Direct Activation Of Macrophages Research Articles

Trichinella spiralis Thioredoxin Peroxidase 2 Regulates Protective Th2 Immune Response in Mice by Directly Inducing Alternatively Activated Macrophages.

Trichinella infection can induce macrophages into the alternatively activated phenotype, which is primarily associated with the development of a polarized Th2 immune response. In the present study, we examined the immunomodulatory effect of T. spiralis thioredoxin peroxidase-2 (TsTPX2), a protein derived from T. spiralis ES products, in the regulation of Th2 response through direct activation of macrophages. The location of TsTPX2 was detected by immunohistochemistry and immunofluorescence analyses. The immune response in vivo induced by rTsTPX2 was characterized by analyzing the Th2 cytokines and Th1 cytokines in the peripheral blood. The rTsTPX2-activated macrophages (MrTsTPX2) were tested for polarization, their ability to evoke naïve CD4+ T cells, and resistance to the larval infection after adoptive transfer in BALB/c mice. The immunolocalization analysis showed TsTPX2 in cuticles and stichosome of T. spiralis ML. The immunostaining was detected in cuticles and stichosome of T. spiralis Ad3 and ML, as well as in tissue-dwellings around ML after the intestines and muscle tissues of infected mice were incubated with anti-rTsTPX2 antibody. Immunization of BALB/c mice with rTsTPX2 could induce a Th1-suppressing mixed immune response given the increased levels of Th2 cytokines (IL-4 and IL-10) production along with the decreased levels of Th1 cytokines (IFN-γ, IL-12, and TNF-α). In vitro studies showed that rTsTPX2 could directly drive RAW264.7 and peritoneal macrophages to the M2 phenotype. Moreover, MrTsTPX2 could promote CD4+ T cells polarized into Th2 type in vitro. Adoptive transfer of MrTsTPX2 into mice suppressed Th1 responses by enhancing Th2 responses and exhibited a 44.7% reduction in adult worm burden following challenge with T. spiralis infective larval, suggesting that the TsTPX2 is a potential vaccine candidate against trichinosis. Our study showed that TsTPX2 would be at least one of the molecules to switch macrophages into the M2 phenotype during T. spiralis infection, which provides a new therapeutic approach to various inflammatory disorders like allergies or autoimmune diseases.

NADPH oxidase and mitochondria are relevant sources of superoxide anion in the oxinflammatory response of macrophages exposed to airborne particulate matter

Exposure to ambient air particulate matter (PM) is associated with increased cardiorespiratory morbidity and mortality. In this context, alveolar macrophages exhibit proinflammatory and oxidative responses as a result of the clearance of particles, thus contributing to lung injury. However, the mechanisms linking these pathways are not completely clarified. Therefore, the oxinflammation phenomenon was studied in RAW 264.7 macrophages exposed to Residual Oil Fly Ash (ROFA), a PM surrogate rich in transition metals. While cell viability was not compromised under the experimental conditions, a proinflammatory phenotype was observed in cells incubated with ROFA 100 μg/mL, characterized by increased levels of TNF-α and NO production, together with PM uptake. This inflammatory response seems to precede alterations in redox metabolism, characterized by augmented levels of H2O2, diminished GSH/GSSG ratio, and increased SOD activity. This scenario resulted in increased oxidative damage to phospholipids. Moreover, alterations in mitochondrial respiration were observed following ROFA incubation, such as diminished coupling efficiency and spare respiratory capacity, together with augmented proton leak. These findings were accompanied by a decrease in mitochondrial membrane potential. Finally, NADPH oxidase (NOX) and mitochondria were identified as the main sources of superoxide anion (▪) in our model. These results indicate that PM exposure induces direct activation of macrophages, leading to inflammation and increased reactive oxygen species production through NOX and mitochondria, which impairs antioxidant defense and may cause mitochondrial dysfunction.

Anti-PD-L1 antibody direct activation of macrophages contributes to a radiation-induced abscopal response in glioblastoma.

