Serum-mediated Immune Cellular Responses to Brucella melitensis IV. Infection of Macrophages Under Anaerobic Conditions

Immune mechanisms active against Brucella were studied under conditions of oxygen deficiency. B. melitensis grew in rabbit serum-Tyrode medium flooded with N₂ and CO₂ gas mixtures. Immune sera from rabbits injected with B. melitensis strain Rev I possessed growth-inhibitory activity that operated in anaerobic environments against Rev I and virulent strain 6015. When mixed with macrophages, immune sera mediated even greater inhibition of bacterial growth and slowed the spread of infection throughout the tissue culture. Although under anaerobic conditions the rate of phagocytosis was reduced, the macrophages in immune serum killed significant percentages of Brucella, suggesting that an antibacterial mechanism had been activated. Sonic extracts of macrophages prepared and tested under anaerobic conditions depressed the growth rate of strain Rev I. The extracts, however, exhibited no immediate killing capacity when tested in Tyrode solution. A factor from serum was required for depression of the growth rate.     

N-acetylcysteine improves in vitro the function of macrophages from mice with endotoxin-induced oxidative stress

Reactive oxygen species (ROS) and proinflammatory cytokines produced by immune cells cause the oxidative stress involved in septic shock induced by endotoxin. This oxidative stress can be controlled to a certain degree by antioxidants, which is specially important for a type of immune cell, i.e. the phagocyte, that uses ROS to kill microorganisms and needs antioxidants in order to support its functions. In a previous study we have observed changes in several functions of peritoneal macrophages from BALB/c mice with lethal endotoxic shock caused by intraperitoneal injection of Escherichia coli lipopolysaccharide (LPS) (100 mg/kg), which were associated with a high production of superoxide anion. N-acetylcysteine (NAC) is a thiolic antioxidant that improves the immune response, and we have observed that when administered intraperitoneally (150 mg/kg) at 30 min after LPS injection it counteracts the effects of LPS on macrophages and lymphocytes. In the present work, we have studied the in vitro effect of several concentrations of NAC (0.001, 0.01, 0.1, 1 and 2.5 mM) on the following functions: adherence to substrate, chemotaxis, ingestion of particles, ROS production and the release of tumor necrosis factor (TNFalpha) of peritoneal macrophages from BALB/c mice at 2, 4,12 and 24 h after LPS injection. The results show that the administration of NAC (especially at 0.1 mM) decreases raised adherence, ingestion, ROS production and TNFalpha levels in macrophages from animals injected with LPS, bringing these functions to values near those of control animals. These effects which seem to be linked to a modulation of NF-kappaB, suggest that the improvement of immune functions observed in previous work after injection of NAC to animals with endotoxic shock could be due to a direct action of this thiol antioxidant on immune cells.     

Effect of orally administered l-tryptophan on serotonin,melatonin, and the innate immune response in the rat

To assess the effects of external administration of L-tryptophan on the synthesis of serotonin and melatonin as well as on the immune function of Wistar rats, 300 mg of the amino acid were administered through an oral cannula either during daylight (08:00) or at night (20:00) for 5 days. Brain, plasma, and peritoneal macrophage samples were collected 4 h after the administration. The accumulation of 5-hydroxytryptophan (5-HTP) after decarboxylase inhibition was used to measure the rate of tryptophan hydroxylation in vivo. Circulating melatonin levels were determined by radioimmunoassay, and the phagocytic activity of macrophages was measured by counting, under oil-immersion phase-contrast microscopy, the number of particles ingested. The results showed a diurnal increase (p < 0.05) in the brain 5-HTP, serotonin (5-hydroxytryptamine, 5-HT), and 5-hydroxyindolacetic acid (5-HIAA) of the animals which had received tryptophan at 08:00 and were killed 4 h later. In the animals which received tryptophan during the dark period, the 5-HT declined but the 5-HT/5-HIAA ratio remained unchanged. There was also a significant increase (p < 0.05) in nocturnal circulating melatonin levels and in the innate immune response of the peritoneal macrophages in the animals which had received tryptophan at 20:00. The results indicated that the synthesis of serotonin and melatonin, as well as the innate immune response, can be modulated by oral ingestion of tryptophan.     

