Effect of modifiers of natural biological response on the functional activity of macrophages (oncological aspect)

Induction of cytotoxicity by biological response modifiers (BRMs) is only one aspect of macrophage activation. After the use of BRMs there were other changes in the functional activity of cells and in particular their increased production or secretion of a number of growth factors. Thus, activation of macrophage antitumor activity induced by BCG vaccine was transitory while activation of growth factor production was more stable in time which finally led to increased proliferation of tumor cells. Combined use of cyclophosphamide and BCG vaccine significantly increased not only the toxicity induced by BCG vaccine but also their liberation of the growth factors. Such macrophages lost their ability to control the growth of a small number of the tumor cells cultivated in their presence. Development of ways for directed activation of macrophages aimed at elimination of the tumor cells which survived the chemotherapy should include evaluation of the combined effect of various BRMs and chemotherapeutics on both antitumor and protumor activity i. e. ability to produce the factors stimulating the tumor growth.     

Metabolic response of macrophages to injury promoted by the activated complement system

In nucleated cells, the swelling promoted by a complement system (CS) attack is not enough to promote cell death, because unlike erythrocytes these cells are able to eliminate cytolytic complement channels from the plasma membrane, by processes that include endocytosis. Several studies have demonstrated that the resistance of nucleated cells to the injury promoted by the CS is related to the cellular metabolism. Despite this, to the present day, no study has clearly related cell survival capacity to injury by the CS to its energetic metabolic status. In macrophages, the challenge imposed by the CS provoked an increase in the total amount of glucose incorporated into fatty acids, including phospholipids and cholesterol; substrates for membrane synthesis. The inhibition of cholesterol synthesis promoted an increase of the cell death rate. These data support the importance of cholesterol metabolism for macrophage resistance to necrosis induced by the activated complement system. Copyright © 1999 John Wiley & Sons, Ltd.     

Liposomes surface conjugated with human hemoglobin target delivery to macrophages

Current strategies to deliver therapeutic molecules to specific cell and tissue types rely on conjugation of antibodies and other targeting ligands directly to the therapeutic molecule itself or its carrier. This work describes a novel strategy to deliver therapeutic molecules into macrophages that takes advantage of the native hemoglobin (Hb) scavenging activity of plasma haptoglobin (Hp) and the subsequent uptake of the Hb-Hp complex into macrophages via CD163 receptor-mediated endocytosis. The drug delivery system described in this work consists of Hb decorated liposomes that can encapsulate any therapeutic molecule of interest, in this case the model fluorescent dye calcein was used in this study. The results of this study clearly demonstrate that this delivery system is specific towards macrophages and demonstrates the feasibility of using this approach in targeted drug delivery.     

Structural development of biological response modifiers based on thalidomide

Thalidomide (N-alpha-phthalimidoglutarimide) is a teratogenic hypnotic/sedative agent which was used widely in the late 1950s and the early 1960s. In spite of its withdrawal from the market because of its severe teratogenicity, there has been a resurgence of interest in the drug in recent years due to its potential usefulness for the treatment of various diseases, including acquired immunodeficiency syndrome (AIDS) and various cancers. It has been revealed that thalidomide elicits pleiotropic effects and is a multi-target drug. Our structural development studies of thalidomide, focusing on tumor necrosis factor-alpha(TNF-alpha) production-regulating activity, anti-androgenic activity, puromycin-sensitive aminopeptidase-inhibiting activity, alpha-glucosidase-inhibiting activity, and inhibitory activities toward some other enzymes, are reviewed in relation to the pharmacological effects of thalidomide.     