Most glioblastomas recur near prior radiation treatment sites. Future clinical success will require achieving and optimizing an "abscopal effect," whereby unirradiated neoplastic cells outside treatment sites are recognized and attacked by the immune system. Radiation combined with anti-programmed cell death ligand 1 (PD-L1) demonstrated modest efficacy in phase II human glioblastoma clinical trials, but the mechanism and relevance of the abscopal effect during this response remain unknown. We modified an immune-competent, genetically driven mouse glioma model (forced platelet derived growth factor [PDGF] expression + phosphatase and tensin homolog loss) where a portion of the tumor burden is irradiated (PDGF) and another unirradiated luciferase-expressing tumor (PDGF + luciferase) is used as a readout of the abscopal effect following systemic anti-PD-L1 immunotherapy. We assessed relevance of tumor neoepitope during the abscopal response by inducing expression of epidermal growth factor receptor variant III (EGFRvIII) (PDGF + EGFRvIII). Statistical tests were two-sided. Following radiation of one lesion, anti-PD-L1 immunotherapy enhanced the abscopal response to the unirradiated lesion. In PDGF-driven gliomas without tumor neoepitope (PDGF + luciferase, n = 8), the abscopal response occurred via anti-PD-L1 driven, extracellular signal-regulated kinase-mediated, bone marrow-derived macrophage phagocytosis of adjacent unirradiated tumor cells, with modest survival implications (median survival 41 days vs radiation alone 37.5 days, P = 0.03). In PDGF-driven gliomas with tumor neoepitope (PDGF + EGFRvIII, n = 8), anti-PD-L1 enhanced abscopal response was associated with macrophage and T-cell infiltration and increased survival benefit (median survival 36 days vs radiation alone 28 days, P = 0.001). Our results indicate that anti-PD-L1 immunotherapy enhances a radiation- induced abscopal response via canonical T-cell activation and direct macrophage activation in glioblastoma.

CPT-11-Induced Delayed Diarrhea Develops via Reduced Aquaporin-3 Expression in the Colon.

While irinotecan (CPT-11) has a potent anti-cancer effect, it also causes serious diarrhea as an adverse reaction. In this study, we analyzed the pathogenic mechanism of CPT-11-induced delayed diarrhea by focusing on water channel aquaporin-3 (AQP3) in the colon. When rats received CPT-11, the expression level of AQP3 was reduced during severe diarrhea. It was found that the expression levels of inflammatory cytokines and the loss of crypt cells were increased in the colon when CPT-11 was administered. When celecoxib, an anti-inflammatory drug, was concomitantly administered, both the diarrhea and the reduced expression of AQP3 induced by CPT-11 were suppressed. The inflammation in the rat colon during diarrhea was caused via activated macrophage by CPT-11. These results showed that when CPT-11 is administered, the expression level of AQP3 in the colon is reduced, resulting in delayed diarrhea by preventing water transport from the intestinal tract. It was also suggested that the reduced expression of AQP3 might be due to the inflammation that occurs following the loss of colonic crypt cells and to the damage caused by the direct activation of macrophages by CPT-11. Therefore, it was considered that anti-inflammatory drugs that suppress the reduction of AQP3 expression could prevent CPT-11-induced delayed diarrhea.

Abstract 641: Interleukin-15 Plays a Role in Atherosclerosis Through Effects on Multiple Inflammatory Cells in ApoE-/- Mice