Modulation of murine macrophage function by N-acetylcysteine in a model of endotoxic shock

In previous studies we have observed changes in several functions of peritoneal macrophages from BALB/c mice with irreversible endotoxic shock caused by intraperitoneal injection of E. coli lipopolysaccharide (LPS) (100 mg/kg), which were associated with a high production of superoxide anion. Since antioxidants, such as N-acetylcysteine (NAC), are free radical scavengers that improve the immune response, in the present work we have studied different functions of peritoneal macrophages from BALB/c mice suffering the endotoxic shock above indicated and administered N-acetylcysteine (150 mg/kg i.p.) at 30 minutes after LPS injection. In the peritoneal macrophages obtained at 2, 4, 12 and 24 h after LPS injection, the following functions were studied: adherence to substrate, mobility, ingestion of particles, and production of superoxide anion and tumour necrosis factor (TNF alpha). The increase in adherence, ingestion and superoxide anion and TNF alpha production shown by macrophages from animals with endotoxic shock was counteracted by NAC injection. Moreover, the survival time of mice with endotoxic shock was increased in the presence of NAC. These data suggest that NAC, administered intraperitoneally, may be useful for the treatment of irreversible endotoxic shock by modulation of the function of macrophages with decreased superoxide anion and TNF alpha production and concomitant increase of survival time.     

In vitro markers and biological activity in mice of seed lot strains and commercial Brucella melitensis Rev 1 and Brucella abortus B19 vaccines.

Four seed lots and fourteen batches of Brucella melitensis Rev 1 and B. abortus B19 living anti-Brucella commercial vaccines obtained from six Spanish laboratories were tested in vitro and in vivo in the reference mouse model for quality control. All the strains tested showed the characteristic morphology of their respective Rev 1 or B19 reference strains with the exception of three Rev 1 strains: seed lot SL2 and commercial vaccine R3, in which giant colonies were predominant, and commercial vaccine R5, in which 5% rough colonies were detected. Strains SL2 and R5 (but not the R3) had a deficient activity when tested in the mouse model. All strains but two (Rev 1 strain R1 and B19 strain B2) had standard resistance/ sensitivity patterns to streptomycin and penicillin G. Strains R1 and B2 had an increased resistance to penicillin when incubated in a 10% CO2 atmosphere and both strains showed an increased residual virulence in mice. As residual virulence and immunogenicity in mice were not always correlated one another nor with the in vitro tests, all tests should be performed to control properly the anti-Brucella live vaccines. A computerized statistical procedure to calculate the residual virulence of vaccines is proposed as an alternative to that used in the current method.     

CD8+ CTL from lungs of Mycobacterium tuberculosis-infected mice express perforin in vivo and lyse infected macrophages

CD8+ T lymphocytes have been implicated in the protective immune response against human and murine tuberculosis. However, the functional role that this cell subset plays during the resolution of infection remains controversial. In this study, we demonstrate the presence of Mycobacterium tuberculosis-specific CD8+ CTL in the lungs and lung-draining lymph nodes of mice infected with M. tuberculosis via the aerosol or i.v. route. These cells expressed perforin in vivo and specifically recognized and lysed M. tuberculosis-infected macrophages in a perforin-dependent manner after a short period of in vitro restimulation. The efficiency of lysis of infected macrophages was dependent upon the time allowed for interaction between macrophage and M. tuberculosis bacilli. Recognition of infected targets by CD8+ CTL was beta 2-microglobulin and MHC class I dependent and was not CD1d restricted. The presented data indicate that CD8+ T cells contribute to the protective immune response during M. tuberculosis infection by exerting cytotoxic function and lysing infected macrophages.     