Whole-cell currents in macrophages: II. Alveolar macrophages

Summary Although an outwardly rectifying K⁺-conductance has been described in murine peritoneal macrophages and a murine macrophage cell line, the expression of this conductance in human monocyte-derived macrophages (HMDMs) is rare. Whole-cell current recordings in this study were obtained from HMDMs differentiated in adherent culture for varying periods of time following isolation and compared to currents obtained in human alveolar macrophages (HAMs) obtained from bronchoalveolar lavage. These studies were undertaken to compare ionic current expression in the in vitro differentiated macrophage to that of a human tissue macrophage. HAMs are the major population of immune and inflammatory cells in the normal lung and are the most readily available source of human tissue macrophages. Of the 974 HMDMs in the study obtained from a total of 36 donors, we were able to observe the presence of the inactivating outward current (I _A ) which exhibited voltage-dependent availability in only 49 (or 5%) of the cells. In contrast, whole-cell current recordings from HAMs, revealed a significantly higher frequency ofI _A expression (50% in a total of 160 cells from 26 donors). In the alveolar cell, there was no correlation observed between cell size and peakI _A amplitude, nor was there a relationship between peakI _A amplitude and time in culture. The current in both cell types was K⁺ selective and 4-aminopyridine (4-AP) sensitive.I _A in both cell types inactivated with a time course which was weakly voltage-dependent and which exhibited a time constant of recovery from inactivation of approximately 30 sec. The time course of current inactivation was dependent upon the external K⁺ concentration. An increase in the time constant describing current decay was observed in elevated K⁺. Current activation was half-maximal at approximately –18 mV in normal bathing solution. Steady-state inactivation was half-maximal at approximately –44 mV. The presence of the outwardly rectifying K⁺ conductance may alter the potential of the mononuclear phagocyte to respond to extracellular signals mediating chemotaxis, phagocytosis, and tumoricidal functions.     

Protective effect of biological response modifiers on murine cytomegalovirus infection

Pretreatment with two biological response modifiers (BRM), OK-432 and PS-K, protected mice from lethal infection by murine cytomegalovirus (MCMV). This was evidenced by an increase in 50% lethal doses and a decrease in titers of infectious viruses replicated in the liver and spleen. Spleen cells from the BRM-treated mice augmented the natural killer (NK) cell activity and suppressed the replication of MCMV in vitro. During MCMV infection, the NK cell activity of the spleen cells was maintained at a high level in the BRM-treated mice, whereas it was severely impaired in untreated mice. The BRM-induced protection was nullified by concomitant administration of antiasialo GM1 antibody. Interferon was neither induced by BRM treatment nor enhanced in BRM-pretreated and MCMV-infected mice. Thus, the protective effect of OK-432 and PS-K seems to be based on activation of NK cells and prevention of MCMV-induced inhibition of the NK cell activity.     

Liposomal delivery of CTL epitopes to dendritic cells

The induction of strong and long lasting T-cell response, CD4+ or CD8+, is a major requirement in the development of efficient vaccines. An important aspect involves delivery of antigens to dendritic cells (DCs) as antigen presenting cells (APCs) for the induction of potent antigen-specific CD8+ T lymphocyte (CTLs) responses. Protein or peptide-based vaccines become an attractive alternative to the use of live cell vaccines to stimulate CTL responses for the treatment of viral diseases or malignancies. However, vaccination with proteins or synthetic peptides representing discrete CTL epitopes have failed in most instances due to the inability for exogenous antigens to be properly presented to T cells via major histocompatibility complex (MHC) class I molecules. Modern vaccines, based on either synthetic or natural molecules, will be designed in order to target appropriately professional APCs and to co-deliver signals able to facilitate activation of DCs. In this review, we describe the recent findings in the development of lipid-based formulations containing a combination of these attributes able to deliver tumor- or viral-associated antigens to the cytosol of DCs. We present in vitro and pre-clinical studies reporting specific immunity to viral, parasitic infection and tumor growth.     

Sensitivity of ovarian tumor cells to effector cells generated by various biological response modifiers