Objective: Interleukin (IL)-15 is a pro-inflammatory cytokine which is essential for the survival and maturation of natural killer (NK) and CD8+ T cells and has recently been reported to directly activate macrophages. In the present study, we used IL-15 overexpressing and IL-15 knockout mice to determine its effect on atherosclerosis in apolipoprotein E knockout (apoE-/-) mice. Approach and Results: Deficiency of IL-15 reduced atherosclerosis while overexpression of IL-15 increased atherosclerosis despite opposite effects on plasma lipoprotein cholesterol levels. Genetic ablation of IL-15 in apoE-/- mice resulted in a lack of NK cells and reduced populations of monocytes and CD8+ T cells, whereas overexpression of IL-15 had the opposite effects. CD8+ T cells were detected in atherosclerotic plaques from apoE-/- mice, were increased in plaques from il-15TgapoE-/- mice and were virtually absent from plaques from il15-/- apoE-/- mice. To test the role of NK, NKT and activated CD8+ T cells in atherosclerosis, NK1.1+ cells were immuno-depleted from apoE-/- mice. Atherosclerosis was reduced but to a lesser extent than in age matched il15-/- apoE-/- mice. Plaques from these mice, however, were almost devoid of CD8+ T cells. ApoE-/- mice that were heterozygous for IL-15 (il15+/-apoE-/-) exhibited normal levels of NK, NKT, CD8T cells and monocytes however atherosclerosis was reduced by 40-50 % compared to control apoE-/- mice. This suggested that IL-15 also affects atherosclerosis through NK/NKT and CD8+ T cell independent pathways. Treatment of macrophages with recombinant IL-15 in vitro induced inflammatory cytokines and foam cell formation. Furthermore, reduced levels of MCP-1 and CD11b immune-reactivity were detected in plaques from il15-/- apoE-/- compared to control apoE-/- mice. Conclusions: IL-15 plays a significant role in promoting atherosclerosis through pathways affecting multiple inflammatory cells including the survival/recruitment of NK, NKT and CD8+ T cells, the modulation of monocyte levels, and direct activation of macrophages.

Abstract 80: Interleukin 15 Plays an Important Role in the Development of Atherosclerosis in Apolipoprotein E Knockout Mice

IL-15 is an essential cytokine for the development of NK and CD8+T cells and also involved in activation of inflammation in macrophages. Therefore, we hypothesized that IL-15 may affect the development of atherosclerosis via regulating immune/inflammatory cells. To test this we generated apoE KO mice that either overexpressed (il-15TgapoE-/-) or lacked either one (il-15+/-apoE-/-) or both copies (il-15-/-apoE-/-) of the IL-15 gene. Deficiency of IL-15 substantially reduced atherosclerosis in spite of increased plasma lipoprotein cholesterol levels. Interestingly, il-15+/-apoE -/- mice exhibited intermediate levels of atherosclerosis. Conversely, overexpression of IL-15 in il-15tg apoE -/- mice resulted in substantially increased atherosclerosis compared to apoE -/- littermates. Deficiency of IL-15 resulted in a lack of NK cells and reduced populations of monocytes and CD8+ T cells. Correspondingly, reduced levels of CD11b+ and CD8a+ cells were detected in atherosclerotic lesions from il-15-/-apoE -/- mice compared to those from apoE -/- mice. To test the role of NK cells in atherosclerosis, NK1.1+ cells were immune-depleted in apoE -/- mice and lesions size was analyzed. Atherosclerosis was reduced but to a lesser extent than in age matched il-15-/-apoE -/- mice. Plaques from these mice were almost devoid of CD8+T cells. Deletion of a single copy of the il-15 gene (il-15+/-) in NK/NKT cell immunodepleted apoE -/- mice resulted in a further reduction in atherosclerosis, suggesting the involvement NK/NKT and CD8-T cell independent pathways participate in IL-15’s proatherogenic effects. Treatment of macrophages with recombinant IL-15 in vitro induced expression/activity of a variety of pro-inflammatory targets. Furthermore, il-15-/-apoE -/- mice exhibited reduced levels of MCP-1 in plaques and reduced circulating levels of SAA and IL-6, whereas circulating levels of SAA and IL-6 were greatly increased in il-15tg apoE -/- mice compared to apoE -/- littermates. In conclusion IL-15 plays a significant role in promoting atherosclerosis through multiple pathways including promotion of the survival/recruitment of NK/CD8+T cells, as well as the modulation of monocyte levels, and/or direct activation of macrophages.

Does orchiectomy enhance the immune-stimulatory effects of melatonin during experimental Chagas’ disease?