Toxicity studies of intravenously administered BCG in baboons

Summary The toxicity of fresh preparations of BCG administered intravenously as single or weekly repeated doses of 1 g, 100 mg, 10 mg, and 1 mg, was studied in 9 adult baboons. Clinical, biochemical, histologic, and bacteriologic examinations were performed. Tissues for histopathologic and bacteriologic examinations were obtained by laparotomy and biopsy, or by autopsy. Four and 5 months after the first injection, the surviving animals were killed purposely for examination. With high doses of BCG (2 g, 1 g, 500 mg) the severe toxicity with death of 2 animals was demonstrated. Diffuse cellular infiltrations of macrophages and lymphocytes in the liver, spleen, lungs, and lymph nodes, with formation of early, typical granulomas and great numbers of cultivable units of BCG were found in these animals. With small doses of BCG, general granulomatous reactions occurred in all organs sampled. Four months after BCG injection, granulomas had regressed, but viable organisms persisted in the lymph nodes. Future investigations may prove feasible the possibility of immunotherapy with small doses of BCG administered intravenously, since live bacilli persist for months after injections, presumably creating the chronic BCGitis necessary for effective immunotherapy. Reprint requests should be addressed to: Institut de Cancérologie et d'Immunogénétique (INSERM)     

Antitumor activity of living or killed Brucella: Modification of the non-specific cytotoxic effector cells

At various times after injection of living or killed smooth (S) or rough (R) Brucella abortus mice received a graft of the semi-allogenic EL4 lymphoma and their survival was studied. In parallel, the NK activity of spleen and peritoneal cells, the level of serum interferon (IFN), and the cytotoxic activity of peritoneal macrophages were investigated. Protection against the lymphoma lasted longer after injection of R organisms than after S. The parallelism between the in vivo resistance to El4 lymphoma and the augmentation of NK and macrophage activity was satisfactory with R but not with S. IFN production did not seem to be correlated with R antitumor activity. The antitumor effect of Brucella cannot therefore be simply explained on the basis of modification of the non-specific cytotoxic effector mechanisms.     

In Vitro Studies on the Mechanism of Acquired Resistance to Tuberculous Infection

The relationship between lymphocytes and macrophages in cellular immunity against tuberculous infection was studied by means of an in vitro cell culture system without addition of streptomycin. The peritoneal macrophages were obtained from normal mice or mice immunized with heat-killed tubercle bacilli in paraffin oil, boosted with live BCG and infected with H37Rv cells in vitro. The infected monolayers of macrophages were cultivated for 48 hr with immune lymphoid cells obtained from immunized mice. The intracellular growth of H37Rv cells 3,5 and 7 days after infection was examined by counting tubercle bacilli within infected macrophages under a microscope. 1) The increase of bacilli within macrophages derived from immunized mice was slightly smaller than that in normal macrophages. 2) The addition of immune lymph node cells to the macrophage monolayers resulted in a marked decrease in the number of bacilli within both normal and “immune” macrophages. Conversely, normal lymph node cells exhibited an enhancing effect on the intracellular bacillary growth. 3) Immune lymph node cells showed a higher capacity to cause macrophages to suppress intracellular growth of bacilli than that of splenic lymphoid cells or thymocytes after addition to macrophage monolayers. 4) The treatment of lymphoid cells with inhibitors of protein synthesis, cycloheximide or streptovitacin A, resulted in a remarkable reduction of the ability of sensitized lymphocytes to cause macrophages to suppress multiplication of intracellular bacilli.     

