction and maintenance of proliferation of macrophages prompted the present experiments.

Key Words: Thymic hormones, thymosin, interleukin 1 and 2, endotoxin, transfer factor, isoprinosine, NPT 15392, levamisole, T cell differentiation, Komuro-Boyse assay, peanut agglutinin, thymocyte proliferation

The formation of distinctive tissue masses known as granulomas is a central aspect of a wide range of diseases affecting both animals and man. Many of these diseases have cutaneous involvement or cutaneous manifestations and are of clinical interest to the dermatologist. These diseases have been depicted in great detail in standard dermatology textbooks and their varying histologies have been described in histopathology texts. This chapter will attempt an analysis of basic cellular and molecular processes underlying granuloma formation. It will not attempt to review the clinical and pathological features of individual disease states. Since the processes involved in granuloma formation are common whether the site of involvement is cutaneous or visceral, the discussion will not be restricted to dermatopathies. The reader is referred to complementary chapters in this text which deal with specific aspects of granulomas as they pertain to various disease states such as Chapters 13 and 19 (granulomatous and allergic vasculitis), 22 (rheumatoid nodules), 29 (chronic granulomatous disease), 33 (sarcoidosis), and 34–37 (infectious granulomas). While the chapter will avoid discussion of malignant lesions which have granulomas (e.g., Hodgkin’s disease, mycosis fungoides, Wegener’s and eosinophilic granuloma, Chapter 42), it is relevant to note that the molecular aspects of granuloma formation which will be developed in this chapter pertain to these lymphoproliferative diseases. One need only recognize that when various secretory lymphoid populations undergo malignant clonal expansion they generally continue to secrete those products which characterize them (e.g., immunoglobulins or lymphokines), which may induce other cell populations to participate in what appears to be a chronic inflammatory response. The focus, then, of this chapter will be the basic cellular and molecular events which underlie the formation of granulomas.

The lymphocyte has provided an important model for the study of the early biochemical events associated with the initiation of cellular proliferation. Resting lymphocytes (G0 or restricted G1 phase), as obtained from human peripheral blood, can be induced in large numbers to undergo clonal proliferation using a variety of mitogens. The two most commonly employed plant lectin mitogens are phytohem-agglutinin (PHA) and concanavalin A (Con A). The initiation process is characterized by rapid, saturable binding of the lectin to cellular receptors (

In the present study we examined the effect of a recently isolated hexacyclodepsipeptide, Cyclomunine (Servier, France), on certain parameters which assess cellular and humoral immunity in the rat and dog. Our data establish Cyclomunine as a potent in vitro inhibitor of 3H-thymidine incorporation by lymphocytes. The concentrations of Cyclomunine required to abrogate mitogen or allogeneic lymphocyte stimulated 3H-thymidine incorporation was greater in the dog than in the rat, 15 micrograms/ml versus 10 micrograms/ml, respectively, to achieve greater than 99% inhibition. Little cytotoxicity was noted at concentrations below 25 micrograms/ml. Oral administration of Cyclomunine to rats (50 mg/kg dissolved in corn oil) did not significantly alter white blood cell count, lymphocyte-polymorphonuclear cell differential, body weight, or histological architecture of the liver, spleen, lymph node or kidney. Our results show that in vitro, Cyclomunine is highly effective in suppressing lymphocyte proliferation. In vivo, Cyclomunine appears to have little myelosuppressive effect a property shared with Cyclosporin A. Cyclomunine may hold promise as a clinically useful immunosuppressive agent.