Melatonin has been reported to play a fundamental role in T-cell immunoregulation. Control of Trypanosoma cruzi parasitism during the acute phase of infection is considered to be critically dependent on direct macrophage activation by cytokines. The aim of this work was to evaluate the influence of exogenous melatonin treatment and the influences exerted by sexual hormones during the acute phase of the experimental Chagas’ disease in rats. With melatonin treatment, orchiectomized animals (CMOR and IMOR) displayed the highest concentrations of IFN-γ and TNF-α. On the 7th day post-infection, untreated and treated orchiectomized animals (IOR and IMOR) showed an enhanced number of peritoneal macrophages. Nitric oxide levels were also increased in untreated and treated orchiectomized (IOR and IMOR) when compared to the other groups, with or without LPS. Our data suggest that melatonin therapy associated with orchiectomy induced a stimulating effect on the immune response to the parasite.

What Fans the Fire

It is now widely accepted that inflammation and immunity play important roles in the pathogenesis of atherosclerosis and diabetes mellitus. Over the past 3 decades, it has been demonstrated that cells of both the innate and adaptive immune systems contribute to the progression of atherosclerosis and modulation of plaque stability.1 Similarly, recent studies in experimental models of obesity and type II diabetes mellitus have shown that not only macrophages, but also T cells and B cells are involved in the modulation of adipose depots and the development of insulin resistance.2–5 However, even with all that is known about the important cellular players in atherogenesis, a clear understanding of the mechanisms by which immune cells influence arterial plaque formation is lacking. Article see p 2933 Macrophages are considered the predominant cell involved in foam cell formation and atherosclerotic plaque development. Numerous studies have linked macrophage activation in atherosclerosis to Toll-like receptors (TLRs) and have demonstrated that “danger signals” such as oxidized low-density lipoprotein (LDL), heat shock proteins, and other products of oxidative stress induce adhesion molecule and chemokine production by cells of the vessel wall and by direct activation of macrophages in the lesion. For example, TLRs such as TLR-4, TLR-2, and TLR-6 (in concert with the scavenger receptor CD36 and TLR-4),6–10 are activated by oxidized LDL and may exacerbate the inflammatory nature of the atherosclerotic plaque. In addition to macrophages, other immune cells, including natural killer (NK) cells, T cells, and a unique group of cells, NK T (NKT) cells, have been identified in atherosclerotic plaques of both humans and mice, and depletion of these cells by either antibodies or genetic approaches has shown that these lymphocytes play important roles in lesion formation.1 Specifically, NK and CD4+ T helper 1 (Th1) …

Immunopharmacological activity of the purified insoluble glucan, zymocel, in mice.

Although it has been established that soluble glucan in fungi is important to host defence against infection, the importance of insoluble glucans is not clear. We have examined the in-vivo immunopharmacological activity of the insoluble glucan, zymocel. Administration of zymocel increased peritoneal exudate cell number and spleen weight, and enhanced: phagocytic activity, hydrogen peroxide production, and nitric oxide production of peritoneal exudate cells; the extravascular release of Evans blue (which might reflect vascular permeability); lipopolysaccharide-triggered synthesis of tumour necrosis factor (TNF); and recovery of white blood cell number in cyclophosphamide-induced leukopenia. Zymocel also showed anti-tumour activity against sarcoma 180 in mice and also enhanced TNF synthesis and hydrogen peroxide production by macrophage-like cell line in-vitro, i.e. resulted in direct macrophage activation. These results show that zymocel shows varied immunopharmacological activity; it is suggested that the administration of insoluble glucan induces the inflammatory response, the subsequent activation of the immune systems via the cytokine network, and direct macrophage activation.

Oral cancer treatment and immune targets – A role for dendritic cells?

Treating a patient suffering from an advanced oral cavity carcinoma by peritumoural injections of mistletoe preparation resulted in a surprising partial response. At the same time an early metastasis, located at the kidney, however remained unaffected. The main difference in treatment being peritumoural versus systematic application supports the hypothesis of immune surveillance. The impact of mistletoe extract in direct contact with the tumour tissue might be explained as activation of macrophage polarization followed by induced cytotoxicity. No direct contact is resulting in no direct macrophage activation. At present there is no clinical trial outlined to test this hypothesis, but as a beginning we would like to encourage submission of case reports with similar clinical experience.