Selective delivery of rifampicin incorporated into poly(DL-lactic-co-glycolic) acid microspheres after phagocytotic uptake by alveolar macrophages, and the killing effect against intracellular Mycobacterium bovis Calmette-Guérin

Macrophages and their phagocytotic abilities play a dominant role for defense against infected organisms. However, Mycobacterium tuberculosis can survive in the phagosomes of macrophages. In this study, the effective delivery of a drug and the killing effect of tubercle bacilli within macrophages were investigated utilizing the phagocytotic uptake of rifampicin (RFP) that had been incorporated into poly(DL-lactic-co-glycolic) acid (PLGA) microspheres. The microspheres were composed of PLGA that had a monomer ratio (lactic acid/glycolic acid) of either 50/50 or 75/25. They had molecular weights from 5000 to 20,000, and diameters of 1.5, 3.5, 6.2 and 8.9 microm. The most significant factor for phagocytotic activity of macrophages was the diameter of the microspheres. By contrast, molecular weight and monomer ratio of PLGA did not influence phagocytosis. The amount of RFP delivered into cells was also investigated. RFP-PLGA microspheres composed of PLGA with a molecular weight of 20,000 and monomer ratio of 75/25 showed the highest amount of delivery (4 microg/1 x 10(6) cells). Fourteen days after infection, the survival rate of treated intracellular bacilli was 1% when compared with untreated cells. There was almost no killing effect of free RFP (4 or 15 microg/ml) on intracellular bacilli. In vivo efficacy of RFP-PLGA was also examined in rats infected with M. tuberculosis Kurono. Intratracheal administration of RFP-PLGA microspheres was shown to be superior to free RFP for killing of intracellular bacilli and preventing granuloma formation in some lobes. These results suggest that phagocytotic activity could be part of a new drug delivery system that selectively targeted macrophages.     

Brucella melitensis 16MΔhfq attenuation confers protection against wild‐type challenge in BALB/c mice

Brucellosis is a globally distributed zoonotic disease that causes animal and human diseases. Although effective, the current Brucella vaccines (Rev.1 and M5‐90) have several drawbacks. The first involves residual virulence for animals and humans and the second is the inability to differentiate natural infection from that caused by vaccination. Therefore, Brucella melitensis 16M hfq mutant (16MΔhfq) was constructed to overcome these drawbacks. Similarly to Rev.1 and M5‐90, 16MΔhfq reduces survival in macrophages and mice and induces strong protective immunity in BALB/c mice. Moreover, these vaccines elicit anti‐Brucella‐specific IgG1 and IgG2a subtype responses and induce secretion of gamma interferon and interleukin‐4. The Hfq antigen also allows serological differentiation between infected and vaccinated animals. These results show that 16MΔhfq is an ideal live attenuated vaccine candidate against virulent Brucella melitensis 16M infection. It will be further evaluated in sheep.     

The amount of thiolic antioxidant ingestion needed to improve several immune functions is higher in aged than in adult mice

With aging there is an increase of oxidative stress due to an imbalance between the oxidant production and the antioxidant levels in favor of the former. Since immune cell functions are specially linked to reactive oxygen species (ROS) generation, the oxidant/antioxidant balance is essential for these cells. Although low levels of antioxidants cause a decrease in immune function, very high levels of antioxidant compounds could show prooxidant effects. In the present work, we have studied the effect of diet supplementation, for 4 weeks, with two different doses of two thiolic antioxidants, namely thioproline (TP) and N -acetylcysteine (NAC), at 0.1% (w/w) and 0.3% (w/w, of each antioxidant) on the main immune system cells, i.e.: macrophages, lymphocytes and natural killer (NK) cells of adult (33 λ±λ1 week old) and aged (75 λ±λ1 week old) female Swiss mice. Two groups of animals, adult and aged mice, fed standard diet were used as controls. The results show that the ingestion of 0.1% doses of thiols improves, in the adult mice, several immune functions such as the chemotaxis capacity of both macrophages and lymphocytes, the phagocytosis of macrophages, the lymphoproliferative response to the mitogen Con A and the NK activity. Moreover, no change was observed in adherence capacity of immune cells, and superoxide production was decreased. By contrast, in aged mice the ingestion of these amounts of antioxidants did not change the immune functions studied with the exception of NK activity, which was stimulated. The ingestion of 0.3% of antioxidants by adult mice only increased some immune functions such as adherence and superoxide production, which are markers of oxidative stress. Other functions such as chemotaxis or lymphoproliferative response decreased. However, the ingestion of these very high amounts of thiols by aged animals increased the phagocytosis, the NK activity and specially the lymphoproliferative response to the mitogen, a function that is very depressed with aging.     