The sensitivity of freshly derived human ovarian tumors (FOT) to various allogeneic cytotoxic effector cells stimulated by recombinant interleukin 2 (rIL-2), recombinant interferon alpha 2 (rIFN-alpha 2), OK-432, and concanavalin A was examined using the 51Cr release assay. Peripheral blood lymphocytes (PBL) of normal female donors were used as source of effector cells. Incubation of PBL with these biological response modifiers for 24 h generated effector cells with high natural killer activity, and only 20% (1/5) of the FOT examined were susceptible to lysis. By contrast, 83% (5/6) of the FOT were sensitive to lymphokine-activated killer (LAK) cells generated by rIL-2. OK-432 and concanavalin A activation of PBL also generated cytotoxic cells, though the cytotoxic activity against FOT was much less than that obtained by LAK cells. The addition of OK-432 to LAK culture medium containing rIL-2 generated effector cells with higher cytotoxicity against FOT than cultures with IL-2 alone. However, the addition of rIFN-alpha 2 in LAK culture medium resulted in the generation of effector cells with lower cytotoxicity. The addition of rIL-2, rIFN-alpha 2, or OK-432 to LAK cells during the in vitro cytotoxicity assay had no significant effect. When FOT target cells were pretreated with OK-432 they became more sensitive to LAK than nontreated tumor cells. However, pretreatment with rIL-2 or rIFN-alpha 2 did not influence cytolysis. These results suggest that the generation of LAK cells in vitro using rIL-2 plus OK-432 may be a more effective way to prepare these cells for adoptive immunotherapy in the treatment of ovarian cancer.     

Response of natural killer cells from dietary tyrosine-and phenylalanine-restricted mice to biological response modifiers

Summary The effect of dietary tyrosine and phenylalanine restriction on splenic natural killer (NK) cell activity was studied in tumor-free B6D2F₁ and NIH nude mice and in B16 bladder-6 (BL6) melanoma-bearing B6D2F₁ mice. This dietary restriction was found to suppress the naturally elevated NK-cell activity of nude mice and to induce a specific lymphocytopenia in B6D2F₁ mice fed the restricted diet for a prolonged period. Baseline NK-cell activity was significantly lower in tumor-free B6D2F₁ mice fed a diet restricted in tyrosine and phenylalanine (restricted diet) than in tumor-free mice fed a basal diet. Similar kinetics of activation after a single i.p. injection of 100 g of polyinosinic:polycytidylic acid (poly I:C) were observed in mice fed both diets. NK-cell activity was not significantly augmented after i.v. inoculation of BL6 melanoma, irrespective of the diet fed; however, it was enhanced in tumor-bearing mice after poly I:C injection. This augmentation was similar to that observed in tumor-free mice. Spleen cells from mice fed either diet were responsive to stimulation of NK-cell activity after in vitro incubation with interleukin-2. These results indicate that dietary restriction of tyrosine and phenylalanine, a potentially useful therapeutic adjunct known to lower NK-cell activity, does not significantly interfere with poly I:C or interleukin-2 induction of NK cells. Our results also demonstrate that, while this dietary restriction causes lymphocytopenia, no effect of the diet could be found on total serum IgG or circulating immune complex levels.     

Psychosocial modifiers of response to stress

The impact of stress upon an organism is far more complex than the simple design of most stress research implies. We offer an expanded model for studying the relation of stressors to pathological outcomes, which takes into account both the adaptive capacity of the organism before the stressor occurs and the defenses marshalled in response to the stressor. The model also distinguishes among the initial responses of alarm, sustained defensive behaviors, and the relatively irreversible endstates which remain after resistance has ended. Realizing that only a multidiscomplinary approach can begin to capture the wholeness of human experience, this research paradigm anticipates that stressors, adaptive capacities, defenses, alarm reactions, and pathologial end-states will take place at the biological, psychological, interpersonal and sociocultural levels simultaneously and successively. Data on life change stress and psychological health outcomes gathered as part of the Air Traffic Controller Health Change Study are analyzed to illustrate the use of the model in identifying psychosocial and biological modifiers of response to stress.     

Immunoregulatory biological response modifiers: effect of cytokines on septic shock

Whole bacteria or bacterial components or their extracts were employed to restore or augment the immune system. Beneficial effects were attained with these agents in treating various diseases. These agents were named biological response modifiers (BRMs) because they regulated certain cellular components of the immune system. The cellular regulation induced by these BRMs was found to be due to cytokines. The cytokines were shown to act directly on the various cellular components and to provide therapeutic benefit in various autoimmune and immune deficiency diseases. Overproduction of specific cytokines however leads to a deleterious effect on the host. Overproduction of tumour necrosis factor (endotoxin, lipopolysaccharide) leads to septic shock. Bacteraemia is the leading cause of overproduction of tumour necrosis factor (TNF). Septic shock in many cases leads to death. Several monoclonal antibodies to lipopolysaccharide (LPS) and anticytokines have demonstrated protection against septic shock.     