Growth hormones therapy in immune response against Trypanosoma cruzi

Growth hormone (GH) is an important hypophyseal hormone that is primarily involved in body growth and metabolism. In mammals, control of Trypanosoma cruzi parasitism during the acute phase of infection is considered to be critically dependent on direct macrophage activation by cytokines. To explore the possibility that GH might be effective in the treatment of Chagas’ disease, we investigated its effects on the course of T. cruzi infection in rats, focusing our analyses on its influences on parasitemia, NO, TNF-α and IFN-γ concentration and on histopathological alterations and parasite burden in heart tissue. T. cruzi-infected male Wistar rats were intraperitoneally treated with 5 ng/10 g body weight/day of GH. Animals treated with GH showed a significant reduction in the number of blood trypomastigotes during the acute phase of infection compared with untreated animals ( P < 0.05). For all experimental days (7, 14 and 21 post infection) of the acute phase, infected and GH treated animals reached higher concentrations of TNF-α, IFN-γ and nitric oxide as compared to untreated and infected counterparts ( P < 0.05) Histopathological observations of heart tissue revealed that GH administration also resulted in fewer and smaller amastigote burdens, and less inflammatory infiltrate and tissue disorganization, indicating a reduced parasitism of this tissue. These results show that GH can be considered as an immunomodulator substance for controlling parasite replication and combined with the current drug used may represent in the future a new therapeutic tool to reduce the harmful effects of Chagas’ disease.

Melatonin enhances pro‐inflammatory cytokine levels and protects against Chagas disease

Pro-inflammatory and modulatory cytokines have an essential role in host defense against human and murine Trypanosoma cruzi infection. Control of T. cruzi parasitism during the acute phase of infection is considered to be critically dependent on direct macrophage activation by cytokines. Melatonin has been proposed to regulate the immune system by affecting cytokine production in immunocompetent cells, enhancing the production of several T helper (Th)1 cytokines. The aims of this work were to evaluate in rats, the influences of exogenous melatonin treatment on T. cruzi-infected host's immune responses. With this in mind, several immunological parameters were analyzed, including tumor necrosis factor-alpha, gamma-interferon, interleukin-12, nitric oxide (NO) and macrophage count. The melatonin therapy was provided in one of two different treatment regimens, that is, either beginning 7 days prior to infection or concomitant with the infection. Both treatments triggered an up-regulation of the immune response, with the concomitant treatment being more effective; in this case all cytokines studied, with exception of NO, displayed enhanced concentrations and there was a higher number of peritoneal macrophages, which displayed reduced concentrations under melatonin therapy. We conclude that melatonin plays a pivotal role in up-regulating the Th1 immune response thus controlling parasite replication.

Newcastle disease virus activates macrophages for anti-tumor activity.

Newcastle Disease Virus (NDV), an agent with interesting immune stimulatory and anti-tumor activity, was investigated for its capacity to activate anti-tumor activity in murine macrophages in vitro and in vivo. Direct macrophage activation was seen under a variety of experimental conditions using two different strains of NDV, different sources of macrophages (spleen and peritoneum) and different strains of mice (DBA/2, C57BL/6, 615). Various macrophage enzymes (ADA, iNOS, lysozyme, acid phosphatase) became upregulated and anti-tumor effector molecules such as nitric oxide (NO) and TNF-alpha were found in the supernatant. NDV activated macrophages performed anti-tumor activity in vitro such as anti-tumor cytostasis and anti-tumor cytotoxicity. The cytotoxic anti-tumor activity was broad and active against all tumor lines tested including mammary carcinoma, lung carcinoma, mastocytoma and immune escape variants (lymphoma). Macrophage activation via BCG/LPS also caused a broad range anti-tumor cytotoxic activity while activation via mixed lymphocyte culture conditioned medium had restricted anti-tumor activity. Anti-tumor activity of NDV activated macrophages could be transfered in vivo. Transfer of macrophages which had not been appropriately activated exerted either no effect or a tumor growth augmenting effect. Repeated intravenous transfer of NDV activated macrophages exerted a significant suppressive effect on pulmonary metastases in a mammary carcinoma tumor model as well as in a lung carcinoma model. Taken together these results demonstrate that NDV can strongly activate macrophages to perform anti-tumor activities in vitro and in vivo.