Role of cytokines and nitric oxide in the induction of tuberculostatic macrophage functions

The aim of this study was to determine phenotypic differences when BCG invades macrophages. Bacilli prepared from the same BCG primary seed, but produced in different culture media, were analysed with respect to the ability to stimulate macrophages and the susceptibility to treatment with cytokines and nitric oxide (NO). Tumour necrosis factor (TNF) activity was assayed by measuring its cytotoxic activity on L-929 cells, interleukin-6 (IL-6) and interferon-gamma (IFN-gamma) were assayed by enzyme-linked immunosorbent assay (ELISA), whereas NO levels were detected by Griess colorimetric reactions in the culture supernatant of macrophages incubated with IFN-gamma, TNF or NO and subsequently exposed to either BCG-I or BCG-S. We found that BCG-I and BCG-S bacilli showed different ability to simulate peritoneal macrophages. Similar levels of IL-6 were detected in stimulated macrophages with lysate from two BCG samples. The highest levels of TNF and IFN-gamma were observed in macrophages treated with BCG-S and BCG-I, respectively. The highest levels of NO were observed in cultures stimulated for 48 h with BCG-S. We also found a different susceptibility of the bacilli to exogenous treatment with IFN-gamma and TNF which were capable of killing 60 and 70% of both bacilli, whereas NO was capable of killing about 98 and 47% of BCG-I and BCG-S, respectively. The amount of bacilli proportionally decreased with IFN-gamma and TNF, suggesting a cytokine-related cytotoxic effect. Moreover, NO also decreased the viable number of bacilli. Interestingly, NO levels of peritoneal macrophages were significantly increased after cytokine treatment. This indicates that the treatment of macrophages with cytokines markedly reduced bacilli number and presented effects on NO production. The results obtained here emphasize the importance of adequate stimulation for guaranteeing efficient killing of bacilli. In this particular case, the IFN-gamma and TNF were involved in the activation of macrophage bactericidal activity.     

Failure of adrenaline to induce hyperglycaemia after fructose injection in young mice.

In control animals a 2-fold increase in liver phosphorylase activity 10min after adrenaline treatment was associated with a 55% increase in plasma glucose (P less than 0.001); at 20 min plasma glucose was 247% of the control value (P less than 0.001). Liver phosphorylase activity was decreased by 74%, 20 min after fructose injection (P less than 0.001), and, although phosphorylase activity increased 5-fold within 5 min of adrenaline injection, no increases in plasma glucose concentration over that found in fructose-injected animals which did not receive adrenaline occurred at either 5, 10 or 20 min. The data confirm inactivation of liver phosphorylase after fructose injection and suggest inhibition of the adrenaline-activated enzyme by the decrease in Pi and elevation of fructose 1-phosphate concentrations produced by the injection of fructose. These findings may be causally related to the hypoglycaemia and the lack of response to glucagon seen in patients with hereditary fructose intolerance after fructose ingestion.     

Lysosomal enzymes and microbicidal capacity of activated macrophage.