Asymmetric Membrane Distribution of Phosphatidylserine in Different Liposomal Preparations

Abstract

We established a quantitative assay (fluorescamine-assay) to determine the distribution of phosphatidylserine (PS) in the outer and inner monolayer of mixed phosphatidylcholine/phosphatidylserine liposomes. Fluorescamine (a non-fluorescent compound) reacts with primary amino groups resulting in a fluorescent product. Fluorescamine is not able to penetrate through the liposomal membrane so only the amount of PS located in the outer monolayer is accessible for the reaction. This assay was used to study the influence of several factors in liposome preparation on liposome asymmetry. An increase in the amount of PS in the liposomal preparation leads to a decrease in the outer to inner PS ratio. This was also the case for unilamellar vesicles of decreasing size. The asymmetry of PS is only slightly influenced by the addition of phos-phatidylinositol or phosphatidylgycerol, whereas small amounts of phosphati-dylethanolamine-rhodamine-B induce a dramatic shift of PS to the inner monolayer. NBD-labeled PS shows a asymmetric distribution different from that of unlabeled PS.     

Cholesteryl ester handling by RAW264 macrophages: response to native and acetylated low density lipoprotein

Summary The effects of LDL and Ac-LDL on the growth properties, morphology, and cholesteryl ester (CE) metabolism of the RAW264 macrophage cell line have been characterized. Cells were grown in media supplemented by a defined media (DM) mixture or fetal bovine serum (FBS). The addition of LDL or Ac-LDL to the culture media did not significantly alter cell growth properties. Cytoplasmic deposition of CE was observed by fluorescence microscopy in macrophages treated with LDL or Ac-LDL but not in untreated controls. Dose-response studies have shown that cholesteryl ester (CE) can accumulate in RAW264 treated with LDL. Cellular cholesterol content saturated at 4 hours with 50 g/ml LDL; this effect may be associated with receptor saturation. Dose-response studies conducted with Ac-LDL in DM have shown dramatic increases in total cell cholesterol content. However, deposition of CE was not observed below Ac-LDL concentrations of 100 g/ml. This indicates that a critical concentration of Ac-LDL must be reached to trigger deposition in DM. In contrast, no critical concentration of Ac-LDL was observed in macrophages grown in medium supplemented with 10% FBS. Cholesterol esterification in response to LDL and Ac-LDL was examined by ¹⁴C-oleic acid incorporation into CE. These results confirmed the mass cellular cholesterol and CE measurements. Kinetic studies conducted with RAW264 cells treated with 50 or 100 g/ml Ac-LDL resulted in a cholesterol efflux from the cells at 6–12 hours of incubation. Therefore, these studies show that (1) the nature of CE deposition is highly dependent upon the incubation media and (2) CE deposition is very sensitive to Ac-LDL concentration under certain conditions.Abbreviations LDL Low Density Lipoprotein - Ac-LDL Acetylated Low Density Lipoprotein - FBS Fetal Bovine Serum - DM Defined Medium - PBS Phosphate Buffered Saline - CE Cholesteryl Ester     

Chitosomes loaded with cranberry proanthocyanidins attenuate the bacterial lipopolysaccharide-induced expression of iNOS and COX-2 in raw 264.7 macrophages