Virulizin® - a review of its antineoplastic activity

Virulizin® (registered trademark of Lorus Therapeutics, Inc.) is a novel biological response modifier (BRM) that enhances cell-mediated immune response to tumour cells by direct macrophage activation. The agent, an aqueous solution containing a 5% (w/v) solid material mixture comprised of inorganic salts (95 - 99% of the dry weight) and organic compounds of molecular weight << 3000 Daltons (1 - 5% of the dry weight), is provided as a sterile, injectable formulation. Virtually all the organic compounds and ionic components in the mixture have been identified by chemical and spectroscopic methods. Virulizin® is obtained from bovine bile by a standardised process involving solvent extraction and heat hydrolysis. In vitro studies have shown that Virulizin® can stimulate blood monocytes, peritoneal macrophages and alveolar macrophages from patients with malignant and non-malignant diseases to mediate a level of tumour cell cytolysis that is equal to, or greater than, that elicited in the same cells by other, more conventional biological activators. Other studies have shown that the stimulation of cytotoxic function by Virulizin® is insensitive to the inhibitory effects of prostaglandins and, hence, is distinct from many other activators. Furthermore, the blood monocytes of patients currently receiving cytotoxic chemotherapy showed activation with Virulizin®. In preclinical studies, Virulizin® showed modest inhibition of tumour growth in a human pancreatic cancer xenograft model (T/C, 0.60; p = 0.012) and increased survival time by 8% (p = 0.046) in a murine melanoma model. Toxicity studies in rats indicated that Virulizin® injected intramuscularly was well-tolerated by the animals. Except for a lower rate of body weight gain in the high dose group, no toxicological effect related to treatment was observed in a chronic 13-week study (three weekly im. injections of up to 1.1 ml/kg). Mutagenicity studies with Virulizin® were negative. Many Phase I/II and Phase II clinical trials were carried out with Virulizin® to evaluate the safety and efficacy of the agent to treat cancer patients. Three of those studies involved the treatment of patients with pancreatic adenocarcinoma. The majority of patients participating in these studies were those who had failed conventional treatment including surgery or chemotherapy. Disease stabilisation was observed in patients treated with Virulizin®, and, based on a meta-analysis of the data from the three studies, the survival of the patients treated with Virulizin® was better than a similar patient population receiving standard chemotherapeutic agent for that disease indication. However, randomised studies may be required to confirm the effect of Virulizin® on disease stabilisation and survival. In the three studies on patients with malignant melanoma, at least 69% treated with Virulizin® showed no additional deterioration or had improved at four weeks with response being only slightly reduced after eight weeks. In the evaluation of safety, assessed overall from these clinical studies in 200 patients treated with Virulizin®, the number and severity of adverse events were small. In conclusion, Virulizin® is a novel, clinically non-toxic biological response modifier that appears to have a clinical effect on disease stabilisation and survival in patients with pancreatic cancer and malignant melanoma. However, additional randomised studies may be required to confirm those effects. Future studies on Virulizin® will focus on the characterisation of the active components in the agent and the development of a synthetic formulation. Subsequently, the preclinical and clinical pharmacokinetics of Virulizin® will be determined and studies to optimise the human dose of the agent will be carried out.

Chlamydial heat shock proteins and disease pathology: new paradigms for old problems?