A relation was sought between acid phosphatase contents and the presence of tubercle bacilli inside the peritoneal exudate cells (PEC) of normal guinea pigs and those immunized with BCG. This was done to investigate the role lysosomal enzymes play in the microbicidal capacity of the cell. In both normal and immune animals tubercle bacilli were present only in those PEC that contained acid phosphatase. Cells without acid phosphatase did not contain bacilli. Thus, only activated cells ingested bacilli. Under the conditions of these experiments, macrophage activation, as indicated by the presence of acid phosphatase, was not related to the immune status of the animal. Similarly, stimulation by ingestion of tubercle bacilli was not significant. Also, the number of acid phosphatase grains/cell did not influence the number of bacilli/cell. Thus, the acid phosphatase content of the cell did not correlate with the number of bacilli inside the cell. It was concluded that acid phosphatase may not be one of the factors that contribute to the microbicidal capacity of the cell.     

Inhibition of antigen presentation by Brucella: many more than many ways

Brucella infection activates the immune system and favors the differentiation of CD4⁺ and CD8⁺ T cells. To persist during a long time inside macrophages evading immune surveillance of these T cells the pathogen must exploit different evasion strategies. We review the mechanisms whereby Brucella, through TLR signaling, inhibits MHC class I and II antigen presentation, allowing infected macrophages to become effective niches for Brucella survival.     

Effect of recombinant interferon-gamma on hydrogen peroxide-releasing capacity of monocyte-derived macrophages from patients with lepromatous leprosy

Monocyte-derived macrophages from 14 patients with lepromatous leprosy respond to rIFN-gamma with an enhanced secretion of H2O2 in a fashion similar to that of cells obtained from normal donors. The activation is not dependent on the cutaneous bacterial index, the length of treatment, or the stage and activity of the disease. H2O2 release can be triggered in these cells both by phorbol myristate acetate and by intact irradiated Mycobacterium leprae. Uptake of M. leprae by both normal donors' and patients' macrophages is proportional to the number of bacilli added. Prior ingestion of M. leprae does not interfere with the ability of macrophages to respond to IFN-gamma by the production of oxygen intermediates. We conclude that the immune defect in lepromatous leprosy probably results from a lack of response to M. leprae by the patients' T cells rather than an inability of mononuclear phagocytes to respond to IFN-gamma.     

Destruction of tumor cells by BCG-activated alqolar macrophages.

Alveolar macrophages obtained from Syrian golden hamsters were tested for their ability to destroy tumor cells. Only macrophages obtained from BCG immune animals rechallenged intratracheally with BCG five days before assay exhibited cytotoxic activity. Maximum destruction of tumor cells occurred after 5 days of incubation. Immunologic activation of macrophages was required to attain cytotoxic alveolar macrophages. Induction of inflammatory lung exudates by a variety of nonspecific irritants did not result in tumor cell destruction by macrophages. These observations may prove useful in designing an approach for immunotherapy of lung cancer.     

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. Initially 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.     

Characteristics of the immunocyte cooperation in 2 strains of mice opposite in their sensitivity to dermaphytoses during the immune response to antigens of the causative agent

C57BL/6 and BALB/c mice, opposite in their sensitivity to dermatophytic infections, show similar activity of phagocytes as regards their capacity for the destruction of injected conidia of dermatophytes, but differ in the changes of this activity after immunization with dermatophytic antigenic complexes (DAC). Shortly after the injection of the antigens, the lymphocyte-mediated suppression of the fungicidal activity of macrophages, caused by the interaction of DAC with intact T-lymphocytes, was detected in the animals of both strains. Later in C57BL/6 mice resistant to mycosis the formation of cell-mediated immunity to DAC occurs, with the simultaneous production of factor stimulating the fungicidal activity of macrophages. In BALB/c mice sensitive to mycosis the injection of DAC induces active antibody production, but not the formation of delayed hypersensitivity with the resulting stimulation of the fungicidal activity of phagocytes. The injection of DAC into mice of the above-mentioned strains induces changes, peculiar to each strain, in the mitogen-induced proliferation of spleen cells and in the character of immune response to sheep red blood cells. Differences in the influence of DAC on the induction of immune response in C57BL/6 and BALB/c mice are realized by cells belonging to the population of T-lymphocytes.