Chitosan binds to negatively charged soy lecithin liposomes by an electrostatic interaction driven by its positively charged amino group. This interaction allows stable covered vesicles (chitosomes) to be developed as a suitable targeted carrier and controlled release system. This study investigated the effect of chitosomes on the activation of cranberry proanthocyanidins (PAC) in Raw 264.7 macrophages. Chitosomes were characterized according to size, zeta potential, PAC-loading, and release properties. Results showed an increase in the net positive charge and size of the liposomes as the concentration of chitosan was increased, suggesting an effective covering of the vesicles by means of electrostatic interactions, as shown by transmission electron microscopy and fluorescence microscopy. About 85% of the PAC that was loaded remained in the chitosomes after release studies for 4 hours in phosphate-buffered saline. Cyclo-oxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) are associated with inflammation. Activated RAW 264.7 macrophages increase the expression of COX-2 and iNOS in response to bacterial infection and inflammation; we, therefore, tested the ability of the PAC-loaded chitosomes to attenuate COX-2 and iNOS expression in LPS (lipopolysaccharide)-stimulated macrophages. Increasing the amount of PAC loaded into the chitosomes caused a dose-dependent attenuation of iNOS and COX-2 expression in LPS-stimulated macrophages. A 2% v/v PAC-loaded chitosomes formulation almost completely attenuated the LPS-induced expression of iNOS and COX-2. PAC-loaded chitosomes were more active than PAC alone, suggesting that the macrophage response to LPS occurs after endocytosis of the PAC-loaded chitosomes.     

Adoptive immunotherapy of cancer: biological response modifiers and cytotoxic cell therapy

Immunotherapy has been developed for the treatment of metastatic cancers refractory to conventional therapies. Immunotherapy utilizes immune cells and/or biological response modifiers (BRMs) to induce an anti-tumor response mediated by the patient's immune system. BRMs, including lymphokines and cytokines, are used as single agents or in combination for cancer therapy. Some BRMs, particularly interleukin 2 (IL-2), can activate and expandin vitro lymphocytes with anti-tumor reactivity which will be adoptively transferred to the patient. To enhance the therapeutic effect of immunotherapy, gene therapy is currently under investigation and involves the insertion of cytokine genes in immune cells or in tumor cells. The development and future of cancer immunotherapy will be discussed in this review.     

The heterogenic properties of monocytes/macrophages and neutrophils in inflammatory response in diabetes

Inflammation is a complicated biological process in response to harmful stimuli, which involves the cooperation of immune system and vascular system. Upon pathogen invasion or tissue injury, resident innate immune cells such as macrophages and dendritic cells are activated and release inflammatory mediators, which result in the vasodilation and recruitment of leukocytes, mainly monocytes and neutrophils. As two of the most important inflammation-mediating immune cells, macrophages and neutrophils have long been regarded to have a pro-inflammatory effect. However, increasing evidences suggest the role of macrophage and neutrophil in inflammation is more complicated and diversified than we thought. Differently activated macrophages and neutrophils lead to diverse even opposite activities. Precise understanding of the role of different subpopulations is critical to achieve the effective treatment for inflammatory diseases. In this review, we discuss the two potentially distinct activation routes of macrophages and neutrophils in obesity and diabetes.     

In vivo and in vitro activation of macrophages with a cyanine photosensitizing dye,platonin

A cyanine photosensitizing dye, platonin, is a potent macrophage-activating agent. Four days after the administration to mice of small amounts of platonin (20–40 ng/mouse), peritoneal macrophages exhibited greatly enhanced Fc-receptor-mediated phagocytic and superoxide-generating capacities. Much higher doses (more than 3000 ng/mouse) did not have this effect. Photodynamic experiments for macrophage activation were performed by exposing mouse peritoneal cells (mixture of macrophages and B and T lymphocytes) to white fluorenscent light (3 J m^–2s^–1) in media containing various low concentrations of platonin. A short exposure to white fluorescent light (5 s, 15 J m^–2) of peritoneal cells in a medium containing 3 ng platonin/ml produced a maximal level of phagocytic capacity of macrophages. Although platonin absorbs light poorly at wavelengths longer than 630 nm, the region of the spectrum in which the tissues are transparent allows reasonable penetration of light. Thus, we designed experiments in which peritoneal cells were exposed to a red fluorescent light (0.5 J m^–2s^–1). In a medium containing 10 ng platonin/ml with 15 J m^–2 red light, a markedly enhanced ingestion activity of macrophages was observed. Photodynamic treatment of peritoneal macrophages alone did not activate macrophages. Thus, participation of nonadherent cells is required for photodynamic activation of macrophages, implying that a macrophage-activating factor is generated within the nonadherent cells and transmitted to macrophages.