The mucosal pathogen Chlamydia trachomatis affects hundreds of millions of people worldwide and is a significant cause of sexually transmitted disease. Although most acute infections can be easily managed, complications often occur that can be especially severe in women. It has been proposed that increased exposure to conserved chlamydial antigens, such as through reinfection or persistent infection, results in chronic inflammation and tissue scarring and contributes to the pathogenesis of endometrial and fallopian tube damage. This immunopathologic damage is believed to be a principal cause of ectopic pregnancy and tubal factor infertility. The chlamydial heat shock protein Hsp60, a homolog of Escherichia coli GroEL, has been identified as one protein capable of eliciting intense mononuclear inflammation. Furthermore, several studies have revealed a correlation between Hsp60 responses and the immunopathologic manifestations of human chlamydial disease. The role of additional antigens in the immunopathologic response to chlamydiae is currently undefined. A prime candidate, however, is the chlamydial GroES homolog Hsp10, which is genetically and physiologically linked to Hsp60. Recent studies provide data to suggest that immune reactivity to Hsp10 is significantly associated with tubal infertility in a chlamydiae-exposed population. Chlamydia pneumoniae is a more recently defined chlamydial species that has been implicated in a variety of ways with chronic disease processes, such as adult onset asthma and atherosclerosis. Evidence indicates that Hsp60 is present in human atheroma and may play a role in lesion development by direct activation of macrophages. Hsp60 causes the elaboration of inflammatory cytokines, the induction of metalloproteinase, and the oxidation of low density lipoprotein. Each of these events is directly associated with the progress of atherosclerosis. Thus, chlamydial heat shock proteins may function in at least two ways to promote chronic disease: first by direct antigenic stimulation and second as signal transducers that result in macrophage activation. These concepts in disease pathology are discussed in the context of chlamydial infections.

Activation of macrophage tumoricidal activity by photodynamic treatment in vitro-indirect activation of macrophages by photodynamically killed tumor cells

Macrophages constitute a major part of natural tumor defense by their capacity to destroy selectively a broad range of tumor types upon specific activation. In the last couple of years, these cells have also been implicated as effector cells in the destruction of tumors by photodynamic therapy. In the present work, the potential role of macrophage-mediated tumor cytotoxicity after photodynamic treatment in vitro has been investigated with respect to photodynamic activation of macrophages for tumoricidal effector functions. Our data show that photodynamic treatment of highly pure murine bone-marrow-derived macrophages with the hematoporphyrin derivative Photosan-3 does not result in activation of these cells for cytotoxicity against YAC-1 tumor cells or secretion of tumor necrosis factor and nitric oxide, irrespective of co-stimulation with interferon-γ, a potent priming agent for macrophage antitumoral activity. On the contrary, treatment with higher photosensitizer doses is found to reduce markedly the viability of the macrophage effector cells. Thus, these results do not lend any support to the hypothesis of direct macrophage activation by photodynamic treatment. However, macrophages are found to be activated for tumoricidal effector functions indirectly by photodynamically killed tumor cells, in a way reminiscent of phagocytosis-inducing stimuli. It is thus suggested that recognition and phagocytosis of photodynamically destroyed tumor cells constitutes the major signal for local activation of macrophages in photodynamically treated tumor tissues, which may be crucial for final, specific eradication by the immune system of tumor cells surviving photodynamic treatment.

Reconstitution of Leishmania immunity in severe combined immunodeficient mice using Th1- and Th2-like cell lines.

Leishmania major disseminates in genetically susceptible BALB/c mice to cause fatal disease. Progressive infection has been linked to the failure of parasite-specific Th1, IFN-gamma-producing, CD4+ T lymphocytes to expand and direct macrophage activation and control of intracellular parasitism. In contrast, Th2 CD4+ cell expansion accompanies disease progression. Immunomodulation using CD4 cell depletion at the time of infection results in control of infection and Th1 CD4+ cell expansion. A Th1-like cell line, H1A, was established from the draining lymph nodes of an anti-CD4-pretreated BALB/c mouse infected with L. major, H1A was CD4, TCR(+)-alpha/beta, and released IL-2 and IFN-gamma in response to parasite Ag. A Th2-like cell line, U1A, was established from the lymph node cells of an infected BALB/c mouse that was also CD4, TCR(+)-alpha/beta but released IL-4 and IL-5 after stimulation. Mice with severe combined immunodeficiency were reconstituted with H1A and U1A before infection with L. major. Non-reconstituted mice were unable to restrict parasite growth. Mice reconstituted with H1A healed infection, whereas mice reconstituted with U1A suffered exacerbation of disease. Analysis of spleen cells by flow cytometry confirmed the reconstitution of CD4+ cells in both instances, and stimulation with mitogen established that the lymphokine profile of the donor cells had been maintained during 6 to 8 wk of infection. Histologic analysis of the lesions confirmed migration of donated cells to sites of infection. Neutralization of IFN-gamma in H1A-reconstituted mice and IL-4 in U1A-reconstituted mice reversed the disease phenotype mediated by the two cell lines. These data demonstrate the capacity of CD4+ T cells alone to modulate both positively and negatively the course of leishmaniasis in a lymphokine-dependent manner.

Correlation between the presence of intracellular OK-432 and antitumor activity of peritoneal macrophages

The mechanism of the generation of tumoricidal macrophages by intraperitoneal administration of OK-432 was investigated. An association between the presence of intracellular FITC-conjugated OK-432 and the increase of macrophage cytostatic activity was observed after i.p. administration of OK-432. When peritoneal exudate cells were fractionated on the basis of cell size by velocity sedimentation, a higher proportion of macrophages and a strong cytostatic activity were shown in the large cell fractions. Furthermore, in these fractions, increased numbers of macrophages with intracellular FITC conjugated and 3H-acetate labelled OK-432 were observed. Thus, the more cytocidal macrophages can be identified by the presence of intracellular microorganisms. A role for OK-432 in the direct activation of macrophages is suggested.

Evidence for granulocyte-mediated macrophage activation after C. parvum immunization

It has been previously demonstrated that at the peak of the peritoneal response to Corynebacterium parvum (Day 4), cytolytic macrophages can be characterized by the presence of intracellular bacteria. In the present study, the role of neutrophils in the activation of peritoneal macrophages by C. parvum was investigated. Inflammatory neutrophils isolated 5 hr after ip administration of C. parvum were transferred to normal, syngeneic mice and the peritoneal macrophages of recipients harvested 4 days later were tested for cytoxicity against HeLa cells. Neutrophils isolated from mice 5 hr after C. parvum immunization were effective in inducing cytolytic macrophages. Less than 100-fold as much bacteria was needed to induce comparable levels of cytotoxic activity when introduced inside granulocytes. Neutrophils obtained from mice 48 hr after C. parvum injection or mononuclear cells were not good macrophage activators. Viable neutrophils were not required as freeze-thawed cells were able to activate macrophages in recipient mice. The intracellular distribution of C. parvum changed dramatically with time. Intitially almost all bacteria were found within neutrophils. By 24 hr, many macrophages contained either bacteria or granulocytes which had ingested C. parvum. Pyridine extracts of C. parvum, which do not activate peritoneal macrophages when injected directly into mice, did not induce neutrophils capable of activating macrophages. The residue of pyridine-extracted C. parvum did induce neutrophils that could activate macrophages when transferred. The results suggest that processing of the bacteria by inflammatory granulocytes may be an obligatory step in macrophage activation by this agent. The peak response occurred earlier than T-cell immunity is usually observed and it is suggested that direct activation of macrophages via ingestion of neutrophils may represent the earliest stage of macrophage activation by C. parvum.

Macrophage Activation by Mycobacterial Water Soluble Compounds and Synthetic Muramyl Dipeptide

The adjuvant effects of mycobacteria can be replaced by more chemically defined isolates of the cell walls including a water soluble fraction (WSA) and by the synthetic analog N-acetyl-muramyl-L-alanyl-D-isoglutamine (MDP), which is the minimal structure required for adjuvanticity. These compounds can directly activate macrophages as determined by an increase in spreading and adherence and by an elevated synthesis of the enzyme collagenase. Moreover, this increase in collagenase production is modulated by enhanced production of prostaglandins that influences intracellular levels of cyclic AMP. In addition, both MDP and WSA induced macrophages to produce a biologically active mediator that triggers quiescent fibroblasts into active proliferation. It thus appears that a mechanism for mycobacterial adjuvant action as determined with MDP and WSA is via activation of macrophages, which may then precipitate a multiplicity of other reactions resulting in enhanced immune phenomena. Furthermore, the granulomatous and fibrotic reactions associated with mycobacterial infection may be a consequence of this direct activation of macrophages.